Paramyxovirus Fusion: Real-Time Measurement of Parainfluenza Virus 5 Virus–Cell Fusion ⁎ Sarah A

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Virology 355 (2006) 203–212 www.elsevier.com/locate/yviro

Paramyxovirus fusion: Real-time measurement of parainfluenza virus 5 virus–cell fusion ⁎ Sarah A. Connolly a, Robert A. Lamb a,b,

a Howard Hughes Medical Institute, Northwestern University, Evanston, IL 60208-3500, USA b Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA Received 7 June 2006; returned to author for revision 30 June 2006; accepted 13 July 2006 Available online 17 August 2006

Abstract

Although cell–cell fusion assays are useful surrogate methods for studying virus fusion, differences between cell–cell and virus–cell fusion exist. To examine paramyxovirus fusion in real time, we labeled viruses with fluorescent lipid probes and monitored virus–cell fusion by fluorimetry. Two parainfluenza virus 5 (PIV5) isolates (W3A and SER) and PIV5 containing mutations within the fusion protein (F) were studied. Fusion was specific and temperature-dependent. Compared to many low pH-dependent viruses, the kinetics of PIV5 fusion was slow, approaching completion within several minutes. As predicted from cell–cell fusion assays, virus containing an F protein with an extended cytoplasmic tail (rSV5 F551) had reduced fusion compared to wild-type virus (W3A). In contrast, virus–cell fusion for SER occurred at near wild-type levels, despite the fact that this isolate exhibits a severely reduced cell–cell fusion phenotype. These results support the notion that virus–cell and cell– cell fusion have significant differences. © 2006 Elsevier Inc. All rights reserved.

Keywords: R18; Paramyxovirus membrane fusion; Parainfluenza virus 5; Simian virus 5; Pyrene; Virus entry; Flourescence

Introduction and Kolakofsky, 2001). Although PIV5 F can mediate fusion of transfected cells in the absence of HN, this fusion is enhanced The entry of enveloped viruses into cells requires fusion of by the addition of HN (Russell et al., 2001) and mutations the viral lipid bilayer with the membrane of a host cell. For the within PIV5 F can impart HN dependence to the fusion process paramyxovirus parainfluenza virus 5 (PIV5, formerly known as (Ito et al., 1997; Paterson et al., 2000). In the context of virus, SV5), fusion requires the concerted action of two glycoproteins; HN is always present with the F protein and thus HN- the receptor binding protein hemagglutinin–neuraminidase independent fusion does not occur during virus–cell fusion. (HN) and the fusion glycoprotein (F). Like other class I viral Paramyxovirus F proteins are synthesized as biologically fusion glycoproteins, F protein is expressed as a metastable inactive precursors (F0) that must be cleaved prior to fusion. protein that is triggered to undergo a protein refolding event that Cleavage of F0 results in the generation of the N-terminus of the induces membrane fusion. Our understanding of the mechanism F1 subunit that contains the hydrophobic fusion peptide (Lamb of paramyxovirus membrane fusion has increased greatly with and Kolakofsky, 2001). After triggering by HN, the fusion the determination of the pre-fusion and post-fusion F atomic peptide intercalates into a target membrane and the F protein structures (Yin et al., 2005, 2006). The mechanism of triggering undergoes a refolding process that drives membrane merger. differs for various class I fusion proteins (Jardetzky and Lamb, Both the fusion peptide and transmembrane regions of the F 2004; Lamb et al., 2006). Whereas influenza virus hemagglu- protein are flanked by heptad repeat regions (HRA and HRB tinin (HA) is triggered by the low pH environment found in the respectively), and these heptad repeats form a stable six-helix endosomal lumen, paramyxovirus F protein is triggered at a bundle upon fusion (Baker et al., 1999; Joshi et al., 1998). neutral pH by HN binding to receptors at the cell surface (Lamb Peptides synthesized to correspond to HRA and HRB sequences can inhibit fusion by trapping F at intermediate states and ⁎ Corresponding author. BMBCB, Northwestern University, 2205 Tech Drive, Hogan 2-100, Evanston, IL 60208-3500, USA. Fax: +1 847 491 2467. preventing completion of the conformational change required E-mail address: [email protected] (R.A. Lamb). for fusion (Russell et al., 2001).

