1 DELIVERY OF ANIMAL DNA INTO THE NUCLEUS

Urs F. Greber Institute of Zoology University of Zürich Winterthurerstrasse 190 CH-8057 Zürich Switzerland Phone: 41 1 635 4841, Fax: 41 1 635 6817, email: [email protected] 1. INTRODUCTION 2. SURFACE BINDING AND UPTAKE Non-enveloped 2.1. : Adenovirus type 2 and type 5 (Ad-2 and Ad-5) 2.2. Papovaviridae: Polyoma virus and Simian virus 40 (SV40) 2.3. : Parvovirus and Adeno-associated virus (AAV) Enveloped viruses 2.4. : virus type 1 (HSV-1) 2.5. : (HBV) 2.6. : Nuclear polyhedrosis virus (NPV) and Granulosis virus (GV) 3. NUCLEAR ENVELOPE TARGETING 3.1. type 1 (HSV-1) 3.2. Nuclear polyhedrosis virus (NPV) 3.3. Other viruses 4. NUCLEAR IMPORT: UNCOATING, DOCKING TRANSLOCATION 4.1. Adenovirus type 2 (Ad-2) 4.2. Simian virus 40 (SV40) 4.3. Herpes simplex virus type 1 (HSV-1) 4.4. virus (HBV) 4.5. Nuclear polyhedrosis virus (NPV) and Granulosis virus (GV) 5. CONCLUSIONS 6. TABLES 7. REFERENCES Abbreviations: AAV: Adeno-associated virus AcNPV: Autographa californic Nuclear polyhedrosis virus Ad: Adenovirus GV: Granulosis virus dHBV: Duck hHBV: Hepatitis B virus HSV: Herpes simplex virus NPV: Nuclear polyhedrosis virus

MMV: Mouse minute virus NLS: Nuclear localization sequence NPC: complex NE: Nuclear envelope SV40: Simian virus 40 c: circular, ds: double stranded, kb: kilo bases, l: linear, pds: partly double-stranded, ss: single stranded 2

1. INTRODUCTION

Viruses are natural carriers of genetic information between cells. They are made up of , nucleic acids and often lipids. Viruses are usually smaller than , but can be as big as a bacterial cell. Their DNA or RNA is tightly packed and well protected with proteins and often enwrapped with a lipid envelope. Within a virus particle, which is outside a organism, the genome is inactive. The genome only becomes activated when the virus interacts with a host cell in a very specific manner. By utilizing the host cell’s mechanisms, a virus particle enters, unpacks the genome and initiates its replication and programs. It finally directs the cell to assemble and release progeny virions into the extracellular milieu for a new round of .

Viruses that infect eukaryotic cells replicate either in the cytoplasm or in the nucleus. Viruses replicating in the nucleus usually contain a DNA or, seldomly, an RNA genome. These viruses take advantage of the nuclear compartment for their DNA synthesis, for generation and processing of their mRNA and in most cases, they also assemble progeny virions within the nucleus (for review, see Fields, et al., 1996). RNA viruses replicating in the nucleus, such as the orthomyxovirus virus, or the RNA , examplified by the human virus (HIV), follow a similar strategy as the DNA viruses, but form their progeny outside the nucleus (for review, see Freed, et al., 1995, Stevenson, 1996, Trono, 1995, Whittaker, et al., 1996). For HIV, the incoming viral RNA is reverse transcribed in the cytoplasm to form linear double stranded DNA, the proviral DNA, which is then imported into the nucleus as a high molecular weight nucleo- complex. The DNA viruses, which do not replicate in the nucleus, such as poxviruses and iridoviruses, do not need nuclear targeting information. They must, however, contain additional genetic information to build up their own DNA and mRNA synthesis factories within the cytoplasm (for review, see Moss, 1996).

The genome of DNA viruses replicating in the nucleus must cross three barriers, the plasma membrane, the cytosol and the nuclear envelope. A virus particle gets across the plasma membrane by attachment with high affinity to a suitable cell surface receptor. The cytosol is traversed either by passive diffusion in the case of small viruses or by exploitation of the cell’s motile functions in the case of large viruses. To overcome the nuclear envelope barrier nuclear DNA or RNA viruses have developed two principal strategies. The onco- wait for the cell to break down its nuclear envelope during cell division. They then integrate their reverse transcribed DNA genome into the host chromatin (for review, see Coffin, 1996). The nuclear DNA- containing viruses, typified by the adenoviridae, parvoviridae, papovaviridae, baculoviridae, hepadnaviridae and herpesviridae have chosen to import their genome into the nucleus across an intact nuclear envelope (see Table 1). Emerging evidence suggests that DNA viruses are using elements of the same pathways, which control transport of cellular proteins and nucleic acids into the nucleus.

In this review, I will survey evidence on different mechanisms of DNA virus entry. I will briefly describe the viruses and their portals of entry into cells, and then discuss various nuclear envelope targeting and DNA import mechanisms. 3 2. CELL SURFACE BINDING AND UPTAKE

The first step in virus infection of a host cell is attachment to the cell surface. Attachment can be mediated by a large variety of cell surface molecules (for review of different virus receptors, see Wimmer, 1994, see also Table 2). Attachment is necessary but not always sufficient for a successful infection, since subsequent steps, such as membrane penetration and disassembly often require virus binding to specialized surface receptors. The presence of specific receptors is often an important determinant of host range and cell and tissue tropism of a virus, but other factors acting in different phases of the replication cycle can be important for productive infection as well. A variety of experimental strategies has been employed over the years to identify specific viral receptors. Most of these studies, however, have not conclusively demonstrated that a putative receptor actually serves to initiate productive virus entry. The most stringent criteria of biological significance of a putative receptor are the transformation of receptor-negative nonpermissive cells to a permissive phenotype by means of recombinant DNA technology and the concomitant block of permissiveness of the engineered cells with a specific antibody applied to the cell surface. This technology is readily applicable if the receptor is encoded by a single gene. If the receptor is a carbohydrate, one can restore the permissiveness by adding exogenous glycolipids of exactly defined composition to nonpermissive cells. In such reconstitution experiments, glycolipids have an advantage over glycoproteins, since they can contain a single carbohydrate chain and can insert themselves into the cell membranes.

