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Mutational Analysis of Receptor Interaction and Membrane Fusion Activity of Sindbis

Jolanda M. Smit Cover illustration: Image reconstruction of Sindbis virus (courtesy of Dr. S. Mukhopadhyay, Purdue University).

ISBN: 90-367-1646-2 RIJKSUNIVERSITEIT GRONINGEN

Mutational Analysis of Receptor Interaction and Membrane Fusion Activity of Sindbis Virus

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. D.F.J. Bosscher, in het openbaar te verdedigen op woensdag 19 juni 2002 om 16.00 uur

door

Jolanda Mariske Smit

geboren op 15 april 1975 te Delfzijl Promotor : Prof. dr. J.C. Wilschut Promotiecommissie : Prof. dr. R. Bittman, City University of New York Prof. dr. D. Hoekstra, Rijksuniversiteit Groningen Prof. dr. W.J.M Spaan, Leids Universitair Medisch Centrum

The study described in the thesis was carried out in the Department of Medical Microbiology, Molecular Section, University of Groningen, as part of a collaborative research programme of Queens College of the City University of New York and the University of Groningen. The project was supported by US National Institutes of Health Grant HL-16660 (Principal Investigator: Professor R. Bittman). Part of the work was carried in the Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, with financial support from the Royal Netherlands Academy of Sciences (KNAW). The Ph.D. programme was carried out under the auspices of the Groningen University Institute for Drug Exploration (GUIDE).

The printing of this thesis was supported by Avanti Polar Lipids (Alabaster, AL, USA), Molecular Probes (Leiden, The Netherlands) and by the Groningen University Institute for Drug Exploration (GUIDE). Contents

Chapter 1 1 General introduction

Chapter 2 29 Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol and sphingolipid-containing liposomes Journal of Virology 73:8476-8484

Chapter 3 53 Adaptation of Alphaviruses to heparan sulfate: Interaction of Sindbis and Semliki Forest virus with liposomes containing lipid-conjugated heparin Journal of Virology, provisionally accepted for publication

Chapter 4 75 Fusion of Alphaviruses with liposomes is a non-leaky process FEBS Letters, in press

Chapter 5 87 A single mutation in the E2 glycoprotein important for neurovirulence influences binding of Sindbis virus to neuroblastoma cells Journal of Virology, in press

Chapter 6 107 The role of N-linked glycosylation of Sindbis virus glycoproteins E2 and E1 in viral infectivity and membrane fusion activity

Chapter 7 121 Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity FEBS Letters 498:57-61 Chapter 8 135 PE2 cleavage mutants of Sindbis virus: correlation between viral infectivity and pH-dependent membrane fusion activation of the spike heterodimer Journal of Virology 75:11196-11204

Chapter 9 157 Summarizing discussion

List of publications 175

Contributing authors 178

List of abbreviations 180

Samenvatting in het Nederlands 183

Curriculum Vitae 189

Nawoord 190

Chapter 1

General Introduction 2 Chapter 1

The alphavirus genus

Sindbis virus (SIN) and Semliki Forest virus (SFV) are positive-strand RNA belonging to the genus Alphavirus of the family Togaviridae (41). The alphavirus genus consists of about 25 different viruses. Alphaviruses are transmitted in nature by anthropod vectors, primarily mosquitoes. SIN has also been isolated from mites and ticks (124). Alphaviruses can infect a wide range of organisms, including birds, insects, mammals, and mosquitoes. The skeletal muscle and fibroblasts are the initial sites of , after which the viruses spread to the central nervous system (41). The alphaviruses Venezuelan equine encephalitis virus (VEE), Western equine encephalitis virus (WEE), and Eastern equine encephalitis (EEE) are known to cause fatal encephalitis in horses and humans, but the incidence particularly in humans is low (124). These viruses are endemic in North and South America and cause periodic outbreaks of encephalitis in horses. Other alphaviruses, like Ross River virus (RR), SFV, and SIN can cause fever, rash, arthralgia, and polyarthritis in humans, but again the incidence is low (41). SFV and SIN are among the least pathogenic alphaviruses for humans, and therefore have been widely used as “prototypes” to study the life cycle of these viruses. These studies take advantage of the fact that alphaviruses grow to high titers in tissue culture cells with high PFU-to-particle ratios, properties which enable a detailed biochemical and structural characterization of the virus particles. In addition, full-length cDNA clones of SFV (77), SIN (107), or VEE (21) have made it possible to study the viral life cycle by site-specific mutagenesis of the viral genome. In this thesis, we primarily used SIN to unravel the molecular mechanisms involved in the cell entry process of alphaviruses.

Structure and characteristics of Sindbis virus

Alphaviruses have a uniform structure with a diameter of about 70 nm (16, 45, 100, 124). The viral genome consists of a single-stranded RNA molecule of positive polarity. The viral RNA is assembled with the protein to form the nucleocapsid (35). The nucleocapsid is surrounded by a lipid bilayer, in which the viral spike proteins are inserted (45). Nucleocapsid. The nucleocapsid contains the single-stranded RNA molecule of approximately 11,7 kb, assembled with 240 copies of the capsid protein (~30 kD). The entire genome of SIN has been sequenced (123). Cryo-electron microscopy (cryo-EM) and image reconstruction analysis have revealed that the nucleocapsid has a symmetrical icosahedral structure with a triangulation number T=4 (16, 100). The individual capsid proteins are arranged as pentamers and hexamers to form a roughly spherical nucleocapsid. Envelope. The SIN envelope consists of a lipid bilayer in which 240 copies each of two transmembrane glycoproteins E1 and E2 are inserted (124). The lipid General introduction 3 bilayer is composed of lipids derived from the plasma membrane of the host cell during budding of the virus. The viral membrane consists of about 25% sphingomyelin, 27% phosphatidylcholine, 19% phosphatidylserine, and 26% phosphatidylethanolamine (4, 132). The cholesterol to phospholipid ratio in the viral membrane is approximately 1:1 (70, 106).

Figure 1. Cryo-EM picture of Sindbis virus. The protruding spikes are clearly visible. Each spike consists of a trimer of E2/E1 heterodimers. (courtesy of Dr. S. Mukhopadhyay, Purdue University).

The 240 copies of the glycoproteins E2 and E1, both with a molecular mass of about 50 kD, extend from the lipid bilayer and are organized in a T=4 lattice (16, 45). The glycoproteins form 80 hetero-oligomeric spikes, a single spike consisting of a trimer of E2/E1 heterodimers. The E2 and E1 glycoproteins are classified as type I membrane proteins, with the amino-terminus facing outward from the membrane. E2 is 423 amino acid residues long, with a carboxy-terminal tail of 33 amino acids. E1 has 439 amino acid residues and a carboxy-terminal tail of only 2 amino acids. Both E2 and E1 are acylated. E2 has three acylation sites in the carboxy-terminal tail and two in the transmembrane region (124). E1 is acylated at one position in the transmembrane region (109). Furthermore, both E2 and E1 are each glycosylated at two positions in the ectodomain of the protein (124). Sequence analysis of the E1 glycoprotein has revealed a hydrophobic domain that extends from residues 75 to 97; this region has been proposed to represent the fusion peptide of the virus (35, 75). Crystallographic and cryo-EM studies revealed that the E1 glycoprotein is folded to an elongated molecule that in many respects resembles the flavivirus E protein (74). SFV E1 is composed of a central (amino-terminal) ß-barrel domain that is flanked by a finger-like projecting domain, containing the fusion peptide, 4 Chapter 1 and a carboxy-terminal Ig-like domain. In the protein crystal E1 forms a head-to- tail linked dimer, which can be fitted in the cryo-EM density map of SFV. In another elegant study, SIN glycosylation mutants were used to localize the carbohydrate moieties on the virus using cryo-EM imaging (104). Both studies revealed that the E1 protein lies approximately parallel to the viral surface, whereas the E2 glycoprotein forms the protruding spikes. These data indicate that the E1 glycoprotein is responsible for the icosahedral scaffold, that organizes the T=4 architecture of the mature virus particle. The E2 glycoprotein is synthesized in the infected cell as a PE2 (PE2 is called p62 in SFV) precursor that is cleaved into E2 and a periphal polypeptide E3 (~11 kDa). E3 is lost in mature SIN particles, but remains associated to the E2/E1 heterodimer of SFV (88). Cryo-EM and image reconstruction analysis of a mutant PE2-containing SIN virus revealed that E3 is positioned as a bulge in the heterodimer localized midway between the center of the spike complex and the tips (100). There is also a small hydrophobic peptide, produced as a linker between E2 and E1, called the 6K protein, that has been found to be associated with the virus in low quantity (7-30 molecules per particle; 33, 82). The 6K polypeptide is palmitoylated at positions 35, 36, 39 (124).

Replication and assembly of Sindbis virus

RNA replication of the viral genome. A schematic representation of the genomic RNA and the translation of the viral proteins is depicted in Figure 2. The genomic RNA of SIN can be divided into two major regions: a non-structural domain encoding the replicase proteins and a structural domain encoding the three major viral proteins. The non-structural proteins are translated as a polyprotein directly from the genomic RNA. This polyprotein is cleaved in an autoproteolytic fashion, resulting in the release of nsP1, nsP2, nsP3, and nsP4. The protease responsible for these cleavages is encoded in the sequences at the C-terminal domain of nsP2 (24, 44, 122). nsP1 appears to be involved in the association of the alphavirus replicase to membranes (3). nsP2 is involved in the regulation of minus- strand RNA synthesis (112) and in the initiation of subgenomic 26S RNA synthesis (126). The function of nsP3 is unknown. nsP4 has been identified as the viral polymerase (7, 43, 73, 111). It appears that the function of the viral polymerase is dependent on an aromatic or histidine residue at the amino-terminal end of nsP4 (115). In summary, the alphavirus non-structural proteins are responsible for replication of both positive-strand genomic RNA and minus-strand RNA, the latter serving as a template for production of the 26S subgenomic mRNA and new positive-strand RNA. General introduction 5

Translation of nonstructural proteins Genomic (+) RNA 5’cap nsP1 nsP2 nsP3 nsP4 capsid PE2 6K E1 3’polyA

(-) RNA 3’ 5’

26S RNA capsid PE2 6K E1 Translation of structural proteins

Figure 2. RNA replication of the SIN genome. See text, for details.

The structural proteins are synthesized as a polyprotein from the 26S subgenomic mRNA, in the order NH2-capsid-PE2-6K-E1-COOH, and are post- translationally processed to produce the individual polypeptides. Figure 3 gives a schematic representation of the translation of the structural proteins, assembly and budding of alphavirus particles. Capsid protein synthesis and nucleocapsid assembly. The capsid protein is autoproteolytically released from the polyprotein precursor by its serine protease activity (6, 76). Once the capsid protein has cleaved itself from the polyprotein chain, the proteinase is no longer active because the active site remains occupied by the C-terminal tryptophan residue (17). Newly synthesized capsid proteins then bind to one or more encapsidation sequences of the genomic RNA, and this 6 Chapter 1

Budding

Nucleocapsid PE2 cleavage

SIN RNA TGN + Capsid proteins Golgi PE2-E1 RNA replication heterodimer ER

Nucleocapsid Capsid translation Translation and translocation release of spike protein subunits

Figure 3. Translation of the structural proteins of SIN, and the assembly of new virus particles. binding is believed to be important for the initiation of nucleocapsid formation (139). For SIN, a signal sequence in the encoding region of nsP1 (from nucleotide 945 to 1076) has been identified as the encapsidation signal (17). Moreover, it was found that a stretch of 18 amino acids, called helix I, in the amino-terminal region of the capsid protein is important for nucleocapsid formation (101). After the binding of a capsid subunit to the RNA, more capsid proteins associate with the complex, until a T=4 symmetry is obtained (36). Glycoprotein synthesis and folding of the viral spike proteins. Once the capsid protein is cleaved off, a signal sequence at the amino-terminus of PE2 targets the remainder of the polyprotein for cotranslational translocation to the rough endoplasmic reticulum (ER). In the ER the polyprotein is cotranslationally cleaved by signal peptidases into PE2, 6K, and E1 (113). Addition of carbohydrates. Mannose groups are added to the PE2 and E1 polypeptides in the lumen of the ER, and these carbohydrate chains are modified during transit of the proteins through the Golgi apparatus (113). The asparagine residues in a consensus sequence Asn-X-Ser/Thr in the ectodomains of the alphavirus glycoproteins are glycosylated and carry either simple or complex oligosaccharide chains. It has been postulated that a glycosylation site remains General introduction 7 simple when the folding of the protein renders the chain inaccessible to cellular processing enzymes during transit through the Golgi apparatus, whereas if the chain is accessible to cellular enzymes it is further modified to a complex type (57). For SIN, the oligosaccharide at E1:139 may be either a complex or a simple type of carbohydrate, while the oligosaccharide at E1:245 appears to be modified to a complex-type. E2 is glycosylated at positions 196 and 318, the oligosaccharide at position 318 being processed to a complex-type of carbohydrate. It is believed that the function of carbohydrate chains at the initial stage of glycosylation is to increase the solubility of the protein and prevent aggregation or side reactions from occurring. However, it appears that glycosylation of the ectodomains of SIN glycoproteins has other distinct functions in the life cycle of alphaviruses (Chapter 6). Addition of fatty acids. Fatty acid acylation of the structural proteins of SIN in vertebrate cells occurs after exit of the ER but probably before arrival in the cis Golgi (10). In most cases the fatty acid, usually palmitic acid, is covalently linked in a thiol ester bond to the cysteine residues of the protein. Mutational analysis of the transmembranal cysteine residues of the glycoproteins E1 (at position 430) and E2 (at positions 388, 390) revealed that these residues are palmitoylated (109). Moreover, it has been shown that the cytoplasmic domain of E2 is palmitoylated at positions 396, 416, and 417 (33, 58). Site-specific mutagenesis of the 6K protein revealed that the cysteines at positions 35, 36, and 39 are palmityolated (32, 33). The role of acylation of the SIN glycoproteins is not quite clear, although it has been shown that deacylation of the carboxy-terminal domain of E2 or the 6K protein affects virus budding. For influenza virus, conflicting reports have been published with regard to the role of envelope glycoprotein acylation in viral fusion (28, 60, 90, 93, 103, 121). In Chapter 7, the influence of acylation of SIN glycoproteins E2 and E1 on the membrane fusion process is investigated. Folding, transport, and PE2 cleavage of the glycoproteins. The folding of the viral glycoproteins begins immediately upon entry of the proteins into the ER. It requires molecular chaperones, folding enzymes, energy, and the formation of disulfide bridges (124). After folding of the glycoproteins, heterodimerization of PE2 and E1 occurs in the ER. The PE2/E1 heterodimer matures further by passing through the Golgi and trans-Golgi network (TGN). During transport of the PE2/E1 heterodimer through the slightly acidic TGN, the uncleaved PE2 is presumed to function as a chaperone, protecting the spike from premature destabilization. The influence of PE2 on the stability of the PE2/E1 heterodimer is further discussed in Chapter 8. In the TGN or in a post-TGN compartment, PE2 is cleaved to form E2 and E3 (88). PE2 cleavage is mediated by a furin-like host protease at the consensus sequence XBXBBX, where X is a hydrophobic and B a basic amino acid (67). After PE2 cleavage the E2/E1 heterodimers are transported to the plasma membrane of the cell, where they are used to assemble 8 Chapter 1 new virus particles. The spikes appear on the cell surface as trimers of E2/E1 heterodimers; it is unclear when exactly these trimers are formed. Spike-nucleocapsid interactions and budding of virus particles. The preformed nucleocapsids in the cytoplasm of the cell interact with the viral spike proteins, located on the plasma membrane of the cell, which results in the budding and release of new progeny virus particles. SFV mutagenesis studies have revealed that particle formation requires co-expression of both the capsid and the spike proteins (127). It has been proposed that alphavirus budding is driven by specific interactions of the carboxy-terminal tail of E2 with the nucleocapsids at the plasma membrane of the cell (16, 36, 71). The carboxy-terminal domain of E2 contains two conserved regions. The first conserved region contains a tyrosine and a leucine residue, and this region is shown to interact with aromatic residues in the capsid protein (54, 72, 98, 119, 143). The second region consists of palmitoylated cysteine residues, flanking the tyrosine-leucine motif, and mutations that disrupt palmitoylation affect virus budding (33, 58). In addition, E2-E1 dimer interactions are important for virus assembly and budding. It was found that the E2 carboxy- terminal tail does not interact with the capsid protein unless the E2 protein is dimerized with E1 (5). Interestingly, it has been observed that pre-assembly of nucleocapsids in the cell cytoplasm is not a prerequisite for budding (30, 118). Moreover, Forsell and co-workers (31) showed that lateral spike-spike interactions are required for virus assembly. It was observed that a capsid mutant with a large deletion (amino acid 40 to 118), which is unable to assemble into nucleocapsids in the cell cytosol, does assemble at the plasma membrane and leads to the release of new virus particles. It was proposed that the role of the capsid proteins is restricted to triggering the spike proteins to undergo lateral spike-spike interactions. This, in contrast to the assumption that spike-capsid interactions would drive virus assembly, suggests that lateral spike-spike interactions are responsible for the envelope formation. Moreover, based on the recent information on the positioning of E1 in the viral envelope of alphaviruses (74, 104), one could argue that lateral spike-spike interactions between the E1 glycoproteins are responsible for envelope formation (34). It has also been shown that the 6K polypeptide is involved in virus budding (32). Deletion analysis of 6K demonstrated that the budding efficiency of the D6K virus was down to 10% when compared to wild-type SFV (77, 79). These observations demonstrate that the 6K polypeptide facilitates virus assembly but that it is not absolutely required. Furthermore, efficient budding of SFV and SIN requires the presence of cholesterol in the cellular membrane (80, 81, 87, 133). Transfection of viral RNA into cholesterol-depleted cells demonstrated that, while RNA replication, spike protein dimerization, and transport to the cell surface were normal, virus budding was dramatically inhibited. General introduction 9

Cell entry of Sindbis virus

Entry of enveloped viruses into a host cell requires fusion of the viral envelope with a cell membrane after attachment of the virus to the cell. Fusion of a viral membrane with a cellular membrane is mediated by the viral spike proteins and occurs either at the plasma membrane of the cell, or from within acidic endosomes after cellular uptake of virus particles through a process of receptor-mediated endocytosis, as depicted in Figure 4. In the process of plasma membrane fusion, the interaction of a virus particle with a cellular receptor triggers conformational changes in the viral spike protein which subsequently results in fusion of the viral membrane with the plasma membrane of the cell. On the other hand, when the virus is taken up by the cell through receptor-mediated endocytosis, the mildly acidic pH in the lumen of the endosomal compartment triggers conformational changes in the viral proteins, that are required for fusion of the viral membrane with the endosomal membrane. There is considerable controversy as to whether SIN infects its host cell by receptor-mediated endocytosis or plasma membrane fusion. Before discussing the evidence for both pathways in detail, we will first describe the receptor binding properties of alphaviruses. Receptor binding. The first step in the entry process of alphaviruses is the interaction of the virus particle with a receptor on the plasma membrane of the cell. It was found that the E2 glycoprotein is primarily responsible for virus- receptor interaction, as evidenced by the ability of anti-E2 antibodies to inhibit binding to cells (12, 84). Anti-idiotypic antibodies, directed against E2-specific antibodies, have been used in attempts to identify putative virus receptors on target cells (131, 137). Furthermore, cryo-EM and image reconstruction analysis of SIN and RR, using Fab fragments of monoclonal antibodies, revealed that the outermost tips of the viral spike protein are involved in interaction with a cellular receptor (120). Identification of specific alphavirus receptors has been difficult and, in retrospect, may have been complicated by the use of virus strains that are adapted to tissue cell culture (69). Because alphaviruses have a wide host range and replicate in a variety of different species as well as in many different cell types, the viruses must use a ubiquitous cell surface molecule or a variety of different molecules as receptors. The first putative receptor identified for SFV was the major histocompatibility complex class I molecule on mouse and humans cells (51). However, cells that do not express class I molecules can still be infected by SFV (95). For SIN, a high-affinity laminin receptor has been proposed as a potential receptor on baby hamster kidney (BHK-21) cells (136). 10 Chapter 1

Receptor binding Receptor binding Out Fusion

In Nucleocapsid Endocytosis release

RNA replication

Fusion + H Nucleocapsid release

Figure 4. Routes of cell entry of enveloped viruses. Left: receptor-mediated endocytosis and fusion from within acidic endosomes. Right: plasma membrane fusion.

Recently, it has been shown that SIN, RR, and VEE interact with heparan sulfate (HS), a ubiquitously expressed glycosaminoglycan on the plasma membrane of the cell, as a cell culture adaptation (9, 12, 13, 49, 69). Passage of non-HS- adapted SIN TR339 on BHK-21 cells results in the generation of virus mutants which bind with high affinity to BHK-21 cells and interact with heparin (69). Positive-charge amino acid substitutions have been identified in the mutant E2 proteins of SIN, RR, and VEE, substitutions which appear to be responsible for interaction with HS (9, 49, 69). With regard to SIN, three loci in E2 have been identified (E2:1; E2:70; E2:114) that mutate during the adaptation of SIN in BHK- 21 cells and can independently confer the ability to the virus to bind to cell-surface HS (69). SIN entry via plasma membrane fusion. A mechanism of SIN entry via plasma membrane fusion has been first suggested on the basis of studies which indicated that weak bases, such as chloroquine and ammonium chloride (which are commonly used to raise the pH of intracellular compartments) have little effect on the formation of SIN infectious centers, as detected by translation of viral RNA in the cell cytosol (14, 18). Accordingly, it was found that SIN was able to infect a mutant Chinese hamster ovary cell line, temperature sensitive for endosome General introduction 11 acidification, at either permissive or non-permissive temperatures (26). Furthermore, it was shown that attachment of SIN to cells leads to the exposure of new transitional epitopes on both glycoproteins, using E1- and E2-specific antibodies (29). In the context of these experiments, Abel and Brown (1) proposed a model for SIN entry in which virus-receptor interaction reduces critical disulfide bridges in the E1 glycoprotein through thiol-disulfide exchange reactions. The reduction of disulfide bridges would reorganize the viral spike protein of SIN, subsequently resulting in fusion of the viral membrane with the plasma membrane of the cell. Consistent with this model, SIN-induced polykaryon formation was enhanced by the presence of 2-mercaptoethanol, which reduces disulfide bonds, while SIN infection was partially inhibited by 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), a thiol-akylating agent and exchange inhibitor (1). Moreover, it has been observed that the infection of mosquito cells by SIN is not inhibited by chloroquine, under conditions such that the drug prevented endosome acidification (55). SIN entry via receptor-mediated endocytosis. There is also evidence that SIN infects its host cell by receptor-mediated endocytosis and fusion from within acidic endosomes. Glomb-Reinmund and Kielian (40) investigated the role of low pH and disulfide shuffling in the entry process of SIN. These authors made a direct comparison between SIN and SFV, a virus which is known to infect cells via receptor-mediated endocytosis and low-pH-induced fusion from within acidic endosomes (50, 52, 53, 59, 64, 85, 86). It was found that both SIN and SFV infection are inhibited in cells treated with ammonium chloride, during entry of the virus, as evidenced by viral RNA translation and infection. Inhibition of viral infection was concentration dependent and correlated with the pH threshold of virus-induced cell-cell fusion. Bafilomycin and concanamycin, two reagents that prevent endosome acidification by a different mechanism than the weak bases, also inhibited cellular infection of SIN and SFV. Furthermore, the authors were unable to detect a specific role for the reduction of disulfide bonds in SIN and SFV infection. In another study, alphavirus infection was studied using cells expressing a dominant-negative mutant of dynamin, which blocks the budding of clathrin- coated pits, and it was found that SIN and SFV infection was inhibited (23). On the basis of these studies it would appear as though SIN, like SFV, infects its host cells by receptor-mediated endocytosis, and that exposure of the virus to the low pH, within the lumen of the endosome, is physiologically important for activation of the viral membrane fusion reaction. The controversy about the route of cell entry of SIN represents the basis for the studies presented in Chapter 2, 3 and 8 of this thesis. Nucleocapsid uncoating. Through fusion of the viral membrane with the cellular target membrane the viral nucleocapsid gains access to the cytoplasm of the cell. It has been demonstrated that after the fusion reaction, the nucleocapsid remains associated with the cytosolic leaflet of the endosomal membrane (117). 12 Chapter 1

The nucleocapsid is uncoated such that the RNA becomes accessible to ribosomes. It has been proposed that the binding of the nucleocapsid to the ribosomes triggers the uncoating process (124). The region from amino acid residue 94 to 106 of the capsid protein appears to be involved in the binding of the protein to the ribosomes (140). This was found to be the same region of the protein that interacts with viral RNA during encapsidation (37, 99). Once the RNA is accessible to the ribosomes, RNA replication and translation of the viral proteins are initiated. This will eventually lead to the production of new virus particles.

Membrane fusion activity of Semliki Forest virus

The basic features underlying the membrane fusion properties of enveloped viruses have been studied extensively in virus-cell and virus-liposome systems. In virus-cell systems, fusion is detected directly, measuring fusion of the virus with the plasma membrane of the cell or, indirectly, on the basis of polykaryon formation. These assays might give a quatitative insight in the membrane fusion reaction, but do not allow kinetic or quantitative analysis of the process. To obtain detailed insight in the membrane fusion process of enveloped viruses, it is necessary to follow the process in a sensitive and continuous fashion. To this end, virus-liposome systems have been developed, which offer a number of advantages over virus-cell assays, including the simplicity of the technique, high sensitivity, possibility to obtain quantitative data, and the opportunity to identify target membrane components required for viral fusion (56). Virus-liposome systems are either based on lipid mixing between the interacting membranes or on intermixing of the interior of the virus and the liposomal lumen. Particulary, the lipid mixing assays provide a kinetic and quantitative insight in the membrane fusion process, while contents mixing meets a very stringent criterion for fusion. Virus-liposome systems have been used extensively to unravel the molecular mechanisms involved in the membrane fusion process of SFV, as discussed in more detail below. General features of SFV-liposome fusion. It has been demonstrated that fusion of SFV with liposomes lacking a protein receptor is strictly dependent on the exposure of the virus to low pH (11, 134, 141). Optimal fusion kinetics were observed at pH 5.5. The pH threshold for fusion activation of SFV was found to be pH 6.2 (11). The in vitro pH dependence of fusion (11) closely correlates with the in vivo pH dependence of virus fusion with the endosomal membrane (40). The efficient fusion kinetics of SFV with protein- and carbohydrate-free liposomes suggests that virus-receptor interaction is not a mechanistic requirement for membrane fusion. SFV fusion is temperature dependent, but fusion can be measured in a temperature range of 4 to 37 °C. The fusion-active conformation of SFV is short-lived, pre-incubation of the virus alone for 30 s at pH 5.5 resulting in a complete loss of membrane fusion activity (11). Fusion of SFV with liposomes General introduction 13 requires cholesterol and sphingolipid in the target membrane, as described in detail below. Lipid dependence of SFV fusion. Fusion of SFV with liposomes composed of a variety of purified lipids revealed that fusion is strictly dependent on the presence of cholesterol (11, 134, 141) and sphingolipid (94, 142) in the target membrane. Optimal fusion kinetics were observed with liposomes containing 35 mol% cholesterol and 2 mol% sphingolipid in the target membrane, with a cholesterol to phospholipid ratio of 1:2. It was found that cholesterol is required for low-pH-induced binding of the virus to the liposomal membrane, whereas sphingolipid is important for the subsequent fusion process (94). Cholesterol has also been found to be required for efficient cellular infection, SFV fusion and infection being dramatically reduced on cholesterol-depleted mosquito cells (see below). To date it has not yet been possible to study the role of sphingolipid during cellular infection, since there are no viable sphingolipid-deficient cell lines available. To unravel the structural features of cholesterol in supporting membrane fusion, SFV-liposome fusion studies were conducted using liposomes composed of a variety of sterol analogs. Three major features of cholesterol are believed to be important in its physical interactions with a membrane: the planar ring structure, the aliphatic side chain at C-17, and the 3ß-hydroxyl group. It was observed that the planar ring structure, and the aliphatic side chain are not required for membrane fusion activity of SFV (63). In contrast, the 3ß-hydroxyl group of cholesterol was found to be essential (63, 102, 134). For example, sterols with a modified 3ß-hydroxyl group such as 3-a-hydroxy cholesterol, cholestanone, 5a- cholestane, cholesterol methyl ether, cholesterol acetate, and chlorocholestene, were inactive in supporting membrane fusion of SFV. The structural features required for sphingolipids to mediate fusion of SFV have been examined in a similar fashion, using liposomes containing a variety of sphingolipid analogs. Efficient fusion of SFV was observed with liposomes containing either sphingomyelin, ceramide, or galactosyl ceramide, demonstrating that the nature of the headgroup of the sphingolipid is not a crucial factor (94). Ceramide is the minimally required sphingolipid still supporting fusion, the sphingosine base being inactive. The length of the acyl chain has no prominent effect on the capacity of ceramide to induce membrane fusion of SFV, since both C8-ceramide and C18-ceramide are active (19). In contrast, the 3ß-hydroxyl group and the 4,5-trans double bond of the sphingosine backbone were found to be critical (19, 46). The sphingolipid requirement is stereospecific, D-threo, L-threo, and L-erythro ceramides or sphingolipids being inactive (92). In conclusion, fusion studies with a wide variety of sphingolipids have demonstrated that the action of sphingolipid in the membrane fusion process of SFV exhibits a remarkable molecular specificity. In Chapter 2, it is demonstrated that fusion of SIN, like that of SFV, also requires sphingolipid in the target membrane. The molecular 14 Chapter 1 specificity of this sphingolipid requirement differs from that of SFV fusion (unpublished observations) and is currently under detailed investigation. E1 conformational changes. Upon exposure of SFV to low pH, conformational changes take place in the viral spike protein, which result in fusion of the viral membrane with the target membrane. The fusion reaction of SFV with liposomes can be slowed down by raising the pH of the medium or by lowering the temperature, through which one can differentiate between different stages in the series of conformational rearrangements that occur. Upon acidification, the E2/E1 spike heterodimer dissociates, as evidenced by the loss of E2-E1 co- immunoprecipitation and co-flotation on sucrose-density gradients (11, 27, 61). After E2/E1 heterodimer dissociation, the E1 glycoprotein undergoes several distinct conformational changes that are independent of E2. Monoclonal antibody binding experiments showed that previously hidden epitopes of E1 become accessible (2, 114). Using these Mabs, it was found that residue E1:157 becomes exposed after low pH treatment of the virus (2). Then, the E1 glycoproteins rearrange to form an E1 homotrimeric structure (134, 135). Treatment of alphaviruses with heat or urea also induces conformational changes within the spike proteins, but there is no formation of an E1 homotrimer under these conditions (38, 91). The formation of an E1 homotrimer is strictly dependent on the incubation of the virus at low pH (134). Analysis of the low-pH-dependent fusion reaction in presence of Zn2+ suggests that the E1 glycoprotein inserts into the target membrane before E1 trimerization (20). Similar results were obtained with the SFV mutant (E1:Gly91Asp; 66). The E1 homotrimer has a very stable configuration: it is found to be resistant to SDS treatment at 30 °C, urea, and trypsin digestion (38, 134). It has been proposed that the energy derived from the formation of the E1 homotrimer could drive the merging of the membranes. This would imply that the formation of the E1 homotrimer occurs concomitantly with membrane fusion. However, kinetic data suggest that E1 trimerization occurs before the onset of membrane fusion (11). Therefore, it appears that additional rearrangements within or between E1 homotrimers are required to establish fusion of the viral membrane with the target membrane. The influence of low pH on the conformation of the spike proteins of SIN is presented in Chapter 2.

Alphavirus mutants and the effect on virus entry and membrane fusion

Adaptation. RNA viruses are known to adapt rapidly to new growth environments as a result of the facile generation and selection of virus mutants. For example, rapid adaptation is the basis for the emergence of new influenza virus variants that have the ability to escape from the immunological response generated by the host against a prior virus variant. Also, adaptation is the basis for General introduction 15 the development of resistance against antiretroviral drugs in HIV-infected individuals. Furthermore, the emergence of new virus variants makes it virtually impossible to design a against HIV that would control the infection by induction of virus-neutralizing antibodies. The rapid generation of RNA virus mutants is directly related to the relatively low fidelity of viral RNA replication, the error frequency being approximately 1 in 10,000 bases (125). Furthermore, viral RNA polymerases generally lack error- correcting mechanisms. This implies for alphavirus RNA replication, that on average each newly synthesized viral genome will contain a base substitution. As these substitutions are inserted randomly throughout the genome, they will generally go unnoticed, either because the substitution remains silent or because it may be lethal. Only when the substitution confers a selective advantage to the corresponding virus mutant, relative to the parent virus, it may become visible. In this case, selective pressure will allow the virus mutant to outgrow and develop into the predominant species (42). These selective conditions may involve immunological pressure within the host, the presence of drugs interfering with replication of the parent virus, changes in culture conditions, etc. The facile emergence of adapted virus mutants has also contributed significantly to our current understanding of the life cycle of RNA viruses. For example, to gain a better insight in the requirement of cholesterol in the fusion and the budding process of SFV particles, cholesterol-independent SFV mutants have been selected by serial passage of the virus on cholesterol-depleted mosquito cells (15, 87, 133). Likewise, Chapter 3 presents a characterization of SIN variants adapted to efficient interaction with HS after passaging of wild-type virus on tissue culture cells. Also, Chapter 8 deals with infectious second-site mutant SIN viruses generated from a poorly infectious PE2 cleavage mutant virus by passaging of the original virus on BHK-21 cells. At the same time, however, one should be aware of the fact that propagation of virus particles in cell culture can lead to unrecognized adaptive mutations, which may result in misinterpretation of results and erroneous conclusions regarding the life cycle of the virus concerned (8). In order to avoid the generation of adaptive mutations, viruses can be used derived from an infectious cDNA clone. Infectious cDNA clones have been developed for SFV (77), SIN (107), and VEE (22). In these clones, the viral genome has been positioned downstream of a promotor encoding an SP6 RNA polymerase. The viral RNA is transcribed in vitro, and transfected into susceptible cells, which subsequently leads to the production of progeny virus. Thus, with this procedure, no additional passage of the virus is required, thereby minimizing the possibility of generating mutant viruses. In the following paragraphs, several alphavirus mutants are described, generated by passage of the virus under selective pressure or by site-specific mutagenesis of the full-length cDNA clone encoding the genome of SFV or SIN. 16 Chapter 1

PE2 cleavage mutants. In SFV, cleavage of p62 (p62 is called PE2 in SIN) was prevented by mutating the p62 cleavage site from Arg-His-Arg-Arg to Arg- His-Arg-Leu (110). The resultant p62-mutant SFV was found to be non-infectious in BHK-21 cells because of impairment of receptor interaction and fusion. Viral infectivity of the p62-containing mutant virus could be restored by in vitro cleavage of p62 with trypsin or by exposure of the virus to extremely low pH (78, 110, 135). Analysis of the conformational changes of the viral spike proteins revealed that the pH threshold for p62/E1 heterodimer dissociation was pH 5.0. This is considerably lower than that for wild-type virus, in which E2/E1 heterodimer dissociation is already observed at pH 6.2. Therefore, it appears that the block in membrane fusion lies at the level of the dissociation of the p62/E1 heterodimer. The role of PE2 cleavage in viral infection has also been investigated for SIN. A chinese hamster ovary cell, RPE.40, defective in PE2 processing, was used to generate PE2-containing SIN particles (138). The produced PE2-containing virus particles were found to be non-infectious on RPE.40 and other cells. Viral infectivity was regained by in vitro cleavage of PE2 by trypsin. In contrast, a PE2-containing mutant of the SIN strain SAAR86 was found to be as infectious as the PE2-cleaving parental virus on RPE.40 cells (108, 138). The PE2 cleavage defect resulted from a single amino acid mutation at E2:1 in which a Ser-to-Asn substitution created a signal for N-linked glycosylation. In a later study, a similar amino acid substitution was introduced in the infectious clones of TRSB (corresponding to a laboratory strain of SIN) and TR339 (containing the consensus sequence of SIN AR339), and it was demonstrated that these mutant viruses were non-infectious on RPE.40 cells and BHK-21 (47, 48, 68). The authors found that infection was blocked downstream of binding of the virus to the cell surface, but before RNA replication. This suggests that these viruses are impaired in their capacity to induce membrane fusion. In Chapter 8, we investigated the infectivity and fusogenic properties of these and other PE2 cleavage mutant SIN viruses with liposomes. Cholesterol-independent SFV and SIN mutants. The presence of cholesterol in the host cell membranes has been found to be required for efficient infection, fusion, and budding of SFV and SIN, as discussed earlier. To gain a better insight in the cholesterol dependence of these viruses, Kielian and co- workers have generated cholesterol-independent mutants of SFV and SIN. Cholesterol-independent mutants of SFV were obtained by passaging wild-type SFV on cholesterol-depleted mosquito cells (15, 87, 133). The resultant mutant, called srf-3 (sterol requirement in function), was found to be more infectious, by about 4 to 5 logs, on cholesterol-depleted cells, when compared to wild-type SFV (133). It was observed that the enhanced growth of the srf-3 mutant virus on sterol-depleted mosquito cells was due to an increased efficiency of and exit. Analysis of the membrane fusion properties of the srf-3 virus with liposomes revealed that both the E1 conformational changes and the membrane fusion General introduction 17 activity of the mutant virus were relatively independent of cholesterol (15). Furthermore, it was found that the srf3 virus, propagated in cholesterol-depleted cells, was less stable to centrifugal force than wild-type SFV, which suggests a possible role for cholesterol in stabilizing the virus particles (133). Sequence analysis of the E1 protein of srf-3 mutant virus revealed a single point mutation from Pro to Ser at position 226. Introduction of this mutation into the full-length clone of SFV resulted in a cholesterol-independent mutant, indicating that the cholesterol dependence of SFV is indeed located at E1:226 (133). The cholesterol dependence of SIN is also located in the E1:226 region, as evidenced by site- specific mutagenesis of the full-length clone of SIN (80). However, other cholesterol-independent mutants of SFV, srf4 and srf5, have been identified which were found to have mutations at E1:44 or E1:178, respectively (62). Thus, it appears that several regions in E1 may be involved in the cholesterol dependence of alphavirus cell entry and membrane fusion. E2 virulence mutants. Sequence analysis of neurovirulent strains of SIN, and the corresponding construction of recombinant viruses, have revealed that E2 and E1 are important determinants of virulence (21, 83, 105). It was found that amino acid changes in E2 which are important for virulence in mice, often affect an early step in viral entry. For one of these mutants (E2:Gly172Arg), the attenuated virulence in mice has been shown to correlate with a decrease in binding to neural cells (128). Other SIN mutants, with a reduced virulence in mice, exhibit an increased penetration rate in BHK-21 cells (21, 89, 96, 97, 108). These mutants were generated by passaging of wild-type SIN virus on tissue culture cells. Sequence analysis revealed that these mutants had positive-charge amino acid substitutions in E2 (E2:Ser114Arg or E2:Glu70Lys). In later studies, it was observed that these amino acid changes confer the ability to the variants to bind to cell-surface HS (69). Indeed, several mutants of SIN, VEE, and RR that bind to HS have been found to have a reduced virulence in mice (9, 49, 68, 69). It has been proposed that in vivo HS-adapted mutants bind non-productively to extracellular membranes and basal laminae, and therefore may be cleared from the blood more rapidly than wild-type viruses (9, 13, 49). Passaging of SIN strain AR339 through neonatal and adult mouse brain resulted in a virus mutant with a profound increase in neurovirulence. Sequence analysis and construction of recombinant viruses revealed that the increased virulence of this mutant is due to a single amino acid substitution, from Glu to His, at E2 position 55 (130). This amino acid change alters viral entry and affects early steps in replication more profoundly in neural cells than in non-neural cells (25, 129). Since the E2 glycoprotein is found to be responsible for virus-receptor interaction, one could argue that the amino acid change in TE influences the virus- receptor interaction at the cell surface of neural cells. This question represents the basis for the study presented in Chapter 5 of this thesis. 18 Chapter 1

SFV fusion mutants. The amino acids 90 to 92 (Gly-Gly-Ala) in E1 are strongly conserved among alphaviruses. These residues lie within the conserved hydrophobic domain (amino acid 79 to 97) of E1, a domain which has been identified as the putative fusion peptide of E1. Mutation from the Gly residue at E1 position 91 to Ala or Asp resulted in virus mutants that were affected in viral fusion and assembly (75, 66, 116). The E1:Gly91Ala mutant was found to have a decreased efficiency in virus-liposome association, E1 trimerization, and fusion when compared to wild-type SFV. Both cell-cell fusion and virus-liposome fusion studies revealed that the pH threshold for fusion of the E1:Gly91Ala mutant is shifted to a more acidic pH. The E1:Gly91Asp mutant is completely blocked in membrane fusion, as evidenced by lipid mixing, content mixing, and polykaryon formation assays. It appears that the block in membrane fusion activity is after binding of the viral to the target membrane and epitope exposure but prior to E1 trimerization. This indicates that the E1 glycoprotein inserts as an E1 monomer in the target membrane and that E1 trimerization occurs upon interaction with the target membrane, as indicated above. Other amino acid substitutions in the fusion peptide of SFV revealed that the membrane fusion activity of the virus is dependent on the length of the fusion peptide, its overall hydrophobicity, and its specific sequence (75, 116). Another SFV fusion mutant, called fus-1, with a lower pH threshold for membrane fusion was isolated by selecting viruses resistant to in vitro fusion with RNase-containing liposomes (65). The acid shift in the membrane fusion activity of fus-1 appears to be a consequence of the more acidic pH threshold for the initial dissociation of the E2/E1 heterodimer (39). Sequences analysis revealed that a single amino acid substitution at E2 position 12 from Thr to Ile is responsible for this fusion phenotype (39). This indicates that the amino-terminal end of E2 stabilizes the E2/E1 heterodimer interaction and regulates the pH dependence of E1-catalyzed fusion by controlling the dissociation of the E1/E2 heterodimer.

Scope of this thesis

Mutational analysis of the viral spike proteins has greatly contributed to our current understanding of the molecular mechanisms involved in the membrane fusion process of alphaviruses. The studies described in this thesis attempt to further unravel the molecular basis of the membrane fusion activity of SIN, by site-specific mutagenesis of amino acids in the E2 and E1 glycoprotein sequences. These mutations are generated in full-length clones of SIN. The receptor interaction and the membrane fusion characteristics of the mutants are analyzed by cell binding assays, on-line lipid mixing assays, content mixing assays, and analysis of the viral spike conformational changes. The first chapters deal with the general characteristics of the membrane fusion process of SIN with liposomes. Subsequently, the influence of post-translational modifications, including General introduction 19 glycosylation, palmitoylation, PE2 cleavage, of the viral glycoproteins on the membrane fusion process of SIN is investigated. In Chapter 2, the basic membrane fusion characteristics of SIN with target liposomes are investigated with emphasis on the issue as to whether receptor interaction or low pH is the crucial factor in triggering the fusion process. Furthermore, the role of cholesterol and sphingolipid in the membrane fusion process of SIN is investigated. Sub-optimal conditions for fusion were used to differentiate between different stages in the virus-liposome fusion process. In Chapter 3, the receptor binding properties of HS-adapted mutant SIN versus non-adapted SIN TR339 are investigated using liposomes in which a lipid- conjugated heparin is incorporated as a specific attachment receptor for HS- adapted SIN. The effect of virus-receptor interaction on membrane fusion activity is analyzed in further depth, using lipid mixing and content mixing assays. Also, the potential adaptation of SFV to cell-surface HS is examined. In Chapter 4, the leakiness of SIN and SFV fusion is investigated by examining the redistribution of fluorescent dyes or radiolabeled contents, entrapped in the liposomal lumen during fusion. It is demonstrated that the fusion reactions are largely non-leaky, supporting the notion that a hemifusion intermediate is involved. In Chapter 5, the cell entry properties of SIN mutants TE and 633 are investigated. A distinction is made between binding and fusion. It is shown that the TE virus is more infectious on neural cells than SIN mutant 633. The enhanced infection efficiency of TE in neural cells is found to be related to an increased ability of TE to bind to these cells rather than to an enhanced membrane fusion capacity of this virus. In Chapter 6, single deglycosylated SIN mutants are used to identify the function of the carbohydrate chains in infectivity and membrane fusion activity of the virus. It is shown that SIN glycosylation mutants have a reduced infectivity on BHK-21 cells. Moreover, the deglycosylated SIN mutants are impaired in their capacity to induce membrane fusion, indicating that the reduced infectivity of the mutant viruses on cells lies at the level of the fusion process. In Chapter 7, the effect of deacylation of the transmembrane domains of SIN glycoproteins E2 and E1 on the viral membrane fusion capacity is investigated. The mutants were generated by site-specific mutagenesis of the infectious cDNA clone Toto1101 of SIN. It appeared that deacylation of the transmembrane regions of E2 and/or E1 has no effect on the membrane fusion characteristics of the virus. In Chapter 8, the role of PE2 in the membrane fusion process of SIN particles is investigated. It is demonstrated that PE2 cleavage mutant viruses are non-infectious on BHK-21 cells because these mutants are impaired in viral fusion. Furthermore, it is shown that second-site resuscitating mutations in PE2 restore viral infection and membrane fusion, despite a sustained lack of PE2 cleavage. 20 Chapter 1

In Chapter 9, the results and conclusions of this thesis are summarized and discussed.