Previously, it has been demonstrated, using cell–cell fusion vesicular stomatitis virus and Semliki forest virus (Pal et al., assays, that mutations within the PIV5 F protein can alter the 1988; Stegmann et al., 1993) in which pyrene is incorporated energy threshold required to trigger refolding into a fusogenic biosynthetically into virus. At high concentration, pyrene forms form. Some mutations, such as extending the length of the excimers that fluoresce at 475 nm. When present in a monomeric cytoplasmic tail, increase the energy threshold required to state, pyrene fluorescence shifts to 396 nm. Fusion of labeled trigger fusion and result in reduced cell–cell fusion (Seth et al., virus membranes with target cell membranes can be measured by 2003; Waning et al., 2004). Other mutations, such as S443P, monitoring pyrene monomer and/or excimer fluorescence as L447F, and L449F, reduce the energy threshold required to fusion progresses and the probe is diluted into the target trigger fusion and result in a hyperfusogenic phenotype membrane. As pyrene is incorporated into both leaflets of the (Paterson et al., 2000; Russell et al., 2003). viral lipid bilayer, both hemi-fusion and complete fusion of the A variety of cell–cell fusion assays have been used to study virus will contribute to the fluorescence signal. fusion mediated by the paramyxovirus F and HN proteins, Pyrene was incorporated biosynthetically into PIV5 W3A including syncytium formation assays, reporter assays including virus, and virus was purified. Pyrene-labeled virions were bound a quantitative luciferase assay, and a fluorescent dye transfer to BHK cells on ice, and virus–cell fusion was examined by assay (Paterson et al., 2000; Russell et al., 2001). Each of these injecting the virus–cell mixtures into 37 °C PBS and observing assays operates on a different time scale, and together they changes in emission spectra over time (Fig. 1A). As expected, provide the basis for our understanding of the fusion mechanism. monomer fluorescence increased and excimer fluorescence While cell–cell fusion assays are useful surrogate methods for decreased over time. To examine fusion kinetics, the increase studying virus fusion, differences between cell–cell fusion and in monomer fluorescence was monitored over 1000 s. To virus–cell fusion exist, and hence the need for direct assays of determine the total amount of pyrene present in each sample, virus–cell fusion. For example, although BHK and HeLa cells Triton X-100 was injected at a final concentration of 1% to are permissive to PIV5 infection, only BHK cells form syncytia. disrupt the membranes and fully dilute the probe. The lack of syncytia in HeLa cells is not due to an inherent PIV5 W3A virus fusion with BHK cells occurred relatively property of these cells, as infection with another paramyxovirus, slowly compared to reports describing low pH-triggered virus human parainfluenza virus 3 (hPIV3) does