Emerging evidence indicates that virus binding to the cell surface is a multistep process for many viruses. Multiple receptors can either act together or sequentially. Binding to a first receptor can cause changes in the virus or the host, which then favor interactions with a second receptor. For blood- or respiratory secretions- born viruses, the initial binding must effect rapid virus docking. In this case, the rate of binding can be more important than the affinity, which is an equilibrium measurement (for review, see Williams, 1991). If the first receptor allows fast binding, the second receptor could be responsible for any slower processes, such as stable binding, or penetration. To understand dynamic interactions between viruses and cells we need to understand questions like, how many molecules are engaged in the interactions and how long do they need to be associated to confer a biological effect. For virus-cell interactions we are just at the beginning of this new phase of understanding.

NON-ENVELOPED VIRUSES

2.1. Adenovirus 2 and 5 (Ad-2 and Ad-5)

Ad-2 and Ad-5 belong to the subgroup C adenoviruses. These viruses are non-oncogenic and widely used as gene transfer vehicles into somatic cells, when rendered replication defective (for review, see Yeh and Perricaudet, 1997). Adenoviruses naturally enter human airway cells and produce progeny virions within the nucleus of the infected cell (for review, see Horwitz, 1990). The Ad-2 particle is icosahedral and has a diameter of approximately 90 nm. It contains at least 11 different structural polypeptides and a linear double stranded DNA molecule, which is condensed with proteins V, VII, µ. The DNA also contains about 10 copies of the p23 and is covalently associated with a terminal protein at its 5’ ends (Mangel, 4 et al., 1993, Stewart, et al., 1993). The chromosome is connected to the inside wall of the via protein VI. The capsid consists of 6 to 7 different proteins. More than 75% of its mass is contributed by the hexon protein. This major protein is held together by homophilic interactions and by the minor protein IX. Protein IIIa is another capsid stabilizing component linking adjacent facets of the icosahedron. The vertices of the capsid are made up of pentameric penton base and protruding trimeric fibers. The fibers are built by a linear shaft and a carboxy-terminal globular head domain of 200 amino acids containing the receptor binding determinant (Louis, et al., 1994, Xia, et al., 1995).

For entry into cells, Ad-2 and Ad-5 use an immunoglobulin gene family protein, termed CAR (, an unrelated RNA virus, and Adenovirus Receptor), as an initial attachment site (Bergelson, et al., 1997, Tomko, et al., 1997). This receptor binds with high affinity to the fiber head domain. The precise nature of the interaction, i.e., the on and off rates or residues making up the interface, are not yet known. The Ig domain is most likely directly involved in fiber binding, since recently, the Ig domain containing MHC class I protein has also been identified as a functional primary receptor for Ad-5 (Hong, et al., 1997). Besides the fiber receptor, the alpha m-beta 2 of hematopoietic cells has also been proposed to function as a high affinity attachment site in the absence of fiber proteins (Huang, et al., 1996). Purified trimeric fiber binds with high affinity to epitheloid cells, but is not endocytozed (Wickham, et al., 1993). For endocytosis, the virus utilizes a secondary receptor, the fibronectin binding alpha v-beta 3/5 integrin, as demonstrated by cDNA experiments in integrin deficient cell lines (Wickham, et al., 1993). DNA transfection experiments in human airway cells or lymphocytes have confirmed that expression of the alpha v-beta 3/5 is essential for efficient virus infection (Goldman and Wilson, 1995, Huang, et al., 1995). It is, however, not established if integrins are true mediators of virus endocytosis or whether they exert an assisting function. Recently, other integrins, such as alpha 5-beta 1 have also been suggested to assist Ad5 internalization thus raising the possibility that multiple related entry pathways could be used by different adenoviruses (Davison, et al., 1997).

For virus uptake, the penton base plays a critical role. Its arginine-glycine-aspartate (RGD)-domain is exposed in five small protrusions at each of the 12 vertices of the icosahedron as indicated by cryo electron microscopy and cristallographic modeling (Stewart, et al., 1997). It engages with alpha v and thus somehow triggers -dependent endocytosis (Bai, et al., 1993, Greber, et al., 1996, Svensson, 1985, Varga, et al., 1991, Wickham, et al., 1994, Wickham, et al., 1993). The RGD motive is present in penton base proteins of four different subgroups (A, B, C, E). Synthetic RGD peptides, which prevent fibronectin binding to integrins, block Ad-2 endocytosis, but not attachment to cells suggesting that the primary receptor binds to fiber independently of integrins. Endocytosis of Ad-2 is highly efficient and accompanied by quantitative shedding of the fibers from the capsid (Greber, et al., 1993). It is possible that fiber detachment is a prerequisite for the virus endocytosis. However, virus uptake via integrins can also occur, if fibers are not quantitatively released from the capsid as demonstrated with the ts1 Ad-2 mutant grown at the nonpermissive temperature (Greber, et al., 1996). This virus lacks functional protease and contains a capsid which is not proteolytically processed (Anderson, 1990, Rancourt, et al., 1995). It presumably binds to the same primary receptor as the wild type virus, is endocytozed into the cells with indistinguishable efficiency and kinetics as wild type virus, but unable to penetrate the endosomal membrane and instead is recycled back to the plasma 5 membrane (Greber, et al., 1996). It is possible that ts1 fibers are shed at only those vertices which mediate cell contact, but left attached on opposite pentons.

With wild type virus, integrin binding to penton base somehow assists capsid penetration of the endosomal membrane, which takes place approximately 15 min after internalization (Greber, et al., 1993, Wickham, et al., 1994). Penetration is enhanced by acidic endosomal pH (Pastan, et al., 1986, Prchla, et al., 1995), but the precise mechanism is unknown. In particular, at high multiplicity of infection, penetration can occur in the absence of acidic endosomal pH (Greber, unpublished results, and Rodriguez and Everitt, 1996). Penton base-integrin interactions together with reducing agents in or the cytosol then reactivate the viral cysteine protease p23 inside the capsid. p23 degrades the internal protein VI (Cotten and Weber, 1995, Greber, et al., 1996). This step is essential to weaken the capsid and to it for final dissociation and DNA import into the nucleus.