References

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Low-pH-Dependent Fusion of Sindbis Virus with Receptor-Free Cholesterol- and Sphingolipid- Containing Liposomes

Jolanda M. Smit, Robert Bittman, and Jan Wilschut

Journal of Virology 73:8476-8484 30 Chapter 2

Abstract

There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH, or uptake by receptor-mediated endocytosis and subsequent low-pH- induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60-85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid in the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike. Low-pH-dependent fusion of Sindbis virus with liposomes 31

Introduction

Sindbis virus (SIN) is the prototype member of the genus Alphavirus of the family of the Togaviridae. Alphaviruses are structurally well-defined viruses which contain three major proteins, the capsid protein C, and two envelope glycoproteins E1 and E2 (27, 54). The glycoproteins are organized in 80 hetero-oligomeric spikes; a single spike consisting of a trimer of E2/E1 heterodimers. In the infected cell, the spike heterodimer is assembled in the endoplasmic reticulum as a PE2/E1 heterodimer in which PE2 is the precursor of the E2. The PE2/E1 heterodimer is subsequently transported through the Golgi and the trans-Golgi network to the plasma membrane. Just before the appearance of the spike on the cell surface, the PE2 precursor is cleaved into E2 and E3 by a cellular furin-like protease, resulting in the formation of the mature E2/E1 form of the heterodimer (33). In some alphaviruses including SIN, the E3 peptide is released from the virus particles (37), whereas in others, such as Semliki Forest virus (SFV), E3 remains associated with the E2/E1 heterodimer. The E2/E1 heterodimer mediates the infectious entry of SIN into its host cell. The initial step in cell entry is the interaction of the virus with a cellular receptor. A high-affinity receptor for binding of SIN to rodent and monkey cells has been identified as the 67-kDa protein laminin (57). Recently, it has been shown that the widely expressed glycosaminoglycan heparan sulfate may be involved in binding of SIN to cells (6, 30). It is the E2 component of the alphavirus spike that is primarily involved in receptor interaction (48, 50). Being an enveloped virus, SIN infects its host cells by a membrane fusion reaction. In principle fusion of enveloped viruses may occur either at the plasma membrane or from within the endosomal cell compartment after internalization of the virus particles through receptor-mediated endocytosis. In the process of plasma membrane fusion, the interaction of the virus with a cellular receptor mediates the conformational changes within the viral spike protein that are required for the fusion reaction, fusion occurring at the neutral pH of the extracellular environment. In the process of virus cell entry through receptor-mediated endocytosis, it is generally the mildly acidic pH within the lumen of the endosomes that triggers the membrane fusion reaction. There is considerable controversy with regard to the cell entry mechanism of SIN. Several lines of evidence suggest that SIN may fuse directly at the cell plasma membrane at neutral pH, mediated by interaction of the viral spike with its cellular receptor. For example, SIN has been observed to infect cells treated with weak bases, like chloroquine and ammonium chloride, which raise the pH of endosomes, as evidenced by the translation of viral RNA in the cell cytosol (7, 8). This suggests that the infection process of SIN does not involve acidic endosomes. Accordingly, SIN has been found to infect a Chinese hamster ovary (CHO) cell mutant, temperature-sensitive for endosome acidification (14), although earlier 32 Chapter 2 observations involving similar CHO cell mutants had suggested that a lack of endosome acidification does inhibit infection (39, 47). Furthermore, Flynn et al. (17) detected conformational changes within the glycoproteins of SIN upon interaction of the virus with cells at neutral pH, and suggested that the virus- receptor interaction induces the fusion-active conformation of the viral spike. Abell and Brown (1) then proposed a model for SIN entry in which the virus-receptor interaction enhances thiol-disulfide exchange reactions reorganizing the viral spike protein, allowing the virus to penetrate cells by direct fusion with the plasma membrane. On the other hand, early (15) and quite recent evidence supports an endocytic mechanism for cell entry by SIN. In fact, while the present study was in progress, DeTulleo and Kirchhausen (12) reported that infection of cells by SIN is inhibited by dominant-negative mutant forms of dynamin, which block the budding of clathrin-coated pits, thus suggesting that SIN infects its host cells by clathrin- mediated endocytosis. Furthermore, Glomb-Reinmund and Kielian (20) published a study also providing evidence for cell entry of SIN through receptor-mediated endocytosis and fusion from within acidic endosomes. These authors made a direct comparison between SIN and SFV, because it is well established that SFV enters cells through an endocytic mechanism (21, 22, 23, 24, 27, 35, 36). Upon exposure of SFV to low pH, the viral E2/E1 heterodimeric glycoprotein dissociates and trypsin-resistant homotrimer of the fusion protein E1 (19, 31) is formed, which presumably represents the fusion-active conformation of the viral spike (4, 25, 29, 55, 56). SFV is also capable of fusing liposomes in a mildly acidic environment, indicating that the sole trigger for fusion is low pH (4, 25, 42, 55, 58, 59). Here, we studied fusion of SIN in a liposomal model system, using virus biosynthetically labeled with pyrene phospholipids (4, 42, 55, 59). It is demonstrated that the virus fuses rapidly with liposomes lacking a specific receptor, indicating that receptor binding is not essential for triggering the fusion reaction. Fusion is strictly dependent on low pH, consistent with cellular entry of SIN, like that of SFV, through acidic endosomes.

Materials and Methods

Lipids. Phosphatidylcholine (PC) from egg yolk, phosphatidylethanol-amine (PE) prepared by transphosphatidylation of egg PC and sphingomyelin (SPM) from egg yolk were obtained from Avanti Polar Lipids (Alabaster, AL). High-purity cholesterol (Chol) was a generous gift from Solvay Pharmaceuticals (Weesp, The Netherlands). The fluorescent fatty acid 16-(1-pyrenyl)hexadecanoic acid (pyrene fatty acid) and 1- hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (pyrPC), were purchased from Molecular Probes (Eugene, OR). Cells and Virus. Sindbis virus strain AR339 was a generous gift of Dr. D.E. Griffin (Johns Hopkins University, Baltimore, MD). The virus was propagated on Baby Hamster Kidney cells (BHK-21). The cells were cultured in Glasgow’s modification of Eagle’s Low-pH-dependent fusion of Sindbis virus with liposomes 33 minimal essential medium (Gibco/BRL, Breda, The Netherlands), supplemented with 5% foetal calf serum, 10% tryptose phosphate broth, 200 mM glutamine, 25 mM Hepes, and 7.5% sodium bicarbonate. Pyrene-labeled SIN was isolated from the medium of infected BHK-21 cells, cultured beforehand in the presence of pyrene fatty acid, essentially as described before for SFV (4, 42, 55, 59). Briefly, BHK-21 cells, prior cultured for 48 h on medium containing 15 µg/ml pyrene fatty acid, were infected with SIN at a multiplicity of infection of 4. At 24 h post-infection, the pyrene-labeled SIN particles were harvested from the medium by ultracentrifugation in a Beckman type 19 rotor, 2.5 h at 100,000 g, 4 °C. The virus particles were further purified on a sucrose density gradient (20-50% w/v) by ultracentrifugation in a Beckman SW41 rotor, 16 h at 100,000 g, 4 °C. [35S]methionine- labeled SIN, and unlabeled SIN were produced in a similar fashion (4, 42, 55, 59). The purity of the SIN particles was evaluated by SDS-PAGE. Viral phospholipid was determined by phoshate analysis (3). Protein concentration was determined according to Peterson (44). The infectivity of the virus preparation was determined by titration on BHK-21 cells in 96-well plates. Liposomes. Large unilamellar vesicles (LUV) were prepared by a freeze/thaw- extrusion procedure (10, 40, 42, 59). Briefly, lipid mixtures were dried from a chloroform/methanol solution under a stream of nitrogen and further dried under vacuum for at least 1 h. The lipid mixtures were hydrated in 5 mM Hepes, 150 mM NaCl, 0.1 mM EDTA, pH 7.4 (HNE), and subjected to five cycles of freezing and thawing. Subsequently, lipid mixtures were extruded 21 times through a Unipore polycarbonate filter with a pore size of 0.2 µm (Nucleopore, Inc., Pleasanton, CA) in a LiposoFast mini-extruder (Avestin, Ottawa, Canada). Smaller liposomes were prepared by an additional 81 times extrusion through two Unipore polycarbonate filters with a pore size of 0.05 µm. The size of the liposomes was determined by quasi-elastic light scattering analysis in a submicron particle sizer model 370 (Nicomp Particle Sizing Systems, Santa Barbara, CA). Liposomes consisted of PC/PE/SPM/Chol (molar ratio, 1:1:1:1.5), PC/PE/SPM (molar ratio, 1:1:1), PC/PE/Chol (molar ratio, 1:1:1), PC/PE (molar ratio, 1:1), or PC/pyrPC/PE/SPM/Chol (molar ratio, 0.85:0.15:1:1:1.5). Trypsin-containing liposomes were also prepared by freeze/thaw-extrusion, but in this case the lipids were dispersed in HNE in presence of 10 mg/ml trypsin (Boehringer, Mannheim, FRG). The liposomes were extruded 21 times through a filter with a pore size of 0.2 µm. The trypsin-containing liposomes were separated from free trypsin by gel filtration on a Sephadex G-100 column in HNE. The phospholipid concentration of liposome preparations was determined by phosphate analysis (3). Fusion assays. Fusion of pyrene-labeled SIN with liposomes was monitored continuously in an AB2 fluorometer (SLM/Aminco, Urbana, IL). Briefly, pyrene-labeled SIN (0.5 µM viral phospholipid) and liposomes (200 µM phospholipid) were mixed in a quartz cuvette of the fluorometer in a final volume of 0.665 ml in HNE, unless indicated otherwise. The content of the cuvette was stirred magnetically and thermostatted at the desired temperature. After 1 min incubation, fusion was triggered by the addition of 35 µl 0.1 M MES, 0.2 M acetic acid, pretitrated with NaOH to achieve the final desired pH. The fusion scale was calibrated such that 0% fusion corresponded to the initial excimer fluorescence value. The 100% fusion value was obtained through the addition of 35 µl 0.2 M octaethyleneglycol monododecyl ether (C12E8; Fluka Chemie AG, Buchs, Switzerland) to achieve an infinite dilution of the probe (4, 42, 55, 59). The initial rate of fusion was 34 Chapter 2 determined from the tangent to the first part of the curve. The extent of fusion was determined 60 s after acidification. Fusion of pyrPC-labeled liposomes with SIN was measured under the same conditions, essentially as described before for SFV (32) and vesicular stomatitis virus (VSV) (41). For these experiments, liposomes were prepared with a diameter of 70 nm (see above). In the fusion reaction, liposomes (2 µM liposomal phospholipids) were mixed with SIN (10 µM viral phospholipid), unless indicated otherwise. Transfer of viral nucleocapsid to the liposomal lumen during SIN-liposome fusion was measured as the degradation of the viral capsid protein by trypsin, initially encapsulated in the liposomes (42, 58, 59). Briefly, a trace amount of [35S]methionine-labeled virus and unlabeled virus (final concentration of 0.5 µM viral phospholipid) were mixed with trypsin- containing liposomes (200 µM phospholipid) in presence of 125 µg/ml trypsin inhibitor (Boehringer, Mannheim, FRG) in HNE. The mixture was acidified, under continuous stirring, to the desired pH with 0.1 M MES, 0.2 M acetic acid, as above. After 30 s, samples were neutralized by the addition of a pretitrated volume NaOH. The samples were further incubated for 1 h at 37 °C and subsequently analyzed by SDS-PAGE. Gels were incubated for 30 min in 1 M sodium salycilate and dried. Protein bands were visualized by autoradiography. Quantification of the capsid protein was done by phosphorimaging analysis using Image Quant 3.3 software (Molecular Dynamics, Sunnyvale, CA). Virus-liposome binding assay. Virus-liposome binding was assessed by a coflotation assay, as described before (4, 9, 40, 42, 55, 59). The reactions were carried out under the same experimental conditions as those in the fusion experiments. A trace amount of [35S]methionine-labeled virus and unlabeled virus (final concentration of 0.5 µM viral phospholipid) were mixed with liposomes (200 µM phospholipid). The mixture was acidified, under continuous stirring, to the desired pH with 0.1 M MES, 0.2 M acetic acid, as above. After 60 s, samples were neutralized through the addition of a pretitrated volume of NaOH. Subsequently, 0.1 ml of the reaction mixture was added to 1.4 ml 50% (w/v) sucrose in HNE. On top of this, 2.0 ml of 20% (w/v) sucrose and 1.0 ml of 5% (w/v) sucrose in HNE were layered. After centrifugation in a Beckman SW50 rotor at 150,000 g for 2 h at 4 °C, the gradient was fractionated in ten samples, starting from the top. The distribution of the viral radioactivity was quantified by liquid scintillation analysis. The radioactivity in the top four fractions, relative to the total amount of radioactivity, was taken as a measure for SIN-liposome binding. Analysis of the conformational changes in the viral spike protein. The conformational changes occurring in the viral spike protein were examined under the same conditions as in the fusion and binding experiments. After low-pH treatment, samples were neutralized by addition of a pretitrated volume NaOH, solubilized in SDS-PAGE buffer, and analyzed by SDS-PAGE. For the appearance of trypsin-resistant forms of E1, samples were incubated in presence of 200 µg/ml trypsin for 15 min at 37 °C. Subsequently, samples were solubilized in SDS-PAGE sample buffer, heated for 4 min at 100 °C, and analyzed by SDS-PAGE. Gels were further incubated for 30 min in 1 M sodium salycilate and dried. Visualization of the bands was done by autoradiography. Quantification was done by phosphorimaging analysis, as described above. Low-pH-dependent fusion of Sindbis virus with liposomes 35

Results

Characterization of pyrene-labeled SIN. In this study we used SIN, biosynthetically labeled with the fluorescent probe pyrene. The labeling procedure involves production of the virus on BHK-21 cells cultured beforehand in the presence of pyrene fatty acid (4, 10, 42, 59). Newly formed virus particles thus carry the pyrene probe in their membrane phospholipids. In order to examine the potential effect of pyrene incorporation in the viral membrane on the infectivity of the virus, the specific infectivities of pyrene-labeled and unlabeled virus were determined. Virus was isolated and purified from the medium of infected pyrene- labeled or unlabeled BHK-21 cells, as described in Materials and Methods. Analysis by SDS-PAGE demonstrated that the virus preparations were pure, as evidenced by the presence of just the three major structural proteins E1, E2, and C (results not shown). Subsequent determination of the viral titer and protein concentration showed that, for pyrene-labeled SIN the infectious unit (IU) to particle ratio was 1/13 under the conditions of the experiment, while for unlabeled control virus the corresponding ratio was 1/12. For the calculation, a theoretical amount of 5.45*10- 17 g protein/virus particle was used. The IU to particle ratios of unlabeled and pyrene-labeled SIN appeared to be very similar, indicating that pyrene labeling has no effect on the infectivity of the virus. Low-pH-dependent fusion of pyrene-labeled SIN with liposomes. Fusion of pyrene-labeled SIN was measured in a liposomal model system (4, 42, 55, 59). The pyrene probe forms excimers with a fluorescence emission maximum at 480 nm, about 100 nm higher than the fluorescence maximum of pyrene monomers. Pyrene excimer formation is dependent on the average distance between the probe molecules. Thus, in the virus membrane, the excimer fluorescence intensity is proportional to the surface density of pyrene-labeled phospholipid molecules (18, 43). Upon fusion of pyrene-labeled virus particles with liposomes, the pyrene phospholipids will be diluted into the liposomes resulting in a decrease in the pyrene excimer fluorescence intensity, which can be monitored continuously. For this assay liposomes were prepared with an average diameter of 200 nm. The diameter of the viral membrane (excluding the glycoproteins) is about 50 nm. Therefore, upon fusion of a virus particle with a liposome the pyrene phospholipids dilute by at least an order of magnitude. In the fusion reaction, pyrene-labeled SIN (0.5 µM viral phospholipid) was mixed with an excess of liposomes (200 µM phospholipid) consisting of PC/PE/SPM/Chol with a molar ratio of 1:1:1:1.5. Figure 1A presents the fusion kinetics of pyrene-labeled SIN with liposomes. At pH 5.0, the virus fused rapidly and efficiently with the liposomes (curve a). By contrast, at pH 7.4, no detectable fusion occurred (curve c). Furthermore, the fluorescence intensity of pyrene-labeled SIN at low pH in the absence of target 36 Chapter 2 liposomes remained constant (curve d), demonstrating that the decrease in excimer fluorescence intensity observed in the presence of liposomes was due to dilution of the probe from the viral into the liposomal membrane. At pH 5.75, an intermediate extent of fusion was observed with the initial rate substantially slower than that at pH 5.0 (curve b). Figure 1B shows the detailed pH dependence of SIN fusion with PC/PE/SPM/Chol liposomes. Optimal fusion in terms of both initial rate (solid circles) and final extents (open squares) was observed at pH 5.0, with a threshold at pH 6.0. Under optimal conditions a decrease of excimer fluorescence intensity by approximately 60% was observed. This corresponds to fusion of a minimum of 60% of the virus particles, since each fusion event is expected to result in a large dilution of the pyrene probe (see above). However, upon fusion of a virus particle with a relatively small liposome, a residual excimer fluorescence intensity may remain. This implies that the level of 60% may represent an underestimate of the actual extent of fusion. The initial rate of fusion at pH 5.0 was very fast, corresponding to 20-25% of the virus particles fusing within the first second after

A a B (%) 60 (%/s) 20 60

15 fusion

(%) 40 40 fusion 10 of b of 20 20 Fusion 5 0 c,d 0 0 Final extent Initial rate 0 20 40 60 4 5 6 7 8 Time (s) pH Figure 1. Low-pH-dependent fusion of pyrene-labeled SIN with liposomes. Fusion was measured on-line at 37 °C as a decrease of viral pyrene excimer fluorescence, as described in Materials and Methods. Final virus and liposome concentrations were 0.5 and 200 µM (membrane phospholipid), respectively. Liposomes consisted of PC/PE/SPM/Chol (molar ratio of 1:1:1:1.5). (A) Curve a, pH 5.0; curve b, pH 5.75; curve c, pH 7.4; curve d, pH 5.0, without liposomes. (B) The initial rate of fusion (solid circles) was determined from the tangent to the first part of the curve. The extent of fusion (squares) was determined 60 s after acidification. the acidification of the virus-liposome mixture. At pH values higher or lower than the optimal pH 5.0, fusion exhibited slower kinetics and lower extents. Fusion required an excess of liposomes, leading to an apparent saturation at 100-150 µM phospholipid (results not shown). In principle, it is possible that with Low-pH-dependent fusion of Sindbis virus with liposomes 37 liposome concentrations increasing beyond the saturating level, a single virus particle will fuse simultaneously with multiple liposomes. However, this is not expected to result in a significant further decrease of the fluorescence signal since, in general a single fusion event already produces a dilution of the pyrene probe by at least an order of magnitude, as indicated above. Fusion of unlabeled SIN with pyrene-labeled liposomes. We also used a reverse variant of the pyrene lipid mixing assay in order to ascertain that the fusion signal seen in the experiments of Figure 1 was not due to a peculiarity of the pyrene-labeled virus used in those experiments. In the reverse assay, an excess of unlabeled SIN was incubated with pyrPC-labeled liposomes, and dilution of the probe from the liposomal into the viral membrane was assessed. For the fusion reaction liposomes were prepared with a diameter of 70 nm. Given the diameter of the viral membrane of 50 nm, fusion of a liposome with a single virus particle thus is expected to result in a 1/3 enlargement of the liposomal membrane, with a concomitant decrease of the pyrene excimer fluorescence by 33%. Figure 2A shows that pyrPC-labeled liposomes consisting of PC/PE/SPM/Chol under low-pH conditions fused efficiently with SIN. At pH 5.0, a decrease in pyrene excimer fluorescence by about 38% in 60 s was observed (curve a). Again, at neutral pH, there was no significant fusion (curve b).

B 40 A a 40 30 30 (%) (%) 20 20 fluorescence of excimer

decrease 10 decrease 10 b Extent

Excimer 0 c 0

0 20 40 60 0 5 10 15 20 Time (s) SIN (µM phospholipid) Figure 2. Low-pH-dependent fusion of SIN with pyrPC-labeled liposomes. Fusion was measured on-line at 37 °C, as a decrease of liposomal excimer fluorescence, as described in the Materials and Methods. Fusion of pyrPC-labeled liposomes (2 µM liposomal phospholipid) with SIN (10 µM viral phospholipid) was measured at pH 5.0, unless indicated otherwise. Liposomes consisted of PC/pyrPC/PE/SPM/Chol (molar ratio 0.85:0.15:1.0:1.0:1.5). (A) Curve a, pH 5.0; curve b, pH 7.4; curve c, pH 5.0, but in the absence of SIN. (B) The final extent of excimer fluorescence decrease (squares) was determined, 60 s after acidification, as a function of the viral phospholipid concentration. 38 Chapter 2

Furthermore, pyrPC-labeled liposomes in the absence of virus did not exhibit any change in fluorescence intensity upon acidification to pH 5.0 (curve c). In Figure 2B the extent of excimer fluorescence decrease was measured as function of the virus concentration. When a fixed concentration of pyrPC-labeled liposomes (2 µM phospholipid, corresponding to 4 x 1010 particles per ml) was incubated with increasing virus concentrations, the extent of the decrease in excimer fluorescence intensity increased in a biphasic manner. At the extrapolated inflection point, the extent of excimer fluorescence decrease was 32%, which is very close to the theoretical value, 33%, expected for one virus particle fusing with a single liposome. The inflection occurred at a virus concentration of 2.5 µM viral phospholipid, corresponding to 1011 particles per ml. Thus, we conclude that on average each liposome fuses at least once with a single virus particle at a virus-to- liposome particle ratio of approximately 2.5. After the inflection point, the extent of excimer fluorescence decrease continued to increase, consistent with one liposome fusing simultaneously with more than one virus particle. Unlike the condition of a labeled virus particle fusing with a considerably larger liposome, fusion of a labeled 70-nm liposome with multiple smaller virus particles is expected to produce a significantly greater dilution of the probe than fusion of one such liposome with a single virus particle. Contents mixing during SIN-liposome fusion. In the above experiments, the detection of SIN-liposome fusion was based on lipid mixing assays. A more stringent criterion for fusion involves the mixing of the internal contents of the virus with the liposomal lumen. To demonstrate contents mixing in the SIN- liposome system, we used an assay involving [35S]methionine-labeled virus and trypsin-containing liposomes (42, 58, 59). Fusion was assayed as the degradation of the viral capsid protein in the presence of an excess of trypsin inhibitor in the medium. As shown in Figure 3A, incubation of SIN with an excess of trypsin- containing PC/PE/SPM/Chol liposomes at pH 5.0 resulted in the degradation of a substantial fraction of the capsid protein (lane a). At neutral pH, no degradation of the capsid protein was detected (lane b). Furthermore, incubation of virus at pH 5.0 with empty liposomes under otherwise the same conditions did not result in degradation of the capsid protein either (lane c). In these controls, the ratio of radioactivity in the capsid band relative to the total radioactivity, as determined by phosphorimaging, was close to 0.4, as expected on the basis of the number of methionine residues in the viral structural proteins (46). To exclude the possibility that the amount of trypsin was limiting under the conditions of the experiment, Triton X-100 was added to the reaction mixture in the absence of trypsin inhibitor. This resulted in complete degradation of the capsid protein, demonstrating that the amount of trypsin was not limiting in the assay (results not shown). Figure 3B presents a quantification by phosphorimaging analysis of the extent of capsid protein degradation as a function of pH. At pH 5.0, approximately 85% of the capsid protein was degraded, while at pH 5.5 and pH 5.75 the corresponding Low-pH-dependent fusion of Sindbis virus with liposomes 39 numbers were 64% and 41%, respectively. It appears, therefore, that qualitatively the pH dependence of the SIN-liposome fusion process is the same in the contents mixing assay and the lipid mixing assay. However, the extent of fusion as assessed by the trypsin assay was consistently slightly higher than that determined from the pyrene assay. This underlines the conclusion that the pyrene assay presumably underestimates the extent of fusion, as indicated above. In addition, there may be small differences in fusion capacity between individual virus batches.

100 A B 80

60 E2/E1 40 20 C Capsid degradation (%) 0 a b c 5.0 5.5 5.75 7.4 pH Figure 3. Transfer of the viral capsid into the liposomal lumen assayed as the degradation of the viral capsid protein. Fusion of [35S]methionine-labeled SIN (0.5 µM viral phospholipid) with trypsin-containing PC/PE/SPM/Chol liposomes (200 µM liposomal phospholipid) in the presence of trypsin inhibitor in the external medium at 37 °C was determined, as described in Materials and Methods. (A) Visualization of the protein bands by autoradiography. Lane a, trypsin-containing liposomes at pH 5.0; lane b, trypsin-containing liposomes at pH 7.4; lane c, empty liposomes at pH 5.0. (B) Quantification of the extent of capsid protein degradation as a result of virus incubation with trypsin-containing liposomes at different pH values as determined by phosphorimaging analysis.

Taken together, the results obtained with the lipid mixing assays involving either pyrene-labeled SIN or pyrene-labeled liposomes, and the results of the contents mixing assay demonstrate conclusively that SIN fuses rapidly and almost quantitatively with receptor-free liposomes in a strictly low-pH-dependent manner. Cholesterol and sphingomyelin are required for low-pH-induced SIN- liposome fusion. A characteristic feature of the fusion mechanism of SFV is the specific requirement of cholesterol and sphingomyelin in the target membrane (4, 26, 42, 45, 55, 58, 59). Because of the similarity between SFV and SIN it was of interest to determine whether SIN exhibits the same lipid dependence. As shown in Figure 4, liposomes of various lipid compositions were examined in order to determine the lipid dependence of SIN fusion. Under low-pH conditions pyrene- labeled SIN fused efficiently with liposomes consisting of PE/PC/SPM/Chol 40 Chapter 2

(curve a). However, SIN was unable to fuse with liposomes consisting of either PE/PC/SPM or PE/PC/Chol (curves b and c). When either PC or PE or both were omitted from the liposomes, SPM and Chol being maintained, sustained fusion activity was observed (results not shown). This demonstrates that the presence of both cholesterol and sphingomyelin in the target membrane are essential for SIN-liposome fusion at low pH.

60 a

(%) 40

20 Fusion

0 b,c

0 20 40 60 Time (s) Figure 4. Effect of the target membrane lipid composition on fusion of pyrene-labeled SIN with liposomes. Fusion of pyrene-labeled SIN with liposomes of different lipid compositions was measured at pH 5.0, 37 °C, as described in the legend to Figure 1. Curve a, PC/PE/SPM/Chol (molar ratio 1.0:1.0:1.0:1.5) liposomes; curve b, PC/PE/SPM (molar ratio 1.0:1.0:1.0) liposomes; curve c, PC/PE/Chol (molar ratio 1.0:1.0:1.0) liposomes.

Low-pH dependent binding of SIN to liposomes. The first step in low-pH- induced SIN-liposome fusion is binding of the virus to the liposomes. Various lipid compositions in the liposomes were examined in order to determine the influence of the target membrane lipids on the binding of SIN to liposomes. Briefly, a mixture of [35S]methionine-labeled SIN, unlabeled virus and liposomes was incubated at pH 5.0 or pH 7.4. After 60 s, the pH of the reaction mixture was neutralized by the addition of NaOH. Liposome-bound virus was separated from unbound virus by flotation on a sucrose density gradient. The results are shown in Figure 5. Binding of SIN to liposomes was strictly dependent on acidic pH. At neutral pH, there was negligible interaction between the virus and the liposomes (open bars a-d). At pH 5.0, we found 72% of the virus particles bound to liposomes consisting of PE/PC/SPM/Chol (bar a). By comparing the fusion data in Figures 1 and 3 with the binding data obtained here, we conclude that all of the particles that bound to the liposomes under these conditions also fused. In the trypsin assay, we even observed a slightly higher extent of capsid protein degradation at pH 5.0 than the extent of virus-liposome binding under the same conditions. This is presumably due to minor differences between individual virus Low-pH-dependent fusion of Sindbis virus with liposomes 41 batches. The association of SIN with PE/PC/Chol liposomes was less efficient, resulting in 25% binding (bar b). However, these virus particles only bound to the liposomes but did not fuse, fusion being undetectable with liposomes lacked SPM (see Figure 4). SIN bound very poorly to liposomes consisting of either PC/PE/SPM (bar c), or PC/PE (bar d). These results indicate that cholesterol promotes binding of the virus to the liposomes but is not sufficient for extensive irreversible binding, as seen under comparable conditions with SFV (40). This indicates that in the case of SIN, both cholesterol and SPM are required for efficient irreversible binding (and fusion) of the virus to the liposomes.

80 pH 5.0 60 pH 7.4

40

Binding (%) 20

0 a b c d Liposomes Figure 5. Influence of cholesterol and sphingomyelin on pH-dependent binding of SIN to liposomes. SIN (trace of [35S]methionine-labeled virus and unlabeled virus, 0.5 µM phospholipid) was incubated with liposomes (200 µM liposomal phospholipid) at pH 5.0 or pH 7.4, at 37 °C. Binding of SIN to liposomes was determined by co-flotation analysis on sucrose density gradients, as described in Materials and Methods. Bar a, PC/PE/SPM/Chol (molar ratio 1.0:1.0:1.0:1.5) liposomes; bar b, PC/PE/Chol (molar ratio 1.0:1.0:1.0) liposomes; bar c, PC/PE/SPM (molar ratio 1.0:1.0:1.0) liposomes; bar d, PC/PE (molar ratio 1.0:1.0) liposomes.

Inactivation of SIN fusion capacity through pre-exposure of the virus alone to low pH. It has been suggested recently that pre-exposure of SIN during freezing of the virus in medium or PBS, which results in a transient lowering of the pH, may activate the viral fusion capacity, thus possibly explaining observations suggesting fusion of the virus under neutral pH conditions at the level of the target cell plasma membrane (12, 16). The implicit assumption underlying this suggestion is that virus activated by pre-exposure to low pH would remain fusion-active for a significant period of time, e.g., several minutes. In this perspective we analyzed whether pre-exposure to low pH of SIN alone results in sustained fusion capacity. Notably, incubation of SFV at acidic pH in the absence of target membranes results in a very rapid loss of the fusion capacity of the virus (4). 42 Chapter 2

100 80

final extent 60 40 20 0 Percentage of 0 30 60 90 120 150 180 Pre-exposure time (s) Figure 6. Inactivation of viral fusion capacity due to the pre-exposure of SIN alone to acidic pH. Fusion was measured on-line at 37 °C, as a decrease of viral pyrene excimer fluorescence, as described in the legend to Figure 1. Pyrene-labeled SIN (0.5 µM viral phospholipid) was incubated at pH 5.0, and at the indicated time periods liposomes (200 µM phospholipid) consisting of PC/PE/SPM/Chol (molar ratio 1:1:1:1.5) in pH 5.0 buffer, were added to the reaction mixture, and subsequently fusion was measured. The final extents of fusion, at 60 s after acidification of the liposomes, were related to the extent of fusion of an untreated control (the absolute extent for fusion of this control was 62%).

Pyrene-labeled SIN was incubated at pH 5.0 at 37 °C in the absence of target liposomes for various periods of time. Subsequently, PC/PE/SPM/Chol liposomes were added and the remaining fusion activity was determined. Figure 6 shows that such a preincubation of the virus alone leads to a rapid loss of fusion activity. Preincubation of SIN at pH 5.0 for 20 s resulted in a 50% reduction of fusion, while a preincubation for 2-3 min resulted in essentially complete loss of fusion capacity. This indicates that fusion activation of SIN triggered by low pH is of a transient nature, resulting in a rapid subsequent irreversible loss of fusion capacity. Next, we exposed SIN to low pH by slowly or rapidly freezing the virus in cell culture medium without Hepes buffer or in PBS, subsequently assessing the capacity of the virus to fuse with liposomes at neutral pH. There was no detectable fusion under these conditions (results not shown). Conformational changes of the viral spike protein under fusion conditions. Finally, we investigated the structural changes occurring in the SIN envelope glycoprotein occurring in the presence of target liposomes under fusion conditions, in an attempt to determine the viral spike conformational requirements for fusion. To this end, the kinetics of fusion and the kinetics of the viral spike conformational changes were determined under comparable conditions. A complicating factor in this respect relates to the extremely high rates of the fusion reaction upon acidification of a SIN-liposome mixture at 37 °C to the optimal pH for fusion (pH 5.0). Therefore, for determination of the relative kinetics of virus- Low-pH-dependent fusion of Sindbis virus with liposomes 43 liposome fusion and the occurrence of spike conformational changes, we chose a pH of 5.75 and a temperature of 20 °C, conditions under which the kinetics of the process are slowed down considerably. Also, the fusion process at pH 5.75 at 20 °C exhibited a lag phase of approximately 9-10 s preceding the onset of fusion (results not shown; see Figure 8). Furthermore, the final extent of fusion was reduced. Figure 7 shows the time course of the structural changes occurring in the viral spike protein. Briefly, mixtures of [35S]methionine-labeled SIN, unlabeled SIN, and liposomes were incubated at pH 5.75 at 20 °C. At the indicated time points, the mixtures were neutralized by the addition of a pretitrated volume of NaOH. Subsequently, the samples were analyzed by SDS-PAGE (Figure 7A). Besides E1 and E2, another protein band became apparent. Determination of the size of the protein, in which a set of marker proteins was used, showed that the protein band had a molecular weight of 150 kD, indicating that a trimer of E1 was formed. To analyze the formation of a trypsin-resistant form of the viral spike protein, we incubated the mixtures in presence of trypsin (200 µg/ml) at 37 °C. After heating of the trypsin-treated samples for 5 min at 100 °C, they were analyzed by SDS- PAGE. Figure 7B shows the time course for the appearance of a trypsin-resistant phenotype of the E1 protein. Since the kinetics of the E1 trimer formation and that of the appearance of the trypsin-resistant phenotype of the E1 were similar, it is likely that it is in fact the trimerization of E1 that renders it trypsin-resistant.

0 2 5 10 15 20 30 60 120 time (s) A E1 trimer

E2/E1 B

E1

Figure 7. Kinetics of the structural changes in the SIN spike protein after incubation at low pH. SIN (trace of [35S]methionine-labeled virus and unlabeled virus, 0.5 µM phospholipid) was incubated with liposomes (200 µM liposomal lipid) consisting of PC/PE/SPM/Chol (molar ratio 1.0:1.0:1.0:1.5) at pH 5.75 at 20 °C. At the indicated time points, samples were neutralized and analyzed for the appearance of E1 trimers (A), and the appearance of a trypsin-resistant form of E1 (B), on SDS-PAGE, as described in Materials and Methods. 44 Chapter 2

Figure 8 presents a kinetic comparison of SIN-liposome binding (squares), E1 trimerization (circles), and fusion (triangles) during the initial 60 s following a pH jump from pH 7.4 to pH 5.75 at 20 °C. The extents of virus-liposome binding, fusion, and E1 trimerization at 60 s were 30, 14, and 56%, respectively. This indicates that not all of the virus bound to the liposomes under these suboptimal conditions also fused. In fact a fraction of the virus initially bound to the liposomes may subsequently dissociate, possibly explaining the relatively high degree of E1 trimerization. We were not able to detect differences between the kinetics of virus- liposome binding and those of E1 trimerization. However, fusion proceeded with a significant delay after the virus-liposome binding and E1 trimerization processes.

100 80 60 40 20 0 Percentage of final extent 0 20 40 60 Time (s) Figure 8. Sequence of events after acidification of a SIN-liposome mixture. The kinetics of SIN-liposome binding (squares), E1 trimerization (circles), and fusion (triangles) are shown after acidification to pH 5.75 at 20 °C. To compare the kinetics of these processes, the final extents of the relative values of the three parameters were set to 100%. The absolute final extents were 30% for SIN-liposome binding, 56% for E1 trimerization, and 14% for fusion. In each case, SIN (0.5 µM viral phospholipid) was incubated with liposomes (200 µM liposomal phospholipid) consisting of PC/PE/SPM/Chol (molar ratio 1.0:1.0:1.0:1.5) at pH 5.75 at 20 °C. SIN-liposome binding was determined as described in the legend to Figure 5. E1 trimerization was determined as described in the legend to Figure 7. Fusion was determined as described in the legend to Figure 1. Discussion

The results presented in this paper indicate that SIN has the capacity to fuse efficiently in a model system involving liposomes as target membranes. SIN- liposome fusion meets a stringent criterion for membrane fusion, i.e. coalescence of the internal encapsulated compartments of the interacting particles and a concomitant mixing of membrane lipids. Coalescence of the viral and liposomal internal contents was assessed on the basis of degradation of the viral core protein by trypsin initially encapsulated in the liposomes (42, 58). Membrane lipid mixing Low-pH-dependent fusion of Sindbis virus with liposomes 45 was monitored continuously using two variants of a fluorescence assay, based on incorporation of pyrene-labeled phospholipids in either the viral or the liposomal membrane. Incorporation of the probe into the viral membrane was achieved through a biosynthetic labeling procedure, involving production of virus from cells cultured beforehand in the presence of pyrene-labeled fatty acid. This methodology has been used before for labeling of SFV (4, 42, 55) and tick-borne encephalitis (TBE) virus (11). The present results demonstrate that the procedure can also be used reliably to produce pyrene-labeled SIN, without affecting the infectivity of the virus. SIN fuses efficiently with liposomes consisting of just phospholipids and cholesterol, lacking a specific protein or carbohydrate receptor for virus binding (Figures 1-4). Furthermore, SIN-liposome fusion is strictly dependent on low pH, fusion being optimal at pH 5.0 and undetectable at neutral pH (Figures 1-3). These characteristics of the fusion process argue strongly in favor of a cell entry mechanism of SIN, involving endocytosis of virus particles into endosomes and subsequent fusion of the viral membrane with the endosomal membrane induced by the mildly acidic pH in the lumen of the endosomes. SIN shares the capacity to fuse to receptor-free target liposomes with a number of other enveloped viruses, such as influenza virus (51, 52, 53), SFV (4, 25, 55, 58), TBE virus (11) and VSV (41). In all cases, low pH appears to be a necessary and sufficient condition for induction of the fusion process. The efficient fusion of low-pH-dependent viruses with receptor-free liposomes suggests that receptor binding is not a mechanistic requirement for expression of membrane fusion activity by these viruses. Receptor binding would appear to be primarily involved in the initial binding of the viruses to the host cell and subsequent endocytic uptake of the virus particles by the cell, although it cannot be excluded that the receptor interaction also influences the detailed characteristics of the subsequent fusion process from within the endosome. In this respect, it is interesting that, in the case of SIN, virus-receptor interaction has been found to result in conformational alterations in the viral envelope glycoprotein, detected on the basis of exposure of specific epitopes recognized by monoclonal antibodies (17, 38). These conformational changes have been suggested to be related to the viral fusion process. Clearly, our present results do not exclude that possibility. However, it would appear that low pH is the essential trigger for fusion of SIN. The conclusion that SIN infects its host cell by entry through receptor- mediated endocytosis and fusion from within acidic endosomes is in agreement with recent observations of Glomb-Reinmund and Kielian (20). These investigators showed that the addition of weak bases during cell entry of the virus efficiently inhibits translation of viral RNA and infection. Previous studies had suggested that weak bases would not inhibit cellular infection by SIN (7, 8). Glomb-Reinmund and Kielian (20) also used balifomycin and concanamycin, two reagents that prevent endosome acidification by a mechanism different from that 46 Chapter 2 of weak bases, and again found that cellular infection by SIN was inhibited, in further support of the conclusion that the infection process involves acidic endosomes. Furthermore, the authors were unable to detect a specific role for reduction of disulfide bridges through thiol-disulfide exchange reactions during the SIN entry process (20). It had been suggested before that disulfide shuffling upon interaction of SIN with its cell surface receptor would reorganize the viral spike to mediate virus cell entry through fusion with the plasma membrane (1, 5). Thus, the observations of Glomb-Reinmund and Kielian (20) argue against fusion with the cell plasma membrane as the physiological infection mechanism of SIN. Recently, DeTulleo and Kirchhausen (12) also came to the conclusion that SIN does not infect cells by plasma membrane fusion but rather through entry via an endocytic pathway. Specifically, these investigators employed expression of dominant- negative mutant forms of dynamin which inhibit clathrin-dependent endocytosis, and showed that these mutant dynamins also inhibit cellular infection by SIN. It is not clear what the explanation is for the above discrepancy between the observations which suggest that SIN enters cells by plasma membrane fusion and those that argue in favor of an endocytic entry mechanism. One possibility, suggested by DeTulleo and Kirchhausen (12) and Ferlenghi et al. (16), involves an undeliberate pre-exposure of the virus to low pH during freezing in cell culture medium or PBS. This would induce a premature conformational change in the viral spike, allowing subsequent fusion of the virus with the cell plasma membrane at neutral pH. Indeed, DeTulleo and Kirchhausen (12) observed that SIN, frozen under such inadequate buffering conditions, has the capacity to enter cells via a clathrin-independent pathway, suggestive of fusion through the plasma membrane. We were unable to detect any fusion of SIN at neutral pH, whether or not the virus had been frozen beforehand in PBS or medium. However, this does nor rule out the possibility of fusion with the plasma membrane at neutral pH of virus frozen under inadequate buffering conditions, since a low degree of fusion (on the order of 1% relative to the control) may well go unnoticed in our assay. The characteristics of SIN fusion in the present liposomal model system in many respects resemble those of SFV-liposome fusion (4, 42, 55, 59). Both viruses fuse with receptor-free liposomes in a low-pH-dependent manner, although we note that the pH optimum (pH 5.0) for fusion of the AR339 strain of SIN used here is lower by about 0.5 pH unit than that of SFV, in agreement with observations of Glomb-Reinmund and Kielian (20). Also, for the Toto 1101 infectious clone of SIN we found a low pH optimum (pH 4.6) for fusion (49). In cell-cell fusion studies the pH optimum for SIN AR339 has been reported to be 5.4 (2). Importantly, SIN, like SFV, requires the presence of both cholesterol and sphingolipid in the target membrane (Figure 4). Recently, in an elegant study, Lu et al. (34) have shown that SIN entry into cells also requires cholesterol. In the liposome system, cholesterol is essential for the initial low-pH-dependent binding of SFV to target liposomes, while the sphingolipid appears to be involved directly Low-pH-dependent fusion of Sindbis virus with liposomes 47 in the subsequent fusion reaction (42, 59). Specifically, extensive irreversible binding of SFV occurs to liposomes consisting of PC/PE/Chol, with fusion being undetectable; on the other hand, virtually no binding (nor fusion) occurs with PC/PE/SPM liposomes (42, 59). SIN appeared to behave in essentially the same manner (Figures 4 and 5), with virus binding to PC/PE or PC/PE/SPM liposomes being at background level and binding to PC/PE/Chol liposomes reaching 25% (Figure 5). On the other hand, the extent of virus binding to PC/PE/Chol liposomes was limited compared to the extent of binding to PC/PE/SPM/Chol liposomes (72%). One explanation would be that the interaction of the virus with PC/PE/Chol liposomes is not completely irreversible, such that in the absence of fusion part of the virus may dissociate. In the presence of Chol and SPM in the liposomes the interaction would become irreversible as a result of fusion subsequent to binding. Indeed, all of the virus that binds to liposomes containing both Chol and SPM appears to be fused as well. Yet, even under optimal conditions a small fraction of the virus does not seem to interact at all with the liposomes. It is possible that, upon exposure of the virus-liposome mixture to low pH, part of the virus may become inactivated so rapidly, that it does not have the opportunity to productively interact with the liposomes. The results shown in Figure 7 demonstrate that under fusion conditions, the E1 component of the SIN spike forms a homotrimeric structure, while at the same time E1 becomes trypsin-resistant. The relative kinetics of the E1 trimer formation and the appearance of the trypsin-resistant phenotype suggest that the E1 homotrimer and the trypsin-resistant form of E1 are in fact identical structures. By analogy to the role of the E1 trimer in SFV fusion (4, 25, 55), we propose that the SIN E1 homotrimer represents the fusion-active conformation of the viral spike. The kinetics of virus-liposome binding and E1 homotrimer formation are indistinguishable (Figure 8). For SFV, on the basis of early results, we have suggested that E1 homotrimer formation precedes virus-liposome binding (4). However, more recent observations involving selective inhibition of SFV E1 trimerization with Zn2+ ions (10) or through a mutation in the E1 protein (28) have shown that dissociation of the E2/E1 heterodimer at low pH suffices for initiation of virus-liposome binding and that E1 trimer formation occurs after the binding of the virus to the liposomes (10, 28). The results shown for SIN in Figure 8 are in agreement with this notion. Therefore, it would appear that E1 trimer formation is strongly facilitated by the association of the virus to the liposomes, occurring without any significant delay after the initial binding process. Under the conditions of the experiment (pH 5.75, 20 °C), fusion then proceeds after a lag period. This lag presumably represents the time required for additional rearrangements within or between homotrimeric E1 spikes. We propose that the target membrane sphingolipid is critically involved in this step, leading to the actual fusion-active structure of the virus. 48 Chapter 2

Acknowledgements

This work was supported by the US National Institutes of Health (grant HL 16660), and by The Netherlands Organization for Scientific Research (NWO) under the auspices of the Foundation for Chemical Research (CW). We thank Dr. Diane E. Griffin (Johns Hopkins University, Baltimore) for generously providing the Sindbis virus AR339 stock.