result in syncytium fusion of tick-borne encephalitis virus, Sindbis virus, and formation (S.A.C., unpublished observations). Semliki forest virus which occurs in seconds (Corver et al., Previous analyses of Sendai virus–cell fusion or virus– 2000; Smit et al., 1999; Waarts et al., 2002). For PIV5 W3A, liposome fusion have yielded conflicting data. In one case, fluorescence was still increasing after 15 min at 37 °C (Fig. 1B) fusion occurred over a 16 h time period (Wharton et al., 2000), and did not reach a plateau even after 30 min (data not shown). whereas in other cases fusion occurred over 10 min (Hoekstra However, the specificity of fusion was confirmed by inhibiting and Klappe, 1986) or 30 min (Ludwig et al., 2003). These fusion with addition of IgG of a neutralizing MAb specific for differences in kinetics may result in part from the different PIV5 F protein (F1a). Fusion was not blocked by addition of a techniques and fluorescent probes used. control MAb IgG (14C2) or F1a IgG denatured by boiling. To compare virus–cell fusion and cell–cell fusion, we Fusion was also inhibited by addition of C1 peptide (corre- employed two virus–cell fusion assays using fluorescent lipid sponding to the amino sequence of HRB), and this inhibition probes. By labeling virus with probes that exhibit concentra- occurred in a dose-dependent manner (Fig. 1C). The C1 peptide tion-dependent fluorescent changes, we were able to monitor prevents fusion by binding to F protein and trapping it in an virus–cell fusion in real time for two PIV5 isolates (W3A and intermediate conformation, most likely at the pre-hairpin inter- SER) and PIV5 containing F mutations. Fusion was specific mediate (Russell et al., 2001). and approached completion within several minutes. As pre- The temperature dependence of W3A fusion was examined dicted from cell–cell fusion assays, the kinetic rate and the final (Fig. 1D). Whereas very little fusion occurred at 25 °C, extent of virus–cell fusion for virus containing an F protein increasing the temperature from 37 °C to 45 °C or 53 °C in- with an extended cytoplasmic tail (rSV5 F551) were reduced creased both the rate and extent of fusion. Conversely, fusion compared to virus containing a wild-type (wt) F protein (W3A). could be prevented by inactivation of W3A virus with high In contrast, virus–cell fusion for the PIV5 isolate SER occurred temperatures (Fig. 1E). Fusion was unaffected by pre-treating at near wt levels, despite the fact that the PIV5 isolate SER the virus for 10 min at temperatures up to 53 °C prior to binding exhibits a severely reduced cell–cell fusion phenotype cells. However, pre-treating the virus at 65 °C or 75 °C com- (Bissonnette et al., 2006; Seth et al., 2003; Tong et al., 2002). pletely inactivated the virus and prevented fusion. This finding supports the notion that virus–cell and cell–cell fusion have significant differences. R18-labeled W3A virus fusion with BHK cells