2.2. Papovaviridae: Polyoma virus and Simian virus 40 (SV40)

Papovaviridae are a family of small non-enveloped viruses with icosahedral . The name is derived from three prototypic members, rabbit papilloma virus, mouse polyoma virus and simian virus 40 (SV40) (earlier called vacuolating virus). The of these tumorigenic viruses are made up of a single circular double stranded DNA molecule, which is replicated in the nucleus. Particularly, polyoma virus and SV40 have been intensely studied by virologists and molecular biologists during the past 30 years (for review, see Cole, 1996). A considerable amount of information is also available about their modes of cell entry. In contrast, much less is known about entry mechanisms of the related papilloma viruses, largely because of difficulties propagating these agents in the laboratory (see zur Hausen and de Villiers, 1994).

Polyoma virus and SV40 capsids have a diameter of approximately 50 nm and consist of three structural proteins Vp1, Vp2, and Vp3 (Stehle, et al., 1996). The capsid enwraps a double stranded closed circular DNA of about 5 kb, which is condensed into a minichromosome by four core , H2A, H2B, H3 and H4 (Hendrix and Garcea, 1994). The major coat protein Vp1 forms a total of 72 pentameric capsomers. Either of the two interiorly located minor capsid proteins, Vp2 and Vp3, is thought to contact both, the minichromosome and the Vp1 pentameric capsomer (Clever, et al., 1993).

Polyoma virus and SV40 infect a large variety of mammalian cells and tissue. The outcome of the infection can be productive or non-productive, largely depending on how the newly synthesized viral large T interacts with the host replication machinery. Infection starts with virus attachment to a surface receptor. Competiton experiments indicated that polyoma virus and SV40 use different receptors for attachment (Basak, et al., 1992). Polyoma virus Vp1 binds to a cell surface determinant (Stehle and Harrison, 1996, Stehle, et al., 1994). This step enhances the tumorigenicity of the virus and is thus thought to be involved in the productive entry pathway. SV40 appears to enter rhesus monkey kidney cells by endocytosis involving attachment to MHC class I molecules at the cell surface as suggested by antibody inhibition experiments (Breau, et al., 1992). SV40 adsorption to lymphoblastoid cells lacking MHC class I expression on the surface due to failure of beta 2 microglobulin or HLA class I expression has not been observed. 6 Different results were, however, obtained with different cells. Polarized monkey kidney cells express HLA class I on both sides, apically and basolaterally, yet SV40 only attaches to the apical membrane (Basak, et al., 1992). These results suggest that MHC class I may be a possible binding determinant for SV40, but there may be additional binding factors present on cellular membranes.

After surface binding, SV40 is internalized via smooth, clathrin-free vesicles, which were identified as caveolae (Anderson, et al., 1996, Stang, et al., 1997). Caveolae are cholesterol-rich, pot-shaped, smooth plasma membrane invaginations of approximately 70 to 90 nm width. Their precise function is unknown, but they have been implicated in endocytosis, transcytosis and intracellular signalling (for reviews, see Dangoria, et al., 1996, Ikonen, 1997, Parton, 1996, Stralfors, 1997). It is possible that caveolae are internalized and targeted to internal membranes, such as the ER. It is interesting that the majority of the incoming SV40 particles are found within vesicular compartments including endosomes and the ER and rarely also the nuclear envelope lumen (Kartenbeck, et al., 1989, Maul, et al., 1978, Yamada and Kasamatsu, 1993). There is, however, no concrete evidence that entry of SV40 into the ER is a productive route of delivering DNA into the nucleoplasm (Greber and Kasamatsu, 1996, Norkin and Anderson, 1996). It is possible that the entry pathway via caveolae and endosomes is far more complex than anticipated. Conceivably, productive SV40 entry could occur by attachment to MHC class I or other receptors in the vicinity of caveolae. Internalization and membrane penetration could then be mediated by some secondary receptor(s) or other trigger factors, perhaps similar to entry of adenovirus.

2.3. Parvoviridae: Parvovirus and adeno-associated virus (AAV)

Parvoviruses are among the smallest of the DNA viruses. Within an icosahedral capsid of about 18 to 26 nm, they contain a linear single stranded DNA molecule of about 5’000 bases (for review, see Berns, 1996). Autonomous parvoviruses, examplified by human , replicate without a , while dependoviruses, such as adeno-associated virus AAV, require functions of helper viruses, like adenovirus or herpes virus, for their replication. Usually, the capsids are made up of three viral proteins, VP1, VP2 and the major capsid protein VP3. Newly synthesized structural proteins and a nonstructural protein NS1 are directed into the nucleus for progeny virus assembly (Nuesch and Tattersall, 1993, Wistuba, et al., 1997). Based on tryptic digests, VP1, VP2 and VP3 seem to be related to each other, perhaps even derived from a common DNA sequence.

Although AAV and parvovirus vectors, together with adenoviruses, are among the most popular vehicles for in vivo gene transfer into non-dividing cells, very little is known about their entry mode. Hemagglutination and antibody inhibition studies together with cryoelectron microscopy suggested that human parvovirus B19 attached to the blood group P antigen, a glycolipid called globoside (Brown, et al., 1993, Chipman, et al., 1996). VP1 of the autonomous mouse minute virus is probably required to initiate a productive infection, but it may not be needed for virus attachment to cells or DNA replication (Tullis, et al., 1993). Perhaps, VP1 is required during virus entry into cells. For entry of AAV into permissive human cell lines, N-linked glycans of a 150 kD glycoprotein have been suggested as a cell surface attachment factor (Mizukami, et al., 1996). To which viral capsid protein this determinant binds and whether it is both, a necessary and sufficient factor for 7 AAV infection is presently unknown. Furthermore, virtually nothing is known about the subsequent steps of nuclear targeting and import of viral DNA.

ENVELOPED VIRUSES

2.4. Herpesviridae: Herpes simplex virus type 1 (HSV-1)

Herpes simplex virus types 1 and 2 are the prototypic human herpes viruses. They are among the most complex but also most intensely studied viruses (for review, see Roizman and Sears, 1996). The HSV particle is made up of four structural elements, an electron dense inner core containing a linear double stranded DNA molecule of about 150 kb, an icosahedral capsid around the core, an amorphous mass called tegument around the capsid, and an outer lipid envelope containing viral membrane proteins. It is estimated that the complete virion contains about 40 or so different proteins, many of which are surface glycoproteins. From numerous studies over the past years it became apparent that incoming virus can use multiple pathways for attachment. There is no single viral surface protein responsible for productive engagement with a surface receptor of nonpolarized epithelial cells.