References

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Adaptation of Alphaviruses to Heparan Sulfate: Interaction of Sindbis and Semliki Forest Virus with Liposomes Containing Lipid-Conjugated Heparin

Jolanda M. Smit, Barry-Lee Waarts, Koji Kimata, William B. Klimstra, Robert Bittman, and Jan Wilschut

Provisionally accepted by Journal of Virology 54 Chapter 3

Abstract

Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycoaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 have been identified (E2:1; E2:70; E2:114), which mutate during adaptation and independently confer the ability to the virus to bind to cell-surface HS (W.B. Klimstra et al., J. Virol. 72:7357-7366, 1998). In this study, we used HS-adapted SIN mutants to evaluate a new model system, involving target liposomes containing heparin-conjugated phosphatidylethanolamine (HepPE) as an HS receptor analog for the virus. HS- adapted SIN viruses, but not non-adapted wild-type SIN TR339, interacted efficiently with HepPE-containing liposomes at neutral pH. Binding was competitively inhibited by soluble heparin. Despite the efficient binding of HS- adapted SIN viruses to HepPE-containing liposomes at neutral pH, there was no fusion under these conditions. Fusion did occur, however, at low pH, consistent with cellular entry of the virus via acidic endosomes. At low pH, wild-type or HS- adapted SIN viruses underwent fusion with liposomes with or without HepPE with similar kinetics, suggesting that interaction with the HS receptor analog at neutral pH has little influence on subsequent fusion of SIN at acidic pH. Finally, Semliki Forest virus (SFV), passaged frequently on BHK-21 cells, also interacted efficiently with HepPE-containing liposomes, indicating that SFV, like other alphaviruses, readily adapts to cell-surface HS. In conclusion, the liposomal model system presented in this paper may serve as a novel tool to study receptor interactions and membrane fusion properties of HS-interacting enveloped viruses. Heparan sulfate interaction and membrane fusion of alphaviruses 55

Introduction

Alphaviruses, such as Ross River virus (RR), Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus (VEE), are enveloped positive-strand RNA viruses belonging to the family of Togaviridae. The viral genome consists of a single-stranded RNA molecule, which is complexed with 240 copies of the capsid protein (50). The nucleocapsid is surrounded by a lipid bilayer in which the spike proteins are inserted. The viral surface contains 80 heterooligomeric spikes, a single spike consisting of a trimer of E2/E1 heterodimers. The E1 and E2 glycoproteins mediate the infectious entry of alphaviruses into cells. The E2 glycoprotein is primarily involved in the interaction of the virus particle with an attachment receptor on the cell surface (7, 28, 49), whereas E1 is required for the subsequent fusion process (19, 53). The spike proteins of RNA viruses are capable of rapid adaptation to their growth environment. Recently, it has been shown that viruses from different families interact with glycoaminoglycans (GAGs), in most cases heparan sulfate (HS), as a cell-culture adaptation. Virus families or genera that exhibit such a GAG-adaptation include alphaviruses (2, 21, 28), flaviviruses (33), pestiviruses (25), picornaviruses (16, 43), and (38, 41). GAGs are highly sulfated polymers of disaccharide repeats, and hence are negatively charged. They are ubiquitously expressed on cell surfaces, but vary with respect to their composition and quantity on different tissues and cell types (3, 52). In the alphavirus genus, positively charged amino-acid substitutions have been identified in the viral spike protein E2 of SIN, RR, and VEE that are responsible for interaction with HS (2, 21, 28). With regard to SIN, three loci in E2 have been identified (E2:1; E2:70; E2:114) that mutate during the adaptation of SIN in BHK- 21 cells and can independently confer the ability to bind to cell-surface HS (28). The sequence XBXBBBX or XBBXBX (where X is any residue and B is a basic residue) is a linear binding motif that allows proteins to attach to HS (9). The positive charge mutation at E2:1 results in the formation (although in opposite orientation) of a linear HS interaction sequence. The HS binding motifs are not present in the E2:70 and E2:114 regions, which suggest that these viruses interact with HS in a conformation-dependent manner. This phenomenon is known to occur in for foot-and-mouth disease virus type O, in which structural studies revealed that heparin makes contacts with all three major capsid proteins VP1, VP2, and VP3 (18). Despite the efficient interaction of the selected mutants of SIN, VEE, and RR with HS, the viruses were found to have an attenuated virulence in animals when compared to wild-type viruses. It has been proposed that HS-adapted mutants could bind to non-productive cellular structures, such as extracellular membranes and basal laminae, and therefore may be cleared from the blood more rapidly than wild-type viruses (2, 8, 21). 56 Chapter 3

In previous studies, membrane fusion activity of SIN, SFV, and Tick-borne encephalitis virus (TBE) has been investigated using liposomes lacking a protein or carbohydrate receptor in the target membrane (5, 13, 46, 47). This suggests that receptor interaction is not a prerequisite for fusion. However, characteristics of virus-liposome fusion in the presence of an attachment receptor have not been studied. In this study, we used HS-adapted SIN mutants to evaluate a new model system, involving target liposomes supplemented with phosphatidylethanolamine- conjugated heparin (HepPE) as an attachment receptor analog for the virus. With HepPE in the target membrane, we were able to directly investigate the role of HS receptor interaction and its potential function in triggering or influencing membrane fusion of HS-adapted SIN with target membranes. It is demonstrated that HS-adapted SIN efficiently interacts with liposomes supplemented with remarkably low concentrations of HepPE in the membrane. Despite the efficient binding at neutral pH, there was no fusion under these conditions. Fusion was observed only at low pH, consistent with cell entry of SIN via acidic endosomes. Finally, it is shown that SFV, either passaged frequently in baby hamster kidney (BHK-21) cells or derived from the BHK-adapted infectious clone pSFV4, interacts efficiently with HepPE-containing liposomes, indicating that SFV like SIN readily adapts to cell-surface HS.

Materials and Methods

Viruses. The viruses were generated from cDNA clones. The construction of the consensus Sindbis virus AR339 clone pTR339 and the HS-adapted SIN virus clones p3970 (called p39K70 in previous articles) and pTRSB has been described previously (28, 29, 36). The construction of the SFV clone pSFV4 has been described before (31). This clone was generated from a laboratory strain of SFV, adapted to growth on BHK-21 cells. A plaque- purified laboratory strain of SFV, also highly adapted to growth on BHK-21 cells, was a generous gift of Dr. Margaret Kielian (Albert Einstein College of Medicine, New York, NY). The viruses were produced by high-efficiency electroporation of BHK-21 cells with in vitro transcripts of linearized cDNA clones as described before (31). Viruses released from the cells at 20 h post-transfection were harvested, and these stocks were subsequently used for the production of pyrene- or [35S]methionine-labeled SIN or SFV particles, as previously described (5, 46). The viruses were characterized by plaque assay on BHK-21 cells (28), phosphate analysis (4), and protein determination (42). The purity of the viruses was confirmed by SDS-PAGE. Liposomes. Large unilamellar vesicles were prepared by n-octyl-ß-D-glucopyranoside (OGP; Calbiochem, Darmstadt, Germany) dialysis followed by a freeze/thaw-extrusion protocol (5, 40, 46). This method is used, instead of the standard extrusion method, because HepPE is not completely soluble in a chloroform-methanol solution. Liposomes consisted of phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SPM), and cholesterol (Chol) or 6-photocholesterol (photoChol) in a molar ratio of 1:1:1:1.5, supplemented with HepPE as indicated. Briefly, PC/PE/SPM/Chol lipid Heparan sulfate interaction and membrane fusion of alphaviruses 57 mixtures were dried from chloroform-methanol and hydrated in 100 mM OGP in 5 mM HEPES, 150 mM NaCl, 0.1 mM EDTA, pH 7.4 (HNE). Subsequently, HepPE, was added to the lipid-detergent mixed micelles and dialysis was initiated against HNE buffer to generate liposomes. The liposomes were subjected to five cycles of freezing/thawing and subsequent extrusion through 0.2-µm polycarbonate filters (Nuclepore Inc., Pleasanton, CA, USA) in a LipoFast mini-extruder (Avestin, Ottawa, Canada). PhotoChol-containing liposomes were prepared in subdued light. The size of the liposomes was determined by quasi-elastic light scattering analysis in a submicron particle sizer model 370 (Nicomp Particle Sizing systems, Santa Barbara, Calif.). The phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL, USA), and cholesterol was from Sigma (St. Louis, Mo, USA). The photoactivatible analog of cholesterol, photoChol, was synthesized as described before (37). The HepPE conjugate, consisting of heparin (from porcine intestinal mucosa, MWav 10,000; Scientific Protein Laboratories, Wannakee, WI) coupled to dipalmitoyl- phosphatidylethanolamine was synthesized and purified as described previously (51). Trypsin-containing liposomes were prepared in a similar manner as outlined above, except that this case the lipids were dispersed in 100 mM OGP in HNE containing 10 mg/ml trypsin (Boehringer, Mannheim, Germany). The trypsin-containing liposomes were separated from free trypsin by gel filtration on a Sephadex G-100 column in HNE. The phospholipid concentration of the liposomes was determined by phosphate analysis (4). Bindings assays. Virus binding to BHK-21 cells and heparin-, bovine serum albumin- agarose beads (both from Sigma) was preformed essentially as previously described (28, 48). In the binding assay, 105 to 106 cpm of [35S]methionine-labeled SIN or SFV (approx. 108 to 109 virus particles) was allowed to attach to monolayers of BHK-21 cells or beads for 1 h at 4 °C. Subsequently, the cells or beads were washed with HNE + 1% FBS buffer. Virus binding was quantified by liquid-scintillation counting. Binding of the virus to liposomes was assessed by a co-flotation assay, as described before (5, 39, 46). Briefly, [35S]methionine-labeled SIN or SFV particles (ranging from 105 to 106 cpm) were mixed with liposomes (100 µM phospholipid) and incubated for 1 h at 4 ºC, unless indicated otherwise. Then, 0.1 ml of the mixture was added to 1.4 ml 50% sucrose (wt/vol) in HNE and loaded onto a discontinuous (60-50-35-20-5%, wt/vol) sucrose gradient. After ultracentrifugation at 4 ºC, the gradient was fractionated into 10 samples, starting from the top. The radioactivity found in the top 4 fractions, relative to the total amount of radioactivity, was taken as a measure of virus-liposome binding. For the heparin binding competition experiments, heparin (MWav 6,000; Sigma) was added to the SIN particles 1 h prior to mixing with liposomes, and the mixture was incubated at 4 ºC. Fusion assays. Fusion of pyrene-labeled SIN or SFV with liposomes was measured on-line at 37 ºC in an AB2 fluorometer (SLM/Aminco, Urbana, USA) at excitation and emission wavelengths of 345 and 480 nm, respectively (5, 46, 48). Briefly, pyrene-labeled SIN or SFV (1 µM phospholipid) and liposomes (100 µM phospholipid) were mixed in 0.665 ml of HNE buffer and stirred magnetically in a quartz cuvette. At t=0 s, fusion was triggered by injection of 35 µl 0.1 M MES (morpholinoethanesulfonic acid) and 0.2 M acetic acid buffer, pre-titrated with NaOH to achieve the final desired pH. Fusion was calibrated such that 0% fusion corresponded with to the initial pyrene-excimer fluorescence and 100 % fusion was obtained after the addition of 35 µl 0.2 M octaethylene glycol monododecyl ether (Fluka, Buchs, Switzerland), to achieve an infinite dilution of the 58 Chapter 3 pyrene probe. The initial rate of fusion was determined from the tangent to the initial phase of the curve. The extent of fusion was determined 60 s after acidification. The mixing of the aqueous contents of interior of virus and the liposomal lumen was determined as the degradation of the viral capsid protein by trypsin, initially encapsulated in the liposomal lumen (54, 46, 47). Briefly, [35S]methionine-labeled SIN particles (ranging from 105 to 106 cpm) were mixed with trypsin-containing liposomes (100 µM phospholipid) in presence of 125 µg of trypsin-inhibitor (Boehringer, Mannheim, Germany) per ml in HNE, at 37 ºC. The mixture was acidified to the desired pH, as described above. After 60 s the reaction mixture was neutralized by the addition of a pre- titrated volume of NaOH and further incubated for 1 h at 37 ºC. Control incubations were carried out with empty liposomes, or in the presence of Triton X-100 and in absence of trypsin inhibitor. All samples were analyzed by SDS-PAGE, the protein bands being visualized and quantified by phosphorimaging analysis using Image Quant 3.3 software (Molecular Dynamics, Sunnyvale, CA, USA). Capsid degradation was determined by relating the intensity of the capsid protein to the intensity of E1 and E2 in a control experiment in which empty liposomes were used. This ratio was used to calculate the expected intensity of the capsid protein from the reaction in which trypsin-containing liposomes were used. The difference between the expected and the found intensity was taken as a measure of the capsid degradation.

Results

Characterization of HS-adapted SIN mutants. In this study we investigated HS interaction and membrane fusion activity of alphaviruses in a liposomal model system using lipid-conjugated heparin. Heparin is a sulfated polysaccharide, which is commonly used as an analog for HS in receptor-ligand assays since ligand interaction with heparin and analogs of HS have little qualitative difference (27).

TABLE 1. Amino acid differences of HS adapted Sindbis viruses Virus nsP3:528 E2:1 E2:70 E1:72

TR339 Arg Ser Glu Ala 3970 Arg Ser Lys Ala TRSB Gln Arg Glu Val

To evaluate the new liposomal model system a comparison was made between the HS-adapted SIN mutants 3970 and TRSB, and the non-adapted SIN TR339 (carrying the consensus sequence of SIN) (28, 29, 36). The HS-adapted SIN Heparan sulfate interaction and membrane fusion of alphaviruses 59 mutant designated 3970 differs from the TR339 clone at position E2:70 (Table 1). The other HS-adapted SIN strain, TRSB, differs from TR339 by a positive charge amino-acid substitution at position E2:1 and a conserved valine for alanine substitution at position E1:72 (Table 1). First, the specific infectivity of each of the viruses was determined by plaque assay on BHK-21 cells (Table 2). In agreement with earlier data, the specific infectivity of TR339 was much lower than the infectivities of the HS-adapted mutants (28, 29). Next, we determined whether the high specific infectivity of 3970 and TRSB was a result of more efficient binding to BHK-21 cells (Table 2). The results show that the HS-adapted mutants 3970 and TRSB bind very efficiently to monolayers of BHK-21 cells, whereas the TR339 virus binds very poorly to these cells (Table 2). Accordingly, TRSB and 3970 virus bound efficiently to heparin- agarose beads (Table 2). In a control, in which albumin-agarose beads were used, none of the viruses bound to the beads (data not shown).

TABLE 2. Characterization of HS adapted Sindbis viruses Virus BHK specific BHK cell HS-agarose infectivitya bindingb beads bindingc

TR339 5 7 10 3970 256 42 50 TRSB 186 51 56

a (PFU/cpm) b % cpm bound c % cpm bound

Binding of HS-adapted SIN to HepPE-containing liposomes. To study the receptor interaction of HS-adapted SIN mutants, we used target liposomes in which HepPE was incorporated in the membrane. Liposomes consisted of PC/PE/SPM/Chol (molar ratio 1:1:1:1.5) with various concentrations of HepPE. In the binding experiment, [35S]methionine-labeled SIN (approx. 108-109 virus particles) was incubated with the liposomes (100 µM phospholipid) at neutral pH, for 1 h at 4 ºC. Subsequently, the liposome-bound virus was separated from non- bound virus by flotation on a sucrose-density gradient. Figure 1A shows the gradient profiles. In the presence of liposomes supplemented with 0.02 mol% HepPE, essentially all SIN 3970 particles floated to the top of the gradient (circles), demonstrating that the virus bound quantitatively to the liposomes. Half- 60 Chapter 3 maximal binding was observed with liposomes supplemented with 0.01 mol% HepPE (diamonds). The virus did not bind to liposomes without HepPE in the membrane (squares). Figure 1B shows the final extent of binding of SIN 3970 to HepPE-containing liposomes, plotted as a function of the molar ratio of HepPE to total phospholipid in the liposomal membrane. Clearly, the binding increased steeply at a ratio of 1 HepPE molecule per 10,000 phospholipid molecules, while binding was maximal at a ratio of 1:5000. A liposome of 200 nm diameter, consisting of phospholipid and cholesterol in a molar ratio of 2:1, has approximately 150,000 phospholipid molecules in its outer membrane leaflet (55). Thus, maximal binding of SIN 3970 to HepPE-containing liposomes was obtained with on average only 30 HepPE molecules exposed on the outer surface of a target liposome, while half-maximal binding occurred with approx. 15 surface-exposed HepPE molecules per liposome. It should be noted that in all likehood the liposomes used in the present study were, on average, smaller than 200 nm, as discussed in more detail below. This implies that the number of HepPE molecules per liposome was proportionately smaller as well.

40000

) A 100 B

cpm 30000 80 ( 60 20000 40

10000 Binding (%) 20

Radioactivity 0 0 1 2 3 4 5 6 7 8 9 10 10-9 10-7 10-5 10-3 10-1 Fraction Ratio HepPE/phospholipid Figure 1. Binding of HS-adapted SIN 3970 to HepPE-containing liposomes. [35S]Methionine-labeled SIN (approx. 108-109 virus particles) was incubated with PC/PE/SPM/Chol liposomes (100 µM liposomal phospholipid) at pH 7.4 for 1 h at 4 °C. Binding was determined by co-flotation analysis on sucrose density gradients, as described in Materials and Methods. (A) Gradient profiles obtained after incubation of virus with control liposomes lacking HepPE (squares), liposomes supplemented with 0.01 mol% (diamonds) or liposomes supplemented with 0.02 mol% HepPE (circles). (B) Extents of binding of SIN to HepPE-containing liposomes as a function of the molar ratio HepPE to total lipid in the liposomes. Bars represent the average of triplicate binding assays.

Next, a comparison was made between the liposome binding capacity of HS- adapted SIN 3970 and TRSB and non-adapted SIN TR339, using liposomes with and without 0.01 mol% HepPE in the membrane. Figure 2 shows the results. For Heparan sulfate interaction and membrane fusion of alphaviruses 61

SIN 3970 and TRSB more than half-maximal binding to the HepPE-containing liposomes was observed, whereas SIN TR339 bound very poorly to these liposomes (shaded bars). There was no binding of any of the viruses to liposomes lacking HepPE in the membrane (solid bars). Figure 2B presents the binding of SIN to liposomes supplemented with 0.01 mol% HepPE after incubation during various periods of time either in the cold or at 37 ºC. Binding of the HS-adapted SIN viruses was fast and efficient at various temperatures. Complete binding of the viruses to HepPE-containing liposomes was observed even after 1 min incubation at 37 ºC. Again, non-adapted SIN TR339 bound poorly to the liposomes, although both at 4 ºC and at 37 ºC there was a detectable degree of binding presumably due to a low affinity of the TR339 virus for HS (confer Table 2).

80 80 without HepPE A B 60 0.01 mol% HepPE 60

40 40 Binding (%)

20 Binding (%) 20

0 0 TR339 3970 TRSB a b c Figure 2. Binding of HS-adapted SIN 3970 or TRSB and non-adapted SIN TR339 to liposomes supplemented with 0.01 mol% HepPE. The extent of binding was determined as described in the legend to Fig.1, unlesss indicated otherwise. (A) Binding for 1 h at 4 °C; solid bars, PC/PE/SPM/Chol liposomes; shaded bars, PC/PE/SPM/Chol liposomes supplemented with 0.01 mol% HepPE. (B) Binding was measured at 1 h 4 ºC (a); 1 min 37 ºC (b); 10 min 37 ºC (c). Solid bars, TR339; shaded bars, 3970; open bars, TRSB. Bars represent the average of triplicate binding assays.

Competition of SIN binding to HepPE-containing liposomes by soluble heparin. To determine whether the HS-adapted SIN viruses interact specifically with the heparin moiety on the liposomal membrane, binding competition experiments were carried out with soluble heparin. SIN was incubated with soluble heparin for 1 h at 4 ºC. Subsequently, HepPE-containing liposomes were added to the reaction mixture and incubation was continued for 1 h at 4 ºC in the presence of the soluble heparin. Liposome-bound virus was separated from non-bound virus by flotation on a sucrose-density gradient..Figure 3 shows the results. In the presence of 5 mg/ml heparin, SIN 3970 virus failed to float with the liposomes to the top of the gradient (squares), while in the absence of soluble heparin efficient 62 Chapter 3 binding of the virus to the liposomes was observed (diamonds). Clearly, soluble heparin blocks binding of the virus to HepPE-containing liposomes, indicating that HS-adapted SIN specifically interacts with the heparin moiety on the liposomal membrane. It is of interest that maximum competition of SIN 3970 binding to HepPE-containing liposomes was achieved only at relatively high concentrations of soluble heparin (5 mg/ml). At lower soluble heparin concentrations, partial competition was observed (results not shown). This indicates that the interaction between HS-adapted SIN and HepPE-containing liposomes is very tight, suggesting that multiple interactions between a single virus and several HepPE molecules on the liposomal membrane are involved.

25000 no heparin ) 20000 5 mg/ml sol heparin cpm 15000

10000

5000 Radioactivity ( 0 1 2 3 4 5 6 7 8 9 10 Fraction Figure 3. Effect of soluble heparin on binding of HS-adapted SIN 3970 to PC/PE/SPM/Chol liposomes supplemented with 0.01 mol% HepPE at pH 7.4, 1 h at 4 °C. Binding was determined as described in the legend to Fig.1. Curves: squares, binding in the presence 5 mg/ml soluble heparin; diamonds, without soluble heparin.

Fusion activity of pyrene-labeled SIN with liposomes with or without HepPE in the target membrane. It has been suggested that receptor interaction, rather than low pH, triggers conformational changes in the viral spike protein of SIN which would subsequently lead to fusion of the viral envelope with the plasma membrane of the cell (1, 17, 24). To study the membrane fusion activity of SIN upon interaction with the HepPE attachment receptor, fusion was measured in a direct manner using pyrene-labeled virus, as described previously (46, 47). Pyrene- labeled SIN 3970 or TRSB (1 µM phospholipid) and PC/PE/SPM/Chol liposomes supplemented with 0.01 mol% HepPE (100 µM phospholipid) were mixed, with continuous stirring, and incubated for 1 min at 37 ºC, pH 7.4, to achieve binding of the virus to the liposomes (see Figs. 1 and 2). While under these conditions with 50-60% of the viruses bound to the HepPE receptor on the liposomal target membrane, there was no detectable fusion (Figure 4, curves b, c). Heparan sulfate interaction and membrane fusion of alphaviruses 63

In a control experiment, in which non-adapted SIN TR339 was used, there was no fusion at neutral pH either (curve a). However, all three viruses fused rapidly and efficiently with HepPE-containing liposomes at pH 5.0. Under these conditions, a decrease of pyrene-excimer fluorescence intensity of 50% was observed. This indicates that SIN bound to HepPE-containing liposomes at neutral pH becomes fusion-active only upon exposure to acidic pH.

25 60 A B a,b,c 20 40 pH 5.0 (%) 15 TR339 3970 TRSB 20 10 Fusion pH 7.4 5 0 a,b,c 0 Initial rate of fusion (%) 0 20 40 60 4 5 6 7 8 Time (s) pH

25 C 20 15 TR339 3970 10 TRSB 5 0 Initial rate of fusion (%) 4 5 6 7 8 pH Figure 4. Low-pH-dependent fusion of HS-adapted SIN mutants with liposomes. On- line fusion experiments were performed at 37 °C, as described in Materials and Methods. The final virus and liposome concentrations were 1.0 and 100 µM (membrane phospholipid), respectively. (A) shows fusion curves SIN with PC/PE/SPM/Chol liposomes supplemented with 0.01 mol% HepPE; curves a, TR339; curves b, 3970; curves c, TRSB. (B) and (C) show the initial rate of fusion, as determined from the tangent to the first part of the curve, of SIN TR339 (diamonds) and HS-adapted 3970 (squares) and TRSB (circles). (B) fusion with PC/PE/SPM/Chol liposomes supplemented with 0.01 mol% HepPE; (C) fusion with control PC/PE/SPM/Chol liposomes without HepPE. All fusion measurements were repeated at least three times. 64 Chapter 3

To further establish that SIN, bound to HepPE in target liposomes, retains the capacity to fuse at low pH, fusion was measured on isolated virus-liposome complexes. Pyrene-labeled SIN 3970 was incubated with HepPE-containing liposomes for 1 h at 4 °C. Then, liposome-bound virus was separated from non- bound virus by flotation on a sucrose-density gradient. Subsequently, the membrane fusion activity of the virus-liposome complex was measured at pH 5.0. Under these conditions, the liposome-bound virus fused rapidly and efficiently, indicating that virus, pre-bound to target liposomes through the interaction with HepPE at neutral pH, remains fully fusion-active when exposed to acidic pH (data not shown). Figure 4B shows the initial rates of fusion of SIN TR339, 3970, and TRSB with liposomes supplemented with 0.01 mol% HepPE as a function of the pH of the medium. Similar fusion kinetics were observed for the HS-adapted SIN 3970 and TRSB versus the non-adapted SIN TR339. Furthermore, using target liposomes without HepPE, we observed indistinguishable fusion kinetics for HS- adapted SIN viruses and non-adapted SIN TR339 (Figure 4C). Clearly, all of the SIN viruses used fuse with liposomes in a strictly low-pH-dependent manner, exhibiting similar fusion kinetics irrespective of the presence of HepPE in the target membrane. The decrease of pyrene excimer fluorescence intensity by approx. 50%, seen in the above measurements, corresponds to 50% fusion under the assumption that when a virus particle fuses with a liposome the pyrene probe is diluted infinitely. However, upon fusion of a virus particle with a comparatively small liposome, a residual excimer fluorescence intensity will remain, implying that in this case the actual extent of fusion may be underestimated in the pyrene assay. In this respect it is important to note that the liposomes produced by the OGP dialysis method, as used in our present experiments, tend to be smaller than the liposomes we use routinely (5, 46, 48). Moreover, inclusion of increasing concentrations of HepPE (> 0.02 mol%) in the membrane results in a further reduction of the size of the liposomes, as judged by a decreasing opalescence of the preparation. As a consequence, it is likely that, using the pyrene excimer fusion assay under these conditions, one in fact underestimates the extent of fusion due to incomplete dilution of the fluorophore. Recently, we have shown that a photoactivatable analog of cholesterol, 6- photocholesterol (photoChol), has the capacity to reversibly quench pyrene excimer and monomer fluorescence intensity (37). With this compound we were able to investigate directly whether the pyrene assay indeed does underestimate the actual extent of fusion. Fusion of pyrene-labeled SIN with photoChol-containing liposomes is not only monitored on the basis of dilution but also on the basis of quenching of the pyrene probe in the target membrane. Figure 5 shows the results. Clearly, at pH 5.0, fusion of SIN 3970 with liposomes consisting of PC/PE/SPM/photoChol with or without 0.01 mol% HepPE (curves a,b) Heparan sulfate interaction and membrane fusion of alphaviruses 65 appeared more rapid and more efficient than fusion with corresponding liposomes containing regular cholesterol (curves c,d). With photoChol-containing liposomes, the initial rate of fusion was extremely fast: 35-40% of the virus particles underwent fusion with the liposomes within the first second after acidification. Furthermore, the apparent extent of fusion was over 70%. Taken together, these results indicate that the extent of fusion of SIN with comparatively small liposomes, as assessed by the regular pyrene assay, represents an underestimation of the actual extent of fusion.

80 a,b 60 c,d 40

Fusion (%) 20

0

0 20 40 60 Time (s) Figure 5. Fusion of pyrene-labeled SIN 3970 with liposomes, containing photocholesterol, at pH 5.0. Fusion was measured on-line at 37 °C as described in the legend to Fig. 4. Curves a, PC/PE/SPM/photoChol liposomes supplemented with 0.01 mol% HepPE; curves b, PC/PE/SPM/photoChol liposomes; curves c, PC/PE/SPM/Chol liposomes supplemented with 0.01 mol% HepPE; curves d, PC/PE/SPM/Chol liposomes. All fusion measurements were repeated at least two times.

Contents mixing during fusion of SIN with trypsin-containing liposomes. To further quantify the extent of SIN-liposome fusion under the conditions of our experiments, we also applied an entirely different fusion assay based on mixing of the interior of the virus with the liposomal lumen. Contents mixing was measured as the degradation of the viral capsid protein by trypsin, encapsulated in target liposomes, in the presence of trypsin inhibitor in the medium (46, 47, 54). [35S]Methionine-labeled SIN, either HS-adapted SIN 3970 or non-adapted SIN TR339, and trypsin-containing supplemented with 0.01 mol% HepPE liposomes (100 µM phospholipid) were incubated for 1 min at 37 ºC, pH 7.4, to allow the virus to bind to the liposomes. Figure 6 shows that there was very little capsid degradation under these conditions (lanes a). This, again, demonstrates that virus-receptor interaction at neutral pH does not induce fusion of the virus 66 Chapter 3

A

E1/E2

C

a b c d e f

B

E1/E2

C

a b c d e f

100 100 C TR339 D

TR339 (%) (%) 80 80 3970 3970 TRSB 60 TRSB 60

40 40

20 20 Capsid degradation Capsid degradation 0 0 pH 7.4 pH 5.0 pH 7.4 pH 5.0

Figure 6. Transfer of the viral capsid into the liposomal lumen assayed as the degradation of the viral capsid protein. [35S]methionine-labeled SIN (approx. 108-109 virus particles) was incubated with trypsin-containing PC/PE/SPM/Chol liposomes supplemented with 0.01 mol% HepPE (100 µM liposomal phospholipid) at 37 °C, and viral capsid protein degradation was determined, as described in Materials and Methods. (A) and (B) show the results for HS-adapted SIN 3970 and non-adapted SIN TR339, respectively. Lanes a and d, trypsin-containing liposomes; lanes b and e, empty liposomes; lanes c and f, trypsin-containing liposomes in presence of TX-100 and absence of trypsin inhibitor in the medium. Lanes a-c, pH 7.4; lanes d-f, pH 5.0. (C) and (D) show the quantification of the extent of capsid protein degradation. Solid bars, TR339; shaded bars, 3970; open bars, TRSB. (C), target liposomes supplemented with 0.01 mol% HepPE; (D), target liposomes without HepPE. All capsid degradation experiments were repeated at least two times. Heparan sulfate interaction and membrane fusion of alphaviruses 67 with target liposomes. On the other hand, when SIN 3970 or SIN TR339 were incubated with the liposomes at pH 5.0, almost all of the capsid protein was degraded (lanes d). In control experiments, in which SIN was incubated with empty HepPE-supplemented liposomes at pH 7.4 or pH 5.0, no capsid degradation was observed (lanes b,e). The ratio of the radioactivity of the capsid band relative to the total amount of radioactivity was close to 0.4, as expected on the basis of the number of methionine residues in the structural proteins of SIN. Complete capsid degradation was observed when Triton X-100 was added to the reaction mixture, in absence of trypsin inhibitor in the medium (lanes c,f). The extent of capsid degradation, as function of the pH, for HS-adapted SIN 3970 and TRSB or non-adapted SIN TR339 after fusion with HepPE-containing liposomes at different pHs is shown in panel 6C. For SIN TR339 (solid bars), little to no capsid degradation was observed at pH 7.4, whereas at pH 5.0 82% of the capsid protein was degraded. For HS-adapted SIN 3970 (shaded bars) and TRSB (open bars), similar results were obtained. Furthermore, in all cases the extent of capsid degradation after fusion of the viruses with liposomes without HepPE was similar to that observed upon fusion with HepPE-containing liposomes (Figure 6D). Interestingly, the extents of fusion measured with the trypsin assay closely correspond to those observed with the pyrene assay in the presence of photoChol in the target membrane (confer Fig. 4). This underlines the above conclusion that the regular pyrene assay underestimates the actual extent of fusion due to incomplete dilution of the probe into the comparatively small dialysis liposomes. Clearly, upon exposure to low pH, under the conditions of our experiments the large majority of the viruses fuse with the liposomes whether or not these liposomes contain the HepPE attachment receptor. Interaction of SFV with HepPE-containing liposomes. Next, we addressed the question whether SFV, another member of the genus Alphaviruses, has the capacity to adapt to HS during passage in cell culture. To this end, we used SFV derived from the infectious clone pSFV4 as well as a strain of virus passaged many times on BHK-21 cells. The pSFV4 clone was generated from a laboratory strain of SFV, which had also been passaged frequently on BHK-21 cells (31). HS adaptation of SFV derived from the infectious clone pSFV4 was evaluated in binding assays. Figure 7A shows that SFV from pSFV4 bound efficiently to monolayers of BHK-21 cells. To assess the specificity of this interaction, we used heparin- versus albumin-agarose beads as cell surrogates in suspension binding assays (28). The results show that SFV bound efficiently to heparin-agarose beads, whereas the virus did not bound to albumin-agarose beads (Figure 7A). Figure 7B shows that pSFV4-derived virus bound efficiently to HepPE- containing liposomes at neutral pH. The extents of binding were very similar to those obtained with HS-adapted SIN (confer Fig. 1A). With liposomes supplemented with 0.02 mol% HepPE, more than 90% of the virus bound, while with 0.01 mol% HepPE in the target liposomes half-maximal binding was 68 Chapter 3 observed. There was no binding to liposomes lacking HepPE in the membrane. Very similar results were obtained with a plaque-purified laboratory-adapted strain of SFV (results not shown). Taken together, these results demonstrate that SFV passaged frequently on BHK-21 cells, is strongly adapted to interaction with HS.

60 100 A B 80 40 60

40 20 Binding (%) Binding (%) 20

0 0 a b c 0 0.01 0.02 HepPE (mole %) Figure 7. Interaction of SFV with BHK-21 cells, heparin-agarose beads and HepPE- containing liposomes. (A) [35S]methionine-labeled SFV particles (approx. 108-109 virus particles) were added to BHK-21 cell monolayers or heparin- or albumin-agarose beads, and binding was measured after incubation for 1 h at 4 °C, as described in Materials and Methods. Bar a, binding to BHK-21 cells; bar b, binding to heparin-agarose beads; bar c, binding to albumin-agarose beads. (B) Binding of [35S]methionine-labeled SFV (approx. 108-109 virus particles) to PC/PE/SPM/Chol liposomes supplemented with various concentrations HepPE (100 µM liposomal phospholipid) at pH 7.4, for 1 h at 4 °C. Binding of SFV to liposomes was assessed as decribed in the legend to Fig. 1. Each bar represents the average of triplicate binding assays.

Finally, we investigated the membrane fusion activity of SFV upon interaction with HepPE-containing liposomes. Figure 8 shows the results. Interaction of pyrene-labeled SFV with HepPE in the liposomal membrane did not result in fusion at neutral pH (curve c). However, the virus fused rapidly and efficiently with HepPE-containing liposomes at pH 5.5 (curve a). Moreover, similar fusion kinetics were observed with or without HepPE in the target liposomes 5.5 (curve a versus b). There was no fusion of SFV with liposomes lacking the HepPE receptor analog at neutral pH (curve d). Heparan sulfate interaction and membrane fusion of alphaviruses 69

60 a,b 40 pH 5.0

20 Fusion (%) pH 7.4 0 c,d

0 20 40 60 Time (s) Figure 8. Low-pH-dependent fusion of pyrene-labeled SFV with liposomes. Fusion of pyrene-labeled SFV with PC/PE/SPM/Chol liposomes with or without 0.01 mol% HepPE (100 µM liposomal phospholipid) at 37 °C was determined as described in the legend to Fig. 4. Curves a, PC/PE/SPM/Chol liposomes supplemented with 0.01 mol% HepPE; curves b, PC/PE/SPM/Chol liposomes; curves c, PC/PE/SPM/Chol liposomes supplemented with 0.01 mol% HepPE; curves d, PC/PE/SPM/Chol liposomes. All fusion measurements were repeated at least three times. Discussion

The study presented in this paper evaluates a new liposomal model system in which a lipid-conjugated heparin (HepPE) is incorporated in the target membrane as an attachment receptor for HS-adapted alphaviruses. The sulfated polysaccharide heparin is commonly used as an analog for HS in receptor-ligand assays, since ligand interaction with heparin and analogous HS have little qualitative difference (27). It is demonstrated here that HS-adapted SIN 3970 and TRSB, at neutral pH, interact efficiently with liposomes supplemented with remarkably low levels of HepPE in the membrane (Figures 1 and 2). Without HepPE in the target membrane the HS-adapted SIN viruses were unable to bind to the liposomes, indicating that the viruses bind specifically to the heparin molecule. Furthermore, SIN strain TR339, which is not adapted to HS, was unable to bind to HepPE-containing liposomes under the same conditions. Moreover, binding competition experiments showed that soluble heparin blocked binding of HS-adapted SIN to HepPE liposomes, further underlining the notion these viruses specifically interact with the lipid-conjugated heparin moiety on the liposomal membrane. Despite the efficient interaction of SIN with HepPE-containing liposomes at neutral pH, there was no fusion under these conditions, as measured with the pyrene- and the trypsin-assay (Figures 4 and 6), as discussed in more detail below. The interaction of HS-adapted SIN with HepPE-containing liposomes is extremely efficient. Half-maximal binding was observed with liposomes containing as little as 0.01 mol% HepPE in the membrane, demonstrating that about 15 70 Chapter 3

HepPE molecules on the outer surface of a liposome are sufficient for efficient binding of the virus to the liposomal membrane. Almost no binding was observed when, on average, 1-2 HepPE molecules were incorporated in a single liposome. Therefore, we hypothesize that a SIN virus particle, after initial binding to a single HepPE molecule in the liposomal membrane, subsequently recruits more HepPE molecules to the site of interaction, resulting in multiple interactions between the virion and most if not all of the HepPE molecules at the liposome surface. This hypothesis is substantiated by the observation that a high concentration of soluble heparin was required for competition. This concentration was at least an order of a magnitude greater than that required for complete competition of binding of HS- adapted SIN to BHK-21 cells (28). An explanation for this difference could be that binding of HS-adapted SIN to cells involves fewer interactions with HS per virus particle than binding to HepPE-supplemented liposomes. This may be related to a limited mobility of HS-carrying core proteins on the cell surface, possibly restricting recruitment of multiple HS moieties to the site of interaction (27). There is convincing evidence to indicate that SIN, like SFV (22, 23, 34, 35), infects its host cell by receptor-mediated endocytosis and subsequent fusion from within acidic endosomes (15, 20, 46, 47). In this regard, it is intriguing to note that recently Hernandez and coworkers (24) published a paper, supporting earlier data (1, 6, 17), suggesting that exposure to an acidic compartment within cells may not be an obligatory step in alphavirus infection. Accordingly, it has been proposed that virus-receptor interaction triggers conformational changes in the viral spike protein, inducing fusion of the viral membrane with the plasma membrane of the cell. The results presented in this study clearly demonstrate that, despite the efficient interaction of SIN with the HepPE receptor in target liposomes at neutral pH, there is no fusion under these conditions. This indicates that the presence in the liposomal membrane of HepPE, an analog of the HS attachment receptor used by cell culture-adapted strains of SIN, has no functional role in triggering membrane fusion activity of SIN at neutral pH. Therefore, it appears that the sole requirement for SIN-liposome fusion, even after interaction with the HS receptor analog, is exposure of the virus to low pH. This is in agreement with earlier data demonstrating that not only SIN, but also SFV, and TBE, fuse efficiently at low pH with liposomes lacking a protein or carbohydrate receptor (5, 13, 46, 47). Formally, it cannot be excluded that initial receptor interaction could influence the detailed characteristics of the pH-dependent membrane fusion process of SIN. However, similar fusion kinetics were observed for HS-adapted SIN versus non- adapted SIN with or without HepPE in the target membrane, indicating that receptor interaction has no influence on the detailed pH dependence of virus fusion in the liposomal model system. Taken together, our present results demonstrate that, even after attachment of SIN to HepPE, as an HS receptor analog, fusion remains strictly dependent on exposure of the virus to a mildly acidic pH. This finding strengthens the notion that SIN infects its host cells via Heparan sulfate interaction and membrane fusion of alphaviruses 71 receptor-mediated endocytosis and low-pH-dependent fusion from within acidic endosomes. It is however, important to note that our present study does not address HS-independent virus-receptor interactions. It is likely that some, if not all alphaviruses have cellular receptors in addition to or instead of HS (7, 8, 28). Newly isolated or unpassaged strains of SIN, RRV and VEE viruses, all members of the alphavirus genus, do not bind to heparin and attach poorly to cells in culture relative to laboratory-adapted strains (2, 21, 28). Passage of non-HS- adapted SIN TR339 on BHK-21 cells resulted in virus mutants which bind with high affinity to BHK-21 cells and interact with HS (28). In vivo, these HS-adapted viruses typically exhibit an attenuated phenotype (8, 28). Similar results were obtained in passaging of RRV and VEE on cells. In the present paper, we demonstrate that SFV, derived from the infectious clone pSFV4 (31), efficiently interacts with cell-surface HS. It was found that this virus efficiently binds to BHK-21 cells. Evidence that the efficient binding of SFV to BHK-21 cells involves cell-surface HS, was obtained from heparin binding experiments. We observed that SFV interacts with immobilized heparin-agarose beads. Furthermore, it is shown that SFV binds to HepPE in liposomal membranes. These results suggest that SFV utilizes HS for infection of cells. Thus, adaptation to HS attachment receptors appears to represent a common cell culture-adaptive mechanism among the alphavirus genus. There is extensive evidence that viruses from different families and genera have the capacity to interact with GAGs, in most cases with HS (2, 7, 10, 11, 12, 14, 26, 28, 30, 32, 33, 44, 56). In several instances, high-affinity binding to HS, as with alphaviruses, has been found to be a cell culture adaptation for a number of viruses, including flaviviruses, pestiviruses, picornaviruses, and retroviruses. In some cases, however, interaction with HS was not found to be a cell culture adaptation. For example, virus type 1 interacts with HS carrying a specific sulfation pattern, which serves as functional receptor or co-receptor for the virus (45). Since numerous viruses interact with HS, the liposomal model system presented here may serve as a novel tool to study basic virus-receptor interactions and membrane fusion properties of viruses from different families or genera.