Results As the optimal conditions for labeling virus with pyrene at excimer concentrations required a high MOI infection and Pyrene-labeled W3A virus fusion with BHK cells. incubation for a maximum of 48 h, the resulting purified virus preparations gave relatively low virus yields (reduced approxi- To study fusion of PIV5 with cells, we adapted a method used mately 10-fold compared to standard conditions). In addition, previously for studying fusion of enveloped viruses, e.g. although it might be expected that the monomer fluorescence S.A. Connolly, R.A. Lamb / Virology 355 (2006) 203–212 205

Fig. 1. Fusion of pyrene-labeled W3A virus with BHK cells. (A) Purified pyrene-labeled W3A was incubated with BHK cells on ice for 30 min, and samples were injected into a cuvette containing 3 mL of PBS pre-warmed to 37 °C. Fluorescence emission spectra were recorded at the initial injection of the sample and after 11 min at 37 °C. (B) Samples were treated as in panel A, and monomeric pyrene formation was monitored continuously at 396 nm for 1000 s. Maximum emission at 396 nm was determined by the addition of Triton X-100 to a final concentration of 1%. Data are expressed as a percentage of maximum emission with background (signal at time zero) subtracted. Pyrene-labeled W3A virions were treated with MAb IgGs prior to binding to cells. F1a is a neutralizing MAb specific for F, and 14C2 is an irrelevant MAb specific for the influenza virus M2 protein. As a control, F1a IgG was boiled prior to binding virus. (C) Pyrene-labeled W3A virions were incubated with the indicated concentrations of C1 peptide prior to binding to BHK cells. Fusion was monitored as in panel B. The initial peptide concentrations listed were diluted 30-fold by injection of samples into 3 mL of PBS. (D) To examine temperature dependence, fusion was monitored as in panel B, except the PBS in the cuvette was held at the indicated temperatures. (E) To determine the temperature of inactivation for W3A virions, pyrene-labeled W3A virions were incubated at the indicated temperatures for 10 min prior to binding to BHK cells on ice. Fusion was monitored as in panel B. would increase to a maximal level immediately after injecting control MAb IgG 14C2. Neuraminidase treatment of BHK cells Triton X-100, we found that the monomer signal took approx- completely prevented fusion. While previous studies raise imately 2 min to achieve a maximum. Thus, to confirm our real- concerns about spontaneous transfer of R18 to target membranes time fusion observations, virus–cell fusion was also monitored (Ohki et al., 1998), these concerns were alleviated in this study using another fluorescent probe, octadecyl rhodamine B (R18). by the lack of R18 transfer to neuraminidase-treated BHK cells R18 associates with membranes rapidly (Hoekstra et al., and the inhibition of fusion by the neutralizing MAb F1a. 1984) and can be used to label intact biological membranes, As shown using the pyrene assay, W3A fusion was permitting the use of high titer virus stocks grown under temperature-dependent (Fig. 2B). While very little fusion standard conditions. At high concentrations, R18 is self- occurred at 25 °C, both the rate and extent of fusion increased quenching and viral fusion can be measured by monitoring at higher temperatures. As the temperature was increased, the R18 dequenching as fusion with a target membrane progresses. fluorescence maximums achieved after the addition of Triton X- Purified PIV5 W3A was labeled with R18 and pelleted through 100 decreased (data not shown). This temperature-related effect a sucrose cushion. After binding virus to BHK cells on ice, on R18 was normalized by expressing the data as percentages of virus–cell fusion was examined by injecting the virus–cell these maximums. mixtures into 37 °C PBS and monitoring R18 dequenching. For each sample, Triton X-100 was injected after 1000 s to R18-labeled W3A virus fusion with RBC ghosts determine a maximum level of R18 dequenching. As found in the pyrene assay, fusion of R18-labeled W3A To investigate the slow kinetics of PIV5 W3A fusion, we (Fig. 2A) was relatively slow. The specificity of fusion was examined virus–cell fusion using RBC ghosts as another target demonstrated by inhibiting fusion with F1a IgG, but not with the cell type. Purified PIV5 W3Awas labeled with R18 and bound to 206 S.A. Connolly, R.A. Lamb / Virology 355 (2006) 203–212

low pH treatment (Fig. 3B). However, PIV5 W3A fusion progressed faster using this technique than compared to the kinetics of fusion when using BHK cells (cf. Figs. 1B, 2A, and

Fig. 2. Fusion of R18-labeled W3A virus with BHK cells. (A) R18-labeled W3A was bound to BHK cells on ice for 30 min. The samples were injected into a cuvette containing 3 mL of PBS pre-warmed to 37 °C. Fluorescence dequenching was monitored at 590 nm for 1000 s. Maximum emission at 590 nm was determined by the addition of Triton X-100 to a final concentration of 1%. Data are expressed as a percentage of maximum emission with background subtracted. As a negative control, BHK cells were treated with neuraminidase to destroy the target cell receptor sialic acid. R18-W3A was pre- incubated on ice with MAb IgG specific for F (F1a) or influenza M2 (14C2). (B) To examine the temperature dependence of R18-labeled W3A virus fusion, fusion with BHK cells was monitored as in panel A, except the PBS in the cuvette was held at the indicated temperatures.