The most prominent among the cellular receptor molecules described for HSV is heparan sulfate, a heparin binding component of the extracellular matrix (Seck, et al., 1994, Spear, 1993). It occurs in limiting amounts and accounts for at least 85% of the virus attachment sites in nonpolarized epithelial cells. Other receptors were proposed for various viral surface glycoproteins and include basic fibroblast growth factor (Baird, et al., 1990, Kaner, et al., 1990), mannose 6-phosphate receptor (Brunetti, et al., 1995), Fc receptors (Johnson, et al., 1988) and HVEM, a herpes virus entry mediator protein of the tumor necrosis factor gene family (Montgomery, et al., 1996, Whitbeck, et al., 1997). Whether all of these surface molecules can separately mediate virus entry or whether they act as co-receptors is under investigation.

After HSV attachment to the cell surface, fusion between viral and cellular membranes is mediated by viral surface glycoproteins. Fusion can occur efficiently at neutral pH suggesting that it may take place at the cell surface rather than in acidic endosomes (for review, see Roizman and Sears, 1996). It is, however, possible that under certain conditions, the virus utilizes the endocytic uptake machinery for passing through the cortical underneath the and then fuses in a pH-independent manner out of endosomes (Marsh and Pelchen-Matthews, 1994). In cases where virus does not enter by endocytosis, it is conceivable that the capsid passes through the cortical actin with the help of an actin-dependent motor protein, such as a member of the myosin family implicated in organellar trafficking (Lin, et al., 1997, Pollard and Ostap, 1996, Welch, et al., 1997). Interestingly, cytochalasin B, which interferes with filamentous actin, has been reported to inhibit HSV-1 entry into neurites of rat dorsal root ganglia (Lycke, et al., 1984).

2.5. Hepadnaviridae: Hepatitis B virus (HBV)

Hepatitis B virus (HBV) is the major cause of human hepatitis (for reviews, see Ganem, 1996, Schaefer and Gerlich, 1995). Among the three virus related particles existing in an infected individual, only the enveloped 8 DNA containing particle, the so called “Dane” particle named after its discoverer, confers infectivity. The genome of “Dane” particles is a relaxed circular, partially duplexed DNA molecule of about 3.2 kb. It encodes surface proteins, the nucleocapsid forming C protein, the DNA-associated polymerase and potentially other polypetides of unknown functions.

Despite a great deal of efforts, little is known about the early steps of infection. This is partly due to difficulties of efficiently reproducing early steps in cultured cells. Most information about the early steps of infection comes from studies of (dHBV). The cell biology of this virus is in many respects similar to that of human hepatitis B virus (hHBV). dHBV efficiently enters freshly isolated primary duck hepatocytes. It is thought that the amino-terminal domain, pre-S1, of the large lipid-anchored surface glycoprotein L contains a receptor binding determinant (Klingmuller and Schaller, 1993, Qiao, et al., 1994). The subsequent fusion between the viral and cellular membrane apparently occurs at neutral pH (Rigg and Schaller, 1992), although the underlying mechanism is unknown (Offensperger, et al., 1991). The nucleocapsid is then released into the cytoplasm and transported to the nucleus, where the genomic DNA is repaired into a closed circular DNA molecule for ensuing RNA polymerase II transcription and packaging of complete RNA into newly assembled nucleocapsids in the cytoplasm. By reverse transcription, RNA within the cytoplasmic capsids is then copied into a double-stranded DNA molecule. Virus particles bud through internal membranes and are finally released by vesicular transport into the extracellular medium.

2.6. Baculoviridae: Nuclear polyhedrosis virus (NPV) and Granulosis virus (GV)

Baculoviruses are a diverse group of large DNA viruses characterized by a circular double stranded DNA genome, which is packaged into a rod-shaped (latin “baculum”) capsid and enveloped with a lipid membrane (for recent review, see Miller, 1996). They occur in two genera, nuclear polyhedrosis viruses (NPVs) and granulosis viruses (GVs). They replicate and produce progeny capsids in the nucleus of insect cells. They are often named according to their host. For example, the commonly used baculovirus vector for protein expression in insect cells is the alfalfa looper nuclear polyhedrosis virus (AcNPV). During their cycle, baculoviruses occur in two forms, an occluded form and an open form. Occluded complete virions are embedded in a characteristic proteinacious matrix in the nucleus of the infected cell. After cell in the infected insect, these inclusions are dissolved in the acidic environment of the midgut lumen and released viruses can be taken up by another epithelial cell. Alternatively, nucleocapsids can somehow exit the nucleus, bud across the plasma membrane and thus aquire a lipid membrane in a nonlytic process. This process may be mechanistically similar to virus budding in mammalian cells, such as HSV-1 budding from human epitheloid cells (see above). Extracellular virus then enters non-infected cells by receptor-mediated endocytosis involving the viral surface protein gp64, passes through acidic endosomes and releases the nucleocapsid after membrane fusion into the cytoplasm (Volkman and Goldsmith, 1985, Volkman and Goldsmith, 1988). Whether a less efficient pH-independent fusion at the plasma membrane can be an alternative infectious pathway is currently uncertain. Likewise, a cell surface receptor and additional stimuli for membrane penetration are not known.

3. NUCLEAR ENVELOPE TARGETING 9 While penetration of cellular membranes can be accomplished by viral capsid proteins without the active assistance of host proteins (for review of enveloped virus fusion mechanisms see White, 1990), the movement of large virus particles through the cytoplasm requires cellular activities. The 90 nm large adenovirus particle, for example, is not transported to the nuclear envelope when cell metabolism is slowed down (Greber and Kasamatsu, 1996). Likewise, passive diffusion of synthetic beads larger than 40 to 50 nm in the cytoplasm does not occur due to steric hindrance and high viscosity, even under physiological conditions (Luby-Phelps, 1994).

Currently, there is evidence for two different modes of virus transport through the cytoplasm, - dependent movement and actin-driven motility. In principle, both mechanisms could be operating on either naked cytoplasmic capsids or lipid-enveloped capsids, such as incoming endosomal or newly synthesized viruses (Cudmore, et al., 1997, Greber and Kasamatsu, 1996). Cell-assisted motility thus allows viruses to rapidly proceed to the intracellular site of replication or find a selective path for egress.