Acknowledgements

This work was supported by the U.S. National Institutes of Health (Grants HL16660 and AI22186-14), by The Netherlands Organization for Scientific Research (NWO) under the auspices of the Foundation for Chemical Research (CW), and by the Royal Netherlands Academy of Arts and Sciences (KNAW) (travel grant to J.M.S.). 72 Chapter 3

References

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Fusion of Alphaviruses with Liposomes is a Non-Leaky Process

Jolanda M. Smit, Gang Li, Pieter Schoen, Jeroen Corver, Robert Bittman, Ke-Chun Lin, and Jan Wilschut

FEBS Letters, in press 76 Chapter 4

Abstract

It has been reported that low-pH-induced fusion of influenza virus with liposomes results in rapid and extensive release of both low- and high-molecular-weight substances from the liposomes (Günther-Ausborn, S., A. Praetor, and T. Stegmann. 1995. J. Biol. Chem. 270:29279-29285; Shangguan, T., D. Alford, and J. Bentz. 1996. Biochemistry 35: 4956-4965). Here, we demonstrate retention of encapsulated water- soluble compounds during fusion of Semliki Forest virus (SFV) or Sindbis virus (SIN) with liposomes at low pH. Under conditions allowing complete fusion of the liposomes, a limited fluorescence dequenching of liposome-encapsulated calcein was observed, particularly for SFV. Also, radioactively labeled inulin or sucrose were largely retained. Freezing and thawing of the virus in the absence of sucrose resulted in an enhanced leakiness of fusion. These results support the notion that the alphavirus fusion event per se is non-leaky and may well involve a discrete hemifusion intermediate. Membrane fusion of alphaviruses is non-leaky 77

Introduction

Alphaviruses, such as Semliki Forest virus (SFV) and Sindbis virus (SIN), belong to the category of enveloped viruses that infect their host cells through receptor- mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes (8, 12, 14, 16, 21). This fusion process is mediated by the E1 component of the viral E2/E1 heterodimeric envelope glycoprotein (4, 9, 11, 18, 23, 28, 35). SFV and SIN fuse efficiently with liposomes in a strictly low-pH-dependent manner (4, 27, 31, 32). Fusion requires the presence of cholesterol (4, 22, 29, 32, 35, 36) and sphingolipids in the target membrane (7, 26, 27, 32). Cholesterol is involved in low- pH-dependent irreversible binding of the virus to target liposomes, while sphingolipids appear to function as a specific cofactor, catalyzing the actual viral membrane fusion process (27). Fusion of SFV with liposomes has been first studied by White and Helenius (36), who used fusion assays based on encapsulation of trypsin or RNase in the liposomes. Efficient degradation of the viral capsid protein or RNA was demonstrated after incubation of SFV with the liposomes at low pH. No RNA degradation was observed when RNase was added to the external medium. Likewise, the presence of an excess of trypsin inhibitor in the external medium did not interfere with degradation of the capsid protein during fusion of SFV or SIN with trypsin-containing liposomes (26, 27, 32, 36). Although the trypsin and RNase assays do not unequivocally prove the non- leakiness of the fusion process, the results of these studies are consistent with the idea that SFV-liposome fusion is not very leaky to large molecules, such as RNase or trypsin inhibitor. Similar observations have been made for influenza virus. For example, White et al. (37), using the above trypsin assay, and Young et al. (38), studying virus interaction with planar bilayers, reported that the fusion process of influenza virus is non-leaky. However, more recent investigations of influenza- liposome fusion (13, 15, 30), strongly suggest that the fusion process is quite leaky. Unlike the study of White et al. (37), these latter investigations are based on the release of reporter molecules from the liposomes during the fusion process. For example, Shangguan and coworkers (30) demonstrated that not only calcein (MW 623) but also 10kD dextran is rapidly released from liposomes upon fusion with influenza virus. These observations prompted us to reinvestigate the leakiness of alphavirus-liposome fusion, using similar reporter molecules encapsulated in the liposomes.

Materials and methods

Chemicals. Phosphatidylcholine (PC) from egg yolk, phosphatidylethanolamine (PE) prepared from egg-PC, sphingomyelin (SPM) from bovine brain, and cholesterol (Chol) were obtained from Avanti Polar Lipids (Alabaster, AL). Calcein and 1-hexadecanoyl-2-(1- pyrenedecanoyl)-sn-glycero-3-phosphocholine (pyrPC) were from Molecular Probes (Leiden, The Netherlands).3H-sucrose and 3H-inulin were from Amersham Pharmacia Biotech 78 Chapter 4

(Buckinghamshire, UK). Viruses. SFV and SIN were produced from baby hamster kidney cells (BHK-21), as described before (4, 32). Virus was purified from cell-culture medium by ultracentrifugation and sucrose-density gradient centrifugation in 5.0 mM HEPES, pH 7.4, containing 0.15 M NaCl and 0.10 mM EDTA (HNE). Unless stated otherwise, fresh virus preparations, that had not been frozen and thawed but just stored in the cold, were used. Storage never exceeded 3 days. Viral phospholipid was determined by phosphate analysis (3) after extraction of the lipids according to Bligh & Dyer (2). Liposomes. Liposomes (large unilamellar vesicles) were prepared by freeze/thaw- extrusion in HNE, as described before (17, 32). The extrusion step was done in a LiposoFast mini-extruder (Avestin, Ottawa, ON, Canada), using first two stacked Nucleopore polycarbonate filters with a pore size of 200 nm and then two stacked filters with a pore size of 50 nm (filters were from Costar Co., Cambridge, MA). The latter extrusion was done 81 times. The mean diameter of vesicles prepared in this fashion was determined by quasi-elastic light scattering in a Model 370 Submicron Particle Sizer (Nicomp, Santa Barbara, CA), and found to be 70 nm with a narrow size distribution. Liposomes consisted of either PC/PE/SPM/Chol/pyrPC (molar ratio, 0.85:1.0:1.0:1.5:0.15), PC/PE/SPM/Chol (1.0:1.0:1.0:1.5), PC/PE/Chol/pyrPC (0.85:1.0:1.0:0.15) or PC/PE/Chol (1.0:1.0:1.0). For encapsulation of calcein, liposomes were prepared in 60 mM calcein (sodium salt), 5.0 mM HEPES, 0.1 mM EDTA, pH 7.4. Non- encapsulated calcein was removed by gel filtration on Sephadex G-50 (Pharmacia, Uppsala, Sweden) in HNE. For encapsulation of 3H-sucrose or 3H-inulin, liposomes were prepared in HNE containing 225 µCi/ml 3H-sucrose (13 Ci/mmol) and 1 mM unlabeled sucrose, or 200 µCi/ml 3H-inulin (0.39 Ci/mmol), respectively. Non-encapsulated sucrose or inulin were removed by gel filtration. Phospholipid contents of the liposome preparations were determined by phosphate analysis (3). Fusion assay. Lipid mixing during virus-liposome fusion was monitored as a decrease of pyrene excimer fluorescence following acidification of mixtures of SFV or SIN with pyrPC- containing liposomes, as described before (32). Virus (10 µM phospholipid) and liposomes (2 µM phospholipid) were mixed in 0.66 ml HNE in a quartz microcuvette. Subsequently, the pH of medium was adjusted to 5.5 (for SFV) or 5.0 (for SIN) by addition of 0.040 ml 0.30 M MES, pretritated to achieve the desired final pH. The time course of pyrene excimer fluorescence was measured at excitation and emission wavelengths of 345 nm and 480 nm, respectively, in the presence of a 475-nm cutoff filter in the emission beam, in an AB2 fluorometer (SLM-Aminco, Urbana, IL). The cuvette holder of the fluorometer was equipped with a magnetic stirring device and maintained at 37 ºC. The fusion scale was set such that the initial excimer fluorescence intensity represented 0% fusion, and the fluorescence intensity at 33% dilution of the probe represented the 100% value. Complete dilution of the probe was induced by addition of 0.035 ml of 0.20 M octaethyleneglycol monododecyl ether (C12E8; Fluka Chemie AG, Buchs, Switzerland). Release of liposome contents during fusion. Release of calcein from the liposomes during fusion with SFV or SIN was determined under conditions as in the fusion assay. Calcein is a water-soluble fluorophore (MW 623) exhibiting fluorescence self-quenching at high concentrations. Thus, leakage of calcein from liposomes containing 60 mM of the probe results in fluorescence dequenching (1). Calcein fluorescence was measured, in the AB2 fluorometer as above, at excitation and emission wavelengths of 460 nm and 520 nm, respectively, in the Membrane fusion of alphaviruses is non-leaky 79 presence of a 495-nm cutoff filter in the emission beam. The fluorescence of completely released calcein was determined after the addition of 0.070 ml of 10% (v/v) Triton X-100 (Fluka) to the cuvette. Leakage of 3H-sucrose was assessed by gel filtration chromatography. Liposomes (10 µM phospholipid) containing 3H-sucrose were mixed with SFV (50 µM phospholipid) in 0.50 ml HNE and acidified as above. After 1 min at pH 5.5 the medium was adjusted to pH 7.8 with 0.018 ml of 0.5 M Tris (pH 9.5). The mixture was loaded on a 24 x 1 cm Sepharose CL-4B column. The column was eluted with HNE, containing 1 mg/ml BSA and 0.1 mM unlabeled liposomes to avoid binding of fusion complexes to the column material. The radioactivity in 0.5 ml fractions was assessed by liquid scintillation counting. Leakage of 3H-inulin during SIN- liposome fusion was determined in a similar manner.

Results

Fusion of SFV or SIN with liposomes assessed by lipid mixing. To determine leakage of liposomal contents during virus-liposome fusion, we used conditions allowing fusion of essentially all of the liposomes in a virus-liposome mixture. This condition is achieved with an excess of virus over liposomes, as shown previously for fusion of SIN with liposomes (32). In this latter study, using 70-nm liposomes, we demonstrated that, at liposome and SIN concentrations of 2 and 2.5 µM phospholipid (corresponding to 4*1010 liposomes and 1011 virions per ml), a liposome fuses on average once with a single virus particle. Given the diameter of the liposomes of 70 nm and that of an alphavirus particle, excluding the external glycoprotein layer, of 50 nm (9), fusion of a liposome with a virion results in a reduction of pyrPC surface density by 33% and thus in a decrease of pyrene excimer fluorescence by 33%, the excimer intensity being proportional to the surface density of the probe (10). Likewise, 100% fusion of the liposomes corresponds to a 33% decrease of the total pyrene excimer fluorescence. This value has indeed been observed previously (32). Now, using pyrPC-labeled PC/PE/SPM/Chol liposomes, again with a diameter of 70 nm, and unlabeled virus at concentrations of 2 and 10 µM, respectively, (corresponding to a liposome:virus particle ratio of 1:10), we observed a decrease of pyrene excimer fluorescence intensity in 1 min of 42% for SFV at pH 5.5 (Figure 1A, curve a) and 37% at pH 5.0 for SIN (Figure 1B, curve a). These values correspond to 127% and 112% fusion, respectively. Therefore, under the condition of these experiments, on average all of the liposomes fuse at least once with a virus particle. The initial rate of SIN-liposome was approx. 2-fold lower than that of SFV-liposome fusion under the condition of the experiment (Figure 1A vs. 1B). Release of calcein from liposomes during fusion with SFV or SIN. Fig. 1A (curve b) shows the result of a calcein dequenching experiment. PC/PE/SPM/Chol liposomes, loaded with 60 mM calcein, were incubated with SFV at pH 5.5, 37 ºC, at a liposome-to-virus particle ratio of 1:10. Clearly, an only limited dequenching of calcein fluorescence occurred, reaching about 15% after 1 min. Virtually no dequenching was observed at pH 7.4 (not shown), or at pH 5.5 with liposomes lacking 80 Chapter 4

SPM (Figure 1A, curve c). We have shown that fusion of SFV requires the presence of SPM in target liposomes (27). Therefore, the limited fluorescence dequenching observed with SPM-containing liposomes at pH 5.5 was due to the fusion process. This dequenching, however, may not be due to release but can possibly be accounted for by redistribution of calcein within the liposome-virus fusion products. When a 70-nm liposome fuses with a 50-nm virion and the fusion product assumes a spherical shape, the internal volume of the fusion product increases almost 2-fold relative to the internal volume of the original liposome. Thus, we compared the fluorescence of 30 mM calcein to that of 60 mM calcein, both encapsulated in liposomes. The residual relative fluorescence intensities were 30% and 17%, respectively. This implies that a 2-fold dilution of the calcein within liposome-virus fusion products would result in an increase of fluorescence intensity of 100x(30- 17)/(100-17)= 16%. The observed dequenching of 15% is similar to this value. Therefore, even though virus-liposome fusion products may not be perfectly spherical, the above argument shows that the observed calcein dequenching is likely to be due, at least in part, to dilution of the probe within virus-liposome fusion products.

A a 120 B 120 a 100 (%) (%) 100 Calcein

Calcein 80 80 60 60 b 40 40 b 20

20 Dequencing Dequencing c Fusion and Fusion and 0 c 0

0 20 40 60 0 20 40 60 Time (s) Time (s) Figure 1. Fusion of SFV (A) or SIN (B) with pyrPC-labeled or calcein-containing liposomes. Fusion was measured on the basis of pyrene excimer fluorescence decrease, and release calcein on the basis of fluorescence dequenching, both at virus and liposome concentrations of 10 µM and 2.0 µM phospholipid, respectively. The fusion scale is calibrated such that 33% decrease of pyrPC excimer fluorescence intensity represents 100% fusion (see text). The calcein dequenching scale is calibrated such that the initial residual fluorescence of the liposomes represents 0% and the fluorescence intensity in the presence of TX-100, inducing lysis of the liposome, 100%. Curves a, fusion of PC/PE/SPM/Chol/pyrPC liposomes with SFV at pH 5.5 or with SIN at pH 5.0; curves b, calcein release from PC/PE/SPM/Chol liposomes upon fusion with SFV at pH 5.5 or SIN at pH 5.0; curves c, calcein release from PC/PE/Chol liposomes at pH 5.5 in the presence of SFV (A) or from PC/PE/SPM/Chol liposomes at pH 5.0 in the absence of SIN (B). Membrane fusion of alphaviruses is non-leaky 81

Fusion of SIN with calcein-loaded liposomes (Figure 1B) was more leaky than SFV-liposome fusion. With SIN we observed approx. 50% dequenching of calcein under conditions where all of the liposomes fuse at least once with a virion (curve b). This implies that, while SFV-liposome fusion appears to be essentially non-leaky, SIN- liposome fusion results in partial release of calcein to the external medium. Retention of radiolabeled sucrose or inulin within virus-liposome fusion products. Final proof for SFV-liposome fusion being essentially non-leaky came from the evaluation of 3H-sucrose release from the liposomes. Liposomes containing 3H- sucrose (MW 344) were incubated with SFV for 1 min at pH 5.5, 37 ºC, at liposome and virus concentrations of 10 µM and 50 µM phospholipid (liposome-to-virus particle ratio of 1:10). Fig. 2A shows the elution profile of 3H-sucrose obtained after gel filtration analysis of the liposome-SFV fusion. It is evident that only a small fraction (approx. 9%) of the 3H-sucrose had leaked out (closed circles). The controls show that unfused liposomes also retained the 3H-sucrose (open circles), while TX100-treated liposomes completely released the probe (triangles). Similar experiments were done with SIN and liposomes containing 3H-inulin. Since from the calcein dequenching experiments (Figure 1B) it was evident that SIN- liposome fusion results in partial release of the probe, we decided to use, rather than sucrose (MW 344) which is smaller than calcein (MW 632), the larger marker inulin (MW 5200). Fig. 2B demonstrates that inulin was largely retained during SIN-liposome fusion, only 11% being released (closed circles).

600 2500 A B )

500 ) 2000

dpm 400 dpm 1500 300 ( 1000 200 Inulin

H- 500

H-Sucrose ( 100 3 3 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Volume (ml) Volume (ml) Figure 2. Release of 3H-sucrose or 3H-inulin from liposomes upon fusion with SFV (A) or SIN (B), respectively. A mixture of SFV or SIN (50 µM) and PC/PE/SPM/Chol liposomes (10 µM) containing 3H-sucrose or 3H-inulin, respectively, was incubated at pH 5.5 (SFV) or pH 5.0 (SIN), 37 ºC, for 1 min and subsequently neutralized. The incubation mixtures were then eluted on a Sepharose CL-4B column, and the fractions were analyzed for radioactivity. Open circles, untreated virus-liposome mixtures at neutral pH; closed circles, liposomes after low-pH-induced fusion with SFV or SIN; triangles, virus-liposome mixtures after addition of TX-100. 82 Chapter 4

Effects of freeze-thawing of the virus on calcein retention. Previous studies (37, 38) have indicated that fusion of SFV or influenza virus with planar bilayers or liposomes becomes increasingly leaky when the virus is damaged by freeze-thawing. In the above experiments, we used fresh virus preparations which had been stored in the cold for no longer than 3 days. In order to assess the effect of freeze-thawing, aliquots of SFV or SIN, in HNE containing 35-40% sucrose, were subjected to freeze- thawing 5 times, subsequently fused to calcein- or pyrPC-containing liposomes. The treatment had no detectable effect on the extents of calcein dequenching (Figures 3A and B, curves b) or fusion in the pyrene assay (results not shown). However, when SFV or SIN were frozen and thawed in HNE in the absence of sucrose as a cryopreservative, the fusion process with liposomes did become leaky, resulting in 55% calcein dequenching with SFV (Figure 3A, curve a). For SIN, similar results were obtained (Figure 3B, curve a), but the effect of freeze-thaw treatment was much less prominent than with SFV. This indicates that freezing and thawing in the absence of a cryopreservative may induce structural defects in the virus membrane, resulting in leakage of liposome contents after fusion of such a damaged virus with liposomes.

60 80 (%) (%) A B a a 60 b,c 40 40 20 b,c 20

0 0 Calcein Dequenching Calcein Dequenching 0 20 40 60 0 20 40 60 Time (s) Time (s) Figure 3: Effect of freezing and thawing on calcein release from liposomes upon fusion with SFV (A) or SIN ( B). Calcein dequenching was measured as in the experiment of Fig. 1, with virus preparations subjected to freezing (in liquid nitrogen) and thawing (in a waterbath of 37 ºC). Curves a, virus frozen and thawed once in HNE; curves b, virus frozen and thawed 5 times in HNE containing 35-40% (w/v) sucrose; curves c, unfrozen virus. Discussion

The results of this study indicate that fusion of alphaviruses with liposomes, particularly fusion of SFV, is a relatively non-leaky process. Early studies on SFV- liposome fusion, employing an assay based on trypsin or RNase encapsulated in the liposomes, had provided circumstantial evidence for the fusion event to be non-leaky Membrane fusion of alphaviruses is non-leaky 83 to large molecules (36). Our present observations, based on direct evaluation of the release of marker molecules from the liposomes, extend this notion by demonstrating that even small molecules, such as calcein and sucrose, are largely retained during the fusion process. The initial integrity of the viral membrane appears to be crucial, as prior freeze-thaw treatment of the viruses in the absence of sucrose resulted in an increased leakiness. This suggests that, when release of marker molecules from target liposomes is observed during alphavirus-liposome fusion, it may not be due to the fusion process per se, but rather the result of the presence of structural defects in the viral membrane. It is interesting that Young et al. (38), studying fusion of SFV with planar lipid bilayers, also concluded that fusion of SFV is a non-leaky process provided that the virus is fresh and the viral envelope undamaged. Furthermore, SFV or SIN have been observed to become hemolytic only when the viruses are subjected a freeze-thaw treatment prior to incubation with the erythrocytes (34). There appeared to be a difference between the degrees of leakiness of SFV- and SIN-liposome fusion, the latter resulting in a more extensive release of calcein (Figure 1). At the same time, freeze-thaw treatment of SIN had a comparatively small effect (Figure 3B). This suggests that the SIN virus preparation, although freshly and carefully isolated, may have acquired more structural defects during the purification process than SFV. Yet, SIN-liposome fusion was essentially non-leaky to the marker molecule inulin (MW 5200). Our results are in apparent disagreement with observations by Spyr et al. (33) and Käsermann et al. (19), indicating that, at low pH, isolated SFV particles become permeable to small molecules, such as propidium iodide. The authors propose that the viral E1 protein forms non-specific pores in the viral membrane under acidic conditions. Accordingly, one would expect that, upon virus-liposome fusion, small molecules initially encapsulated in the liposomes would be released to the external medium through these pores. However, since we did not observe such release, it would appear that the pores are either not or only transiently formed under the conditions of our experiments or that they are not accessible, possibly due to blocking by the viral nucleocapsid. It is remarkable that, while alphavirus-liposome fusion is essentially tight, influenza-liposome fusion has been found to be leaky not only to calcein but also to 10kD dextran (13, 15, 30). It is unclear what the basis for this difference is. However, the following points can be made. There is convincing evidence to indicate that fusion mediated by the influenza hemagglutinin (HA) involves a hemifusion intermediate, a so-called stalk (5, 6, 20, 25). Conceptually, a distinct hemifusion intermediate, in which the outer leaflets of the interacting membranes have merged while the inner leaflets are still separate, would provide a very plausible explanation for a fusion process being non-leaky. Therefore, the observed leakiness of influenza-liposome fusion (15, 30) may in fact not be due to leakiness of the fusion process per se, but rather to preexisting structural defects in the viral membrane, as discussed above, or to a secondary effect of virus-liposome fusion. It is interesting that influenza virus fusion 84 Chapter 4 is strongly hemolytic (24) whereas the fusion event with erythrocytes per se, involving a hemifusion intermediate, may well be non-leaky. At the same time, the relative non-leakiness of alphavirus-liposome fusion supports the notion that this fusion involves a distinct hemifusion intermediate. In this regard, we have characterized the effects of lysophosphatidylcholine and free fatty acids on SFV-liposome fusion and observed that these effects indeed strongly suggest that the fusion reaction proceeds via a stalk mechanism (Ortiz et al., to be submitted).

Acknowledgements

This work was supported by the Beijing Medical University (Travel Grant to G.L.) and by the Netherlands Organization for Scientific Research (NWO) under the auspices of the Council for Chemical Sciences (CW) and the Technology Foundation (STW) (Research Grants supporting P.S. and J.C.). The expert technical assistance of Liesbeth Bijl is gratefully acknowledged.

References

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A. Salminen, B. U. Barth, H. Zhao, K. Forsell and M. Ekstrom. 1994. Assembly and entry mechanisms of Semliki Forest virus. Arch Virol. 9Suppl.:329-338. 13. Gibson, S., C. Y. Jung, M. Takahashi, and J. Lenard. 1986. Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities. Biochemistry 25:6264-6268. 14. Glomb-Reinmund, S. and M. Kielian. 1998. The role of low pH and disulfide shuffling in the entry and fusion of Semliki Forest virus and Sindbis virus. Virology 248:372-381. 15. Günther-Ausborn, S., A. Praetor, and T. Stegmann. 1995. Inhibition of Influenza-induced membrane fusion by lysophosphatidylcholine. J. Biol. Chem. 270:29279-29285. 16. Helenius, A., J. Kartenbeck, K. Simons, and E. Fries. 1980. On the entry of Semliki Forest virus into BHK-21 cells. J. Cell. Biol. 84:404-420. 17. Hope, M. J., M. B. Bally, G. Webb, and P. R. Cullis. 1985. Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and the ability to maintain a membrane potential. Biochim. Biophys. Acta 812:55-65. 18. Justman, J., M. R. Klimjack, and M. Kielian. 1993. Role of spike protein conformational changes in fusion of Semliki Forest virus. J. Virol. 67:7597-7607. 19. Käsermann, F., and C. Kempf. 1996. Low pH-induced pore formation by spike proteins of enveloped viruses. J. Gen. Virol. 77:3025-3032. 20. Kemble, G. W., T. Danieli, and J. M. White. 1994. Lipid-anchored Influenza hemaglutinin promotes hemifusion, not complete fusion. Cell 78:383-391. 21. Kielian, M. 1995. Membrane fusion and the Alphavirus life cycle. Adv. Virus. Res. 45:113-151. 22. Kielian, M., and A. Helenius. 1984. Role of cholesterol in fusion of Semliki Forest virus with membranes. J. Virol. 52:281-283. 23. Kielian, M., M. R. Klimjack, S. Ghosh, and W. A. Duffus. 1996. Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus. J. Cell. Biol. 134:863-872. 24. Maeda, T., and S. I. Ohnishi. 1980. Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes.FEBS Lett. 122:283-287. 25. Melikyan, G. B., J. M. White, and F. S. Cohen. 1995. GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes. J. Cell Biol. 131:679-691. 26. Moesby, L., J. Corver, R. K. Erukulla, R. Bittman, and J. Wilschut. 1995. Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner. Biochemistry 34:10319-10324. 27. Nieva, J. L., R. Bron, J. Corver, and J. Wilschut. 1994. Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane. EMBO J. 13:2797-2804. 28. Omar, A., and H. Koblet. 1988. Semliki Forest virus particles containing only the E1 envelope glycoprotein are infectious and can induce cell-cell fusion. Virology 166:17-23. 29. Phalen, T., and M. Kielian. 1991. Cholesterol is required for infection by Semliki Forest virus. J. Cell. Biol. 112:615-623. 30. Shangguan, T., D. Alford, and J. Bentz. 1996. Influenza-virus-liposome lipid mixing is leaky and largely insensitive to the material properties of the target membrane. Biochemistry 35:4956- 4965. 31. Smit, J. M., R. Bittman, and J. Wilschut. 2001. Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity. FEBS Lett. 498:57-61. 32. Smit, J. M., R. Bittman and J. Wilschut. 1999. Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes. J. Virol. 73:8476-8484. 33. Spyr, C. A., F. Käsermann, and C. Kempf. 1995. Identification of the pore forming element of Semliki Forest virus spikes. FEBS Lett. 375:134-136. 34. Väänänen, P, and L. Kääriäinen. 1979. Haemolysis by two alphaviruses: Semliki Forest and Sindbis virus. J. Gen. Virol. 43:593-601. 86 Chapter 4

35. Wahlberg, J. M., R. Bron, J. Wilschut, and H. Garoff. 1992. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein. J. Virol. 66:7309-7318. 36. White, J., and A. Helenius. 1980. pH-dependent fusion between the Semliki Forest virus membrane and liposomes. Proc. Natl. Acad. Sci. USA 77:3273-3277. 37. White, J., J. Kartenbeck, and A. Helenius. 1982. Membrane fusion activity of Influenza virus. EMBO J. 1:217-222. 38. Young, J. D. E., G. P. H. Young, Z. A. Cohn, and J. Lenard. 1983. Interaction of enveloped viruses with planar bilayer membranes: observations on Sendai, Influenza, Vesicular Stomatitis, and Semliki Forest viruses. Virology 128:186-194. Chapter 5

A Single Mutation in the E2 Glycoprotein Important for Neurovirulence Influences Binding of Sindbis Virus to Neuroblastoma Cells

Peiyu Lee, Ronald Knight, Jolanda M. Smit, Jan Wilschut, and Diane E. Griffin

Journal of Virology, in press 88 Chapter 5

Abstract

The amino acid at position 55 of the E2 glycoprotein (E2:55) of Sindbis virus (SIN) is a critical determinant of SIN neurovirulence in mice. Recombinant virus strain TE (E2:55= histidine) differs only at this position from virus strain 633 (E2:55= glutamine), yet TE is considerably more neurovirulent than 633. TE replicates better than 633 in a neuroblastoma cell line (N18), but similarly in BHK- 21 cells. Immunofluorescence staining showed that most N18 cells were infected by TE at a multiplicity of infection (MOI) of 50-500 and by 633 only at an MOI of 5,000 while both viruses infected essentially 100% of BHK-21 cells at an MOI of 5. When exposed to pH 5, TE and 633 viruses fused to similar extents with liposomes derived from BHK-21 or N18 cell lipids, but fusion with N18-derived liposomes was less extensive (15-20%) than fusion with BHK-21-derived liposomes (~50%). Binding of TE and 633 to N18, but not BHK-21, cells was dependent on the medium used for virus binding. Differences between TE and 633 binding to N18 cells were evident in Dulbecco’s modified Eagle medium (DMEM), but not in RPMI. In DMEM, the binding efficiency of 633 decreased significantly as the pH was raised from 6.5 to 8.0 while that of TE did not change. The same pattern was observed in RPMI when the ionic strength of RPMI was increased to that of DMEM. TE bound better to heparin-Sepharose than 633, but this difference was not pH-dependent. Growth of N18 and BHK-21 cells in sodium chlorate to eliminate all sulfation decreased virus-cell binding, suggesting the involvement of sulfated molecules on the cell surface. Taken together, the presence of glutamine at E2:55 impairs SIN binding to neural cells under conditions characteristic of interstitial fluid. We conclude that mutation to histidine participates in or stabilizes the interaction between the virus and the surface of neural cells contributing to greater neurovirulence. Cell binding and neurovirulence of Sindbis virus 89

Introduction

Sindbis virus (SIN), the prototype member of the genus Alphavirus in the family Togaviridae, causes age-dependent mortality in mice (23). In newborn mice, the AR339 strain of SIN causes acute fatal encephalomyelitis while in older mice the disease is mild and nonfatal. A neuroadapted strain of Sindbis virus (NSIN) isolated after serial intracerebral passages of SIN causes high mortality in older mice associated with severe encephalomyelitis resulting in kyphoscoliosis and hindlimb paralysis (24). Fatal infection correlates with the induction of neuronal death, the primary target cells of SIN infection in the nervous system (34). SIN is a plus-strand RNA virus with a cell-derived lipid bilayer membrane containing heterodimers of the two viral envelope glycoproteins E2 and E1 (51). Entry of SIN into host cells involves association of the virus with the cell surface, interaction with specific receptors, clathrin-mediated endocytosis, and acid- induced fusion of SIN with the endosomal membrane to release the nucleocapsid into the cytoplasm (29). Coordinated association and dissociation of the E2 and E1 glycoproteins during interaction with the cellular membrane and exposure to acidic pH are required for alphaviruses to deliver genomic RNA into the cytoplasm (19). The E2 glycoprotein is the major mediator of attachment while the E1 glycoprotein carries membrane fusion activity (5, 46, 51, 57). Many studies suggest that the attachment of SIN to the cell surface is mediated by a cell surface protein (51). Tissue culture-adapted SIN strains become highly dependent on the proteoglycan heparan sulfate (HS) for initial virus attachment (7, 32) and it is likely that multiple cellular and viral factors influence SIN attachment and entry into cells in vivo and in vitro. RNA viruses often adapt quickly to new environments due to the generation and selection of mutants facilitated by the lack of proofreading of the viral RNA- dependent RNA polymerase. Alterations at the viral entry steps, which include receptor recognition, virus attachment/penetration and membrane fusion, could influence the rate of virus growth and tissue tropism, two major determinants in . Studies of SIN, Venezuelan equine encephalitis (VEE), Semliki Forest (SFV) and Ross River (RRV) viruses have demonstrated that mutations in the envelope proteins are important determinants of virus virulence (12, 21, 55, 56) and the mechanisms of virulence have begun to be elucidated. For instance, rapid penetration of BHK-21 cells is negatively correlated with virus virulence for SIN and VEE (11, 12, 39) and binding to HS contributes to the attenuation of SIN infection in mice (8, 32). Major determinants of the neurovirulence of the neuroadapted strain of SIN have been mapped to the glycoprotein regions of the genome and, in particular, the change from glutamine to histidine at position E2:55 is rapidly acquired during replication in the nervous system and plays a critical role in the acquisition of neurovirulence (35, 54). Recombinant viruses TE and 633 were constructed on the same genomic background and differ only at the 90 Chapter 5

E2:55 position, where TE has a histidine and 633 has a glutamine. In 2-week old mice inoculated intracerebrally, TE causes close to 100% mortality, whereas 633 causes no mortality (54). Neuronal death occurs more often in TE-infected than 633-infected mice (34). In tissue culture, both TE and 633 replicate equally well in non-neural cell lines, such as BHK-21 cells. However, in mouse brain and in N18 mouse neuroblastoma cells, replication is markedly different (14). TE replicates more rapidly and grows to higher titer than 633. Therefore, replication of SIN in N18 cells may reflect the molecular and cellular basis of neurovirulence in vivo. Results of infectious-center assays indicate that more N18 cells are infected with TE than with 633 at the same multiplicity of infection (MOI) (14), but differences in the initial interactions between these viruses and the N18 cell surface did not appear to account for the profound differences in infection (53). Knowledge of the interactions between the viral envelope and cellular receptor(s) could substantially facilitate the understanding of the process of cell entry. In this study, we have extended the studies of infection of N18 cells to identify determinants of the differential infection by TE and 633. We show that the lipids of N18 cells are much less conducive to SIN fusion than the lipids of BHK-21 cells and that the ionic strength of the virus diluent alters binding of 633 to the N18 cell surface in a pH-dependent fashion, while binding of TE is unaffected.

Materials and Methods

Cell culture, virus stocks, and plaque assays. BHK-21 cells and the N18 clone of the C1300 neuroblastoma cell line (1) were maintained in Dulbecco's modified Eagle medium (DMEM) (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS), 50 µg/ml gentamicin and 2 mM glutamine (GIBCO). Plaque forming units (PFU) were determined on BHK-21 monolayers and MOIs were calculated based on these data. To prepare [35S]-labeled TE and 633, BHK-21 cells were grown to confluence and infected at an MOI of 1. At 5 h after infection, the medium was replaced with methionine/cysteine-free DMEM containing 2% FBS and 33 µCi/ml TRANS35S-LABEL (~1,000 Ci/mmol; ICN, Irvine, Calif.). After overnight incubation, the culture supernatant fluid was clarified and virus was concentrated by polyethylene glycol precipitation, purified by centrifugation through a 15 to 45% (wt/vol) sucrose gradient, and concentrated through a 15% sucrose cushion (15). The final pellet was resuspended in DMEM containing 1% FBS and stored in aliquots at –80 °C. To prepare virus stocks for infection, BHK-21 cells were infected at an MOI of 0.1 and incubated overnight. The supernatant fluid was clarified and virus concentrated either by PEG precipitation or by centrifugation at 32,000 rpm in a Beckman SW-41 rotor at 4 °C for 90 minutes. Pellets were resuspended in DMEM containing 1% FBS and stored in aliquots at –80 °C. Cell binding and neurovirulence of Sindbis virus 91

Pyrene-labeled TE and 633 viruses were produced on BHK-21 cells, essentially as described before for SFV and SIN (5, 38, 46, 57). Briefly, BHK-21 cells were cultured in medium containing 15 µg/ml 16-(1-pyrenyl)-hexadecanoic (Molecular Probes, Eugene OR). At sub-confluence, viral infection was initiated with an MOI of 4. At 24 h post- infection, the pyrene-labeled TE or 633 viruses were harvested from the medium by ultracentrifugation in a Beckman type 19 rotor for 2.5 h at 100,000 x g, 4 °C, and further purified on a sucrose density gradient (20-50% w/v) by ultracentrifugation in a Beckman SW41 rotor for 16 h at 100,000 x g, 4 °C. The phospholipid content of the viruses was determined, after lipid extraction according to Bligh and Dyer (3), by phosphate analysis (4). Immunofluorescence staining and microscopy. BHK-21 and N18 cells were seeded at 3-4 x 104 cells/well in 8-well Permanox chamber slides (Nalge Nunc, Naperville, Ill.) and allowed to grow for 24 h. Cells were washed once in the specified binding medium and infected with 200 µl of virus diluted in the same binding medium. After 1 h at 37 °C, the virus inoculum was replaced with 200 µl of DMEM/2% FBS and 2 h later 200 µl of DMEM/2% FBS containing 20 mM ammonium chloride was added to each well. After an additional 3 h of incubation, the cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 5 min, washed and incubated at room temperature overnight with mouse anti-E2c monoclonal antibody (MAb) 209 (36, 49) diluted 2,000 in PBS/2% FBS. After 3 washes in PBS, Alexa 488-conjugated goat-anti-mouse IgG (H+L) (Molecular Probes) diluted 1/200 in PBS/2% FBS was added for 30 min, followed by 3 washes in PBS. To stain the nuclei, 1 µg/ml propidium iodide (Sigma, St. Louis, MO) in PBS containing 0.1% Triton X-100 was added to the cells for 1 min. After 5 washes in PBS and 1 wash in H2O, the slides were mounted in PermaFluor aqueous mounting medium (Immunon, Pittsburgh, PA), and examined with a Nikon Eclipse E800 microscope. RNA synthesis. N18 cells were seeded in 12-well plates at 3 x 105 cells per well and cultured overnight. TE and 633 were diluted either in DMEM/1% FBS or in RPMI containing 20 mM HEPES (pH 7.55) and 0.5% bovine serum albumin (BSA). After 1 h of incubation at 37 °C in the presence of 5% CO2, the virus inoculum was aspirated, the cells were overlayed with DMEM/2% FBS containing 1 µg/ml actinomycin D (Calbiochem, San Diego, Calif.) and incubated at 37 °C. To label newly synthesized viral RNA, DMEM/2% FBS with 20 µg/ml actinomycin D and 150 µCi/ml 3H uridine (50 Ci/mmol; ICN, Boston, Mass.) was added to the cells and incubated for 1 h. Cell lysates were prepared in 1% SDS with 200 µg/ml proteinase K and precipitated in 10% trichloroacetic acid (TCA) at 4 °C. The precipitates were harvested onto GF/C glassfiber filters (Whatmann, Maidstone, Kent, UK) which were washed 2 times with 5% TCA (4 °C) and once in cold 95% ethanol. The filters were counted in BetaMax (ICN) in a Beckman LS 6500 scintillation counter. Virus-cell binding assays. A variety of binding buffers were used and were based either on DMEM, RPMI-1640 or Dulbecco’s PBS (8.5 mM Na2HPO4, 1.8 mM KH2PO4 , 2.7 mM KCl, 137 mM NaCl, pH7.5) as the basic salt solution. DMEM with sodium bicarbonate (3.7 mg/ml) (DMEM+SB), DMEM without sodium bicarbonate (DMEM- SB), and DMEM+ SB without glucose (Gibco BRL) were supplemented with combinations of 1% FBS, 20 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, pH 7.55 (HEPES; Gibco BRL). RPMI without sodium bicarbonate (RPMI-SB) (Gibco BRL) 92 Chapter 5 was supplemented with 0.5 % bovine serum albumin (BSA) and 20 mM HEPES (20). PBS was supplemented with 1mM MgCl2, 1mM CaCl2, and 0.5%BSA. To make binding buffers of different pH values, 20 mM phosphate buffers (diluted from 1M sodium phosphate made with appropriate ratios of 1M NaH2P04 and 1M Na2HP04) with different pH values were used to replace HEPES. For the compositions of binding buffers used for specific experiments, see figure legends. The virus-cell binding assays were carried out essentially as previously described (7, 52). For chlorate treatment, the cells were dissociated in trypsin and incubated in F-12 HAM medium (Sigma) supplemented with 10 mM sodium chlorate, 2% dialysed FBS, 50 µg/ml glutamine, and 10,000 U/ml penicillin for one additional day. Cells were washed once and detached by incubation in PBS containing 1 mM EDTA for 15 min. Cells were washed in the specified binding buffer (4 °C), and resuspended at 4-7 x 105 cells per ml. Subsequently, 290 µl of the cell suspension was mixed with 10 µl of diluted virus (10,000 cpm) in a 1.7-ml microfuge tube. The tubes were rocked at 4 °C for 30 to 180 min and centrifuged for 5 min at 4,500 rpm at 4°C. The cells were washed twice in 600 µl of the binding buffer and lysed in 100 µl 1% SDS. The tubes were rinsed once with 100 µl water. The lysate and the rinse were combined and mixed with Liquiscint (National Diagnostics, Atlanta, GA) and counted. Binding of virus to BHK-21 cells was carried out under similar conditions with cell monolayers in 12-well tissue culture plates. At 95-100% confluence, cells were washed twice with 300 µl of the binding buffer (4 °C). [35S]-labeled TE or 633 was diluted to 10,000 cpm in 300-µl of the appropriate binding buffer and added to each well. The plates were rocked at 4 °C for 30-180 min, washed twice with 600 µl binding buffer and lysed in 300 µl of 1% SDS. Cell lysates were transferred to scintillation vials together with 300 µl of H2O used to rinse the well and counted. All of the binding assays were carried out in triplicate. Three separate aliquots of the viruses used were counted to determine total cpm and calculate % bound. Binding to HS was determined by heparin-Sepharose chromatography as previously described (7). Preparation of liposomes. Phosphatidylcholine (PC) from egg yolk, phosphatidylethanolamine (PE) prepared by transphosphatidylation of egg PC and egg sphingomyelin (SPM) were obtained from Avanti Polar Lipids (Alabaster, AL). High- grade cholesterol (Chol) was from Solvay Pharmaceuticals (Weesp, The Netherlands). Alternatively, lipids were extracted from N18 and BHK-21 cells according to Bligh and Dyer (3). Large unilamellar vesicles (LUV) were prepared by a freeze/thaw-extrusion procedure. Briefly, lipid mixtures were dried from a chloroform/methanol (2:1) solution under a stream of nitrogen and further dried under vacuum for at least 1 h. The lipids were hydrated in 5 mM HEPES, 150 mM NaC1, 0.1 mM EDTA, pH 7.4 (HNE), and subjected to five cycles of freezing and thawing. The vesicles were then extruded through a Unipore polycarbonate filter with a pore size of 0.2 µm (Nucleopore, Pleasanton, CA) in a LiposoFast mini-extruder (Avestin, Ottawa, Canada). Liposomes consisted of N18 cell lipids, BHK-21 cell lipids, or a PC/PE/SPM/Chol mixture (molar ratio, 1:1:1:1.5). Phospholipid content of the liposomes was determined by phosphate analysis (4). Fusion assay. Fusion of pyrene-labeled TE or 633 viruses with liposomes was monitored continuously as a decrease of pyrene-excimer fluorescence intensity due to dilution of pyrene-labeled lipids from the viral envelope into the target liposomes (5, 38, Cell binding and neurovirulence of Sindbis virus 93

46, 57). Briefly, pyrene-labeled virus (0.5 µM viral phospholipid) and liposomes (200 µM phospholipid) were mixed in a cuvette of an AB2 fluorometer (SLM/Aminco, Urbana, IL) in a volume of 0.665 ml HNE. The content of the cuvette was magnetically stirred and maintained at 37 °C. Fusion was initiated by injection of 35 µl 0.1 M MES, 0.2 M acetic acid, pretitrated with NaOH to achieve the final desired pH. Pyrene excimer fluorescence intensity was monitored at excitation and emission wavelengths of 345 and 480 nm, respectively. The fusion scale was calibrated such that 0% fusion corresponded to the initial excimer fluorescence intensity and 100% fusion to the fluorescence intensity at infinite probe dilution obtained by addition of 35 µl 0.2 M octaethyleneglycol monododecyl ether (C12E8; Fluka Chemie AG, Buchs, Switzerland).

Results

Susceptibility of N18 cells to infection by TE and 633. As demonstrated by infectious center assays, TE infects more N18 cells than 633 at an MOI of 10 to 50 (14). There are at least two possible explanations for this observation. A subpopulation of N18 cells (such as cells at a particular stage of the cell cycle) may be resistant to infection by 633, but not TE, or all of the N18 cells may be susceptible to infection by both viruses, but infection with 633 is much less efficient than infection with TE. To test these hypotheses, N18 cells were infected with TE and 633 at MOI of 50, 500, and 5,000, and incubated for 2 h to allow virus entry before adding ammonium chloride to block secondary virus infection. Cells were fixed 5 h after infection and stained with an anti-E2 MAb to detect the expression of E2 on the cell surface. Individual cells were identified by propidium iodide staining of the nuclei (Figure 1). At an MOI of 50, TE infected more N18 cells than did 633 (70 % vs 7 %) (Figures 1A and D). At an MOI of 500, 80% of the N18 cells were infected with TE, but only 39% were infected with 633 (Figures 1B and E). However, at an MOI of 5,000, almost all N18 cells were infected with 633 (91%) as well as with TE (97%) (Figures 1C and F). Therefore, the whole population of N18 cells was susceptible to infection by 633, but 633 was less efficient at establishing infection than TE. However, N18 cells were much less susceptible than BHK-21 cells to infection with either virus. At an MOI of 5 essentially 100% of BHK-21 cells were infected by either TE or 633 (Figures 1J and K). To be sure that the SIN glycoprotein on the N18 cell surface reflected virus replication at these high MOIs, viral RNA synthesis was examined (Figure 2). At an MOI of 5,000, similar levels of 3H-uridine were incorporated into a TCA- precipitable form in TE- and 633-infected N18 cells at 5 to 6 h after infection. There was less viral RNA synthesis in 633- than in TE-infected N18 cells at MOIs of 50 (P = 0.03) and 500 (P = 0.02). These results imply that as long as 633 and TE can enter the N18 cells to initiate virus infection, viral RNA synthesis does not differ. 94 Chapter 5

A B C

D E F

G H I

J K L

Figure 1. Susceptibility of N18 and BHK-21 cells to TE and 633 infection. N18 (A-I) and BHK-21 (J-L) cells were infected with TE or 633 at BHK-21 MOIs of 5, 50, 500 or 5000. The virus inoculum was diluted in either DMEM+SB supplemented with 1% FBS or RPMI-SB supplemented with 20 mM HEPES and 0.5% BSA. At 2.5 h after infection, 20 mM ammonium chloride was added and incubation continued for an additional 3 h. Cells were fixed in paraformaldehyde and stained with an E2-specific monoclonal antibody (209). Alexa 488- conjugated goat anti-mouse antibody (green) was used as the secondary antibody. Visualization of individual cells was facilitated by a propidium iodine nuclear stain (red). Cells, viruses, medium and MOI are as follows: N18 plus 633 in DMEM at MOI = 50 (A), 500 (B) or 5000 (C); N18 plus TE in DMEM at MOI = 50 (D), 500 (E) or 5000 (F). N18 plus 633 (G) or TE(H) in RPMI at MOI = 50; N18 plus TE in DMEM, control without primary Ab (I). BHK- 21 + TE(J) or 633(K) in DMEM at MOI = 5. BHK-21 + TE in DMEM, control without primary Ab (L). Cell binding and neurovirulence of Sindbis virus 95

30 mock TE ) 4 20 633 * , 10 incorporation cpm ( 10 * uridine H- 3 0 0 50 500 5000 multiplicity of infection Figure 2. Viral RNA synthesis in N18 cells infected with TE and 633. N18 cells were incubated for 1 h at 37 °C in the presence of 5% CO2 with TE and 633 diluted in DMEM+SB/1% FBS to contain BHK-21 MOIs of 0, 50, 500 or 5,000. The cells were maintained in DMEM+SB/2% FBS for 5 h before the addition of 150 mCi/ml of [3H]-uridine, 20 mg/ml actinomycin D. After 1 h TCA-precipitable counts in the cells were determined. *, P < 0.005, Student’s t test.

Fusion of 633 and TE to N18 and BHK-21 cell-derived liposomes. Since a substitution of threonine for isoleucine at E2:12 of SFV, a related alphavirus, leads to decreased virus-cell fusion by lowering the pH required for the initial E1-E2 dimer dissociation (22), the effects of the mutation at E2:55 on fusion were examined. Liposomes were prepared using lipid extracts from N18 or BHK-21 cells and used as target membranes for fusion of pyrene-labeled TE and 633 (Figure 3). At pH 7.4, there was no fusion between the viruses and liposomes. On the other hand, at pH 5.0, TE and 633 fused with BHK-21- or N18-derived liposomes with similar kinetics and to similar levels. Also, the detailed pH dependences of fusion of TE and 633 were very similar (results not shown). Although TE and 633 did not differ in fusion, both viruses fused more extensively with BHK-21-derived liposomes (~ 50% fusion) than with N18-derived liposomes (17-20% fusion). Fusion of the viruses with BHK-21-derived liposomes was very similar to fusion with PC/PE/SPM/Chol liposomes in terms of both kinetics and final extent (results not shown). Effect of buffer composition on binding of TE and 633 to N18 cells. TE and 633 were previously demonstrated to bind to N18 cells to similar extents. However, the conditions for testing binding were different (RPMI-SB/0.5% BSA/20 mM HEPES at 4 °C without CO2) than those used routinely for infection (DMEM+SB/1% FBS at 37 °C in 5% CO2) (7, 53). Therefore, binding of [35S]- labeled TE and 633 to N18 cells at 4 °C was compared with viruses diluted in RPMI-SB, PBS or DMEM+SB (Figure 4A). TE and 633 bound to N18 cells with 96 Chapter 5

A TE, B 633 50 20 pH 5.0 663 TE 40 pH 5.0 30 10 20 Fusion (%) Fusion (%) pH 7.4 TE 10 pH 7.4 TE, 0 633 0 633

0 20 40 60 0 20 40 60 Time (s) Time (s) Figure 3. Fusion of TE and 633 with liposomes prepared from N18 or BHK-21 cell lipids. Lipids were extracted from N18 (A) and BHK-21 (B) cells and used to prepare liposomes. Pyrene-labeled TE or 633 viruses were mixed with liposomes at 37 °C. At = 0 sec, the buffer pH was adjusted to 5.0 or maintained at 7.4, after which the change in pyrene excimer fluorescence intensity at 480 nm, due to dilution of the pyrene-labeled viral lipids into the liposome membrane, was monitored continuously. similar kinetics and to similar extents when diluted in RPMI-SB-based buffer. TE showed somewhat improved binding in PBS and DMEM compared to RPMI while 633 showed lower binding than TE when diluted in PBS (P < 0.03 at all time points) and almost no binding when diluted in DMEM+SB (P < 0.0005 at all time points). Under the same conditions, no consistent diluent-dependent differences were detected in TE and 633 binding to BHK-21 cells (Figure 4B). Consistent with previous observations (53) both viruses were able to bind to BHK-21 cells more efficiently than to N18 cells when diluted either in RPMI (P = 0.0004 for TE; P = 0.0054 for 633) or in DMEM (P < 0.0001 for TE and 633). Since in our previous studies (14) and in the above experiments (Figures 1 and 2) comparing TE and 633 entry, and replication in N18 cells, virus was diluted in DMEM+SB, it was important to determine whether TE and 633 would replicate similarly in N18 cells if virus was diluted in RPMI rather than DMEM. N18 cells were infected with TE or 633 diluted in RPMI-SB at an MOI of 50. Infection of N18 cells with TE and 633 in RPMI led to similar high proportions (47% and 68%) of infected cells at 5 h (Figures 1G and H). Sensitivity to pH of TE and 633, binding to N18 cells in DMEM. To investigate whether binding of TE and 633 diluted in DMEM to N18 or BHK-21 cells is sensitive to pH, DMEM-SB supplemented with 1% FBS was adjusted to pHs between 6.5 and 8.5 with 20 mM phosphate (Figure 5A). Binding to BHK-21 cells did not change over this pH-range, while binding of 633 to N18 cells Cell binding and neurovirulence of Sindbis virus 97

N18 cells BHK-21 cells 100 100 A TE/PBS B 75 TE/RPMI 75 TE/DMEM bound 50 50 633/PBS cpm

% 25 633/RPMI 25 633/DMEM 0 0 0 50 100 150 200 0 50 100 150 200 Time (min) Time (min) Figure 4. Effects of binding buffer on binding of 633 and TE to N18 and BHK-21 cells. N18 (A) and BHK-21 (B) cells were incubated with [35S]-labeled TE or 633 diluted in DMEM+SB supplemented with 20 mM HEPES, pH 7.3 and 1% FBS (circle). RPMI-SB supplemented with 20 mM HEPES (pH 7.3) and 0.5% BSA (square) or PBS (pH 7.3) containing 1 mM MgCl2, 1 mM CaCl2, and 0.5% FBS (triangle) at 4 °C. Virus binding was assessed after rocking the virus-cell mixture for 10, 30, 90 and 180 min at 4 °C. Binding of 633 to N18 cells was significantly lower than that of TE when viruses were diluted in PBS (P = 0.0036, 0.005, 0.004 and 0.025 at 10, 30, 90 and 180 min respectively) or DMEM (P < 0.0005 at all time points). decreased at pHs above 7.0. Binding of TE to N18 cells decreased only above pH 8.0. Binding of 633 was significantly lower than that of TE at pHs of 6.95 and above (P < 0.005). pH-dependent binding differences to N18 cells were not observed when these viruses were diluted in RPMI (results not shown). Effects of glucose concentration and ionic strength on binding of TE and 633 to N18 cells. The composition of RPMI and DMEM differ in a number of ways including glucose concentration (11 mM and 25 mM), ionic strength (10,800 µS and 11,970 µS) and Ca2+ concentration (0.4 mM and 1.8 mM). To determine the relevance of these differences each parameter was adjusted independently and binding assessed. DMEM+SB without glucose was supplemented with 11 or 25 mM glucose and 1% FBS. TE bound to N18 cells better than 633 at both concentrations of glucose (Figure 5C) so lowering the glucose concentration of DMEM to that RPMI did not improve 633 binding. The ionic strength of RPMI (pH 7.5) was systematically increased by addition of NaCl (Figure 5B), resulting in a steady decline in virus binding to N18 cells that was greater for 633 than TE. Binding to BHK-21 cells was not affected. The conductivity of RPMI was adjusted to that of DMEM, requiring the addition of about 14 mM NaCl and binding assessed at different pHs (Figure 5D). Binding to N18 cells, but not to BHK-21 cells of TE and 633 diluted in RPMI of increased ionic strength differed significantly in a pH-dependent manner (Figure 5D) and 98 Chapter 5

A DMEM B RPMI

80 TE/BHK-21 80

60 633/BHK-21 60 bound bound 40 TE/N18 40 cpm cpm % % 20 633/N18 20 0 0 6.5 7.0 7.5 8.0 8.5 0 10 20 30 40 pH mM increase in [NaCl]

C D TE 633 40 80

30 60 bound bound 20 40 cpm cpm %

% 10 20

0 0 25mM 11mM 6.5 7.0 7.5 8.0 [glucose] Buffer pH

Figure 5. Binding of 633 in DMEM to N18, but not BHK-21, cells is more sensitive to pH changes than binding of TE. (A) BHK-21 and N18 cells were mixed with [35S]-labeled TE and 633 in DMEM-SB supplemented with 1% FBS and 20 mM phosphate buffer at pH 6.5, 6.95, 7.4, 7.85, and 8.5 for 90 min at 4 °C. Binding of 633 was lower than binding of TE to N18 cells at pHs of 6.95 (P = 0.0013) and above (pHs 7.4 and 8.5, P < 0.0001; pH 7.85, P = 0.0037). (B) The glucose concentration of DMEM without glucose was adjusted to 11 mM and 25 mM. N18 cells were mixed with [35S]-labeled TE and 633 for 120 min at 4 °C. (C) The ionic strength of RPMI-SB containing 0.5% BSA (conductivity = 10,800 µS) was adjusted to that of DMEM-SB containing 1% FBS (conductivity = 11,970 µS) by raising the NaCl concentration approximately 14 mM. Phosphate (20 mM) was used to adjust the pH to 6.6, 7.15, 7.5, 7.85, and 8.10. N18 and BHK-21 cells were incubated with [35S]-labeled TE and 633 for 150 min at 4 °C. (D) The NaCl concentration of RPMI-SB supplemented with 0.5% BSA and 20 mM phosphate (pH 7.5) was raised by 0, 5, 10, 15, 17.5, 25, 35 mM (final conductivities were 12,500, 12970, 13380, 13730, 14550, 15340 µS, respectively) and binding of TE and 633 assessed as above. the pattern was similar to that observed for virus diluted in DMEM (Figure 5A). Lastly, increasing the Ca2+ concentration of RPMI to that of DMEM did not decrease binding of either virus to N18 cells, but did enhance the difference between TE and 633 possibly due to the concomitant increase in ionic strength. There was no effect on binding to BHK-21 cells (results not shown). Cell binding and neurovirulence of Sindbis virus 99

The role of sulfated molecules in TE and 633 binding to N18 and BHK- 21 cells. HS, a highly negatively charged molecule, is a determinant of SIN binding to the cell surface. HS binding increases the efficiency of infection of CHO and BHK-21 cells (7, 32). To determine if the differences in binding of TE and 633 to N18 cells and the effect of pH on that binding could be explained by the relative strength of binding to HS we examined binding to heparin-Sepharose and to cells differing in the levels of sulfated molecules on the cell surface. Each virus was bound to a heparin-Sepharose column at a NaCl concentration of 50mM and then eluted with a 50-500 mM NaCl gradient prepared at either pH 6.5 or 7.5 (5 mM phosphate). The results show that 633 bound less tightly to HS than TE at both pH values, TE required 403 and 320 mM NaCl, respectively; 633 required 346 and 302 mM NaCl, respectively. To investigate whether HS or other sulfated molecules participated in the SIN- N18 cell binding that is ionic strength- and pH-sensitive, N18 and BHK-21 cells were grown in low sulfate medium in the presence of 10 mM sodium chlorate, a potent inhibitor of ATP-sulfurylase, the first enzyme in the biosynthesis of the cosubstrate for sulfation (2, 28). Binding assays were carried out in DMEM-SB containing 1% FBS and 20 mM phosphate, pH 6.5 or 7.5, conditions at which TE and 633 showed the least and the most significant differences in binding to N18 cells (Figure 5A). At pH 6.5, binding of TE and 633 to N18 cells was decreased similarly by the chlorate treatment, indicating that binding of both viruses to N18 cells is affected by the desulfation of the cells (Figure 6A). At pH 7.5, TE reacted well with sulfated molecules on the surface of N18 cells, but 633 did not. Binding of TE (P = 0.0045) and 633 (P = 0.0025) to the chlorate-treated N18 cells was lower than to untreated N18 cells (Figure 6A), but differences between TE and 633 were still observed. Chlorate treatment decreased binding of TE and 633 to BHK-21 cells at pH 7.5 from >70 % to approximately 10% (Figure 6B), similar to the binding of both viruses to CHO cells lacking HS (results not shown).

Discussion

The substitution of the amino acid histidine for glutamine at residue 55 of the E2 glycoprotein has a profound effect on the neurovirulence of SIN. This is reflected in the relative abilities of recombinant viruses containing either histidine (TE) or glutamine (633) to replicate in mouse brain and in N18 neuroblastoma cells (34, 54). In the current studies we have shown that all N18 cells can be infected by both viruses, but infection is 10- to 100-fold less efficient for 633 than TE. Both viruses infect N18 cells less efficiently than BHK-21 cells, presumably as a result of decreased binding to the cell surface combined with a limited ability of 100 Chapter 5

N18 cells BHK-21 cells 80 A TE 80 B 633 60 60

bound 40 bound 40 cpm cpm

% 20 % 20

0 0 pH 6.5 6.5 7.5 7.5 pH 6.5 6.5 7.5 7.5 Chlorate - + - + Chlorate - + - +

Figure 6. Sulfated molecules appear to be involved in the ionic strength- and pH- sensitive binding of TE and 633 to N18 cells. N18 (A) and BHK-21 (B) cells were grown in Ham’s medium containing a low concentration of sulfate with (+) or without (-) 10 mM sodium chlorate overnight. Binding was performed in DMEM-SB containing 1% FBS and 20 mM phosphate at pH 6.5 or 7.5. the lipids in the plasma membrane of N18 cells to support SIN fusion. Both TE and 633 bound to HS, a major determinant of binding to BHK-21 and CHO cells (32). Binding of the viruses to N18 cells differed in that 633, but not TE, was exquisitely sensitive to the composition of the diluent used in the binding assay and decreased substantially as ionic strength and pH increased to physiologic levels. Treatment of N18 cells with sodium chlorate to inhibit sulfation decreased the binding of TE at pH 7.5 to approximately the binding of 633 to N18 cells not treated with chlorate. The limited binding of 633 was further decreased by chlorate treatment. The data suggest that BHK-21 cells have a sulfated molecule to which TE and 633 bind efficiently to initiate infection and that this molecule is either not present on N18 cells or present in an altered form. The interaction of SIN with the primary receptor on N18 cells is affected by a glutamine at E2:55 in a way that impairs binding at physiologic conditions of salt concentration and pH. Different types of cells exhibit different degrees of susceptibility to SIN infection. Clearly, both TE and 633 infect N18 cells much less efficiently than BHK-21 cells. An MOI of 50-500 (determined on BHK-21 cells) was required to establish synchronous infection in N18 cells with TE, while 633 required an MOI of 5,000 (Figure 1). Both viruses bind less efficiently to N18 cells than to BHK-21 cells (Figure 4). Furthermore, the viruses fuse less extensively with N18-derived liposomes than with BHK-21-derived liposomes (Figure 3). It is well established that the lipid composition of the target membrane has a profound effect on the membrane fusion characteristics of enveloped viruses (41, 44, 50). For alphaviruses, studies in both liposomal model systems as well as in Cell binding and neurovirulence of Sindbis virus 101 cells have shown that membrane interaction and fusion are dependent on cholesterol (5, 30, 40, 57, 59). Furthermore, fusion requires the presence of sphingolipid in the target membrane (10, 37, 38). Cholesterol appears to be primarily involved in the initial low-pH-dependent binding of the virus to the target membrane lipids, while spingolipids presumably play a catalytic role in the subsequent fusion event itself (38). There is no reason to suspect that in our present study the N18 liposomes were deficient in either cholesterol or sphingolipid: the plasma membrane of N18 cells has a high cholesterol-to- phospholipid ratio, and sphingomyelin is a major component of the phospholipid fraction (9). The detailed fusion characteristics of TE or 633 in the liposomal model system (Figure 3) indicate that with N18-derived liposomes a smaller fraction of the virus fuses than with BHK-21-derived liposomes or liposomes consisting of an artificial PC/PE/SPM/Chol mixture. Yet, the fraction of virus that does fuse exhibits very similar fusion kinetics in either case. In view of the high cholesterol content of the liposomes, it is unlikely that the low extent of fusion is due to a low degree of binding. It thus appears that with N18-derived liposomes part of the liposome-bound virus remains unfused. This is suggestive of a degree of heterogeneity at the surface of the liposomes, such that sites or domains that do support efficient fusion coexist with other areas that do not. Although recent experiments have shown that the presence of cholesterol/sphingolipid domains in target liposomes is neither required nor inhibitory to alphavirus membrane fusion (B. L. Waarts and J. Wilschut, unpublished observations), the fusion of TE and 633 with N18-liposomes is reminiscent of SIN fusion with liposomes containing domains with a relatively high degree of fatty acid saturation resulting in tight lipid packing. Cultured neuroblastoma cells generally contain low concentrations of polyunsaturated fatty acids, unless specific fatty acid-supplemented medium is used (42). In addition, N18 cell membranes contain N-acylphosphatidylethanolamine and N- acylethanolamine (25) which may contribute to a tighter lipid packing and a decreased susceptibility to SIN fusion. While there was a distinct difference between N18-derived and BHK-21- derived liposomes in terms of fusion with TE or 633, paired comparison showed that both viruses fused equally well. Apparently, substitution of His for Gln at E2:55 has little effect on the viral fusion process, suggesting that an improved interaction of the TE virus with target cell membrane lipids does not account for the increased neurovirulence of TE relative to that of 633. Fusion of alphaviruses is mediated by the E1 glycoprotein, presumably in a homotrimeric configuration (5, 46, 57) while receptor interaction is a function of E2 (47). Therefore, it is likely that amino acid changes in E1 primarily affect fusion with the neuronal cell membrane, while changes in E2 are expected to have their principal effects on virus binding. 102 Chapter 5

Significant differences in binding to N18 cells were observed between TE and 633 diluted in DMEM, but not in RPMI, while binding to BHK-21 cells was similar. These differences were explained primarily by the higher ionic strength of DMEM, which was associated with a loss of 633 binding to N18 cells at physiologic pH. Binding of both TE and 633 to N18 and BHK-21 cells was diminished after treatment with chlorate, suggesting that the binding of SIN to N18 cells is mediated primarily by sulfated molecules which are highly negatively charged with ligand interactions that are often sensitive to ionic strength and pH (6). Binding of 633 to BHK-21 cells was not sensitive to changes in pH and ionic strength, implying that the sulfated groups on the surface of BHK-21 and N18 cells with which SIN is interacting are different. The sulfated molecules on BHK- 21 cells could provide more stable interactions to the SIN glycoproteins than those on N18 cells and therefore would be less sensitive to the interference by high salt concentration. Alternately, the density of sulfated molecules on BHK-21 cells could be more abundant than on N18 cells, providing stronger binding interactions. Therefore, increased efficiency of SIN infection of BHK-21 cells compared to N18 cells is likely to represent a combination of better initial binding to sulfated molecules including HS on the cell surface and the fusogenicity of the lipids present in the plasma membrane. Both of these parameters are suboptimal in N18 cells so that selection of viral variants with improved attachment and/or fusion would be predicted to increase neurovirulence. Binding of proteins to glycoaminoglycans (GAG) has been considered to be primarily electrostatic, but recent studies indicate that this interaction is both more complicated and more specific than solely charge-determined interactions would imply. Proteoglycan expression is regulated in both developmental and in tissue- specific ways (18, 27, 31). Proteoglycans vary in core proteins, the types, locations and numbers of GAG chains attached, and in the degree of sulfation. This variability allows relatively specific binding of a wide range of ligands (27). Of the HS proteoglycans, syndecan-2 is most abundant in liver and kidney and syndecan- 3 is highly expressed in neonatal, but not adult brain (45). Little is known about the proteoglycans expressed on neurons and the sulfated molecule on N18 cells involved in SIN binding to the cell surface is not clear. Tyrosine sulfation is a relatively common posttranslational modification of proteins (26) and sulfation of CCR5 at an N-terminal tyrosine residue has been implicated as an important factor in cytokine-CCR5 binding and in HIV entry into cells (17). The region of the E2 glycoprotein we investigated in the present study is important for virulence in other alphaviruses as well. A mutant with a deletion of amino acid 55-61 of E2 of RRV generates small plaques and is less virulent in mice (55). Whether E2:55 is involved in binding to the sulfated molecules directly or indirectly is not known. Glutamine is not charged at neutral pHs, while histidine is titratable with pKR' at pH 6.0. At higher pH, histidine becomes less protonated Cell binding and neurovirulence of Sindbis virus 103 which would be predicted to decrease the affinity of E2 for negatively charged sulfate groups. When histidine has been implicated directly in HS binding this interaction is improved at pH 6.5 (6). However, in DMEM, binding of TE to N18 cells remained unaffected when the pH was raised between 7 and 8, suggesting that E2:55 is not directly involved in the charge interactions between E2 and the cell surface. It is possible that additional receptors are present on N18 and BHK-21 cells that are cell-specific and their interactions are facilitated by the virus-sulfate molecule interactions. Other viruses use different cell receptors on different cells. Mouse hepatitis virus uses isoforms of the murine carcinoembryonic antigen gene family (16) and herpes simplex viruses are able to use a number of different receptors alone or in combination to infect various cell types such as epithelial and neuronal cells (48, 58). From the results of the binding assays, the attachment of 633 virus to N18 cells appears more susceptible to changes in the environment than TE. The association between the change from glutamine to histidine at E2:55 and SIN isolates with higher degrees of neurovirulence has been repeatedly detected in our laboratory (33), suggesting that virus with a histidine at E2:55 has a strong selective advantage in mouse brain. Altered kinetics of virus entry into the cell could affect virus growth rate and virulence by influencing the establishment or spread of the virus. Efficiency of virus spread is a critical determinant for neurovirulence of polytropic murine retroviruses, rabies virus and mouse hepatitis virus (13, 43). Considering the ionic strength and pH of extracellular fluid, histidine at E2:55 would facilitate virus spread in mouse brain. Therefore, we hypothesize that TE has an envelope that interacts stably with the neuronal receptor in the microenvironment of the CNS and is able to initiate more rounds of replication before the onset of the immune response, thus increasing virulence.

Acknowledgements

We would like to thank Andrew Byrnes for many helpful discussions and Marcia Lyons for technical assistance. This work was supported by research grants RO1 NS18596 (DEG), and R01 HL1666 (JW) and training grant T32 AI 07417 (RK) from the National Institutes of Health.

References

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The Role of N-Linked Glycosylation of Sindbis Virus Glycoproteins E2 and E1 in Viral Infectivity and Membrane Fusion Activity

Jolanda M. Smit, Suchetana Mukhopadhyay, Richard J. Kuhn, and Jan Wilschut 108 Chapter 6

Abstract

The ectodomains of Sindbis virus (SIN) glycoproteins E2 and E1 are glycosylated at aspargine residues in a consensus sequence Asn-X-Ser/Thr. Here, we studied the biological role of the N-linked oligosaccharides at positions E2:196 and E1:139, using SIN mutants with an asparagine-to-glutamine substitution at either one of these positions. It was observed that the removal of a single glycosylation site dramatically affects the infectivity of the virus on baby hamster kidney cells (BHK-21). Moreover, deglycosylated SIN mutants were impaired in their ability to fuse with liposomes under mildly acidic conditions, indicating that the reduced infectivity of the virus mutants on BHK-21 cells lies at the level of the fusion process. We suggest that the membrane fusion process of deglycosylated SIN mutants is affected by subtle changes in the conformation of the viral spike proteins, influencing the stability of the E2/E1 heterodimer. Membrane fusion of Sindbis virus glycosylation mutants 109

Introduction

Sindbis virus, the prototype member of the alphavirus genus, has a highly structured envelope composed of a host-cell derived lipid bilayer in which 80 hetero-oligomeric spikes are assembled (42). A single spike consists of a trimer of E2/E1 heterodimers. The E2 and E1 glycoproteins, which mediate the infectious entry of the virus into its host cell, are type I transmembrane proteins with the amino-terminus facing the external surface of the virus particle. The carboxy- terminal domain of E2 interacts with the viral nucleocapsid, which contains the positive-strand genomic RNA associated with 240 copies of the capsid protein. The structural proteins of alphaviruses are translated as a single polypeptide in the order NH2-capsid-PE2-6K-E1-COOH from the 26S subgenomic RNA (42). Upon folding of the capsid protein, it cleaves itself off in an autoproteolytic fashion, and the remainder of the polypeptide is translocated to the rough endoplasmatic reticulum (ER). As soon as the nascent polypeptide enters the lumen of the ER, it is cleaved in three distinct polypeptides (PE2, 6K, and E1) by ER resident signal peptidases. The PE2 (PE2 is the precursor protein of E2) and E1 polypeptides start to fold immediately, disulfide bonds are formed and the proteins become glycosylated. The polypeptides contain oligosaccharide chains linked to the asparagine residues at positions E2:196, E2:318, E1:139, and E1:245 (40). It is believed that N-linked glycosylation, at this stage, increases the solubility of the folding intermediates and prevents the formation of protein aggregates (5, 8). In addition, molecular chaperones such as calnexin and calreticulin (16) bind to the oligosaccharide chains of PE2 and E1, and assist the folding of the proteins (31, 32). During the folding of the spike proteins in the ER, the PE2 and E1 subunits associate to form a PE2/E1 heterodimer. Then, the PE2/E1 heterodimer continues its folding and maturation while passing through the Golgi-apparatus. If the folding of the protein renders the oligosaccharide chains accessible to cellular processing enzymes, the mannosidic side chains are further modified to a complex-type oligosaccharide (48). Complex N-linked glycans were observed at position E2:318, and E1:245, an mannosidic side chain at position E2:196, and a mixture of both types at position E1:139. However, the modification of oligosaccharide chains to a complex-type was found to be dependent on the host cell (17, 18, 28). Not only the spike proteins of SIN, but in fact many viral envelope proteins have been found to be glycosylated. These include the envelope glycoproteins of HIV-1, hepatitis C virus, dengue virus, influenza virus, and respiratory syncytial virus (14, 21, 24, 30, 50). A variety of functions have been ascribed to the N- glycans of viral glycoproteins, including the folding and transport of the proteins (5, 7, 12), the stability of the protein conformation (11, 14, 35), the activity or the immunological properties of the protein (9, 12, 36, 44), and host cell tropism (2, 3). For example, glycosylation of the asparagine residues in the stem region of 110 Chapter 6 influenza hemaglutinin (HA) has been found to stabilize the metastable form of HA required for fusion activity (35). On the other hand, the N-linked oligosaccharide chains attached to the HA ectodomain in close proximity to the receptor-binding site appear to control the receptor binding specificity and affinity (13, 15, 36). Furthermore, glycosylation of HA at antigenic epitopes has been shown to interfere with the access of antibodies (33, 44), and may therefore contribute to the antigenic drift of influenza virus (43). In contrast, little is known about the function of N-linked glycosylation of the spike proteins of alphaviruses. Moreover, the oligosaccharide chains show large variation in location, structure, and number among the alphavirus genus. Earlier studies showed that removal of a single glycosylation site in the ectodomain of SIN glycoproteins E2 or E1, resulted in virus mutants with reduced growth properties on baby hamster kidney cells (BHK-21), when compared to wild-type virus (38). A further decrease of the growth rate was observed with SIN mutants in which more than one glycosylation site were eliminated. In this study, we examined the role of the oligosaccharide chains, linked to asparagine residues E2:196 and E1:139 of the spike proteins of SIN, in the infectious cell entry and membrane fusion activity of the virus. To this end, we used the infectious SIN clone TE12, and two single deglycosylated mutants, in which the asparagine residues at position E2:196 or at position E1:139 were substituted by glutamines. It is demonstrated that the removal of either one of these glycosylation sites dramatically affects the infectivity of the virus on BHK-21 cells. Furthermore, the fusogenic properties of the mutant viruses were evaluated in a liposomal model system, as described previously (45, 46). There was no detectable fusion of the single deglycosylated SIN mutants in a pH range from 4.5 to 7.4, indicating that the reduced infectivity of these viruses lies at the level of the fusion process.

Materials and Methods

Viruses. All viruses were generated from cDNA clones. The construction of SIN clone TE12, and the single deglycosylated SIN virus clones E2:196 and E1:139 has been described previously (27, 38). The viruses were produced by high-efficiency electroporation of BHK-21 cells with in vitro transcripts of linearized cDNA clones, as described before (26). Viruses released from the cells at 20 h post-transfection were harvested, and these stocks were used directly for the production of pyrene-labeled SIN particles, as previously described (6, 45, 46). Labeling of SIN with the pyrene-probe does not affect the infectivity of the virus (45, 46). The viruses were characterized by plaque assay on BHK-21 cells (22), phosphate analysis (4), and protein determination (37). The purity of the viruses was confirmed by SDS- PAGE. The pyrene concentration of the labeled virus preparations was determined by measuring the emission spectrum of the pyrene probe in an AB2 fluorometer (SLM/Aminco, Urbana, IL) at an excitation wavelength of 345 nm. Membrane fusion of Sindbis virus glycosylation mutants 111

Liposomes. Large unilamellar vesicles (LUV) were prepared by a freeze/thaw- extrusion procedure, as previously described (6, 45). Liposomes were prepared with an average size of 200 nm. Liposomes consisted of phosphatidylcholine (PC) from egg yolk, phosphatidylethanolamine (PE) prepared by transphosphatidylation of egg PC, sphingomyelin (SPM) from egg yolk, and cholesterol (Chol) in a molar ratio of 1:1:1:1.5. The lipids were obtained from Avanti Polar Lipids (Alabaster, AL). The phospholipid concentration of liposome preparations was determined by phosphate analysis (4). Fusion assay. Fusion of pyrene-labeled SIN with liposomes was monitored continuously in an AB2 fluorometer, at excitation and emission wavelengths of resp. 345 and 480 nm (6, 45, 49). Briefly, pyrene-labeled SIN (1.0 µM viral phospholipid) and liposomes (100 µM phospholipid) were mixed in a magnetically stirred and thermostatted (37 °C) quartz cuvette of the fluorometer in a final volume of 0.665 ml in HNE. At t= 0 sec, fusion was initiated through the addition of 35 µl 0.1 M MES, 0.2 M acetic acid, pretitrated with NaOH to achieve the final desired pH. The fusion scale was calibrated such that 0% fusion corresponded to the initial excimer fluorescence value and 100% fusion to infinite dilution of the probe (6, 45, 49). The initial rate of fusion was determined from the tangent to the first part of the curve. The extent of fusion was determined 60 s after acidification.

Results

Characterization of pyrene-labeled deglycosylated SIN viruses. To directly evaluate the potential role of spike protein glycosylation in the infectivity and cell entry of SIN, virus mutants derived from the clone TE12 were used in which the asparagine residue at E2:196 or E1:139 had been substituted for a glutamine (Table 1). Pyrene-labeled stocks of the parental virus and both mutants

TABLE 1. Amino acid differences of deglycosylated Sindbis viruses Virus E2:196 E1:139 TE12 Asn Asn E2:196 Gln Asn E1:139 Asn Gln

were generated to allow fusion measurements, as described below. However, first, the purified pyrene-labeled virus preparations were characterized by plaque assay on BHK-21 cells. It was observed that the plaque titer of the single deglycosylated SIN viruses was at least 3 logs reduced, compared to the parental TE12 virus 112 Chapter 6

(Table 2). On the other hand, analysis of the protein and phosphate contents of the virus preparations demonstrated that the total amounts of virus produced were similar for the parental TE12 virus, and the E2:196 and E1:139 mutants (data not shown). Calculation of the number of virus particles showed that about 1.0*1012 particles per ml were present in all three purified virus preparations (data not shown). For the calculation, a theoretical amount of 5.45 *10-17 g of protein or 4.6 *10-20 mol of phosphate per viral particle was used (see also corresponding numbers for SFV; 23). Determination of the PFU-to-particle ratio revealed that almost all of the produced SIN TE12 particles were infectious on BHK-21 cells, the PFU-to-particle ratio being less than 1:10 under the conditions of the experiment (Table 2). By contrast, the PFU-to-particle ratios of both single deglycosylated SIN mutants were found to be greater than 1 to 40.000 (Table 2). Thus, it appears that elimination of a glycosylation site in the ectodomain of SIN glycoprotein E2 or E1 dramatically affects the infectivity of these viruses. Next, we investigated whether the high PFU-to-particle ratio of the single deglycosylated SIN mutants was related to the membrane fusion activity of these viruses.

TABLE 2. Characterization of deglycosylated SIN viruses Viral infectivity PFU to particle PFU to particle Virus on BHK-21 cells ratio based on ratio based on (PFU/ml) protein phosphate TE12 6*1011 1:8 1:4 sM139 1*108 1:82.000 1:41.000 sM196 5*107 1:164.000 1:64.000

Low-pH-dependent fusion of pyrene-labeled deglycosylated SIN viruses with liposomes. Fusion was evaluated in a liposomal model system on the basis of lipid mixing, using pyrene-labeled SIN, as described previously (45, 46). Pyrene- labeled SIN (1 µM phospholipid) and PC/PE/SPM/Chol liposomes (100 µM phospholipid) were mixed, with continuous stirring, and incubated for 1 min at 37 ºC. Then, fusion was triggered by acidification of the medium to pH 5.0. Figure 1 shows the results. At pH 5.0, SIN TE12 fused rapidly and efficiently with liposomes (curve a). At 60 s after acidification, an extent of fusion of approximately 52% was observed. However, under the same conditions, there was no detectable fusion observed with SIN mutants E2:196 and E1:139 (curve b, c). Membrane fusion of Sindbis virus glycosylation mutants 113

60 a 40 (%)

20 Fusion

0 b,c

0 20 40 60 Time (s) Figure 1. Fusion of pyrene-labeled deglycosylated SIN viruses with PC/PE/SPM/Chol liposomes at pH 5.0. Fusion was measured on-line at 37 °C, as described in Materials and Methods. Final viral and liposome concentrations were 1.0 and 100 µM membrane phospholipid, respectively. All fusion measurements were repeated at least three times. Curves: a, TE12; b, E2:196; c, E1:139.

To investigate whether deglycosylation of SIN influences the detailed pH- dependent fusion properties, fusion was evaluated in a pH range from pH 4.5 to 7.4. Figure 2A presents the initial rate of fusion of SIN TE12 (circles), E2:196 (triangles), and E1:139 (squares) as function of the pH. At pH 5.0, 22% of the SIN TE12 virus particles fused with the liposomes within the first second after acidification. Higher or lower pH values than pH 5.0, resulted in a slower rate of fusion. The pH threshold of fusion was 6.5, similar to that of other SIN strains (45, 46). The initial rates of fusion for the single glycosylated SIN mutants were undetectably low, in the entire pH range from 4.5 to 7.4. Figure 2B shows the extent of fusion, determined 60 sec after acidification. Again, throughout the entire pH range no fusion was observed for the single deglycosylated SIN mutants E2:196 (triangles) and E1:139 (squares), whereas the parental SIN TE12 virus (circles) showed high extents of fusion with an optimum at pH 5.0.

Discussion

The present analysis of the SIN clone TE12 and two SIN glycosylation mutants revealed that oligosaccharide chains coupled to the E2 and E1 viral envelope proteins strongly facilitate the membrane fusion activity of the virus. It is demonstrated that the removal of a glycosylation site within the ectodomain of either E2 or E1 dramatically affects both infectivity and low-pH-dependent membrane fusion activity of the virus, indicating that the reduced infectivity of the 114 Chapter 6

25 60 A B 20 (%/s) 15 40

fusion 10

of 20 5

0 Extent of fusion (%) 0 Initial rate 4 5 6 7 8 4 5 6 7 8 pH pH Figure 2. Detailed pH-dependent fusion of pyrene-labeled deglycosylated SIN viruses with liposomes. Fusion of SIN TE12 (circles), E2:196 (triangles), and E1:139 (squares) virus with liposomes was determined at different pH values, as described in the legend to Figure 1. All fusion measurements were repeated at least two times. (A) The initial rates of fusion were determined from the tangents to the first part of the curve. (B) The extents of fusion were determined 60 s after acidification. mutant viruses lies at the level of the membrane fusion process occurring in acidic endosomes. How can N-linked glycans influence the membrane fusion process of SIN? One possibility is that the oligosaccharide chains are directly involved in the fusion reaction. However, it seems unlikely that an oligosaccharide chain in E2 plays a direct role in the fusion process of SIN, since alphavirus fusion is mediated by the E1 glycoprotein (6, 20, 45, 49). Moreover, preliminary characterization of a double deglycosylated SIN mutant carrying a second-site resuscitating mutation in E2 revealed that membrane fusion is partially restored, despite a sustained lack of glycosylation (unpublished results). Another possibility is that the N-linked glycans are required for the proper folding of the viral glycoproteins. Elimination of a glycosylation site could induce a conformational change within the viral spike that may not be tolerated in the stringent lattice of the icosahedral SIN particle. However, detailed cryo-EM analysis demonstrated no detectable conformational differences between the parental SIN TE12 virus and the SIN glycosylation mutants, other than those directly associated with the lack of oligosaccharide chains (38). This indicates that deglycosylation of E2 or E1 does not result in major conformational alterations of the viral spike. Therefore, we suggest that the elimination of a single glycosylation site in either E2 of E1 induces an only subtle change in the conformation of the viral spike protein, as discussed in more detail below. This conformational change could affect the stability of the E2/E1 heterodimer, and therefore influence the structural rearrangements of the viral glycoproteins during the membrane fusion reaction. Membrane fusion of Sindbis virus glycosylation mutants 115

Inhibition of glycosylation by glucosidase inhibitors or mutagenesis of the glycosylation sites has been shown to cause misfolding and aggregation of several viral glycoproteins, resulting in the retention of these proteins within the ER (7, 29). Although, it has been observed that individual N-linked oligosaccharides differ in terms of their importance for folding of the glycoprotein in the ER, some N- glycans can be eliminated with little or no consequence while others appear to be essential (1, 19, 34, 35, 41). For SFV, the glucosidase inhibitor castanospermine blocked binding of p62 (corresponding to PE2 in SIN) and E1 to calnexin and calreticulin, and as a result, 80% of the folding intermediates ended up in protein aggregates and were retained within ER (32). Apparently, the removal of a single glycosylation site in E2 or E1 does not dramatically affect the folding of the protein. This may suggest that calnexin and/or calreticulin still assist the folding of the protein, albeit perhaps to lesser extents. On the other hand, the reduced binding capacity of the molecular chaperones might allow subtle changes in the folding intermediate of the protein, which could result in a slightly different conformation of the spike heterodimer. A role for N-glycans in the stability of the viral glycoproteins has also been proposed for a number of other enveloped viruses, including influenza virus, dengue virus, HIV-1, and human respiratory syncytial virus (9, 10, 11, 14, 35, 50). For example, treatment of HIV-1 infected cells with N-butyldeoxynojirimycin (NB-DNJ), a a-glucosidase inhibitor, blocks formation and the formation of infectious virus (9). Although binding to CD4 occurs, the conformational shift and cleavage of gp120 that results in the exposure of gp41 does not (11). Thus, viral entry is blocked by the increased stability of gp120 in the viral envelope. Moreover, it has been observed that in the presence of NB-DNJ there is a regional misfolding in the V1/V2 loops of gp120 (10). On the other hand, deglycosylation of the stem region of influenza HA resulted in a higher pH threshold for fusion, indicating that the oligosaccharides preserve the metastable form of HA required for triggering fusion (35). Also, the removal of the glycosylation site in the dengue virus E protein resulted in virus mutants with a higher pH threshold for fusion (14). In a later study, it was shown that this oligosaccharide chain protects the fusion peptide of the virus, which suggests that the N-linked glycan stabilizes the E dimer contacts within the viral envelope (39). Although the overall fold of alphavirus E1 is similar to that of flavivirus E, the alphavirus E1 protein is not glycosylated at this position (25, 38, 39). Recently, the three-dimensional localization of the oligosaccharide moieties on SIN have been revealed by cryo-EM image difference map analysis of a panel of deglycosylated SIN mutants (38). It was shown that the asparagine residue at position E2:196 lies at the external side of the spike trimer that forms the receptor binding motif of alphaviruses (47). The glycosylation site at E2:318 lies immediately downstream of the conserved Trp-Ile-Val region, a region that is assumed to interact with the fusion peptide of E1. Residues E1:139 and E1:245 lie 116 Chapter 6 within the viral “skirt” around the base of each spike. A crystallographic dimer of the ectodomain of SFV E1 shows that the E1:139 residue lies within the central domain of the protein, whereas the E1:245 residue lies within the dimerization domain of the protein (25). The location of the glycosylation sites would suggest that the individual sites have different effects on the stability of the E2/E1 heterodimer. For example, one could argue that the oligosaccharide chain at E2:318 is involved in E2-E1 dimer interactions. Deglycosylation of the protein at this position could thus destabilize the E2/E1 heterodimer, which might result in a higher pH threshold for fusion. On the other hand, the removal of the glycosylation sites in E1 could induce a subtle change in the protein conformation, such that it cannot rearrange to a fusion competent complex at mildly acidic pH. We are currently investigating the influence of N-linked glycans on the stability of the E2/E1 heterodimer.