3.1. Herpes simplex virus (HSV-1)

Targeting of HSV-1 to the nucleus is one of the best studied intracellular movements of an incoming virus particle, since this process is important for cell to cell spread of infectivity within the neuronal system of both, animals and . HSV infects epithelial cells of the mucosa and can be latent or lytic in a variety of neurons in the periphery and the central nervous system (Roizman and Sears, 1996). It is thought to enter neuronal cells at the presynatic membrane. Transport to the then occurs via retrograde movement of the capsid along (Bosem, et al., 1990, Kristensson, et al., 1986, Lycke, et al., 1984, Topp, et al., 1994). Electron microscopy and indirect immunofluorescence studies in fibroblastic Vero cells have recently demonstrated that incoming HSV-1 capsids are closley associated with microtubules (Sodeik, et al., 1997). In these studies, cytoplasmic dynein has been found in the vicinty of HSV-1 capsids in close proximity to microtubules. In many cells, dynein occurs in the dynactin complex as a large protein assembly of about 1.2 MDa implicated in transporting cellular vesicles along microtubules (Rickard and Kreis, 1996, Schroer, 1994, Vallee and Sheetz, 1996). When most of the microtubules in Vero cells were depolymerized with nocodazole or cholchicine, capsid targeting to the nucleus was slowed down, but not completely blocked. It is possible that those viruses, which attached to the plasma membrane proximal to the nucleus, arrived at the nuclear envelope independently of microtubules. Alternatively, it is conceivable that nocodazole-resistant microtubules still served as tracks for nuclear targeting. When microtubules were collapsed into paracristalline arrays around the nucleus by treating cells with vincristine, nuclear targeting of HSV was inhibited similar to the nocodazole results. The data would thus suggest that the retrograde-directed motor protein dynein could carry HSV-1 along microtubules. Which of the capsid or tegument proteins is contacting the dynein/dynactin complex and if dynein is sufficient to generate retrograde HSV movement remains to be determined.

3.2. Nuclear polyhedrosis virus (NPV) 10 Morphological studies on Autographa californica NPV entry into insect cells have suggested that cytoplasmic viral capsids induced the formation of actin cables (Charlton and Volkman, 1993). Actin polymerization did not require protein synthesis and was only observed after the capsid penetrated the endosomal membrane. Often times, virion capsids appeared to be located at the tips of actin filaments. Two virion derived proteins have been implicated so far in the regulation of actin polymerization, an actin-binding and perhaps actin-polymerizing protein and an actin degrading viral protease (Lanier, et al., 1996). Whether capsid motility within the cytoplasm is entirely derived from polymerizing actin similar to the mobile bacteria Shigella and Listeria (Pollard, 1995), or whether microtubules are also involved in driving NPV through the cytoplasm will be important to determine. Interestingly, treatment of insect cells with the microtubule- stabilizing agents taxol was reported to delay virus replication, but colchicine had no effect (Volkman and Zaal, 1990).

3.3. Other viruses

Much less is known about nuclear targeting of other DNA viruses. Purified Ad-5 has been shown to associate in vitro with polymerized microtubules, which contained high molecular weight microtubule- associated proteins (Luftig and Weihing, 1975, Weatherbee, et al., 1977). Electron microscopy in human epithelial cells has visualized incoming Ad-2 and Ad-5 particles in association with microtubules (Dales and Chardonnet, 1973, Miles, et al., 1980). Often times, distinctive globular projections of 10 to 20 nm were seen at the vertices of the capsids in these images. Whether these projections represent a physiological motor or a cytoplasmic linker protein is unknown.

The mechanisms, by which SV40 is transported through the cytoplasm are controversial. Electron microscopy and measurement of early in CV-1 cells suggested that disruption of microtubules with a variety of inhibitors blocked virus transport to the nucleus (Shimura, et al., 1987). However, when purified SV40 was microinjected into fibroblastic cells, disruption of microtubules by nocodazole treatment had little effect on capsid protein targeting to the nucleus (for review, see Greber and Kasamatsu, 1996). It is possible that SV40 naturally enters cells by passing through endosomes and that transport of virions within endosomes towards the nucleus requires microtubules or, alternatively, that nocodazole resistant microtubules were mediating nuclear targeting. Interestingly, receptor-mediated uptake of the SV40-related bovine papilloma virus into primary hepatocytes required functional dynamic microtubules and actin microfilaments as suggested by taxol and cytochalasin B inhibition experiments (Muller, et al., 1995, Zhou, et al., 1995).

4. NUCLEAR IMPORT: UNCOATING, DOCKING, TRANSLOCATION

Nuclear targeting of a virus capsid is generally not sufficient for import of the genomic DNA into the nucleoplasm. The capsid around the DNA must be broken up, the DNA released and translocated across the nuclear envelope. All nuclear DNA viruses studied so far, appear to be using nuclear import pathways that converge at nuclear pore complexes (NPCs). 11 NPCs are large molecular assemblies of about 124 MDa embedded in the double lipid bilayer of the nuclear envelope (Reichelt, et al., 1990). In an average HeLa cell nucleus, about 3000 NPCs are evenly distributed over approximately 300 µm2 (Maul, 1977). The NPC is an eight fold symmetrical structure with peripheral nuclear and cytoplasmic ring elements and a central plaque-spokes complex (for review, see Davis, 1995). The plaque-spokes complex harbours two types of channels, eight peripherally located aqueous channels for passive diffusion of small proteins and solutes (Hinshaw, et al., 1992), and a central channel harboring a so called transporter complex. The peripheral diffusional channels are thought to allow passage of small molecules up to about 50 kDa. The maximal NPC diameter is thought to be located in the center of the NPC. It functions in an energy-dependent manner and can - in exponentially growing cells - accommodate colloidal gold particles as large as 27 nm (Feldherr and Akin, 1991). Filaments radiating from the nuclear rings inward are forming a basket-like structure with a terminal ring element. Cytoplasmic filaments etending from the cytoplasmic rings outward are somewhat thicker in appearance than the nuclear filaments and are thought to provide a binding site for incoming cargo. NPCs are anchored in the membrane by the pore membrane domain, a specialized subdomain of the nuclear envelope (NE), which lines the pore and links the outer and inner nuclear membranes (for reviews see Davis, 1995, Panté and Aebi, 1996). The pore membrane domain is an integral part of the scaffolding framework and contains transmembrane proteins, such as the major NPC glycoprotein gp210 or Pom121 (for review see Gerace and Foisner, 1994).