References

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Deacylation of the Transmembrane Domains of Sindbis Virus Envelope Glycoproteins E1 and E2 Does Not Affect Low-pH-Induced Viral Membrane Fusion Activity

Jolanda M. Smit, Robert Bittman, and Jan Wilschut

FEBS Letters 498:57-61 122 Chapter 7

Abstract

The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390). Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Toto1101 infectious clone), in which the E1 C430 and/or E2 C388/390 residues had been substituted for alanines. Both the E1 and E2 mutant viruses, as well as a triple mutant virus, fused with liposomes in a strictly low-pH-dependent manner, the fusion characteristics being indistinguishable from those of the parent Toto1101 virus. These results demonstrate that acylation of the transmembrane domain, of Sindbis virus E1 and E2 is not required for expression of viral membrane fusion activity. Membrane fusion of Sindbis virus deacylation mutants 123

Introduction

Sindbis virus (SIN), the prototype member of the genus Alphavirus of the family Togaviridae, contains three major structural proteins: the capsid protein, C, and two envelope glycoproteins, E2 and E2 (15, 27, 33). The E1 and E2 proteins, which mediate the infectious host cell entry of the virus, are exposed on the viral surface as 80 trimeric spikes each consisting of three E1/E2 heterodimers. E1 and E2 are type-I integral membrane proteins with a single transmembrane anchor sequence and a very small C-terminal domain located at the internal half of the viral membrane. The transmembrane sequences of E1 and E2 contain a number of cysteine residues, which are conserved among most of the members of the alphavirus genus and represent potential sites for covalent attachment of long- chain fatty acids. Indeed, in SIN, these cysteine residues, at E1 position 430 and E2 positions 388 and 390, appear to be palmitoylated (26). Not only the spike proteins of alphaviruses, but in fact many transmembrane glycoproteins of animal viruses have been shown to be acylated (11, 28, 31). These include the G protein of vesicular stomatitis virus (37), the hemagglutinin (HA) of influenza virus (19, 20, 29, 32, 34) and the transmembrane subunit of the envelope glycoproteins of human and simian immunodeficiency viruses (39). Although modification with long-chain fatty acids thus appears to be a common phenomenon among viral transmembrane proteins, the biological function of this acylation remains elusive. Several investigators have used site-directed mutagenesis in order to selectively replace the cysteine residues that provide sites for acylation. Such modified proteins have been studied after expression in cultured cells or in systems generating recombinant viruses. However, conflicting results have been obtained with regard to the role that spike protein acylation plays in the life cycle of the viruses involved. Particularly, the potential function of fatty acids in the membrane fusion activity of viral spikes, such as the influenza virus hemagglutinin, has remained controversial (7, 14, 17, 19, 24, 29, 32, 40). With regard to SIN, studies involving site-specific mutagenesis have demonstrated that deacylation of the transmembrane domains of the E1 and/or E2 spike glycoproteins slows down virus growth early in infection (26). Furthermore, these deacylation mutant viruses are more sensitive to treatment with detergent as compared to wild-type SIN (26). Little is known about the potential effect of spike protein deacylation on the membrane fusion activity of SIN. However, the fact that the E1 and/or E2 deacylation SIN mutants do infect a variety of cell types (26) suggests that the viral life cycle, including the membrane fusion step, is unlikely to be grossly affected by lipid modification. Here, we studied the effect of deacylation of the transmembrane domains of SIN glycoproteins E1 and E2 on the viral membrane fusion capacity. Fusion of SIN virus derived from the clone Toto1101 and several acylation mutants was evaluated in a liposomal model system on the basis of both lipid 124 Chapter 7 mixing and contents mixing. It is demonstrated that SIN fuses rapidly and efficiently with liposomes in a strictly low-pH-dependent manner. Moreover, deacylation of the transmembrane domains of E1 and/or E2 had no effect on the membrane fusion characteristics of the virus.

Materials and Methods

Acylation mutant SIN viruses. A cDNA, containing the Toto1101 infectious clone of SIN virus (25), as well as three acylation mutant cDNAs, were generously provided by Dr. Milton Schlesinger (Washington University, St. Louis, MO, USA). In the first acylation mutant, the transmembranal cysteine at E1 position 430 had been substituted for an alanine (E1:C430A), which results in an almost complete lack of palmitoylation of E1 and a minor reduction in palmitoylation of E2 (26). In the second mutant, the cysteines at E2 positions 388 and 390 had been substituted for alanines (E2:C388A- C390A), resulting in a reduction of E2 palmitoylation by about 70% and a reduction in E1 palmitoylation by about 50% (26). The third mutant, with all three transmembrane cysteine mutations in E1 and E2 (E1:C430A/E2:C388A-C390A), lacks E1 palmitoylation completely and has a residual 30% palmitoylation of E2 (26). The residual fatty acid binding to E2 in the presence of the C388A and C390A mutations is presumably due to acylation at other cysteine residues in the cytoplasmic domain of the protein (12). Production and characterization of virus particles. For the production of virus particles, RNA was synthesized and transfected into baby hamster kidney cells (BHK-21) by electroporation, as described previously (16). Viruses released from cells at 20 h post- transfection were harvested, and these stocks were subsequently used directly for the production of pyrene- and [35S]methionine-labeled viruses, essentially as described before for Semliki Forest Virus (SFV) or SIN (2, 21, 30). The concentration of the virus preparations was determined by lipid phosphate (1) and protein (23) analysis. The purity of the virus particles was confirmed by SDS-PAGE. Viral infectivity was determined by titration on BHK-21 cells in 96-well plates. Preparation of liposomes. Liposomes (large unilamellar vesicles) were prepared in 5 mM Hepes, 150 mM NaCl, 0.1 mM EDTA, pH 7.4 (HNE) by subjection of lipid mixtures, dried from chloroform solution, to five cycles of freezing and thawing and subsequent extrusion (10) through 0.2 µM filters (Nuclepore Inc., Pleasanton, CA, USA) in a LiposoFast mini-extruder (Avestin, Ottawa, Canada). Liposomes consisted of phospholipids (Avanti Polar Lipids, Alabaster, AL, USA) and cholesterol (Sigma Chem. Co., St Louis, MO, USA). The phospholipids were phosphatidylcholine (PC) derived from egg yolk, phosphatidylethanolamine (PE) prepared by transphosphatidylation of egg-PC, and sphingomyelin (SPM) from egg yolk, mixed with cholesterol (Chol) in a molar ratio of 1:1:1:1.5. Trypsin-containing liposomes were prepared likewise, only in this case lipids were dispersed in HNE containing 10 mg/ml trypsin (Fluka Chemie Ag, Buchs, Switzerland). The trypsin-containing liposomes were separated from free trypsin by gel filtration on a Sephadex G-100 column in HNE. The phospholipid concentration of the liposome preparations was determined by phosphate analysis (1). Fusion assays. Fusion of pyrene-labeled SIN with liposomes was monitored on-line in an AB2 fluorimeter (SLM/Aminco, Urbana, IL), as described previously (30). Briefly, Membrane fusion of Sindbis virus deacylation mutants 125 pyrene-labeled SIN (0.5 µM viral phospholipid) and liposomes (200 µM phospholipid) were mixed in a volume of 0.665 ml in HNE, in a quartz cuvette, magnetically stirred and maintained at 37 °C. At t=0 s, fusion was initiated by the addition of 0.035 ml 0.1 M MES, 0.2 M acetic acid, pretitrated with NaOH to achieve the final desired pH. The fusion scale was calibrated such that the initial pyrene excimer fluorescence at 480 nm represented 0% fusion. The 100 % fusion value was set after addition of 0.035 ml 0.2 M octaethyleneglycol monododecyl ether (Fluka Chemie AG). The initial rate of fusion was determined from the tangent to the first part of the curve. The extent of fusion was determined 60 s after acidification. Fusion of SIN with liposomes was also assessed using a contents mixing assay based on degradation of the viral capsid protein by trypsin, initially encapsulated in the liposomes (18, 30, 36). Briefly, [35S]methionine-labeled virus (0.5 µM phospholipid) was incubated with trypsin-containing PC/PE/SPM/Chol liposomes (200 µM liposomal phospholipid) in presence of 125 µg/ml trypsin inhibitor (Boehringer, Mannheim, Germany) in the external medium, at 37 °C. The mixture was acidified, under continuous stirring, to the desired pH with 0.1 M MES, 0.2 M acetic acid, as described above. After 30 s, samples were neutralized by the addition of a pretitrated volume of 0.1 M NaOH, and further incubated for 1 h at 37 °C. Subsequently, samples were analyzed by SDS- PAGE, the protein bands being visualized by autoradiography. Quantification of the viral proteins was done by phosphorimaging analysis using Image Quant 3.3 software (Molecular Dynamics, Sunnyvale, CA, USA).

Results

Low-pH-dependent fusion of pyrene-labeled SIN with liposomes. Fusion of SIN was measured on-line in a liposomal model system, using a lipid mixing assay based on pyrene excimer fluorescence (2, 21, 30). In order to exclude any potential effects of the fluorescence labeling procedure on the biological properties of the virus, we first evaluated the specific infectivities of pyrene-labeled Toto1101 and acylation mutant SIN virus preparations. Viral infectivity was determined by titration on BHK-21 cells and related to the number of virus particles present based on biochemical analyses (30). In all cases a particle to infectious unit ratio of 4-5 was found, similar to that seen in unlabeled virus (data not shown). These results demonstrate that neither the fluorescence labeling procedure nor the presence of the acylation mutations in E1 and/or E2 had any significant effect on the specific infectivity of the viruses. The pyrene fusion assay relies on a decrease of pyrene excimer fluorescence owing to dilution of pyrene-labeled phospholipids from the viral into the liposomal membrane. This decrease can be translated directly to the extent of fusion, since each individual fusion event results in a large dilution of the probe and, thus, in an essentially complete disappearance of the excimer fluorescence intensity of the virus particle involved. Figure 1 presents the fusion kinetics of pyrene-labeled Toto1101 SIN virus with PC/PE/SPM/Chol liposomes. At pH 126 Chapter 7

4.6, the virus fused rapidly and efficiently with the liposomes, the extent of fusion being approximately 55% at 10 s after acidification of the virus-liposome mixture (curve a). With increasing pH, fusion became slower and less extensive (curves b, c). At pH 7.4 there was no detectable fusion (curve d). We also measured fusion of pyrene-labeled Toto1101 SIN virus with liposomes lacking either SPM or Chol or both, and observed that fusion was absent (data not shown). These results indicate that the membrane fusion activity of SIN derived from the Toto1101 infectious clone is triggered by a mildly acidic pH and exhibits a similar overall lipid dependence as fusion of wild-type SIN (laboratory-adapted strain AR339) (30) or SFV (4, 21).

60 a b 40

20 c Fusion (%)

0 d

0 20 40 60 Time (s) Figure 1. Low-pH dependent fusion of pyrene-labeled SIN derived from cDNA clone Toto1101 with liposomes. Fusion of pyrene-labeled SIN Toto1101 with PC/PE/SPM/Chol liposomes was measured on-line at 37 °C as described in Materials and Methods. Final concentrations of virus and liposomes corresponded to 0.5 µM and 200 µM phospholipid, respectively. Curves: a, pH 4.6; b, pH 5.0; c, pH 5.75; d, pH 7.4.

Fusion characteristics of SIN acylation mutants. Figure 2 presents the fusion kinetics of the three acylation mutant SIN viruses, E1:C430A, E2: 388A/C390A, and E1:C430A/E2:C388-C390A, in comparison with the original Toto1101 virus. Clearly, the fusion kinetics of the acylation mutants were indistinguishable from those of the unmodified virus. Importantly, none of the viruses had any significant fusion activity at neutral pH. Figure 3 compares the detailed pH dependence of Toto1101 (open triangles) and the acylation mutant virus E1:C430A/E2:C388A-C390A (closed triangles). Panel A shows the initial rate of fusion as a function of the pH, determined from the tangent to the first part of the fusion curves. Rates were very similar for unmodified and acylation mutant viruses in the entire pH range from pH 4.0 to pH 7.4. Figure 3B presents the extent of fusion as function of the pH, measured 60 s after acidification. Again, throughout the pH range from 4.0 to 7.4, no Membrane fusion of Sindbis virus deacylation mutants 127 differences were observed between the unmodified and acylation mutant SIN. Under optimal conditions, 18-20% of the virus particles fused within the first second after acidification, with an extent of fusion of 60% at 60 s post- acidification. The pH threshold for fusion was 6.2 for both viruses. The acylation mutant viruses E1:C430A and E2:C388A-C390A gave essentially identical results in terms of initial rate and extent of fusion (data not shown).

60 a-d pH 4.6 40

20 Fusion (%) pH 7.4 0 a-d

0 20 40 60 Time (s) Figure 2. Low-pH dependent fusion of pyrene-labeled acylation mutant SIN viruses with liposomes. Fusion of pyrene-labeled acylation mutant viruses with liposomes was measured at pH 4.6 and 7.4, as described in the legend to Figure 1. Curves: a, SIN; b, E1:C430A; c, E2:C388A/C390A; d, E1:C430A/E2:C388A/C390A .

20 A 60 B

40 10 20

0 Extent of fusion (%) 0 Initial rate of fusion (%/s)

4 5 6 7 4 5 6 7 pH pH Figure 3. Comparison of the pH-dependent of fusion of pyrene-labeled acylation mutant and SIN Toto1101 viruses. Fusion of SIN Toto1101 (open triangles) and the acylation mutant virus, E1:C430A/E2:C388A/C390A (closed triangles) was determined at different pH values, as described in the legend to Figure 1. (A) The initial rates of fusion were determined from the tangent to the first part of the curve. (B) The extents of fusion were determined 60 s after acidification. 128 Chapter 7

Contents mixing of [35S]methionine-labeled SIN with trypsin-containing liposomes. In the above experiments fusion was evaluated on the basis of lipid mixing. Another, very stringent, criterion for fusion involves the coalescence of the interior of the virus with the liposomal lumen. Contents mixing was assayed as the degradation of the viral capsid protein by trypsin, initially encapsulated in the liposomes. Figure 4A shows the capsid degradation of SIN Toto1101 and the acylation mutant virus E1:C430A/E2:C388A-C390A. In both cases, incubation of the virus with trypsin-containing liposomes at pH 4.6 resulted in the

A

E1/E2

C

a b c d e f

80 B 60

40

20

Capsid degradation (%) 0 4.6 5.0 5.5 7.4 pH Figure 4. Degradation of the viral capsid protein by trypsin, initially incorporated in liposomes. The trypsin assay was carried out as described in the Materials and Methods. Final concentrations of [35S]methionine-labeled virus and (trypsin-containing) PC/PE/SPM/Chol liposomes corresponded to 0.5 µM and 200 µM phospholipid, respectively. (A) Viral structural proteins of SIN Toto1101 (lanes a-c) and acylation mutant E1:C430A/E2:C388A/C390A (lane d-f), visualized by autoradiography. Lanes: a and d, trypsin-containing liposomes at pH 4.6; b and e, trypsin-containing liposomes at pH 7.4; c and f, empty liposomes at pH 4.6. (B) Quantification of the extent of capsid protein degradation on the basis of the ratio C/[C+E1+E2] by phosphorimaging analysis. The ratios C/[C+E1+E2] of the controls, in which empty liposomes were incubated with the viruses under otherwise identical conditions, were taken as the 100% values. Bars: shaded, SIN; open, E1:C430A/E2:C388A/C390A. Membrane fusion of Sindbis virus deacylation mutants 129 degradation of a substantial fraction of the capsid protein (lane a, d). At pH 7.4 no capsid degradation was observed (lane b, e). The controls, in which the viruses were incubated with empty liposomes at pH 4.6, did not show degradation of the capsid protein either (lane c, f). The ratio of the radioactivity of the capsid band relative to the total radioactivity (C+E1/E2) of the samples incubated at pH 7.4 and the control samples with empty liposomes was close to 0.4, as expected on the basis of the number of methionine residues in the SIN structural proteins (33). Complete degradation of the capsid protein was observed when Triton X-100 was added to the reaction mixture, in the absence of trypsin inhibitor (results not shown). Figure 4B shows the extent of capsid degradation as function of the pH, quantified by phosphorimaging. For SIN Toto1101, incubated at pH 4.6, approximately 70% of the capsid protein was degraded, while at pH 5.0 and pH 5.5 the corresponding numbers were 41% and 19%, respectively. With the triple acylation mutant virus, E1:C430A/E2:C388A-C390A, we detected an extent of capsid degradation of 72% at pH 4.6, 45% at pH 5.0, and 25% at pH 5.5. Similar results were obtained with the separate E1 or E2 acylation mutant viruses (results not shown). The extents of capsid protein degradation seen in the pH range from 4.6 to 5.5 (Figure 4B) are very similar to the corresponding extents of lipid mixing observed in the pyrene fluorescence assay (Figure 3B).

Discussion

The results presented in this paper demonstrate that SIN derived from the infectious clone Toto1101 fuses rapidly and efficiently with receptor-free cholesterol- and sphingolipid-containing liposomes in a strictly low-pH-dependent manner. Fusion was evaluated on the basis of both membrane lipid mixing and internal contents mixing between the virus and the liposomes. The pH dependence of fusion of the Toto1101 virus is very similar to that of the wild-type, laboratory-adapted, SIN strain AR339 (30) and argues strongly for a cell entry mechanism of SIN involving receptor-mediated endocytosis and acid-induced fusion of the viral envelope with the endosomal membrane. This is entirely consistent with recent observations of Glomb-Reinmund and Kielian (8). These investigators showed that infection of BHK-21 cells by SIN is inhibited by agents that interfere with endosomal acidification, such as NH4Cl, balifomycin or concanamycin. Further support for a cell entry mechanism of SIN involving receptor-mediated endocytosis has been provided by DeTulleo and Kirchhausen (5), who observed that BHK-21 cell infection by SIN is affected by a mutated form of dynamin which inhibits the budding of clathrin-coated vesicles. In the light of this fairly convincing evidence, it is intriguing that quite recently (9) Brown and coworkers have presented data which support their previous suggestion (3, 6) that exposure to an acidic pH may not be an obligatory step in the infection of cells by alphaviruses. These investigators observed that the 130 Chapter 7 infection of mosquito cells by SIN was not blocked by choloroquine under conditions such that the drug did raise the pH of the endosomal compartment of the cells (9). Although it is possible that alphaviruses use different routes of infection in vertebrate and insect cells, our previous (2, 21, 30, 35) and present studies in model systems, designed to specifically address the issue of alphavirus fusion activation, strongly argue for exposure to a mildly acidic pH being an essential step in the triggering of the fusion process. The principal result of this study demonstrates that acylation of the transmembrane domains of the SIN glycoproteins E1 and E2 is not required for expression of viral membrane fusion activity. Indeed, deacylation of E1 and/or E2 has no effect on the kinetics or the detailed pH dependence of the fusion process. This conclusion is in agreement with similar results obtained for other enveloped viruses, such as vesicular stomatitis virus (37) and human and simian immunodeficiency viruses (39). The potential role of acylation in the membrane fusion activity of influenza HA however, remains controversial. HA-mediated fusion has been studied extensively in cultured cells expressing the isolated HA, with erythrocytes or erythrocyte ghosts serving as target membranes. In such systems, deacylation of HA does not affect membrane lipid mixing, as measured by fluorescence dequenching of the fluorophore R18 (7, 24). Likewise, deacylation has been observed to have little effect on syncytia formation mediated by HA expressed on the cell surface (32, 34). By contrast, other investigators have reported distinct effects of deacylation of HA on various stages of the fusion process, including initial fusion pore flickering (17) and late fusion pore dilation as assessed by syncytia formation (7, 19). It is possible that this apparent discrepancy is, at least in part, a consequence of the fusion process being primarily studied in HA-expressing cells rather than with virus. In these cell systems the surface density of HA may vary considerably and also its biological activity may be affected when it is expressed in the absence of the M2 protein (22). It is a major advantage of our present study that it involves the use of whole virus derived from a cDNA clone, rather than SIN envelope glycoproteins expressed on the surface of cultured cells. Similar approaches followed for influenza, involving reverse genetics and the generation of mutant virions however, have again resulted in conflicting conclusions. Zurcher et al. (40) reported that deacylation of HA affects virus assembly, whereas little effect on either assembly or infectivity was observed by others (13, 14, 38). With regard to alphaviruses, SIN in particular, the lack of effect of E1 and/or E2 deacylation on viral membrane fusion activity, reported here, and the limited effects on virus assembly and release (28) explain the observation made by us in the present study and by others before (28) that deacylation mutant viruses are fully infectious on cultured cells, exhibiting specific infectivity values. On the other hand, the conserved nature of the cysteine residues in the transmembrane domains of the E1 and E2 envelope glycoproteins suggests that fatty acid modification of Membrane fusion of Sindbis virus deacylation mutants 131 these proteins has an important function in the life cycle of alphaviruses. Our present results suggest that this biological function is not at the level of the low- pH-induced virus membrane fusion process.

Acknowledgments

This work was supported by the US National Institutes of Health (grant HL 16660), and by The Netherlands Organization for Scientific Research (NWO) under the auspices of the Foundation for Chemical Research (CW). We would like to thank Prof. Dr. M. Schlesinger for providing us the cDNAs of the acylation mutant viruses.

References

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15. Kielian, M. 1995. Membrane fusion and the alphavirus life cycle. Adv. Virus Res. 45:113-151. 16. Liljestrom, P., S. Lusa, D. Huylebroeck, and H. Garoff. 1991. In vitro mutagenisis of a full-length cDNA clone of Semliki Forest virus: The small 6,000-molecular weight membrane protein modulates virus release. J. Virol. 65:4107-4113. 17. Melikyan, G. B., H. Jin, R. A. Lamb, and F. S. Cohen. 1997. The role of the cytoplasmic tail region of Influenza virus hemaglutinin in formation and growth of fusion pores. Virology 235:118-128. 18. Moesby, L., J. Corver, R. K. Erukulla, R. Bittman, and J. Wilschut. 1995. Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner. Biochemistry 34:10319-10324. 19. Naeve, C. W., and D. Williams. 1990. Fatty acids on the A/Japan/305/57 influenza virus have a role in membrane fusion. EMBO J. 9:3957-3978. 20. Naim, H. Y., B. Amarneh, N. T. Ktistakis, and M. G. Roth. 1992. Effects of altering palmitylation sites on biosynthesis and function of the Influenza virus hemaglutinin. J. Virol. 66:7585-7588. 21. Nieva, J. L., R. Bron, J. Corver, and J. Wilschut. 1994. Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane. EMBO J. 13:2797-2804. 22. Ohuchi, M., A. Cramer, M. Vey, R. Ohuchi, and H. D. Klenk. 1994. Rescue of vector expressed fowl plaque virus hemaglutinin in biologically active form by acidotrophic agents and coexpressed M2 protein. J. Virol. 68:920-926. 23. Peterson, G. L. 1977. A simplification of the protein assay method of Lowry et al which is more generally applicable. Anal-Biochem. 83:346-356. 24. Philipp, H. C., B. Schroth, M. Veit, M., Krumbiegel, A. Herrmann, and M. F. G. Schmidt. 1995. Assesment of fusogenic properties of Influenza virus hemaglutinin deacylated by site-directed mutagenisis and hydroxylamine treatment. Virology 210:20-28. 25. Rice, C. M., R. Levis, J. H. Strauss, and H. V. Huang. 1987. Production of infectious RNA transcripts from Sindbis virus cDNA clones: Mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J. Virol. 61:3809-3819. 26. Ryan, C., L. Ivanova, and M. J. Schlesinger. 1998. Effects of site directed mutations of transmembrane cysteines in Sindbis virus E1 and E2 glycoproteins on palmitylation and virus replication. Virology 249:62-69. 27. Schlesinger, S. and M. J. Schlesinger., Eds. 1986. The Togaviridae and Flaviviridae, Plenum, New York.. 28. Schlesinger, M. J., M. Veit, and M. F. G. Schmidt. 1992. in: Lipid Modifications of Proteins (M. J. Schlesinger, Ed.) pp. 1-20, CRC Press, Boca Raton, Florida. 29. Simpson, D. A. and R. A. Lamb. 1992. Alterations to Influenza virus hemagglutinin cytoplasmic tail modulate virus infectivity. J. Virol. 66:790-803. 30. Smit, J. M., R. Bittman, and J. Wilschut. 1999. Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes. J. Virol. 73:8476-8484. 31. Schmidt, M. F. G. 1982. Acylation of viral spike glycoproteins: a feature of enveloped RNA viruses. Virology 116:327-338. 32. Steinhauer, D. A., S. A. Wharton, D. C. Wiley, and J. J. Skehel. 1991. Deacylation of the hemaglutinin of Influenza A/Aichi/2/68 has no effect on membrane fusion properties. Virology 184:445-448. 33. Strauss, J. H., and E. G. Strauss. 1994. The alphaviruses: gene expression, replication, and evolution [published erratum appears in Microbiol Rev 1994 Dec;58(4):806]. Microbiol. Rev. 58:491-562. 34. Veit, M., E. Kretzschmar, K. Kuroda, W. Garten, M. F. G. Schmidt, H. Klenk, and R. Rott. 1991. Site specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of Influenza virus hemaglutinin. J. Virol. 65:2491-2500. Membrane fusion of Sindbis virus deacylation mutants 133

35. Wahlberg, J. M., R. Bron, J. Wilschut, and H. Garoff. 1992. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein. J. Virol. 66:7309-7318. 36. White, J., and A. Helenius. 1980. pH-dependent fusion between the Semliki Forest virus membrane and liposomes. Proc. Natl. Acad. Sci. USA 77:3273-3277. 37. Whitt, M. A., and J. K. Rose. 1991. Fatty acylation is not required for membrane fusion activity or glycoprotein assembly into VSV virions. Virology 185:875-878. 38. Yang, C., and R. W. Compans. 1996. Palmitoylation of the murine leukemia virus envelope glycoprotein transmembrane subunits. Virology 221:87-97. 39. Yang, C, C. P. Spies, and R. W. Compans. 1995. The Human and Simian Immunodeficiency virus envelope glycoprotein transmembrane subunits are palmitoylated. Proc. Natl. Acad. Sci USA 92:9871-9875. 40. Zurcher, T., G. Fluo, and P. Palese. 1994. Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation. J. Virol. 68:5748-5754. 134 Chapter 7 Chapter 8

PE2 Cleavage Mutants of Sindbis Virus: Correlation Between Viral Infectivity and pH-Dependent Membrane Fusion Activation of the Spike Heterodimer

Jolanda M. Smit, William B. Klimstra, Kate D. Ryman, Robert Bittman, Robert E. Johnston, and Jan Wilschut

Journal of Virology 75:11196-11204 136 Chapter 8

Abstract

The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin- like protease. Previous work has shown that SIN mutants, impaired in PE2 cleavage, are non-infectious on BHK-21 cells, the block in infection being localized at a step after virus-receptor interaction but prior to RNA replication. Here, we studied the membrane fusion properties of SIN PE2 cleavage mutants and observed that these viruses are impaired in their ability to form an E1 homotrimer and to fuse with liposomes at a mildly acidic pH. The block in spike rearrangement and fusion could be overridden by exposure of the mutant viruses to very low pH values (pH < 4.5). Cleavage mutants with second-site resuscitating mutations in PE2 were highly infectious for BHK-21 cells. The ability of these viruses to form E1 homotrimers and to fuse at a mildly acidic pH was completely restored, despite a sustained lack of PE2 cleavage. Membrane fusion of Sindbis virus PE2 cleavage mutants 137

Introduction

Alphaviruses, such as Sindbis virus (SIN) and Semliki Forest virus (SFV), are enveloped viruses which contain three major structural proteins, the capsid protein, C, and two envelope glycoproteins, E2 and E1 (38). The glycoproteins are organized on the surface of the virion in 80 hetero-oligomeric spikes, which mediate the infectious entry of these viruses into cells. The E2 glycoprotein is primarily involved in the interaction of a virus particle with cell surface attachment receptors (37), whereas the E1 glycoprotein is necessary for the subsequent membrane fusion process (8, 40). Very recent crystallographic and cryoelectron microscopy studies have revealed that E1 in many respects resembles the flavivirus E glycoprotein. The alphavirus E1 protein appears to lie flat on the viral surface which drives lateral spike interactions, whereas E2 forms the spike protrusions (21, 30). In the infected cell, the alphavirus structural proteins are translated as a large polyprotein. Once the C protein is cleaved off, the NH2-terminus of PE2, the precursor protein of E2 (PE2 is called p62 in SFV), serves to direct the cotranslational translocation of the remaining polyprotein to the endoplasmic reticulum (ER) (38). This polyprotein is processed by signal peptidase within the ER, and the two envelope proteins associate to form PE2/E1 heterodimers (38). The PE2/E1 heterodimer matures further while passing through the Golgi and trans-Golgi network (TGN). In the TGN or in a post-Golgi compartment PE2 is cleaved close to its amino-terminus to form E2 and a residual peptide E3 (24). PE2 cleavage is mediated by a furin-like host protease at a consensus recognition sequence XBXBBX close to the amino-terminus, in which B is a basic and X a hydrophobic amino acid (18). The peptide E3, which contains the XBXBBX sequence, is retained on the SFV spike, but it is released in the case of SIN (24, 28). Several investigators have shown that the efficiency of PE2 cleavage can be influenced by amino-acid changes within the cleavage site (5, 13, 14, 19, 22, 34, 39). Thus, a number of PE2 cleavage mutants of SIN, SFV, and Venezuelan Equine Encephalitis virus (VEE) have been identified, and these viruses appear to have defects in one or more biological functions due to the presence of uncleaved PE2. For example, in SFV, cleavage of p62 was prevented by replacement of Arg by Leu at position -1 of E2 (34). This p62 cleavage mutant virus (mL) was found to be non-infectious on BHK-21 cells. The block in infection appeared to involve both the virus-receptor interaction and fusion activity of the mutant virus. Viral infectivity on BHK-21 cells was restored by in vitro cleavage of p62 with trypsin or by exposure of the virus to very low pH values (22, 34, 40). Likewise, several SIN PE2 cleavage mutants have been shown to be non-infectious. For example, replacement of the Arg or Ser residues at position 1 of E2 (the last amino acid of the XBXBBX cleavage site) by an Asn residue within the context of the infectious clones TRSB (corresponding to a laboratory-adapted strain of SIN) or TR339 138 Chapter 8

(containing the consensus sequence of wild type SIN) almost completely blocked viral infectivity (13, 14, 19, 25). SIN cleavage mutants in which part of the cleavage site was deleted were also found to be essentially non-infectious for BHK-21 cells (19). Interestingly, it has been found that the lethality of PE2 cleavage mutation in TRSB-N (Arg-to-Asn substitution at position 1 of E2) could be reversed by resuscitating second-site mutations in PE2 (14). These investigations showed that after infection with TRSB-N virions occasionally small plaques were formed. Sequence analysis of the purified plaques revealed that some of these viruses carried second-site mutations in PE2, but yet remained cleavage-deficient. While these PE2 cleavage mutants with a resuscitating mutation in PE2 were found to be infectious on BHK-21 cells, the viruses appeared to have an attenuated virulence in CD-1 mice, as compared to the parental TRSB virus. Recent data showed that the block in infection of PE2 cleavage-deficient SIN viruses without second-site resuscitating mutations, is not at the level of the initial virus-receptor interaction (19). By contrast, SIN PE2 cleavage mutants with an intact cleavage site, bind very efficiently to BHK-21 cells, even better than the parental TR339 virus. It has been shown that, the presence of the basic XBXBBX sequence mediates an efficient interaction with heparan sulfate (HS), which is abundantly expressed on BHK-21 cells and thus acts as a receptor for the virus (4, 20). Furthermore, it has been shown that the presence of uncleaved PE2 in virions does not influence RNA replication, or virus assembly and release (14, 19). Taken together, these data indicate that the block in infection of these SIN PE2 cleavage mutants lies downstream of the interaction of a virus particle with a cellular receptor but prior to RNA replication, suggesting that these mutant viruses are impaired in their membrane fusion properties. Here, we studied the fusogenic properties of PE2 cleavage mutants, based on the infectious clone TR339, using a liposomal model system. PE2 cleavage mutants in which either the Ser residue at position 1 of E2 was replaced by Asn (E2:N1) or from which the BXBB sequence within the PE2 cleavage site was deleted (FDF) were used. The results show that PE2 cleavage mutants were unable to fuse with liposomes at pH 5.0, indicating that the block in infection lies at the level of the fusion process. Incubation of PE2 cleavage mutants at low pH demonstrated that the viruses were impaired in their ability to form an E1 homotrimer, the fusion- active conformation of the viral spike protein. The block in rearrangement and fusion could be overridden by exposure of the virus to very low pH values (pH < 5.0). Furthermore, E2:N1 cleavage mutants with resuscitating mutations in PE2 were generated, analogous to those identified in TRSB-N, and found to be infectious in BHK-21 cells. These PE2 resuscitated mutant viruses do form E1 homotrimers at a physiologically relevant acidic pH, despite the lack of PE2 cleavage. Accordingly, their membrane fusion activity appeared to be completely restored. Membrane fusion of Sindbis virus PE2 cleavage mutants 139

Materials and Methods

Constructs. The construction of the cDNA clones pTR339, pE2:N1 (called p39N1 in previous articles), and pFDF has been described previously (19, 20). The “p” prefix indicates the cDNA form of the virus clone. The cDNA pE2:N1/E3:R25 was constructed by substitution into pE2:N1 of an AatII-to-StuI fragment from pTRSB-E3R25 (14). The cDNA clones pE2:N1/T191 and pE2:N1/G216 were constructed by substitution into pE2:N1 of an StuI-to-BssHII fragment from pTRSB-NE2T191 resp. pTRSB-NE2G216 (14). The introduced mutations were confirmed by DNA sequence analysis at the University of North Carolina at Chapel Hill Automated Sequencing Facility with a 373A DNA sequencer with the Taq DyeDeoxy terminator cycle sequencing kits (Applied Biosystems). Production and characterization of pyrene- and [35S]methionine-labeled virus particles. Viruses were produced on BHK-21 cells. The cells were cultured in Glasgow’s modification of Eagle’s minimal essential medium (Gibco/BRL, Breda, The Netherlands), supplemented with 5% foetal calf serum (FCS), 10% tryptose phosphate broth, 200 mM glutamine, 25 mM Hepes, and 7.5% sodium bicarbonate. For the production of pyrene- labeled virus particles, BHK-21 cells, prior cultured for 48 h on medium containing 10 µg/ml 16-(1-pyrenyl)hexadecanoic acid (pyrene fatty acid; Molecular Probes, Eugene, OR, USA), were transfected, by electroporation, with in vitro transcripts of linearized cDNA clones. Pyrene-labeled SIN particles released from the cells at 20 h post-transfection were harvested and purified from the medium, as described before (3, 27, 36). For the production of [35S]methionine-labeled virus particles, BHK-21 cells were transfected with in vitro transcribed viral RNA by electroporation, essentially as previously described (3, 27, 36). Briefly, at 3-4 h post-transfection, the culture medium was replaced by methionine-free DMEM (Gibco/BRL) supplemented with 5% FCS and 200 mM glutamine. After 2 h starvation, 200 µCi/5 ml [35S]methionine was added to the medium and incubation was continued overnight. At 20 h post-transfection [35S]methionine-labeled viral particles were harvested and purified from the medium, as described before (3, 27, 36, 43). The purity of the produced virus particles was analyzed with SDS-PAGE. Visualization of the protein bands and quantification of the PE2 content in virions were done by phosphorimaging analysis using Image Quant 3.3 software (Molecular Dynamics, Sunnyvale, CA, USA). The percentage PE2 in virions was determined, by relating the intensity of PE2 to the total intensity of E1, PE2 and E2, corrected for the contribution of E1 on the basis of the relative numbers of methionine residues in the E1, E2 and PE2 proteins (19, 32). The specific infectivity (PFU/cpm) was calculated for all batches [35S]methionine-labeled viruses by plaque assay on BHK-21 cells (20). Binding assays. Virus attachment to BHK-21 cells and heparin- and bovine serum albumin (BSA)-agarose beads (both from Sigma Chemical Co, St. Louis, MO, USA) was performed, essentially as described previously (19, 20). Briefly, BHK-21 cells (cultured in a 12-well plate; Costar, Cambridge, MA, USA) were washed two times with cold 5 mM Hepes, 150 mM NaCl, 0.1 mM EDTA, pH 7.4 (HNE) + 1% fetal calf serum (HNE+). Subsequently, 150 µl [35S]methionine-labeled virus particles were added to the cells (ranging from 106 to 107 cpm ~ approx. 109 to 1010 virus particles) and incubation was continued at 4 °C for 2 h with gentle agitation. The cells were then washed twice with HNE+ and trypsinized with 1*Trypsin/EDTA (Gibco/BRL). For binding to heparin-agarose and 140 Chapter 8

BSA-agarose beads, 1 ml of beads was washed with HNE+ three times, and resuspended in 1 ml HNE+. Fifty microliters of beads were added to 50 µl virus particles, similar to that of the cell binding assay, and incubated for 2 h at 4 °C with gentle agitation. Subsequently, the mixtures were washed three times with HNE+, and resuspended in 0.6% triton (Sigma) in HNE. Viral radioactivity was quantified by liquid scintillation analysis. Control reactions with no added virus were performed for each binding experiment, the cpm counted was subtracted from total cpm bound. Preparation of liposomes. Liposomes (large unilamellar vesicles) were prepared by freeze/thaw extrusion as described before (3, 27, 36, 43). Briefly, lipid mixtures, dried from chloroform/methanol, were hydrated in HNE and subjected to five cycles of freezing and thawing. Subsequently, the suspension was extruded 21 times through two 0.2 µM filters (Nucleopore, Inc., Pleasanton, CA, USA) in a LiposoFast mini-extruder (Avestin, Ottawa, Canada). Liposomes consisted of phosphatidylcholine (PC)/phosphatidylethanolamine (PE) /sphingomyelin (SPM)/Cholesterol (Chol) with a molar ratio of 1:1:1:1.5. The phospholipids were obtained from Avanti Polar lipids (Alabaster, AL, USA) and cholesterol from Sigma. The phospholipid concentration of the liposomes was determined by phosphate analysis (2). Fusion assay. Fusion of pyrene-labeled SIN with liposomes was monitored on-line in an AB2 fluorimeter (SLM/Aminco, Urbana, IL, USA), at 37 ºC (3, 27, 36, 43). In the fusion reaction, pyrene-labeled SIN (approx. 1010 particles) was continuously mixed with an 5-fold excess of liposomes in a final volume of 0.665 ml in HNE. At t=0 s, fusion was initiated by the addition of 35 µl 0.1 M MES (morpholinoethanesulfonic acid)-0.2 M acetic acid, pretitrated with NaOH to achieve the final desired pH. The fusion scale was calibrated such that 0% fusion corresponded to the initial pyrene-excimer fluorescence level and 100% fusion to the fluorescence value obtained after the addition of 35 µl 0.2 M octaethyleneglycol monododecyl ether (Fluka Chemie AG, Buchs, Switzerland) (3, 27, 36, 43). The extent of fusion was determined 60 s after acidification. Analysis of the conformational changes in the viral spike protein. The conformational changes occurring in the viral spike protein were examined under the same conditions as in the fusion experiments (3, 36). After the indicated pH treatment, samples were neutralized by addition of a pretitrated volume NaOH, solubilized in SDS-PAGE sample buffer, and analyzed by SDS-PAGE. Running gels were further incubated for 30 min in 1 M sodium salicylate and dried. Visualization and quantification of the E1 trimer was done by phosphorimaging analysis, as mentioned above, by relating the intensity of the E1 trimer to the total intensity of E1, PE2, E2 and E1 trimer, corrected for the contribution of PE2 or E2 on the basis of the relative numbers of methionine residues in the E1, E2 and PE2 proteins.

Results

Characterization of PE2 cleavage mutant SIN viruses. In this study we investigated the membrane fusion activity of a number of SIN PE2 cleavage mutant viruses. PE2 cleavage mutations were introduced into the background of the infectious SIN clone TR339. Two mutants were chosen that have previously been shown to affect PE2 cleavage efficiency (Table 1; 19). In the mutant Membrane fusion of Sindbis virus PE2 cleavage mutants 141 construct, designated E2:N1, the Ser residue at position 1 of E2 is replaced by an Asn residue. This residue creates an N-linked glycosylation signal, and the glycosylation of the Asn residue at position 1 of E2 is proposed to interfere with furin activity (19). SDS-PAGE analysis of the E2:N1 mutant showed that indeed PE2 cleavage was completely inhibited. Furthermore, the virus was poorly infectious, as determined by plaque assay on BHK-21 cells. Likewise, the FDF mutant, in which the BXBB sequence was deleted, incorporated 100% uncleaved PE2 and also had a very low specific infectivity (Table 1).

TABLE 1. Characterization of PE2 cleavage mutant Sindbis viruses Virus Amino acids at PE2 Furin cleavage % PE2 BHK specific cleavage site site infectivitya TR339 G R S K R S Intact 4 18 FDF G - - - - S Deleted 100 0.5 E2:N1 G R S K R N Intact 100 1.5

a (PFU/cpm)

The first step in viral entry is the interaction of a virus particle with a cellular receptor. Consistent with previous reports (20), we found that TR339 virus does not bind very efficiently to BHK-21 cells (Figure 1A). The FDF mutant bound equally poorly to BHK-21 cells. On the other hand, the E2:N1 virus did bind efficiently to BHK-21 cells. Recent data have demonstrated that SIN viruses with an intact cleavage site interact with HS, a cellular receptor which is abundantly expressed on BHK-21 cells (19). Accordingly, we found that the E2:N1 mutant bound efficiently to heparin-agarose beads (Figure 1B). In a control, in which albumin-agarose beads were used, none of the viruses bound to the beads (data not shown). Since the two mutant viruses, as compared to TR339, bind similarly (FDF) or much better (E2:N1) to BHK-21 cells, it is clear that the block in infection for these mutants lies after binding of the virus particle to the cell surface, presumably at the level of the viral membrane fusion process. Fusion activity of pyrene-labeled PE2 cleavage mutant SIN viruses. To study the fusogenic properties of the PE2 cleavage mutants, we used SIN variants biosynthetically labeled with the fluorescent probe pyrene. Because the mutant viruses are almost non-infectious, RNA transcripts derived from the cDNA were transfected directly into BHK-21 cells, cultured beforehand in the presence of pyrene fatty acid. At 20 h post-transfection the pyrene-labeled virus particles were 142 Chapter 8

100 80 BHK-21 cells A Heparin-beads B 80 60 (%) (%) 60 40 bound bound 40

20 20 Virus Virus 0 0 TR339 FDF E2:N1 TR339 FDF E2: N1 Figure 1. Binding of PE2 cleavage-deficient SIN viruses to BHK-21 cells or heparin- agarose beads. Approx. 1010 [35S]methionine-labeled virus particles were added to the cells or beads and binding was measured after 2 h incubation at 4 °C, as described in Materials and Methods. Each bar represent the mean of triplicate binding assays. Each set of triplicates was repeated three times. The error bars represent standard deviations. (A) Binding to BHK-21 cells. (B) Binding to heparin-agarose beads. harvested and purified from the medium (3, 27, 36). With this procedure no additional passage of the virus is involved, thereby minimizing the possibility of generating revertant viruses. Subsequently, fusion of pyrene-labeled SIN was measured in a liposomal model system (3, 27, 36). Upon fusion of pyrene-labeled virus particles with target liposomes, the pyrene phospholipids are diluted into the liposomal membrane, resulting in a decrease of pyrene excimer fluorescence intensity. For this assay, liposomes were prepared with an average diameter of 200 nm. Figure 2 presents the results. SIN derived from the infectious clone TR339 fused rapidly and efficiently with the liposomes at pH 5.0 (curve a), 60% of the virus fusing within 60 s after acidification. No fusion was seen at neutral pH (data not shown). In contrast to TR339, PE2 cleavage mutant E2:N1 hardly fused with the liposomes at pH 5.0 (curve b). At 60 s after acidification to pH 5.0, an extent of fusion of only 7% was observed. Like E2:N1, PE2 cleavage mutant FDF, was unable to fuse at pH 5.0 (curve c). Construction and characteristics of viable E2:N1 revertants. Using SIN PE2 cleavage mutant TRSB-N, Heidner et al. (14) showed that after infection of cells with TRSB-N virions occasionally small plaques were formed. These viruses were found to be infectious revertants either with a restored PE2 cleavage phenotype or with second-site mutations in PE2 along with retention of the PE2 cleavage defect. Some of the second-site resuscitating mutations, identified in TRSB-N, were now introduced into the E2:N1 PE2 cleavage mutant virus, i.e. in the TR339 background. In the first mutant, E2:N1/T191, the Pro residue (CCG) at position 191 of E2 was replaced by a Thr (ACG). In the second mutant, E2:N1/G216, the Membrane fusion of Sindbis virus PE2 cleavage mutants 143

60 a

40 (%)

20 Fusion b c 0 0 20 40 60 Time (s) Figure 2. Fusion of PE2 cleavage-deficient SIN viruses with liposomes at pH 5.0. On- line fusion experiments, with pyrene-labeled viruses, were performed at 37 °C, as described in Materials and Methods. All fusion measurements were repeated at least three times. Approx. 1010 virus particles were mixed with a 5-fold excess of liposomes, consisting of PC/PE/SPM/Chol (molar ratio 1:1:1:1.5). Curve a, TR339; curve b, E2:N1; curve c, FDF.