Nuclear import of most cellular proteins depends on nuclear localization signals (NLSs) and signal decoding machineries (for different models, see Duverger, et al., 1995, Görlich and Mattaj, 1996, Melchior and Gerace, 1995, Panté and Aebi, 1996, Pollard, et al., 1996, Rexach and Blobel, 1995). Classical NLSs have been defined by the SV40 large T antigen sequence PKKKRKV and the bipartite nucleoplasmin motif KRPAATKKAGQAKKK (capital letters representing single amino acid code: P=Proline, K=Lysine; R=Arginine; V=Valine; A=Alanine; T=Threonine; G=Glycine; Q=Glutamine) (Dingwall and Laskey, 1991, Kalderon, et al., 1984). These sequences are recognized in the cytosol by karyopherin/importin alpha. The alpha receptor then docks with the cargo to the karyopherin/importin beta at the cytoplasmic filaments of the NPC. O-linked glycoproteins of the NPC are directly or indirectly involved in this docking process (Adam and Adam, 1994). Recently, additional NLSs have emerged, such as the glycine rich M9 sequence first identified in the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 (Siomi and Dreyfuss, 1995). A cytosolic M9 receptor, termed transportin, has been identified in mammalian cells, and also in yeast cells, termed Kar104p (Aitchison, et al., 1996, Pollard, et al., 1996). Translocation of the signal bearing molecule then occurs via the central transporter region of the NPC. This step is GTP- and temperature-dependent and requires the small GTP-binding protein Ran/TC4, and other cytosolic factors.

Lumenal factors of the NE also have a role in maintaining the functionality of the NPC. The NE cisternae are major intracellular calcium stores (for review see Pozzan, et al., 1994). Depletion of calcium from internal stores by calcium ionophores or the calcium ATPase inhibitor thapsigargin blocked passive diffusion and signal-mediated transport across the nuclear envelope in somatic mammalian cells and frog oocytes (Greber and Gerace, 1995, Stehno-Bittel, et al., 1995, Sweitzer and Hanover, 1996). The inhibition is most likely due to a sterical block of the central and peripheral NPC transport channels, as suggested by atomic force microscopy in Xenopus nuclear envelopes (Perez-Terzic, et al., 1996). Such a transport block can be 12 bypassed by elevating the cytosolic calcium concentrations. Under these conditions, nuclear import of classical NLS-bearing proteins became calmodulin-dependent and no longer required Ran-TC4, but still operated through NPCs in an ATP dependent manner (Sweitzer and Hanover, 1996).

4.1. Adenovirus type 2 (Ad-2)

A number of electron microscopy studies indicated that numerous incoming adenovirus capsids attach to the cytoplasmic side of NPCs, but never was a capsid found within the nucleus (for reviews, see Dales, 1973, Greber and Kasamatsu, 1996, Horwitz, 1990). Quantitative electron microscopy of thin sectioned epitheloid HeLa cells indicated that about 29% of the capsids, which were located within 2 capsid diameters at the NE, were attached to NPCs (Greber, et al., 1996). This number is probably even underestimating the real extent of capsid association with NPCs, since it is possible that thin sections crossed through a capsid at an NPC, but spared the underlying NPC. Interestingly, only partially disassembled capsids, but not so called “empty” capsids were found in association with NPCs,. It is possible that the weakly stained “empty” particles observed at the NE in earlier studies represented partially disassembled capsids (Dales, 1973).

The first adenovirus capsids arrive at the nuclear envelope of HeLa cells about 20 min post infection, depending on the distance between the portal of entry at the plasma membrane and the nuclear envelope (NE). At 60 min post infection, the majority of incoming capsids were found at the NE as indicated by fluorescently labeled virions (Greber, et al., 1997). Cytoplasmic capsids contained fewer proteins than the extracellular particles and mainly consisted of the major hexon protein (present in 720 copies per intact virion) and 15 to 40% of the penton base (about 10 to 30 copies) (Greber, et al., 1993). During entry capsid proteins were shed in a stepwise process. The first protein released was the primary cell surface attachment factor, fiber, followed by the capsid-stabilizing proteins IIIa and VIII. Protein IX, a cementing factor keeping the hexons together was removed between 30 and 60 min after entry. Removal of protein IX coincided with the onset of capsid disassembly, but was not sufficient for disassembly. Capsid disassembly also required the activity of a virus-resident cysteine protease, p23 (Cotten and Weber, 1995, Greber, et al., 1996, Weber, 1995). If p23 was inactivated either chemically or by mutagensis, capsid disassembly was inhibited and cell infection impaired. During virus entry, p23 gets reactivated by at least two different triggers, virus attachment to cell surface integrins and reducing intracellular milieu. Activated p23 degraded an internal capsid protein, VI, and thus enabled the detachment of the viral DNA from the shell. The loss of stabilizing proteins including protein VI degradation, was still not sufficient for capsid disassembly and DNA import into the nucleus. The incoming capsid must also associate with the NPC thus triggering DNA release (Greber, et al., 1997). This was demonstrated by specific inhibitors of O-linked glycoproteins of the NPC, the RL1 antibody and wheat germ agglutinin. In living cells, these inhibitors blocked virus attachment to the NE and also capsid disassembly. It can be speculated that some surface-exposed determinants of either hexon or penton base are involved in attaching the capsid to the NPC. Protein IX is less likely to be involved in attachment and triggering disassembly, since it does not appear to be located at the capsid surface (Stewart, et al., 1993). Whether capsid disassembly is triggered by an integral NPC component directly or together with a peripherally attached cytosolic component is not known yet. 13 After capsid disassembly, DNA and associated protein VII were imported into the nucleus through NPCs. This was demonstrated by immunocytochemistry and sensitive fluorescent in situ hybridization protocols combined with confocal laser scanning microscopy (Greber, et al., 1997). NPC function was blocked by depletion of calcium from internal stores using ionophores or a calcium ATPase inhibitor, thapsigargin. Calcium store depletion did not affect virus targeting to NPCs or capsid disassembly, but excluded incoming viral DNA from the nuclear interior. This data directly implies that functional NPCs are required for nuclear import of incoming viral genome through NPCs. A classical NLS of the terminal protein p55, which is covalently attached to the incoming viral DNA, has been identified (Zhao and Padmanabhan, 1988). Whether this signal is involved in threading the linear DNA through the NPC is not known. p55 is, however, required for directing the viral DNA to specific sites of the nuclear matrix and for initiation of viral transcription (for review, see Shenk, 1996).