Glu residue (GAA) at position 216 of E2 was replaced by a Gly (GGA). In the third mutant, E2:N1/E3:R25, the Cys residue (TGT) at a position corresponding to position 25 of E3 was replaced by an Arg (CGT). We were interested in whether the introduced mutations promoted viral viability and membrane fusion capacity, with retention of the PE2 cleavage-defective phenotype.

TABLE 2. Characterization of PE2 cleavage mutant Sindbis viruses Virus Substitution % PE2 BHK specific mutation infectivitya E2:N1 100 1.5 E2:N1/T191 E2 position 191 Pro ® Thr 100 230 E2:N1/G216 E2 position 216 Glu ® Gly 100 136 E2:N1 E3:R25 E3 position 25 Cys ® Arg 100 160 a (PFU/cpm)

First, PE2 cleavage of each of the mutants was determined by SDS-PAGE analysis of [35S]methionine-labeled protein from purified particles (Table 2). In agreement with earlier observations on the corresponding TRSB-N revertants (14), all of the second-site mutant viruses maintained the defect in PE2 cleavage. On the other hand, the specific infectivities of the mutants were increased by a factor of 144 Chapter 8 approx. 150-200, compared to the parental E2:N1 virus (Table 2). Thus, all three resuscitating mutations restored virus viability while retaining the PE2 cleavage- deficient phenotype. It is noteworthy, that the specific infectivities of the resuscitated mutants (Table 2) were significantly higher than the specific infectivity of the TR339 virus (Table 1). To determine whether the high specific infectivity of the resuscitated mutants was due to an improved binding of these viruses to BHK-21 cells, cell-binding assays were performed. Figure 3A shows the results. As indicated above (Figure 1), the E2:N1 virus binds efficiently to BHK-21 cells. Likewise, the resuscitated mutants E2:N1/T191, E2:N1/G216, and E2:N1/E3:R25 also bound very efficiently to BHK-21 cells. Clearly, all PE2 cleavage-deficient mutants, retaining the XBXBBX sequence, bind better to BHK-21 cells than TR339, explaining the higher specific infectivity of the resuscitated mutants. Since the resuscitated mutants are still PE2 cleavage-deficient, retaining the XBXBBX furin cleavage site, it is likely that these viruses bind to HS on BHK-21 cells (19). To investigate this

100 80 BHK-21 cells A Heparin-beads B 80

(%) (%) 60 60 40 bound 40 bound 20 Virus 20 Virus

0 0 a b c d a b c d Figure 3. Binding of PE2 cleavage-deficient second-site resuscitated SIN viruses to BHK-21 cells or heparin-agarose beads. Binding was measured after 2 h incubation at 4 °C, as described in the legend to Figure 1. Bars: a, E2:N1; bars b, E2:N1/T191; bars c, E2:N1/G216; bars d, E2:N1/E3:R25. (A) Binding to BHK-21 cells. (B) Binding to heparin- agarose beads. directly, we determined the binding capacity of the resuscitated mutants for heparin- versus albumin-agarose beads. All viruses appeared to bind efficiently to the heparin-beads (Figure 3B). None of the viruses bound to albumin-beads (data not shown). Taken together, PE2 cleavage-deficient resuscitated viruses are infectious, and have a high specific infectivity on BHK-21 cells, presumably due to an interaction of the uncleaved PE2 with HS on the cell surface. The high infectivity of the resuscitated mutant viruses, as compared to the low infectivity of the parent E2:N1 virus, suggests that the second-site mutations somehow restore the fusion capacity of these viruses. This question was addressed subsequently. Membrane fusion of Sindbis virus PE2 cleavage mutants 145

Fusion activity of pyrene-labeled PE2 cleavage mutant SIN viruses carrying a resuscitating mutation in PE2. Figure 4 presents the fusion kinetics of pyrene-labeled PE2 resuscitated cleavage mutant SIN viruses with liposomes at pH 5.0. The E2:N1/T191 mutant fused efficiently with liposomes, the extent of fusion being about 55% within 60 s (curve a). The E2:N1/G216 and E2:N1/E3:R25 mutants also fused efficiently with liposomes at pH 5.0 (curves b, c). Again, the E2:N1 virus was unable to fuse at pH 5.0 (curve d). Thus, it appears that, as a result of a single amino-acid substitution, the non-infectious PE2 cleavage-deficient E2:N1 virus regains infectivity due to restoration of its membrane fusion competence, despite retention of the uncleaved PE2 phenotype.

60 a b,c 40 (%)

20 Fusion d 0

0 20 40 60 Time (s) Figure 4. Fusion of PE2 cleavage-deficient SIN viruses with liposomes. On-line fusion experiments were performed at 37 °C, as described in the legend to Figure 2. Curve a, E2:N1/T191; curve b, E2:N1/G216; curve c, E2:N1/E3:R25; curve d, E2:N1.

PE2 cleavage mutant SIN viruses show an acidic pH-shift in the activation of membrane fusion. Previous work, based on SFV, showed that the block in infection of the p62 cleavage mutant mL (Arg-to-Leu substitution at position –1 of E2) could be overridden by exposure of the virus to a very low pH (34). Therefore, we were interested in whether incubation at a very low pH could activate fusion activity of PE2 cleavage mutant SIN. Figure 5A presents the fusion kinetics of several pyrene-labeled PE2 cleavage mutants at pH 4.0 in the liposomal model system. As expected, the resuscitated mutant E2:N1/T191 (curve a) fused efficiently, with fusion kinetics identical to those of the TR339 virus (curve b). Importantly, however, under these extreme pH conditions the non-infectious E2:N1 and FDF mutants were able to fuse as well (curve c, d). As a control, the different pyrene-labeled SIN viruses were incubated at pH 4.0 in the absence of liposomes. In all cases, the fluorescence intensity remained constant, demonstrating that, also under these extreme pH conditions, 146 Chapter 8 the decrease in pyrene excimer fluorescence intensity observed in the presence of liposomes was due to dilution of the fluorescent probe from the viral membrane into the liposomal membrane (data not shown). These results demonstrate that the PE2 cleavage mutants FDF and E2:N1 are fusion-competent, but only when the viruses are exposed to an unphysiologically low pH. Figure 5B shows the detailed pH dependence of fusion of SIN PE2 cleavage mutants with liposomes. For TR339, optimal fusion was observed at pH 5.0 (squares). The threshold for fusion was pH 6.25, similar to that of the laboratory- adapted SIN strain AR339 (36). The PE2 cleavage mutants FDF (diamonds) and E2:N1 (circles) showed a clear-cut downward shift in the pH dependence of fusion, with a threshold at pH 5.0. Only at very low pH values fusion of these viruses was fast and extensive. Interestingly, an intermediate pH dependence was observed for the resuscitated mutant viruses E2:N1/T191 (upward triangles) and E2:N1/G216 (downward triangles). The other resuscitated mutant virus, E2:N1/E3:R25, showed a comparable pH dependence to that of the E2:N1/T191 and E2:N1/G216 mutants (data not shown). The threshold for fusion was very close to pH 5.75 for all cleavage-deficient SIN viruses with a resuscitating mutation in PE2. Taken together, SIN PE2 cleavage mutants exhibit an acidic pH-shift in their ability to fuse with liposomes, and resuscitating mutations in PE2 partly reverse this pH-shift.

60 A a,b 60 B

c (%) 40 d 40 (%) fusion

20 of 20 Fusion

0 Extent 0 0 20 40 60 4.0 4.5 5.0 5.5 6.0 6.5 7.5 Time (s) pH Figure 5. Low-pH dependent fusion of PE2 cleavage-deficient SIN viruses with liposomes. On-line fusion experiments were performed, as described in the legend to Figure 2. (A) Curve a, E2:N1/T191; curve b, TR339; curve c, E2:N1; curve d, FDF. (B) The extent of fusion was determined 60 s after acidification. Line (n) TR339; line (s) E2:N1/T191; line (t) E2:N1/G216; line (l) E2:N1; line (u) FDF. Membrane fusion of Sindbis virus PE2 cleavage mutants 147

Rearrangement of the spike heterodimer is impaired in PE2 cleavage mutants. Earlier studies on SFV and SIN have demonstrated that, under low-pH conditions, the E2/E1 heterodimer dissociates and a trypsin-resistant E1 homotrimer is formed. (3, 16, 29, 36, 40, 41). It is believed that the E1 homotrimer is the fusion-active conformation of the viral spike protein (16, 40). To determine whether the PE2 cleavage mutants were blocked in their ability to rearrange to the fusion-active conformation, the conformational changes of the viral spike proteins were studied. [35S]Methionine-labeled virus was incubated at low pH in the presence of liposomes. At 60 s after acidification the mixture was neutralized and the appearance of the E1 homotrimer was analyzed on SDS-PAGE. Figure 6 shows the results. The spike protein of TR339 already rearranged to form an E1 homotrimer at pH 5.75. Under optimal conditions for fusion (pH 5.0), 76% of the TR339 E1 protein was converted to the trimeric configuration. The PE2 cleavage mutants FDF and E2:N1 showed a large pH shift in the formation of the E1

pH 7.4 5.75 5.5 5.0 4.5 4.0 E1 trimer

TR339 E2/E1

E1 trimer PE2 FDF E1

E1 trimer PE2 E2: N1 E1

E1 trimer PE2 E2:N1/T191 E1

Figure 6. E1 trimerization of PE2 cleavage-deficient SIN viruses at 37 °C and the indicated pH values. Approx. 1010 [35S]methionine-labeled SIN particles were incubated with an 5-fold excess of liposomes consisting of PC/PE/SPM/Chol (molar ratio 1:1:1:1.5). After 60 s, samples were neutralized and analyzed for the appearance of E1 trimers on SDS-PAGE, as described in Materials and Methods. 148 Chapter 8 trimer. Clearly, with these viruses, at pH values of 5.0 or above no significant trimer formation occurred. Only when these mutants were incubated at a very low pH, the E1 spike protein rearranged to its trimeric configuration. By contrast, the resuscitated cleavage mutant E2:N1/T191 did convert to an E1 homotrimer at pH 5.0. The extent of E1 trimerization increased by incubation at pH values lower than pH 5.0. Similar results were obtained with the other resuscitated mutants, E2:N1/G216 and E2:N1/E3:R25 (data not shown). These results suggest that the PE2/E1 heterodimer of the PE2 cleavage mutants FDF and E2:N1 is stable at pH 5.0, rearranging to the fusion-active state only at very low, non-physiological, pH values. On the other hand, the PE2/E1 heterodimer of the resuscitated viruses is less stable than that of the parental E2:N1 virus and rearranges to a fusion-active conformation at a physiological mildly acidic pH (pH 5.0). Figure 7 presents a comparison of E1 trimerization and fusion for the PE2 cleavage mutant SIN viruses at different pH values. Clearly, there is a distinct correlation between the appearance of the E1 homotrimer and the ability of the virus to fuse with liposomes. Under optimal conditions for fusion, the extent of E1 trimerization and fusion of TR339 at 60 s after acidification were 76 and 59%, respectively. At pH 5.75, already 64% of the E1 glycoprotein had rearranged to the fusion-active conformation and half-maximal fusion was observed. On the other hand, the PE2 cleavage mutant FDF, at pH 5.0, showed extents of E1 trimerization and fusion of only 5% and 3%, respectively. At pH 4.0, the FDF mutant did become fusogenic, 70% of the E1 protein converting to a homotrimer, and 40% of the virus particles fusing with liposomes, under these conditions. Likewise, the PE2 cleavage mutant E2:N1 showed little E1 trimerization and fusion at pH 5.0 (6 and 8%, respectively), whereas at pH 4.0 the extents of E1 homotrimer formation and fusion for this virus were 70% and 43%, respectively. At pH 5.0, the resuscitated mutant E2:N1/T191 showed extents of E1 trimerization and fusion of 38% and 49%, respectively. At a pH lower than 5.0, the extent of E1 trimerization and fusion further increased. At pH 5.75, only a very small amount (2%) of the E1 glycoprotein was converted to a homotrimer, and accordingly fusion was negligible. Similar results were obtained for the other resuscitated mutants, E2:N1/G216 and E2:N1/E3:R25 (data not shown). The pH- threshold of fusion for TR339 is 6.2 (Figure 5B), which is 0.5 pH unit higher than that of the resuscitated viruses. The resuscitated mutants clearly showed a pH shift in the formation of an E1 homotrimer and fusion as compared to TR339. Taken together, the lower stability of the PE2/E1 heterodimer of the resuscitated mutants, as compared to the parental E2:N1 mutant, enables the virus to become fusion-active under physiological pH conditions, consistent with the restored infectivity of these viruses in BHK-21 cells. Membrane fusion of Sindbis virus PE2 cleavage mutants 149

A 100 C 100 80 80 60 60 40 40 20 20 0 0

final extent 100 B final extent 100 D 80 80 % of % of 60 60 40 40 20 20 0 0 4.00 4.50 5.00 5.50 5.75 7.40 4.00 4.50 5.00 5.50 5.75 7.40 pH pH Figure 7. Relative pH-dependence of E1 trimerization and fusion for several PE2 cleavage-deficient SIN viruses. The extents of E1 trimerization (shaded bars) and fusion (open bars) are shown at 60 s post-acidification to the indicated pH values. To compare E1 trimerization and fusion, the final extents of these processes at pH 4.0, were set to 100%. (A) TR339 (absolute extents: 76% E1 trimerization, 55% fusion). (B) FDF. (absolute extents: 70% E1 trimerization, 40% fusion). (C) E2:N1. (absolute extents: 70% E1 trimerization, 43% fusion). (D) E2:N1/T191. (absolute extents: 70% E1 trimerization, 56% fusion). E1 trimerization was determined as described in the legend to Figure 6. Fusion was measured as described in the legend to Figure 2. Discussion

In this paper, we provide evidence that the block in viral infectivity of SIN PE2 cleavage mutant viruses on BHK-21 cells lies at the level of the membrane fusion activity of the viral envelope glycoprotein (Figures 2,6-7). Earlier studies on similar SIN PE2 cleavage mutants had shown that viral infection in vertebrate cells was not blocked at the level of virus-receptor interaction, RNA replication or virus assembly (14, 19). Our present results show that SIN PE2 cleavage mutants are impaired in their fusion activity because of the inability of the immature PE2/E1 heterodimer to rearrange to an E1 homotrimer, at a physiological acidic pH. The E1 trimer has been shown to represent the fusion-active conformation of the alphavirus spike (3, 11, 16, 36, 40). The lethality of SIN PE2 cleavage defect could be overridden by second-site resuscitating mutations in PE2 (Table 2), which destabilize the PE2/E1 heterodimer interaction. Thus, the spike regains the ability 150 Chapter 8 to form a fusion-competent E1 trimer at a physiologically relevant acidic pH (Figures 4-7). Among members of the alphavirus genus, PE2 cleavage has been associated with different biological functions of the viruses involved. Recent work has shown that SIN PE2 cleavage mutants with an intact cleavage site (XBXBBX) interact electrostatically with HS, a glycosaminoglycan which is abundantly expressed on BHK-21 cells (19). These findings are in agreement with our results, showing that the E2:N1 mutant (with an intact cleavage site) binds efficiently to BHK-21 cells and heparin-beads, as compared to the PE2 cleavage mutant FDF (with a deleted cleavage site) (Figure 1). PE2 cleavage mutant E2:N1 binds much more efficiently to BHK-21 cells than the parental SIN TR339, confirming that the TR339 virus does not interact directly with HS (20). For SFV, p62 cleavage mutants have been generated by mutation of the cleavage site from RHRR to RHRL (mL mutant) (22, 34) or SHQL (1, 39). In contrast to our PE2 cleavage-deficient SIN viruses, these SFV mutants show a large reduction in binding capacity to BHK-21 cells, as compared to wild-type SFV. However, in the case of SFV the cleavage site was disrupted to create the p62 cleavage-deficient viruses. Therefore it is difficult to interpret these results in the context of our present data on SIN mutants which are known to bind to cells through the furin cleavage site. Despite the efficient binding of the E2:N1 PE2 cleavage mutant to BHK-21 cells, the virus was still found to be non-infectious, due to its inability to fuse (Table 1; Figure 2). However, after introduction of resuscitating mutations in PE2 E2:N1 regained fusion competence and infectivity on BHK-21 cells, as discussed in more detail below. Interestingly, the specific infectivity of these viruses on BHK-21 cells was found to be very high, even 10-fold higher than that of the parental TR339 (Table 2). Our results demonstrate that the resuscitated PE2 cleavage mutants are more infectious than TR339, very likely as a result of the more efficient interaction of these viruses with HS (Figure 3). The inability of cleavage mutants E2:N1 and FDF to fuse at a physiologically relevant acidic pH appears to be caused by a downward shift in the pH dependence of the viral membrane fusion reaction (Figure 5B). Within the overall process of low-pH-dependent alphavirus membrane fusion, the E2/E1 heterodimer first dissociates, followed by formation of a trypsin-resistant E1 homotrimer (3, 11, 16, 36, 40). In the present study, it became clear that the PE2/E1 heterodimer of the cleavage mutant viruses is too stable to rearrange to the fusion-active E1 homotrimeric conformation, at a physiologically relevant acidic pH (Figures 6-7). However, when the viruses were incubated at pH 4.0, the spike did form an E1 homotrimer, resulting in expression of full membrane fusion activity (Figures 6-7). In conclusion, the low specific infectivity of SIN PE2 cleavage mutants is the result of an impairment of the viral spike to rearrange to the fusion-active conformation at a mildly acidic pH, due to the high stability of the immature PE2/E1 heterodimer. This is in agreement with observations on the SFV p62 cleavage- Membrane fusion of Sindbis virus PE2 cleavage mutants 151 deficient mL mutant, showing that viral infectivity on BHK-21 cells was restored by exposure of the virus to pH 4.5 (34). Several investigators have identified second-site resuscitating mutations in the E3, E2 or E1 glycoproteins which promote infectivity of PE2 cleavage-deficient alphaviruses (5, 14, 39). Some of these resuscitating mutations, found with the TRSB-N SIN clone (14), were now introduced in the E2:N1 virus to investigate the role of these mutations in viral infectivity versus the stability of the spike heterodimer. These E2:N1 mutants with resuscitating mutations in PE2 were indeed infectious on BHK-21 cells (Table 2), and at the same time exhibited a restored membrane fusion capacity at a physiologically relevant acidic pH (Figures 4-5). In this respect it is important to note that viruses which do cleave PE2 and interact efficiently with the cell attachment receptor HS are more infectious on BHK-21 cells than the resuscitated PE2 cleavage-deficient viruses (20). This indicates that SIN cleavage mutants with resuscitating mutations in PE2 have an intermediate infectivity on BHK-21 cells, which in fact correlates precisely with the intermediate pH dependence of their membrane fusion activity with liposomes (Figure 5B). For example, the TR339 virus with a PE2 cleavage-deficient phenotype, is already fully fusion-competent at pH 5.5, whereas the resuscitated PE2 cleavage mutant viruses express full membrane fusion activity only at pH values of 5.0 or below. Analysis of the conformational changes occurring in the viral spike protein showed that the PE2/E1 spikes of the resuscitated mutants were able to form an E1 homotrimer at a physiological acidic pH, in contrast to the heterodimer of the E2:N1 virus (Figure 6). In conclusion, the presence of the resuscitating mutations in PE2 destabilizes the PE2/E1 heterodimer such that the viral spike protein regains the ability to undergo conformational changes with formation of a fusion-active E1 trimer at a physiological relevant pH, despite retention of the uncleaved PE2 phenotype. The maturation of the alphavirus spike heterodimer through cleavage of the PE2 precursor of E2 has a distinct function in the viral life cycle. During transport of the heterodimer from the ER to the surface of the infected cell, the uncleaved PE2 protein is presumed to function as a chaperone, protecting the spike from premature destabilization within the acidic TGN (9). Subsequently, after cleavage of PE2 in a post-TGN compartment by a furin-like protease, the mature viral spike protein is primed for expression of membrane fusion activity when exposed to a mildly acidic pH within the endosomal compartment of a new target cell. The results of this study indicate that, while cleavage maturation is a common phenomenon in viral assembly, it is not absolutely required for viral infection. SIN PE2 cleavage mutants with resuscitating mutations in PE2 were found to be infectious on BHK-21 cells, despite retention of the uncleaved PE2 phenotype. Apparently, the resuscitating mutations in PE2 destabilize the PE2/E1 heterodimer to such an extent that the spike has the capacity to rearrange to the fusion-active conformation at the lumenal pH of endosomes, while at the same 152 Chapter 8 time the heterodimer is stable enough to survive the mildly acidic lumen of the TGN (pH ~6.0 (35)) during transport to the cell surface. Interestingly, another alphavirus PE2 cleavage mutant called S12 (Ser-to-Asn substitution at position 1 of E2), derived from SAAR86, was found to be fully infectious on BHK-21 cells (31, 33). In the light of our present results, we would argue that the PE2/E1 heterodimer of the S12 mutant is less stable than that of the E2:N1 virus, despite the fact that both viruses carry the same amino-acid substitution at E2 position 1. Differences in the genetic background of S12 and E2:N1, outside the PE2 cleavage region, could account for the different spike stability, in agreement with our present observation that distant mutations in the E2:N1 mutant have the capacity to restore viral infectivity by destabilization of the spike heterodimer. We postulate that the S12 spike heterodimer has a very precise pH dependence, so that it survives the TGN during maturation, but that it does become fusion competent at acidic pH in endosomes. The stability of the spike heterodimer and its fusion competence at low pH values of SAAR86 or S12 remain to be determined. The results in Figures 6 and 7 show that there is a clear correlation between expression of low-pH-dependent membrane fusion activity of SIN and formation of the E1 homotrimeric conformation. However, close scrutiny of the data reveals that mutant SIN viruses with a relatively low pH threshold for fusion require a lower extent of trimer formation to reach a certain level of fusion than the TR339 virus with a high pH threshold for fusion. This suggests that there are different levels of pH control of viral fusion activation and that, besides E1 trimerization, other pH-dependent conformational alterations are involved. In previous studies on SFV (3) and SIN (36), we have shown that, kinetically, fusion in the liposomal model system occurs with a distinct delay after virus-liposome binding and E1 trimerization. This lag phase prior to the onset of fusion may well involve the rearrangement of several E1 trimers into a fusion complex. Interestingly, the duration of the lag phase decreases with decreasing pH (3, 36). Thus, our present observation suggesting that, at a relatively high pH, a relatively high extent of E1 trimerization is required for fusion, may well reflect the comparatively long lag phase between E1 trimerization and the onset of fusion under these conditions. The present results suggest that there is a direct correlation between the infectivity of SIN on BHK-21 cells and low-pH-dependent membrane fusion activity of the virus with liposomes. Not only is SIN-liposome fusion clearly dependent on low pH, it also appears that subtle shifts in the pH dependence of fusion have a profound effect on viral infectivity. This result provides additional support for the notion that exposure to a mildly acidic pH is an obligatory step in the infectious entry of SIN into its host cell. This conclusion is in agreement with recent observations of Glomb-Reinmund and Kielian (12) and DeTulleo and Kirchausen (6), indicating that SIN enters cells by receptor-mediated endocytosis and fusion from within acidic endosomes. On the other hand, very recently Membrane fusion of Sindbis virus PE2 cleavage mutants 153

Hernandez et al. (15) provided evidence, based on investigation of mosquito cell infection by SIN, suggesting that exposure to an acidic pH may not be an obligatory step in alphavirus cell entry. Clearly, it can not be excluded that alphaviruses use different routes of cell entry in mosquito and vertebrate cells. Also, our observations do not rule out the possibility that interaction of SIN with cell-surface components induces conformational alterations priming the spike to subsequently rearrange to its fusion-active structure (7, 26). Nevertheless, from our present results it would appear that low pH is a very important factor in alphavirus spike heterodimer destabilization and thus in the activation of viral membrane fusion activity. This conclusion is completely consisted with recent structural data on the SIN spike (30). Furthermore, SIN closely resembles SFV in terms of both spike organization (21, 30) and membrane fusion characteristics (3, 27, 36). For SFV it has been clearly demonstrated that it infects cells via receptor-mediated endocytosis and fusion from within acidic endosomes (6, 10, 12, 17, 23, 42).

Acknowledgements

This work was supported by the US National Institutes of Health (grants 2R01HL16660-27 and R01AI22186-14), by The Netherlands Organization for Scientific Research (NWO) under the auspices of the Foundation for Chemical Research (CW), and by the Royal Netherlands Academy of Arts and Sciences (KNAW) (Travel Grant to J.S.).

References

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Summarizing Discussion 158 Chapter 9

Summary

The studies presented in this thesis address the molecular mechanisms involved in the membrane fusion process of Sindbis virus (SIN). There is considerable controversy regarding the route of cell entry of SIN (see Chapter 1). Several lines of evidence suggest that SIN enters cells via receptor-mediated endocytosis and fusion from within acidic endosomes. However, in other reports evidence is presented indicating that SIN infection is mediated by virus-receptor interaction at the cell surface. Therefore, the first objective of the present study was to elucidate the role of receptor interaction and/or low pH in triggering or influencing membrane fusion of SIN (Chapter 2 and 3). We decided to use a liposomal model system because such a system allows highly sensitive fusion measurements, thus providing qualitative and quantitative insight in the membrane fusion process of the virus involved. In Chapter 2, we demonstrate that SIN fuses efficiently with receptor-free liposomes in a pH-dependent manner, consistent with fusion from within acidic endosomes. The fusion process of SIN with liposomes can be subdivided in several stages, including dissociation of the E2/E1 heterodimer, binding of the virus to the liposomal membrane, and the formation of a trypsin-resistant E1 homotrimer. It is demonstrated that low-pH-dependent fusion of SIN, similar to that of SFV, requires the presence of sphingolipid and cholesterol in the target membrane. Cholesterol is primarily involved in low-pH- dependent binding of the virus to the target membrane, whereas sphingolipid is required for the subsequent fusion process. The liposomes that were used in this study consisted of just lipids, which suggests that virus-receptor interaction is not a mechanistic requirement for fusion. To investigate the role of virus-receptor interaction more directly, we used liposomes supplemented with heparin-conjugated phosphatidylethanolamine (HepPE), as a functional attachment receptor for the virus (Chapter 3). It had been shown previously that cell-culture-adapted SIN binds efficiently to BHK-21 cells through interaction with heparan sulfate (HS), and that adapted viruses also bind to heparin (44). In Chapter 3, it is demonstrated that cell-culture-adapted SIN efficiently interacts with liposomes supplemented with HepPE in the membrane at neutral pH, whereas non-adapted SIN does not bind to these liposomes. Despite the efficient binding of HS-adapted SIN to these liposomes, there was no fusion at neutral pH. These results demonstrate that virus-receptor interaction does not support fusion of the viral membrane with the liposomal membrane. Fusion of SIN with HepPE-containing liposomes was observed, however, when the virus-liposome mixture was acidified. This indicates that the exposure of the virus to low pH is the sole requirement for triggering membrane fusion of SIN, consistent with cellular entry via receptor-mediated endocytosis and fusion from within acidic endosomes. Summarizing discussion 159

In Chapter 4, it is shown that fusion of alphaviruses with liposomes is a non- leaky process. Fusion of SFV was non-leaky to molecules as small as calcein (MW 623) or sucrose (MW 342) initially encapsulated in the liposomes. SIN fusion was found to be more leaky than that of SFV. Yet, during SIN-liposome fusion inulin, with an average molecular weight of 5.2 kD, was retained. These results indicate that membrane fusion of alphaviruses is a tight process, and suggest the involvement of a hemifusion intermediate. In Chapter 5, the binding and fusion characteristics of SIN mutants TE and 633 were investigated. SIN mutant TE differs at only one amino acid position, E2:55, from 633. Yet, TE is considerably more virulent in mice, and replicates better in neuroblastoma (N18) cells (18, 77, 78). The data presented in Chapter 5 demonstrate that the TE virus is 10- to 100-fold more infectious on N18 cells than SIN mutant 633. The enhanced infection efficiency of TE in N18 cells was found to be related to an increased ability of TE to bind to these cells. Treatment of N18 cells with a sulfation inhibitor decreased the binding of TE to approximately that of 633, indicating that TE interacts with a sulfated molecule expressed on the cell surface of N18 cells. Moreover, it was observed that both TE and 633 bind efficiently to BHK-21 cells, through interaction with HS. Taken together, the results suggest that the sulfated receptor molecule, to which TE and 633 bind on BHK-21 cells, is not present or exposed in an altered form on N18 cells. Both TE and 633 fused equally well with liposomes derived from BHK-21 cell or N18 cell lipids. However, the viruses fused significantly better with liposomes derived from BHK-21 cell lipids. In conclusion, it appears that the increased infection efficiency of TE in N18 cells is related to the binding properties of the virus to these cells, rather than the membrane fusion process. In Chapter 6, we demonstrate that N-linked oligosaccharides located at position E2:196 and E1:139, support membrane fusion of SIN. Removal of one of these glycosylation sites was found to strongly affect the infectivity of the mutant virus on BHK-21 cells. Moreover, evaluation of the fusion properties in a liposomal model system revealed that the single deglycosylated SIN mutants were impaired in their capacity to induce membrane fusion. This indicates that the reduced infectivity of the mutant viruses on cells lies at the level of the fusion process. In Chapter 7, it is shown that fatty acid acylation of the transmembrane regions of SIN glycoproteins E2 and E1 is not required for membrane fusion. The fusion characteristics of SIN derived from the infectious clone Toto1101 and three deacylated SIN mutants were determined in a liposomal model system using both lipid and content mixing assays. Very similar fusion kinetics were observed for the parental virus and the three deacylated SIN mutants in a pH range from 4.0 to 7.4. In Chapter 8, we investigated the fusogenic properties of PE2 cleavage mutant SIN viruses. It is demonstrated that PE2 cleavage mutants are non-infectious on BHK-21 cells, because these mutants are impaired in membrane fusion. Analyses 160 Chapter 9 of the spike conformational changes revealed that the block in membrane fusion was caused by the inability of the PE2/E1 heterodimer to rearrange to an E1 homotrimer. Rearrangement of the viral spike glycoprotein, with formation of an E1 homotrimer, and membrane fusion activity were observed, however, upon the exposure of the virus to non-physiological pH conditions (pH < 4.5). Second-site resuscitating mutations were identified that destabilize the PE2/E1 heterodimer through which these viruses regain the ability to rearrange to a fusion-competent E1 homotrimer at a physiological relevant acidic pH.

Cell entry of alphaviruses

Numerous studies have been conducted to unravel the cell entry properties of alphaviruses, SFV and SIN in particular. More than two decades ago, it was demonstrated that SFV infects cells via receptor-mediated endocytosis and fusion from within acidic endosomes (34). This result has been confirmed by a number of other studies (33, 38, 40, 53, 54). Furthermore, SFV fusion studies in model systems have revealed that the virus fuses rapidly and efficiently with receptor-free liposomes at a mildly acidic pH (5, 39, 58, 83, 86). Although SIN is an alphavirus similar to SFV, several lines of evidence suggest that SIN infects its host cell by a mechanism independent of low pH (1, 6, 9, 13, 19, 22, 36). Instead, fusion would be triggered by virus-receptor interaction at neutral pH, resulting in fusion of the viral membrane with the plasma membrane of the cell. In Chapter 2 and 3 we investigated, in a direct manner, the role of low pH and receptor interaction in triggering the membrane fusion process of SIN. It was found that SIN, like SFV, fuses rapidly and efficiently with receptor-free liposomes in a low-pH-dependent manner. This indicates that receptor interaction is not a mechanistic requirement for fusion of SIN. Furthermore, as shown in Chapter 3, the interaction of SIN with liposomes supplemented with lipid-conjugated heparin, as a functional attachment receptor for the virus, did not result in fusion of the viral membrane with the liposomal membrane at neutral pH. Fusion of SIN with these liposomes was observed, however, after exposure of the virus-liposome mixture to low pH. Moreover, the data presented in Chapter 8 indicate that there is a direct correlation between the infectivity of SIN in BHK-21 cells and low-pH-dependent fusion with liposomes. These results demonstrate that the sole requirement for membrane fusion of SIN is exposure of the virus to low pH, consistent with cellular entry from within acidic endosomes. This is entirely in agreement with reports of Glomb-Reinmund and Kielian (27) and DeTulleo and Kirchhausen (17). These studies showed that infection of BHK-21 cells by SIN is inhibited by agents that interfere with endosomal acidification or by inhibition of the budding of clathrin-coated vesicles, respectively. What then could be the explanation for the above discrepancy? It has been proposed that in their studies, Brown and coworkers used virus preparations that Summarizing discussion 161 had been frozen in phosphate-buffered medium (17, 20). The pH of phosphate- buffered medium decreases upon freezing, through which the viral spike proteins would possibly rearrange to their fusion-active conformation. After thawing, the virus would then be able to fuse directly with the plasma membrane of the cell at neutral pH. This, however, does not seem to be a very plausible explanation. First, in a recent paper Brown and coworkers have denied the use of virus prior frozen in phosphate-buffered medium (36). Second, virus exposed to low pH in the absence of target membranes becomes rapidly fusion-inactive and, moreover, does not subsequently express fusion activity at neutral pH (5, Chapter 2). As an alternative explanation, Glomb-Reinmund and Kielian (27) suggested that the types of cell-cell fusion assays, as used by Brown and coworkers, could be the basis for the discrepancy. In cell-cell fusion assays, fusion is evaluated by morphological changes of the cell that occur after the primary fusion event. Therefore, it would be questionable whether the observations that were made are related to fusion or are in fact caused by secondary effects on the morphological reorganization of the cells. Yet, it remains difficult to explain positive observations of fusion under conditions where endocytic entry is inhibited on the basis of such a reasoning. Furthermore, quite recently, Brown and coworkers further examined the effects of weak bases on the entry and replication of SIN in mosquito cells (36). The authors showed that chloroquine inhibits the acidification of endosomes but does not block the infection of mosquito cells by SIN. These data support their previous results, suggesting that exposure of the virus to an acid environment within the cell may not be required for viral infection of cells by SIN. Taken together, it is not clear what the explanation is for the above discrepancy. It could be possible that SIN uses different pathways for infection of vertebrate versus insect cells. To conclude, we believe, on the basis of our own studies (Chapters 2, 3 and 8) and those of Glomb-Reinmund and Kielian (27) and DeTulleo and Kirchhausen (17), that alphaviruses infect their host cell by receptor-mediated endocytosis and fusion from within acidic endosomes. It would appear that the virus-receptor interaction is primarily needed for the efficient binding of the virus to the host cell and for the endocytic uptake of the virus by the cell. After the endocytic uptake, the mildly acidic pH in the lumen of the endosomes triggers fusion of the viral membrane with the endosomal membrane, and this membrane fusion reaction can occur without involvement of the virus receptor.

The involvement of cholesterol and sphingolipid in membrane fusion of alphaviruses

It has been shown that fusion of SFV and SIN requires the presence of sphingolipid and cholesterol in the target membrane (5, 58, 86, 49, 64, Chapter 2 and 7). Cholesterol is primarily involved in low-pH-dependent binding of the virus 162 Chapter 9 to the liposomal membrane, whereas sphingolipid is important for the subsequent fusion process (58, 87, Chapter 2). Interestingly, detergent-resistant domains enriched in sphingolipid and cholesterol, known as rafts, have been identified in cellular membranes (7, 8, 66, 74). Rafts were found to be involved in a number of important cellular processes, including trafficking of membrane proteins and signal transduction. Moreover, it has been shown that budding of influenza virus, measles virus, and HIV occurs selectively via sphingolipid-cholesterol domains (59, 69, 72, 81, 88). Also, for SV40 and HIV, a role for rafts in virus entry has been demonstrated (3, 30, 51, 60, 63). It has been observed that sphingolipid-cholesterol domains exist in early and recycling endosomes (57). Since alphavirus fusion occurs from within acidic endosomes and is strictly dependent on the presence of cholesterol and sphingolipid in the target membrane, one could argue that fusion may involve raft- like domains in the target membrane. However, studies in our laboratory on fusion of SFV or SIN have revealed that raft formation is not essential for fusion (82). In these studies highly efficient fusion of SIN and SFV was observed with liposomes containing a variety of sphingolipid analogs that do not interact with cholesterol to form detergent-resistant domains. Furthermore, cholesterol-independent mutants of SFV retain the requirement of sphingolipid to support membrane fusion, indicating that cholesterol and sphingolipid act independently (10). On the other hand, when raft-like domains are present in the target membrane, fusion is not inhibited (82). Therefore, during infection of cells, the virus could take advantage of the presence of rafts in the endosomal membrane and therefore fuse at these domains. Initial interaction of the virus with the attachment receptor HS could bring the virus particles in close proximity to the raft domain. Upon exposure of the virus to low pH in the endosomal lumen, the virus could interact and fuse with these domains. Accordingly, it has been observed that a proteolytically truncated ectodomain of E1, E1*, interacts efficiently with sphingolipid-cholesterol domains in liposomal target membranes (2). It appears that this interaction is mediated by the fusion peptide of E1. In contrast to this result, however, it was found that the envelope glycoproteins of whole SFV virus particles do not associate with raft domains. Taken together, although the fusion peptide of E1* efficiently interacts with sphingolipid-cholesterol domains in the liposomal membrane, the presence of these domains is not absolutely required for viral fusion. Fusion of the viral membrane with a target membrane requires the presence of both sphingolipid and cholesterol in the membrane, but these lipids do not have to be organized in rafts.

Model of alphavirus membrane fusion

Knowledge of the molecular mechanisms involved in the fusion process of alphaviruses is far from complete. In this section we will try to compose a model. Summarizing discussion 163

A hypothetical schematic view of the low-pH-dependent membrane fusion process of alphaviruses is depicted in Figure 1. The course of events, with emphasis on spike protein conformational changes and the hypothetical roles of cholesterol and sphingolipid will be described in further detail below. E2/E1 heterodimer dissociation. The first step in the fusion process of alphaviruses involves low-pH-induced conformational changes in the E1 and perhaps in the E2 glycoprotein that result in the dissociation of the E2/E1 heterodimer (5, 47, 84). Upon dissociation, the E2 glycoproteins move away from the center of the spike, through an as yet unidentified mechanism. At the same time, the E1 subunits would appear to move from their peripheral position in the spike towards the center, thus preparing for the subsequent homotrimer formation (see below). Recent structural data on the alphavirus spike strongly support this idea (25, 46, 65). The E1 protein is composed of an amino-terminal b-barrel domain, which is flanked by a carboxy-terminal Ig-like domain and a finger-like projection containing the fusion peptide. The molecule has an elongated shape resembling in many respects the overall fold of the E protein of tick-borne encephalitis virus, belonging to the family Flaviviridae. The alphavirus E1 protein, like the flavivirus E protein, lies almost flat on the virus membrane (46). Interestingly, from the structure it would appear as though E1-E1 interactions mediate the formation of a network stabilizing the virus, in agreement with the observation that lateral spike-spike interactions suffice for building the virus particle during assembly and budding (23). During fusion activation, these same spike-spike interactions are weakened. E1-E1 interaction during assembly and weakening of this interaction during cell entry thus provides a possible molecular explanation for the assembly-disassembly paradox in the viral life cycle (25). Binding to the target membrane. After E2/E1 heterodimer dissociation, the E1 glycoprotein associates with the target membrane, presumably via membrane insertion of the putative fusion peptide located between amino acids 79 and 97 of E1 (2, 40, 42). This fusion peptide is located in the finger-like projection flanking the central b-barrel domain. It is plausible that upon exposure to low pH the E1 protein not only moves from its position rather parallel to the viral membrane to a more perpendicular position, so as to allow the fusion peptide to interact with the target membrane. It has been found that efficient binding of SIN to liposomes requires the presence of cholesterol in the target membrane (Chapter 2). It is likely that virus-liposome binding does not require trimerization of E1, but rather that E1 trimerization occurs after binding of the virus to the target membrane, as evidenced by Zn2+ inhibition experiments (15). Analysis of the spike conformational changes in the presence of Zn2+ showed normal E2/E1 heterodimer dissociation and binding, whereas E1 trimerization was completely inhibited. Moreover, characterization of a fusion peptide mutant of SFV 164 Chapter 9

pH 5.0 E2 E1 1 2 3

4 5 6

7

Figure 1. Model of alphavirus fusion. Step 1: exposure of the virus to low pH results in dissociation of the spike heterodimer. Step 2: binding of the E1 fusion peptide to cholesterol in the target membrane. Step 3: cholesterol facilitates the formation of an E1 homotrimer. Step 4: clustering of E1 homotrimers, mediated by sphingolipid in the target membrane. Step 5: the clustered E1 homotrimers induce hemifusion. Step 6: formation and dilation of a fusion pore. Step 7: end stage of the fusion process. (For further details, see text).

(Glu91Asp) revealed that this mutant was blocked in E1 trimerization, while normal E2/E1 heterodimer dissociation and binding to liposomes was observed (41). E1 trimerization and formation of a fusion complex. We propose that upon the interaction of the E1 glycoprotein with cholesterol in the target membrane, cholesterol facilitates the formation of an E1 homotrimer. This hypothesis is substantiated by the observation that incubation of alphaviruses with Summarizing discussion 165

PC/PE/Chol liposomes results in efficient formation of E1 homotrimers (14, own unpublished results). The formation of E1 homotrimers is strongly reduced with liposomes lacking cholesterol in the membrane. Despite the efficient formation of E1 homotrimers with PC/PE/Chol liposomes, these E1 homotrimers are unable to support membrane fusion. For the formation of fusion-competent E1 homotrimers, the presence of both cholesterol and sphingolipid in the target membrane is required. Under suboptimal conditions for fusion in terms of pH and temperature, there may be a considerable lag phase between the formation of E1 homotrimers and the onset of the actual fusion reaction (5, Chapter 2). The occurrence of a lag phase implies that additional rearrangements within or between the E1 homotrimers are required for the initiation of the fusion process. We hypothesize that sphingolipids play an important role in this rearrangement. Specific interactions of the E1 homotrimer with sphingolipid molecules in the target membrane could mediate clustering of E1 homotrimers, thereby facilitating the formation of a fusion-competent complex. The prediction that clustered E1 trimers represent the fusion-active state of the virion is substantiated by the observation that higher-order oligomers are formed upon acidification of a virus- liposome mixture (own unpublished results, 26). Moreover, it has been observed that membrane fusion mediated by the influenza virus hemaglutinin (HA) requires the concerted action of at least three HA trimers (16). Hemifusion, formation of a fusion pore, and membrane merging. Upon the formation of a fusion complex a hemifusion (“stalk”) intermediate is presumably formed. This means that the monolayers of the interacting membranes merge, while the inner monolayers remain separate. The formation of a hemifusion intermediate in alphavirus fusion has been studied using lipids such as lysophosphatidylcholine or free fatty acids that, upon incorporation in the outer monolayers of the interacting membranes, inhibit or promote stalk formation, respectively (11, 12, 29). Indeed, it has been shown that SFV fusion is inhibited by lysophosphatidylcholine at a stage after the formation of an E1 homotrimer (62). On the other hand, SFV fused very efficiently with liposomes supplemented with free fatty acids. These data support the notion that the membrane fusion process of alphaviruses proceeds via the formation of a hemifusion intermediate. Accordingly, the data presented in Chapter 4 demonstrate that the fusion process of alphaviruses is essentially non-leaky. It is reasonable to assume that a membrane fusion process, in which the merging of the outer membrane leaflets and that of the inner ones are separated in time, remains tight throughout. We hypothesize that at the end of the fusion process, the glycoproteins adopt a final conformation that places the fusion peptide and the transmembrane domain at the same end of the molecule. This conformation has been demonstrated for influenza virus HA (85). At this moment, there is no data available that support this conformation in fusion process of alphaviruses. Finally, as a result of pore dilation, membrane 166 Chapter 9 merging is completed, which in the context of the host cell leads to the delivery of the viral nucleocapsid to the cell cytosol.