4.2. Simian virus 40 (SV40)

In contrast to adenoviruses, SV40 executes a much less efficient entry program in cultured fibroblastic cell lines. Only one out of 200 particles is able to produce progeny in these in vitro systems (Cole, 1996). It is not entirely clear what the limiting step(s) is. Possibly, many of the isolated particles are damaged and thus not infectious. When SV40 particles were microinjected into the cytoplasm or the nucleus, every 10th or even every single particle, respectively, lead to the expression of large T antigen (Diacumakos and Gershey, 1977, Gersey and Diacumakos, 1978). This data would suggest that damaged particles were retained at the earliest barriers, the plasma membrane and the cytoplasm. Indeed, cytoplasmically injected chromosal SV40 DNA devoid of capsid proteins was not imported into the nucleus, although it was complexed with NLS-bearing histones (Nakanishi, et al., 1996). Conversely, microinjected particles lacking DNA readily entered the nucleus suggesting that the capsid contained some nuclear targeting information.

Mutagenesis experiments on individual capsid proteins have identified classical NLSs in the structural proteins Vp1, Vp2 and Vp3 (Ishii, et al., 1996). The NLS of Vp1 is a bipartite signal comprising the amino- terminal 19 residues, in which two clusters of basic residues are independently important for nuclear localization activity. In the intact virion, there are 360 Vp1 NLSs hidden in the capsid near the minichromosome (Liddington, et al., 1991). The classical NLS of Vp2 and Vp3 is located close to the carboxyl end of the both proteins, but the precise location of the Vp2 and Vp3-NLS in the interior of the virion is not known (Ishii, et al., 1996).

SV40 particles injected into the cytoplasm accessed the nucleus via NPCs (Clever, et al., 1991, Yamada and Kasamatsu, 1993). Most likely, this route of entry represents the natural infectious pathway, since cytoplasmically administered antibodies against capsid proteins or NPC inhibitors blocked the expression of T antigen (Nakanishi, et al., 1996). In electron microscopy studies, the presence of electron dense virion-like particles in the nucleus has been noted (Yamada and Kasamatsu, 1993). Whether these particles truely represent incoming capsids and contain the viral genome is unknown. In addition, it is not clear how the 50 nm large SV40 capsid is transported through the NPC channel, which can maximally accommodate gold particles of 27 nm diameter (Davis, 1995). One possibility is that conformational changes alter the shape of 14 the cytoplasmic capsids, as suggested by in vitro uncoating experiments (Colomar, et al., 1993, Frost and Bourgaux, 1975). It is possible that such changes expose a latent NLS on some SV40 capsid protein mediating capsid association with the NPC and finally nuclear import. In what kind of a configuration the viral DNA enters the nucleus is, however, not known. It is possible that subsequent to nuclear import some additional disassembly steps are necessary for viral transcription, as suggested by nuclear microinjections of antibodies against the Vp3 capsid protein (Nakanishi, et al., 1996). It is, however, uncertain if all papova viruses are taken into the nucleus for disassembly or if disassembly could mainly occur in the cytoplasm. Bovine papilloma virus capsid proteins, for example, are imported into the nucleus via classical NLSs, but incoming capsids have not been observed in the nucleoplasm supporting the notion that the virus capsid dissociated before reaching the nucleoplasm (Zhou, et al., 1995).

4.3. Herpes simplex virus type 1 (HSV-1)

As with adenoviruses, incoming HSV-1 capsids have been seen in large numbers at the nuclear envelope in association with NPCs (for review, see Roizman and Sears, 1996). Interestingly, also so called “empty”, electron luscent capsids were frequently seen at NPCs. It is, however, not known if these particles are a true intermediates of the DNA release process or if they still contain DNA, perhaps in a different configuration than the native capsids. Studies with the HSV-1 mutants tsB7 and 50B indicated that one or several viral functions are involved in DNA release from the capsids (Batterson, et al., 1983, Tognon, et al., 1981), analogous to the adenovirus protease p23, required for capsid dissociation and DNA release (Greber, et al., 1996). At the restrictive temperature, the tsB7 capsids at the nuclear envelope have an electron dense appearance, but become electron luscent after lowering the temperature suggesting that some changes in the capsids have occurred (Batterson, et al., 1983). Unfortunately, we do not know exactly which capsid components are affected in the tsB7 or B50 mutants. Whether the DNA is released and then transported through the NPC also remains to be determined.

4.4. Hepatitis B virus (HBV)

The viral capsid, which comprises DNA, complexed core protein, and polymerase, is released into the cytoplasm, and somehow transported to the nuclear envelope. Within the nucleus, genome replication occurs via an RNA polymerase II-generated RNA intermediate. (Ganem, 1996). Thus, DNA must be imported into the nucleus to initiate the infectious cycle.

Mutagenesis studies on the core protein have identified a classical NLS (Eckhardt, et al., 1991). Since it is thought that this NLS could overlap with the nucleic acid binding domain, it may not be functional on a nucleocapsid (for references, see Kann, et al., 1997). In vitro binding studies have shown that phosphorylation of the core protein by a protein kinase C (PKC) activity supresses RNA binding (Kann and Gerlich, 1994). Since a PKC-like activity was found in isolated human HBV (Gerlich, et al., 1982, Kann and Gerlich, 1994), it is possible that core phosphorylation exposes an NLS on the surface of the capsids. Such an NLS then might mediate capsid association with the nuclear envelope. A nuclear membrane association of incoming capsid-containing DNA was suggested by biochemical fractionation experiments using the hepatic 15 HepG2 cells (Qiao, et al., 1994). It is, however, not known if capsid association with the nuclear membrane was entirely due to attachment at NPCs or if the nuclear membrane interaction was of indirect nature in these experiments. In addition, nothing is known about the configuration of the capsid associated with the nuclear envelope.

How nuclear import of the DNA actually occurs is still under investigation. HBV DNA-polymerase complex isolated from woodchock liver was apparently sufficient for nuclear import in permeabilized cultured cells as assayed by semi-quantitative polymerase chain reaction (Kann, et al., 1997). Nuclear import of DNA- polymerase complex was ATP- and cytosol-dependent and in this respect, similar to signal-mediated nuclear protein import. These data would suggest that capsid proteins were not essential for DNA translocation. Redundancy in the involved signals is, however, still possible, since small amounts of capsid protein could have been present in the isolated polymerase-DNA complexes. It will be interesting to determine the signals and mechanisms of DNA translocation through the NPC.

4.5. Nuclear polyhedrosis virus (NPV) and Granulosis virus (GV)

As with the other viruses described here, association of the nucleocapsid of baculoviruses with the NPC has been described by electron microscopy without many functional biochemical or genetic studies (Miller, 1996, Summers, 1971). Even though so called “empty” capsids were found attached to NPCs, it is not clear if these capsids represented true intermediates in the release process of the DNA or whether they were not related to the actual import process at all. Based on the dimensions of the capsids (approximately 50 nm), it can be expected that the rod-like nucleocapsids are dissociated at least to some extent before the DNA transits across the NE.

5. CONCLUSIONS

Most DNA viruses replicate their genomes in the cell nucleus. The mechanisms of DNA delivery to the nucleus are different for different virus families and may differ even among members within a family. Common steps for nuclear delivery include virus attachment to a suitable cell surface receptor, penetration of the plasma or endosomal membrane, targeting to the nuclear envelope and finally translocation across the nuclear pore complex (see Tables 1 and 2). As the virus passes from one step to the next, its structure is altered. Completion of one entry step is often necessary to facilitate a correct excecution of a subsequent step.

If we want to figure out, how DNA viruses manage to overcome the barriers separating the nucleus from the extracellular milieu we will have to understand the interdependent virus entry steps in correlation with a detailed structural map of the virus particle. We have to extend the large body of existing literature on morphological aspects of virus entry by functional cell biological and genetic assays, and directly measure the efficiency of a particular entry step. With the advent of sensitive fluorescent DNA in situ hybridization protocols detecting single copies of incoming DNA genomes, as described for adenovirus, some of the tools are now readily available for addressing these burning questions. If we understand the mechanisms 16 regulating intracellular trafficking of viral genomes, we will not only expand our knowledge of normal and pathological cellular function, but will also know better how to use viruses for protocols in molecular medicine.

ACKNOWLEDGEMENT

Work from the author’s laboratory was supported by the Kanton of Zurich and a grant from the Swiss National Science Foundation. 17

6. TABLES

Table 1: Animal DNA viruses replicating in the nucleus

Virus Familiy Genome Genome Nucleocapsid Prototypic Virus Host (type) (kb) (nm) Non-enveloped Adenoviridae l-ds 34 90 Adenovirus type 2 or 5 (Ad- Human 2, Ad-5) Papovaviridae c-ds 5-8 40-50 Papilloma virus Rabbit Polyoma virus Mouse Simian virus 40 (SV40) Monkey Parvoviridae l-ss 5 18-26 Adeno-associated virus Human (AAV) Parvovirus B19 Human Enveloped Baculoviridae c-ds 90-200 ca 40 x 400 Nuclear polyhedrosis virus Insects (e.g. (NPV) alfalfa looper) Hepadnaviridae c-pds 3.2 25 Hepatitis B virus (HBV) Human Duck Herpesviridae l-ds 150 100 Herpes simplex virus type 1 Human (HSV-1) 18

Table 2: Virus-cell interactions in the delivery of DNA to the nucleus

Event Cellular mediator Virus References

Surface attachment Ig domain (CAR, MHC I) Ad-2, Ad-5 (Bergelson, et al., 1997, Hong, et al., 1997, Tomko, et al., 1997) Sialic acid Polyoma virus (Stehle, et al., 1994) MHC class I SV40 (Breau, et al., 1992) Glycolipid Parvovirus B19 (Brown, et al., 1993, Chipman, et al., 1996) N-linked glycans MMV (Mizukami, et al., 1996) Heparan sulfate and HSV-1 (Brunetti, et al., 1995, Montgomery, additional factors et al., 1996, Roizman and Sears, 1996, Spear, 1993, Whitbeck, et al., 1997) Factor X binding to gp64 AcNPV (Volkman and Goldsmith, 1988)

Plasma membrane fusion ? HSV-1 (Roizman and Sears, 1996) (pH independent) ? dHBV (Ganem, 1996) ? AcNPV (Miller, 1996)

Endocytosis pH dependent penetration Integrin αv β5 and ? Ad-2 (Greber, et al., 1993, Nemerow, et al., 1994, Pastan, et al., 1986, Varga, et al., 1991) pH independent penetration ? AcNPV (Miller, 1996) Caveolae SV40 (Anderson, et al., 1996, Stang, et al., 1997)

Nuclear targeting Microtubules HSV-1 (Lycke, et al., 1984, Penfold, et al., 1994, Sodeik, et al., 1997, Topp, et al., 1994)

Microtubules? Ad-2 (Dales and Chardonnet, 1973, Luftig and Weihing, 1975, Miles, et al., 1980, Weatherbee, et al., 1977) Microtubules? SV40 (Greber and Kasamatsu, 1996, Shimura, et al., 1987) Microtubules/Actin Papilloma virus (Muller, et al., 1995, Zhou, et al., 1995) Actin polymerization? AcNPV (Charlton and Volkman, 1993)

Capsid disassembly NPC Ad-2 (Greber, et al., 1997) Nucleoplasmic factor(s)? SV40 (Cole, 1996)

Nuclear import NPC Ad-2 (Greber and Kasamatsu, 1996) NPC? Polyoma virus (Zhou, et al., 1995) NPC SV40 (Clever, et al., 1991, Yamada and Kasamatsu, 1993) NPC? HSV-1 (Batterson, et al., 1983, Tognon, et al., NPC? dHBV 1981) NPC? AcNPV (Kann, et al., 1997) (Miller, 1996, Summers, 1971) 19 7. REFERENCES

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