Post-translational modifications of the spike proteins of alphaviruses

Glycosylation. The E2 and E1 proteins undergo N-linked glycosylation within the lumen of the ER. The oligosaccharide chains exposed on the outer surface of the glycoprotein are further modified within the Golgi (see Chapter 1). The N- linked glycosylation sites, each characterized by an Asn-X-Ser/Thr motif, are located at residues Asn196 and Asn318 in E2, and at residues Asn139 and Asn245 in E1 (68). In Chapter 6, we evaluated the biological role of N-linked oligosaccharides in the membrane fusion process of SIN, using SIN mutants lacking a glycosylation site at residue E2:196 or at residue E1:139 (65). The results show that the resulting deglycosylation reduces viral infectivity by 3-4 orders of a magnitude, when compared to the parental SIN TE12 virus. It appears that the block in infection lies at the level of the fusion process. At the same time there does not appear to be any significant defect in the assembly of the glycosylation mutant viruses. At this point it is not clear how N-linked oligosaccharide chains may specifically affect the membrane fusion process of SIN. We hypothesize that the addition of oligosaccharide chains is required for the proper folding and conformation of the spike proteins on the viral envelope (4, 21, 35). Specifically, removal of a glycosylation site could induce a subtle conformational change within the viral glycoproteins that influences the stability of the E2/E1 heterodimer. Indeed, it has been observed that deglycosylation of the stem region of the influenza HA or the ectodomain of the dengue virus E glycoprotein affects the stability of the viral spike, such that a higher pH threshold for fusion is obtained (28, 61). We are currently investigating the effects of N-linked glycosylation on the stability and functional integrity of the SIN E2/E1 spike heterodimer. Fatty acid acylation. The glycoproteins of SIN are palmitoylated at the cysteine residues within the transmembrane and cytoplasmic domains (see Chapter 1). In Chapter 7, we investigated the role of fatty acid acylation of the transmembrane domains of the glycoproteins E2 and E1 on the membrane fusion activity of SIN with liposomes. To this end, deacylated SIN mutants were used, in which the transmembranal cysteines at either E2 positions 388 and 390 or E1 position 430 or in both glycoproteins were replaced by alanine residues. The data presented in Chapter 7 clearly demonstrate that deacylation of the transmembrane domains of the SIN glycoproteins has no effect on the membrane fusion process of the virus in a liposomal model system. Very similar fusion kinetics and characteristics were observed for the parental virus and the deacylated SIN mutants. These results indicate that acylation of the transmembrane regions of SIN Summarizing discussion 167 glycoproteins E2 and E1 is not required for expression of viral membrane fusion activity. The conserved nature of the cysteine residues in E2 or E1 among the alphavirus genus would suggest that acylation of the viral glycoproteins has an important function in the life cycle of alphaviruses. What then could be the physiological function of this acylation phenomenon? It has been proposed that palmitoylation of proteins is one of the factors that target membrane proteins to detergent-resistant raft domains (55). Accordingly, the palmitoylated envelope glycoproteins of influenza virus and HIV appear to be associated with rafts (55, 56, 69, 73). Targeting of influenza HA to rafts requires palmitoylation of all three cysteine residues (55, 88). Moreover, it has been found that budding of influenza virus and HIV occurs selectively via raft domains. Therefore, it appears that palmitoylation of influenza HA and HIV gp160 targets these proteins to rafts to mediate trafficking and targeting of the envelope glycoproteins and selective recruitment of raft domains into the budding virus particles. With regard to SIN, deacylation of the cytoplasmic domain of the E2 glycoprotein has been shown to affect virus budding (24, 37). In contrast to influenza virus and HIV, however, analysis of the presence of detergent-resistant lipid domains within the SFV envelope revealed that the glycoproteins of SFV are not associated with rafts (73). However, deacylation of the transmembrane domains of SIN glycoproteins E2 and E1 showed that these mutants are more sensitive to treatment with Triton X-100 than the parental virus (70). This suggests that the lack of palmitoylation increases the solubility of the viral envelope in detergent. In agreement with this view, DPH fluorescence polarization experiments have shown that the membrane of SFV has a comparatively limited fluidity (73). Therefore, we postulate that it is the palmitoylation of the SIN glycoproteins that causes the viral membrane to become less fluid. During budding of a new virus particle, the saturated acyl chains of acylated glycoproteins might alter the lipid composition in the surrounding regions and thereby facilitate a more rigid membrane microenvironment. Indeed, analysis of the lipid composition of the membrane of SFV cultured on BHK-21 cells, revealed that the cholesterol to phospholipid ratio within the viral membrane is significantly increased, compared to that of the plasma membrane of BHK-21 cells (45, 67, 79). Perhaps, during virus assembly, the saturated palmitic acid chains coupled to the viral spike proteins, recruit cholesterol resulting in a locally reduced membrane fluidity. This may be beneficial for an ordered arrangement of the viral spike proteins at the site of budding (23). In this respect, it is interesting to note that cholesterol is required for efficient assembly of SFV and SIN (49, 50, 52, 80). PE2 cleavage. In the infected cell, the E2 glycoprotein is synthesized as a precursor protein, called PE2 in SIN or p62 in SFV (see Chapter 1). PE2 is presumed to act as a chaperone, protecting the spike from premature destabilization during transport of the PE2/E1 heterodimer through the mildly 168 Chapter 9 acidic trans-Golgi network (TGN). Just before the appearance of the spike proteins on the plasma membrane of the cell, PE2 is cleaved by a furin host protease to form the mature E2 and a small peptide E3 (48). It has been demonstrated that this cleavage is necessary for the production of infectious virus particles (31, 43, 71). Likewise, the PE2 cleavage mutants of SIN, studied in Chapter 8, are non-infectious on BHK-21 cells. It is shown that these viruses are impaired in membrane fusion capacity at a physiologically relevant acidic pH. The reason appeared to be that the PE2 cleavage mutants exhibit a significantly more acidic pH threshold for the formation of an E1 homotrimer and the initiation of membrane fusion. These results are in agreement with observations of Salminen and coworkers (71), who showed that the infectivity of a cleavage mutant of SFV on BHK-21 cells was restored by exposure of the virus to pH 4.5. Clearly, the presence of PE2 stabilizes the PE2/E1 heterodimer. More importantly, these observations support the notion that PE2 acts as a chaperone to protect the spike from premature destabilization within the slightly acidic TGN. Yet, the data presented in Chapter 8 indicate that PE2 cleavage itself is not absolutely required for viral infectivity. SIN mutants with second-site resuscitating mutations in PE2 were found to be highly infectious on BHK-21 cells, despite a sustained lack of PE2 cleavage. Apparently, the second-site resuscitating mutations in PE2 destabilize the PE2/E1 heterodimer to such an extent that the spike rearranges to a fusion-competent E1 homotrimer within the mildly acidic endosomal compartment of the cell. At the same time the heterodimer remains stable enough to survive the acidic pH in the TGN during the process of spike maturation. In conclusion, PE2 stabilizes the PE2/E1 heterodimer, during passage of the spike through the TGN, by protein-protein interactions. These protein-protein interactions are weakened by PE2 cleavage, through which the viruses are primed to rearrange to a fusion-active conformation at a mildly acidic pH. Apparently, the protein-protein interactions that stabilize the PE2/E1 heterodimer may also be weakened by second-site resuscitating mutations in PE2. The second-site mutations act to destabilize the PE2/E1 heterodimer enough for the spike to rearrange to its fusion-competent conformation at a physiologically relevant acidic pH, but not so much that the spike would presumably rearrange in the acidic TGN during maturation in the infected cell.

Perspectives

In recent years there has been a revolutionary increase in our understanding of the molecular mechanisms involved in viral membrane fusion. By far the best understood is the HA of influenza virus (76). HA was the first viral envelope glycoprotein for which the structure was resolved at the atomic level. More recent crystallographic studies have revealed that the structures Summarizing discussion 169 of several other viral membrane fusion proteins closely resemble that of influenza HA. These include the envelope glycoproteins of retroviruses, filoviruses and paramyxoviruses (75, 85). It is likely that these viruses all use a similar mechanism of membrane fusion to penetrate their host cells, a key feature being the conversion of a triple helix into a six-helical bundle. The identification of this common structural conversion has led to the development of small peptides that strongly interfere with the formation of the six-helical bundle. These peptides inhibit the membrane fusion process of these viruses. Interestingly some of these molecules are currently in clinical evaluation as potential antiviral drugs, for example in patients infected with human immunodeficiency virus. Quite recently, as discussed in Chapter 1 and above, the structure of the E1 membrane fusion protein alphaviruses has been resolved at the atomic level (46). Remarkably, this protein does not at all resemble the influenza HA. Rather, the alphavirus E1 protein appears to be similar in many respects to the E fusion protein of tick-borne encephalitis (TBE) virus, a member of the family Flaviviridae (32). This family includes, besides TBE virus, several other major human , such as hepatitis C virus, Dengue virus and yellow-fever virus. Based on the similarity between their envelope glycoproteins, it is, again, quite plausible that alphaviruses and flaviviruses utilize a similar membrane fusion mechanism. At the same time, it is likely that this mechanism will be different from that used by HA and HA-related fusion proteins. Based on this reasoning, membrane fusion mediated by HA and HA-related proteins is being referred to as “class I” fusion and fusion mediated by alphavirus or flavivirus membrane fusion proteins as “class II” fusion (46). We hope that the present study on the membrane fusion properties of Sindbis virus, which is by itself a minor human , will contribute to a further understanding of “class II” viral membrane fusion in general and thus, possibly, to the development of antiviral drugs interfering with cell entry of other, highly pathogenic, viruses belonging to the “class II” category.

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87. Wilschut, J., J. Corver, J. L. Nieva, R. Bron, L. Moesby, K. C. Reddy, and R. Bittman. 1995. Fusion of Semliki Forest virus with cholesterol-containing liposomes at low pH: a specific requirement for sphingolipids. Mol. Membr. Biol. 12:143-149. 88. Zhang, J., A. Pekosz, and R. A. Lamb. 2000. Influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike proteins. J. Virol. 74:4634-4644. List of publications

Contributing authors

List of abbreviations 176 List of publications

List of publications

1. Moor, A. C. E., A. E. Wagenaars-van Gompel, R. C. A. Hermans, J. Van der Meulen, J. M. Smit, J. Wilschut, A. Brand, T. M. A. R. Dubbelman, and J. Van Steveninck. 1999. Inhibition of various steps in the replication cycle of vesicular stomatitis virus contributes to its photoinactivation by AlPcS4 or Pc4 and red light. Photochem. Photobiol. 69:353-359.

2. He, L., H. Byun, J. M. Smit, J. Wilschut, and R. Bittman. 1999. Enantioselective synthesis of novel trans double bond ceramide analogue via catalytic asymmetric dihydroxylation of an Enyne. The role of the trans double bond of ceramide in the fusion of Semliki Forest virus with target membranes. J. Am. Chem. Soc. 121:3807-3903.

3. Smit J. M., R. Bittman, and J. Wilschut. 1999. Low-pH-dependent fusion of Sindbis virus with receptor free cholesterol- and sphingomyelin-containing liposomes. J. Virol. 73:8476-8484.

4. Smit J. M., R. Bittman, and J. Wilschut. 2001. Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity. FEBS Lett. 498:57-61.

5. Smit J. M., W. B. Klimstra, K. Ryman, R. Bittman, R. E. Johnston, and J. Wilschut. 2001. PE2 cleavage mutants of Sindbis virus: correlation between viral infectivity and pH-dependent membrane fusion activation of the spike heterodimer. J. Virol. 75:11196-11204.

6. Lee, P., R. Knight, J. M. Smit, J. Wilschut, and D. E. Griffin. 2002. A single mutation in the E2 glycoprotein important for neurovirulence influences binding of Sindbis virus to neuroblastoma cells. J. Virol., in press.

7. Smit, J. M., L. Gang, P. Schoen, J. Corver, R. Bittman, and J. Wilschut. 2002. Fusion of Alphaviruses with liposomes is a non-leaky process. FEBS Lett., in press.

8. Smit J. M., B.-L. Waarts, K. Kimata, W. B. Klimstra, R. Bittman, and J. Wilschut. 2002. Adaptation of Alphaviruses to heparan sulfate: Interaction of Sindbis and Semliki Forest virus with liposomes containing lipid-conjugated heparin. J. Virol., provisionally accepted for publication. List of publications 177

9. Smit J. M., B.-L. Waarts, R. Bittman, and J. Wilschut. 2002. Liposomes as a model system in the study of viral membrane fusion mechanisms. Meth. Enzymol., in press. 178 Contributing authors

Contributing authors

Robert Bittman, Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, NY, USA

Jeroen Corver, Department of Medical Microbiology, Molecular Virology Section, University of Groningen, Groningen, The Netherlands Present affiliation: Department of Virology, Leiden University Medical Center, Leiden, The Netherlands

Diane E. Griffin, Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

Robert E. Johnston, Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Koji Kimata, Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Japan

William B. Klimstra, Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Ronald Knight, Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

Richard J. Kuhn, Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

Peiyu Lee, Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA Present affiliation: Farming Intelligene Corp., Taipei, Taiwan

Gang Li, Department of Biophysics, Beijing Medical University, Beijing, China.

Ke-Chun Lin, Department of Biophysics, Beijing Medical University, Beijing, China.

Suchetana Mukopadhyay, Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Contributing authors 179

Kate D. Ryman, Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Pieter Schoen, Department of Medical Microbiology, Molecular Virology Section, University of Groningen, Groningen, The Netherlands Present address: HaemoProbe, L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands

Barry-Lee Waarts, Department of Medical Microbiology, Molecular Virology Section, University of Groningen, Groningen, The Netherlands

Jan Wilschut, Department of Medical Microbiology, Molecular Virology Section, University of Groningen, Groningen, The Netherlands 180 List of abbreviations

List of abbreviations

BHK-21 baby hamster kidney cells BSA bovine serum albumin CHO Chinese hamster ovary Chol cholesterol Cryo-EM cryo-electron microscopy DMEM Dulbecco’s modified Eagle medium DTNB 5,5’-dithiobis(2-nitrobenzoic acid) EEE Eastern equine encephalitis ER endoplasmic reticulum FBS fetal bovine serum FCS fetal calf serum GAG glycoaminoglycan HA hemaglutinin HepPE heparin-conjugated phosphatidylethanolamine HNE 5 mM Hepes, 150 mM NaCl, 0.1 mM EDTA, pH 7.4 HS heparan sulfate IU infectious unit LUV large unilamellar vesicles MAb monoclonal antibody MES morpholinoethanesulfonic acid MOI multiplicity of infection N18 neuroblastoma cell line NB-DNJ N-butyldeoxynojirimycin OGP n-octyl-ß-D-glucopyranoside PBS phosphate-buffered saline PC phosphatidylcholine PE phosphatidylethanol-amine PFU plaque forming units PhotoChol 6-photocholesterol Pyrene fatty acid 16-(1-pyrenyl)hexadecanoic acid PyrPC 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3- phosphocholine RR Ross River virus SIN Sindbis virus SFV Semliki Forest virus SPM sphingomyelin srf sterol requirement in function TCA trichloroacetic acid TGN trans-golgi network TBE Tick-borne encephalitis virus List of abbreviations 181

VEE Venezuelan equine encephalitis virus WEE Western equine encephalitis virus 182 List of abbreviations Samenvatting in het Nederlands

Curriculum Vitae

Nawoord 184 Nederlandse samenvatting

Inleiding

Sindbis virus (SIN) behoort tot de genus “Alphavirussen” binnen de familie Togaviridae. Er zijn in totaal 25 verschillende alphavirussen geïdentificeerd. Alphavirussen kunnen verschillende diersoorten infecteren, waaronder vogels, knaagdieren en zoogdieren. Sommige alphavirussen veroorzaken een fatale encefalitis (ontsteking van de hersenen) bij bijvoorbeeld paarden, terwijl andere alphavirus-infecties veel mildere symptomen laten zien. Ook mensen kunnen worden geïnfecteerd door alphavirussen, alhoewel dit echter relatief weinig voorkomt. De symptomen van een alphavirus-infectie bij mensen variëren van griepachtige verschijnselen tot een ernstige encefalitis. Alphavirussen behoren tot de arbo (arthropod-borne) virussen; dat wil zeggen dat de infectie in de natuur wordt overgedragen door insecten, zoals teken en muggen. Als een besmette mug bloed zuigt bij een dier of mens, komt het virus in de bloedbaan terecht. Het SIN virus is één van de minst gevaarlijke alphavirussen voor de mens en wordt daarom vaak gebruikt als een modelvirus in wetenschappelijk onderzoek. Alphavirussen zijn bolvormige deeltjes met een grootte van 70 nm (Hoofdstuk 1, figuur 1). In de kern bevindt zich het nucleocapside. Het nucleocapside bestaat uit 240 kopieën van het capside-eiwit en één RNA molecuul met een grootte van circa 11.000 basen (het virale genoom). Het nucleocapside wordt omgeven door een laag van lipiden (membraan) waarin zich een tweetal zogenoemde spike- eiwitten, het E2 en het E1, bevinden. In totaal zijn er 80 virale spikes aanwezig op een virusdeeltje, waarbij iedere spike bestaat uit een trimeer (drieling) van E2/E1 heterodimeren. De E2 en E1 eiwitten steken door de virale membraan heen. Dit houdt in dat het eiwit uit drie verschillende regio’s bestaat, het cytoplasmatische domein (bevindt zich binnenin het virusdeeltje), het transmembraan-domein (dit gedeelte zit in de membraan) en het ecto-domein (bevindt zich buiten de membraan). Het E2 eiwit heeft een lengte van 423 aminozuren met een cytoplasmatisch domein van 33 aminozuren. Het E1 eiwit is 439 aminozuren groot en heeft een cytoplasmatische staart van slechts 2 aminozuren. Zowel het E2 als het E1 eiwit zijn geglycosyleerd en geacyleerd; dat wil zeggen dat beide eiwitten aminozuren bevatten waaraan respectievelijk suikergroepen en vetzuren gekoppeld zijn. Membraanvirussen kunnen in principe op twee verschillende manieren een cel infecteren. Ze kunnen rechtstreeks fuseren met de plasmamembraan van de cel. Daarnaast is het ook mogelijk dat de virusdeeltjes eerst worden opgenomen door de cel via receptor-gemedieerde endocytose en pas daarna fuseren vanuit zure blaasjes (endosomen) (Hoofdstuk 1, figuur 4). In het geval van plasmamembraan- fusie bindt het virus aan een receptor op het celoppervlak waarbij deze interactie er op zichzelf voor zorgt dat de virale membraan en de plasmamembraan van de cel met elkaar versmelten (fusie). Virusdeeltjes die worden opgenomen door receptor- gemedieerde endocytose binden ook eerst aan een receptor op het celoppervlak. Receptor interactie en membraanfusie-activiteit van Sindbis virus 185

Vervolgens worden de virus-receptor complexen ingesloten door instulping en afsnoering van een deel van de plasmamembraan; uiteindelijk komt het virus in endosoom terecht. De inhoud van de endosomen verzuurt, waardoor het virus verandert van structuur en zo het vermogen krijgt te fuseren met het endosomale membraan. Er bestaat in de literatuur grote onduidelijkheid over de route van infectie van het SIN virus. Sommige studies laten zien dat SIN de cel infecteert via plasmamembraan-fusie, terwijl andere onderzoekingen aangeven dat SIN fuseert vanuit zure endosomen. Vast staat dat het E2 eiwit een belangrijke rol speelt bij receptor-interactie en E1 verantwoordelijk is voor het fusieproces. Hoe dan ook, na het samensmelten van de virale membraan met de plasmamembraan dan wel de endosomale membraan komt het nucleocapside vrij in de cel. Het nucleocapside wordt vervolgens ontmanteld waarna de synthese van nieuwe virusdeeltjes begint. Het virale RNA wordt afgelezen in het cytoplasma van de cel, wat leidt tot de vorming van nieuwe eiwitten en RNA moleculen. Uiteindelijk wordt er een groot aantal nieuwe virusdeeltjes gevormd door een proces van “budding” (knopvorming).

Resultaten en Conclusies

Het onderzoek beschreven in dit proefschrift richt zich op het ontrafelen van het infectieproces van SIN. Hoofdstuk 2, 3 en 4 beschrijven de basale karakteristieken van SIN fusie in een modelsysteem. In Hoofdstuk 3 en 5 t/m 8 zijn aminozuur-veranderingen (mutaties) aangebracht op specifieke plaatsen in het E2 en het E1 eiwit. Vervolgens is het effect van deze mutaties op de receptor- interactie en membraanfusie-activiteit van SIN onderzocht. In Hoofdstuk 2 is gekeken naar de invloed van lage pH op het fusieproces van SIN. Het fusieproces van SIN is bestudeerd met behulp van een modelsysteem. In dit systeem worden kunstmatige lipidemembranen (liposomen) gebruikt als doelmembraan. Membraanfusie wordt gemeten met behulp van de fluorescerende stof pyreen, die wordt ingebouwd in het virusmembraan. Fusie van een pyreen-gemerkt virusmembraan met een liposomaal membraan resulteert in een uitverdunning van de pyreen-moleculen in de liposomale membraan waardoor de fluorescentie intensiteit van de merker daalt. Met deze methode kan het fusieproces continu gevolgd worden. De resultaten laten zien dat fusie strikt afhankelijk is van het blootstellen van het virus aan lage pH. Optimale fusie wordt waargenomen bij pH 5.0. Onder suboptimale condities kan er onderscheid worden gemaakt tussen verschillende fasen in het fusieproces. Onder invloed van lage pH valt de virale E2/E1 heterodimeer uit elkaar en bindt het virus aan het liposomale membraan. Vervolgens ondergaat het E1 eiwit allerlei veranderingen waarbij een trypsine- resistente E1 trimeer wordt gevormd. Dan treedt er na een korte pauze fusie op. 186 Nederlandse samenvatting

De aanwezigheid van deze “lag phase” zou kunnen betekenen dat er nog andere veranderingen in het E1 trimeer plaatsvinden die nodig zijn voor fusie. Het is nog niet bekend welke structuur het E1 eiwit heeft op het moment van fusie. Wel is het duidelijk dat de aanwezigheid van cholesterol en sfingolipiden in het doelmembraan noodzakelijk is voor de membraanfusie-activiteit van het virus. SIN is niet in staat te fuseren met liposomen waarin één van deze lipiden ontbreekt. Cholesterol speelt een belangrijke rol bij de binding van het virus aan het liposoom, terwijl sfingolipiden verantwoordelijk zijn voor het fusieproces zelf. Uit de bovengenoemde resultaten blijkt dat het blootstellen van het virus aan lage pH essentieel is voor het initiëren van het fusieproces. Dit ondersteunt de gedachte dat SIN de cel infecteert via receptor gemedieerde-endocytose en fusie vanuit zure endosomen, en niet door directe fusie met de plasmamembraan. In Hoofdstuk 3 is de rol van virus-receptor interactie in het membraanfusie- proces van SIN nader onderzocht. Hierbij werden liposomen gebruikt waarin lipide-gekoppeld heparine, als een specifieke receptor voor aangepast SIN, is ingebouwd. Uit een eerdere studie was gebleken dat het SIN virus na vermenigvuldiging op cellen is aangepast aan de receptor heparan-sulfaat en bindt aan heparine. De resultaten beschreven in dit hoofdstuk laten zien dat het aangepaste SIN virus goed bindt aan de heparine-lipide receptor, ingebouwd in liposomen, bij neutrale pH. Desondanks treedt er geen fusie op onder deze condities. Fusie wordt alleen waargenomen na aanzuring van het medium. Samenvattend kan er geconcludeerd worden dat de virus-receptor interactie geen belangrijke rol speelt in het fusieproces van SIN met liposomen. Het blootstellen van het virus aan lage pH daarentegen is essentieel voor membraanfusie, opnieuw suggererend dat fusie van SIN in cellen plaatsvindt vanuit zure endosomen. In Hoofdstuk 4 is gekeken of het fusieproces van alphavirussen met liposomen gepaard gaat met lekkage van ingesloten moleculen. Hiertoe werden kleine water-oplosbare moleculen (sucrose en inuline) ingesloten in liposomen. Uit de resultaten blijkt dat tijdens membraanfusie deze stoffen nauwelijks weglekken. Dit geeft aan dat de fusiereactie nauwkeurig georganiseerd is en ondersteunt de gedachte dat het proces plaatsvindt via een zogenoemd hemifusie-intermediair. Dit betekent dat de buitenste monolagen van de beide membranen eerst met elkaar versmelten, pas in een wat later stadium gevolgd door de binnenste monolagen. In Hoofdstuk 5 zijn de bindingskarakteristieken en membraanfusie- eigenschappen van twee SIN varianten (SIN TE virus en SIN 633 virus) met elkaar vergeleken. Deze virussen verschillen in slechts één aminozuur in het E2 eiwit. Desondanks is het TE virus veel meer pathogeen in muizen en vormt meer virusdeeltjes in zenuwcellen dan het 633 virus. De resultaten laten zien dat de infectiviteit van het TE virus hoger is in zenuwcellen omdat het virus beter bindt aan deze cellen dan het 633 virus. Daarnaast is er gekeken naar de membraanfusie- activiteit van beide virussen met liposomale membranen. De liposomen werden gemaakt van lipiden afkomstig van zenuwcellen of niercellen. Er zijn geen Receptor interactie en membraanfusie-activiteit van Sindbis virus 187 verschillen waargenomen tussen de fusiekarakteristieken van het TE en het 633 virus. Echter, beide virussen fuseren beter met liposomen afkomstig van niercellen dan van zenuwcellen. Samenvattend kan er gezegd worden dat de verhoogde infectiviteit van het TE virus voor zenuwcellen is gerelateerd aan de bindingskarakteristieken van het virus en niet aan de membraanfusie-activiteit. In Hoofdstuk 6 is de invloed van glycosylering van de virale spike eiwitten op infectie van cellen door SIN bestudeerd. In SIN zijn twee suikergroepen gekoppeld aan zowel het ecto-domein van het E2 als het E1 eiwit. Het is in het algemeen bekend dat suikergroepen een belangrijke rol kunnen spelen bij de vouwing van membraaneiwitten. In dit hoofdstuk wordt aangetoond dat het verwijderen van een suikergroep in het E2 of in het E1 eiwit tot gevolg heeft dat het virus nauwelijks infectieus is voor cellen. Daarentegen heeft de afwezigheid van een enkele suikergroep in E2 of E1 geen grote gevolgen op de vouwing van de spike eiwitten en op de vorming van virusdeeltjes. De blokkade in infectie lijkt dus te liggen in de eerste stappen van het infectieproces. Uit de resultaten blijkt dat de mutante virusdeeltjes niet in staat zijn te fuseren met liposomen onder lage pH condities, wat suggereert dat het infectieproces is geblokkeerd op het niveau van de fusiereactie. Onze hypothese is dat de afwezigheid van suikergroepen subtiele veranderingen in de spike eiwitten teweeg brengt waardoor het virus niet de conformationele veranderingen kan ondergaan, die nodig zijn voor membraanfusie. In Hoofdstuk 7 wordt gekeken naar het effect van acylering van de transmembraan-domeinen van de spike eiwitten op het fusieproces van SIN. De transmembraan-domeinen van het E2 en het E1 eiwit bevatten respectievelijk twee en één vetzuurketens. De aanwezigheid van vetzuurketens is sterk geconserveerd bij verschillende alphavirussen, wat impliceert dat vetzuurketens een belangrijke rol vervullen in de levencyclus van deze virussen. Er wordt in dit hoofdstuk aangetoond dat de aanwezigheid van vetzuurketens niet belangrijk is voor membraanfusie. Dezelfde fusiekarakteristieken werden waargenomen voor het wild-type virus en drie virusmutanten waarbij de vetzuurketens in het E2 en/of het E1 eiwit waren verwijderd. In Hoofdstuk 8 zijn de fusogene eigenschappen van immature SIN virusdeeltjes bepaald. In een geïnfecteerde cel wordt het E2 eiwit gevormd als een voorloper eiwit, het zogenoemde PE2. In een van de laatste stappen van de vorming van het virusdeeltje wordt het PE2 eiwit gemodificeerd tot het mature E2. In deze studie zijn SIN virussen gebruikt waarbij het PE2 niet meer wordt omgezet naar E2. Dit betekent dat de virale spike bestaat uit een trimeer van PE2/E1 heterodimeren. Eerdere studies hebben aangetoond dat deze virussen niet infectieus zijn en dat de blokkade in infectie na receptor-interactie, maar vòòr RNA replicatie ligt. Inderdaad, de resultaten laten zien dat het infectieproces stagneert op het niveau van de fusiereactie. Er is gevonden dat PE2-bevattende SIN virussen niet in staat zijn te fuseren met liposomen onder mild-zure condities. 188 Nederlandse samenvatting

Vervolgens zijn de verschillende fasen in het virus-liposoom fusieproces onderzocht. Uit de resultaten blijkt dat PE2-bevattende virussen niet in staat zijn een E1 trimeer te vormen. Kortom, PE2-bevattende virussen zijn niet infectieus in cellen omdat de spike te stabiel is om conformationele veranderingen te ondergaan en fusie-actief te worden. Vermenigvuldiging van PE2-bevattende virusdeeltjes op cellen leidt tot de generatie van virusmutanten die wel infectieus zijn in cellen. Na het karakteriseren van deze mutanten bleek dat de virussen nog steeds PE2 bevatten in de spike. Echter, de mutanten blijken een enkele mutatie in het PE2 eiwit te hebben. Deze mutatie destabiliseert de virale spike waardoor het virus onder mild-zure condities in staat is een E1 trimeer te vormen en te fuseren met liposomen. In Hoofdstuk 9 worden de resultaten samengevat en in een algemene context bediscussieerd. Slotopmerkingen

De laatste jaren is er veel bekend geworden over de moleculaire mechanismen van virale membraanfusie. Vooral onderzoek naar de fusie van het influenza virus (griepvirus) heeft sterk bijgedragen aan dit verbeterde inzicht. Recent is gebleken dat het membraanfusie-eiwit van het influenza virus, het hemagglutinine (HA), in structureel opzicht sterke overeenkomsten vertoont met de fusie-eiwitten van een aantal andere membraanvirussen, waaronder het humane immunodeficientie-virus (HIV). En het lijkt erop dat al deze virussen in principe gebruik maken van een overeenkomstig membraanfusiemechanisme om hun onderscheiden doelwitcellen te infecteren. Deze nieuwe inzichten, die -nogmaals- vooral zijn gebaseerd op onderzoek aan het griepvirus, hebben geleid tot de ontwikkeling van specifieke moleculen die interfereren met dit fusieproces. Een aantal van deze fusieremmers wordt momenteel geëvalueerd in klinische studies bij HIV-patienten. Ook de structuur van de fusie-eiwitten van alphavirussen is recentelijk in detail in kaart gebracht. Het blijkt dat deze eiwitten in het geheel niet lijken op het HA van het influenza virus, maar wel op de fusie-eiwitten van membraanvirussen binnen de familie van de Flaviviridae. Tot deze familie behoort een aantal zeer belangrijke pathogenen, zoals het hepatitis C virus, het dengue-virus, het gele koorts virus en het teken-encefalitisvirus. De overeenkomst tussen de alphavirus en flavivirus fusie-eiwitten heeft ertoe geleid dat het fusieproces dat deze eiwitten mediëren nu wordt geklassificeerd als klasse II membraanfusie (tegenover klasse I fusie gemedieerd door het HA en aanverwante virale membraanfusie-eiwitten). Het is waarschijnlijk dat klasse II virale membraanfusie in alle gevallen in principe op dezelfde wijze verloopt. Hopelijk draagt het onderzoek aan een relatief onbelangrijk humaan pathogeen als het SIN virus, zoals beschreven in dit proefschrift, uiteindelijk bij aan een beter inzicht in klasse II virale membraanfusie in het algemeen en, op die wijze, aan de ontwikkeling van antivirale middelen tegen zeer belangrijke humaan-pathogenen. Curriculum Vitae 189

Curriculum Vitae

Persoonlijke gegevens

Naam : Jolanda Mariske Smit Geboortedatum : 15 april 1975 Geboorteplaats : Delfzijl

Opleiding

1987 – 1991 : MAVO D 1991 – 1994 : Middelbaar Laboratorium onderwijs, afstudeerrichting “Chemisch” 1994 – 1997 : Hoger Laboratorium onderwijs, afstudeerrichting “Biologisch” Hoofdvakken: Moleculaire Biologie, Immunologie en Genetica, Microbiologie. Stage/afstudeerperiode: vakgroep Fysiologische Chemie, Faculteit der Medische Wetenschappen, Rijksuniversiteit Groningen

Werkervaring

1997 – 2001 : Assistent in opleiding bij de vakgroep Fysiologische Chemie (1997-1999) en bij de afdeling Medische Microbiologie, Sectie Moleculaire Virologie (1999-2001), Faculteit der Medische Wetenschappen, Rijksuniversiteit Groningen

Sept.– Nov. : Werkbezoek aan de Universiteit van North Carolina in Chapel 1999 Hill, Verenigde Staten; afdeling Microbiologie en Immunologie

2001 – 2004 : Onderzoeker bij de afdeling Medische Microbiologie, Sectie Moleculaire Virologie, Faculteit der Medische Wetenschappen, Rijksuniversiteit Groningen 190 Nawoord

Nawoord

Het is alweer bijna zes jaar geleden dat ik voor het eerst bij de –toen nog- vakgroep Fysiologische Chemie kwam binnen lopen. Het eerste jaar was ik aan het werk als stagiaire van de hogere laboratorium opleiding. Ik raakte al snel enthousiast voor het vak en toen de mogelijkheid zich voordeed om te blijven als aio twijfelde ik dan ook geen moment. De daarop volgende jaren zijn voorbij gevlogen! Ik kan het daarom nog haast niet geloven maar ik kan nu met recht zeggen: “Ik ben …bijna klaar!!” Zoals de meeste mensen in mijn familie zeggen: “Jolanda gaat nu echt bijna afstuderen.” Nu ben ik het niet helemaal eens met deze woordkeuze maar vooruit dan maar. Het feit dat ik zover ben gekomen heb ik aan een aantal mensen te danken. Als allereerste wil ik mijn promotor Jan Wilschut bedanken. Er zijn zoveel dingen waarvoor ik jouw moet bedanken dat ik vast de helft vergeet. Ten eerste wil ik je bedanken voor de kans die je mij gegeven hebt om gelijk na mijn Hbo- opleiding aio te worden. We wisten allebei niet precies hoe het zou gaan lopen, maar ik ben blij dat we de gok gewaagd hebben. Natuurlijk hebben we allemaal wel eens iets aan te merken op de “directe baas”, maar ik vind (met alle lof) dat je de beste begeleider bent die een aio zich kan wensen. Je was altijd erg enthousiast over ons werk en hoe druk je het ook had ik kon altijd bij je terecht. Door jouw perfectionisme in het schrijven van Engelstalige wetenschappelijke artikelen is mijn schrijftalent gevorderd van slecht tot matig of misschien wel tot redelijk goed. Al heeft dat ons heel wat energie gekost, ik ben blij dat je (steeds maar weer) de tijd hebt genomen om mij het proces van artikelen schrijven te leren. Zonder jouw inzet (op alle fronten) was het nooit zover gekomen. Bedankt! Het motto van de MMB luidt: “Ik werk gelukkig bij de Medische Microbiologie!” Dat geldt voor mij zeker! Wij hebben een ontzettend leuke groep waar het altijd erg gezellig is. Voor mij persoonlijk is er veel veranderd toen de aio- kamer werd ingesteld. De vele uurtjes die ik daar heb doorgebracht met Laura Bungener, Diana Spierings en Barry-Lee Waarts waren erg gezellig. Inmiddels zitten we allemaal niet meer bij elkaar op de kamer, maar gelukkig is het contact er niet minder om. Het volgende aio-etentje is bij mij! Barry, bedankt dat je mijn paranimf wilt zijn! Natuurlijk wil ik ook alle andere kamerbewoners (Gerben Koning, Jolanda Busscher, Jeroen Visser, Koen Glazenburg en de vissen) bedanken voor de sfeer en gezelligheid. Ook op het lab is het altijd erg gezellig, iedereen bedankt! Wouter wil ik graag nog bedanken voor de periode dat je bij mij stagiaire bent geweest. Ik vond het leuk om jouw destijds te begeleiden. Ook wil ik iedereen van het GUIDE-buro bedanken. Ik vond het altijd erg leuk om actief mee te denken bij de GUIDELines en GUIDE early summer meeting vergaderingen en om een voordracht te geven in Kloostenburen. Tip top met chauffeur. Ik durf het haast niet meer te zeggen, maar jullie financiële Nawoord 191 ondersteuning voor cursussen, congressen en werkbezoeken heb ik altijd erg gewaardeerd! Daarnaast wil ik iedereen van het GAIOO bedanken voor het lekkere eten, de gezellige vergaderingen en de gigantische hoeveelheid e-mails! I would like to thank Prof. Robert Bittman for our collaboration. My boyfriend and I really enjoyed the visit to your lab (including the spectacular view over Manhattan and walking around China town). Also, I would like to thank you for carefully reading our manuscripts. The collaboration with Prof. Robert Johnston, Dr. William Klimstra, and Dr. Kate Ryman was of great importance for my thesis. I learned a lot during my short-term working period at your lab, thanks for your great hospitality. The barbecue was lots of fun! We miss the beautiful weather of North Carolina, but we are glad that we do not have hurricanes here. Also, I would like to thank Prof. Richard Kuhn and Dr. Suchetana Mukhopadhyay. Tuli, I am really glad that we met last year in Paris and later again in Tilton. I really enjoy our scientific and social discussions via e-mail! I think that the results we have already obtained are just the beginning of something spectacular. I hope to see you again in the near future! Furthermore, I would like to thank Prof. Diane Griffin and Dr. Peiyu Lee. Peiyu, I enjoyed the time your were working in our lab. Finally, I wish to thank Jeroen Corver, Koji Kimata, Ronald Knight, Ke-Chun Lin, Gang Li, and Pieter Schoen for their contribution to this thesis. Natuurlijk was het nooit zover gekomen als mijn lieve papa en mama mij niet op deze wereldbol hadden gezet! Ik heb een fantastische jeugd gehad in Uitwierde, waar ik alles had wat mijn hartje begeerde. Dat we vorig jaar nog weer vijf maanden met plezier thuis hebben gewoond, laat maar weer eens zien dat we een zeer hechte band met elkaar hebben. Bedankt voor ALLES!! Tevens wil ik graag Erwin zijn ouders bedanken. Ook jullie staan altijd voor ons klaar. Op de hondjes passen, meehelpen met wat dan ook, een belletje en jullie staan er weer. Ik vond het heel leuk om jullie nog beter te leren kennen, in de periode dat jullie bij ons hebben gewoond. Dat was erg gezellig. Graag wil ik ook alle broers en zussen, zwagers en schoonzussen, neefjes en nichtjes bedanken. We hebben inmiddels al een hele grote familie! Wij zien jullie allemaal veel te weinig, maar ik hoop dat daar straks meer tijd voor is. Jan Pieter, als mijn twee na grootste broer, wil ik nog speciaal bedanken voor zijn show in rokkostuum! Bedankt dat je mijn paranimf wilt zijn! En dan valt er nog één iemand te bedanken: de liefde van mijn leven! Geukie, samen lachen, samen huilen, samen staan we sterk, samen kunnen we de hele wereld aan! De afgelopen maanden zijn een aaneenschakeling geweest van deadlines, bedankt voor je geduld, de peptalks, je kokkerellen en de verwennerij! De laptop gaat nu voor een lange tijd uit! Ik hou ontzettend veel van je. Altijd en eeuwig! Jolanda Stellingen

behorende bij het proefschrift

“ Mutational analysis of receptor interaction and membrane fusion activity of Sindbis virus”

1. De waarde van een liposomaal modelsysteem voor het ontrafelen van de fundamentele aspecten van virusfusie mag niet worden onderschat. Dit proefschrift.

2. De zinsnede: “exposure to an acid environment within the cell may not be an obligatory step in the process of infection of cells by alphaviruses” is onjuist. Hernandez et al., 2000, J. Virol. 75:2010-2013.

3. De stelling van Hubbard en Ivatt dat: “the main outlines of the asparagine- linked oligosaccharide synthesis are now clear” is een vereenvoudiging van de werkelijkheid. Hubbard and Ivatt, 1981, Ann. Rev. Biochem. 50:555-583.

4. Alhoewel flavi- en alphavirussen beide zijn geclassificeerd als klasse II membraanfusie-virussen, wil dit niet zeggen dat de fusiereacties van deze virussen identiek aan elkaar verlopen, aangezien alleen de membraanfusie- activiteit van alphavirussen strikt afhankelijk is van sphingolipiden en cholesterol in het doelmembraan. Lescar et al., 2001, Cell 105:137-148.

5. Adaptatie van het Sindbis virus aan de cellulaire receptor heparan sulfaat heeft geen gevolgen voor de membraanfusie-aktiviteit van het virus. Dit proefschrift.

6. Als Fiedler en Simons hun stelling: “Among the molecular constituents of cells, carbohydrates have been overshadowed by their more glamorous neighbors, the nucleic acids and proteins” anno 2002 hadden geformuleerd zouden ze waarschijnlijk de nog minder tot de verbeelding sprekende lipiden niet ongenoemd hebben gelaten. Fiedler and Simons, 1995, Cell 81:309-312.

7. Het tot stand komen van een proefschrift is als de voorbereiding op de Olympische Spelen: 4 jaren intensief werken, gevolgd door een uiteindelijke ultieme inspanning. 8. “Een blik deskundigen opentrekken….” Tijdens het inblikken van zogenaamde deskundigen, die ogenblikkelijk na een gebeurtenis vaak vruchteloos terugblikken, moet een selectievere blik geworpen worden op hun houdbare (w)eetbaarheid.

9. Het kleinere aantal parkeerplaatsen aan de Oostersingel zal, met het sluiten van het Bodenterein, leiden tot een grotere incidentie van ochtendhumeur.

10. Het schrijven van medisch gerelateerde krantenartikelen dient voorbehouden te worden aan deskundigen.

11. De naamgeving: “Are you surprised” van een recovered tekstbestand van msword verhoogt het frustratiegevoel van een gestreste promovendus en zou vervangen moeten worden door: “I am sorry for this inconvenience.”

12. De faculteit of universiteit zou de drukkosten van een proefschrift volledig moeten vergoeden.

Jolanda Smit Groningen, 19 juni 2002