RBC ghosts on ice. R18 dequenching was monitored as before, and Triton X-100 was injected after 200 s to determine the maximum levels of dequenching. Data were expressed as a percentage of these maximums. Unlike in the previous assays, the initial values were not subtracted because light scattering upon injection of the 100 μg of RBC ghosts resulted in large Fig. 3. Fusion of R18-labeled W3A virus with RBC ghosts. (A) R18-labeled spikes in the readings (Fig. 3). These large spikes preclude an W3A was bound to RBC ghosts on ice for 30 min. The samples were injected estimation of the initial rate of fusion. For viruses that have low- into a cuvette containing 3 mL of PBS pre-warmed to 37 °C. Readings were pH triggered fusion, virus and target membranes can be mixed at initiated 20 s prior to the sample injection, and fluorescence dequenching was 37 °C prior to injection of acid pH and it is relatively simple to monitored at 590 nm for 200 s. Maximum emission at 590 nm was determined measure the initial rates of fusion (Fig. 3B). However, for viruses by the addition of Triton X-100 to a final concentration of 1%. Data are expressed as a percentage of maximum emission at 590 nm. As a negative that fuse at neutral pH and where incubation at 37 °C is the control, RBC ghosts were treated with neuraminidase to destroy the target cell trigger for fusion, there is no alternative but to bind the virus to receptor sialic acid. R18-W3A virus was pre-incubated on ice with MAb IgG the ghosts at 4 °C and to inject the virus–ghosts into pre-warmed specific for F (F1a) or influenza M2 (14C2). (B) For comparison, R18-labeled PBS in the fluorimeter. It is not possible to hold the labeled virus influenza virus was treated as in panel A. To trigger the pH-dependent fusion of bound to RBC ghosts at 4 °C in the fluorimeter cuvette and to influenza virus, the pH of the solution was lowered to 5 at 80 s. (C) To examine temperature dependence, the virus–cell fusion assay was performed as in panel heat the sample instantaneously to precisely 37 °C at time zero. A with the PBS in the cuvette held at the indicated temperatures during fusion. PIV5 W3A virus fusion with RBC ghosts (Fig. 3A) was Fusion with neuraminidase-treated RBC ghosts was monitored as a negative relatively slow compared to influenza virus fusion induced after control. S.A. Connolly, R.A. Lamb / Virology 355 (2006) 203–212 207

3A). As in the previous assays, MAb F1a or neuraminidase treatment inhibited fusion (Fig. 3A) and fusion was found to be temperature-dependent (Fig. 3C). The temperature of fusion inactivation was also examined using this assay. As in the pyrene assay, viral fusion was inactivated after pre-treatment at 65 °C for 10 min, whereas fusion was not inactivated by pre-treatment at 53 °C (data not shown).

Mutations in F glycoprotein affect virus–cell fusion

The R18 virus–cell fusion assay was used, with BHK cells or RBC ghosts as target membranes, to compare the fusion of PIV5 W3A and another PIV5 viral isolate, SER (Fig. 4A). SER is a porcine isolate of PIV5 that grows to titers equivalent to W3A but, unlike W3A, does not form syncytia in infected cells (Bissonnette et al., 2006; Seth et al., 2003; Tong et al., 2002). The F protein of SER has nine residue differences as compared to W3A F and a 22 amino acid extension to its cytoplasmic tail (Tong et al., 2002). Previous studies suggest that this extended tail forms a structure that inhibits fusion (Seth et al., 2003; Waning et al., 2004). One of the SER mutations is a serine to proline mutation at residue 443. In previous studies, introducing an S443P mutation into W3A F caused a hyperfusogenic phenotype with cell–cell fusion occurring at 22 °C (Paterson et al., 2000). It is thought that S443P destabilizes the pre-fusion form of the F protein, rendering F activated at lowered temperature (Paterson et al., 2000; Yin et al., 2006). In addition to SER, W3A virus fusion was compared to PIV5 mutants containing F mutations in the W3A background (Fig. 4A). The 22 amino acid tail extension of SER was added to the W3A F protein, and a virus carrying this protein (rSV5 F551) was generated by reverse genetics (Waning et al., 2004). W3A virus carrying the hyperfusogenic S443P mutation was also generated (rSV5 S443P), as well as W3A virus carrying both S443P and the SER tail extension (rSV5 F551-S443P). Based on fusion studies in cells transfected with these F constructs (Waning et al., 2004), we expected to see reduced levels of fusion for SER and rSV5 F551, increased fusion for rSV5 S443P, and intermediate fusion for rSV5 F551-S443P. All of the purified virus preparations showed similar levels of purity when analyzed by silver stain (Fig. 4B and data not shown). When R18 virus–cell fusion assay was performed with the mutant viruses, using BHK cells or RBC ghosts as target cells, it was found that W3A and SER fusion levels were similar (Figs. 4C, D), whereas rSV5 F551 fusion was reduced. As found for W3A, SER and rSV5 F551 fusion was eliminated by treatment of cells with neuraminidase (Figs. 4C, D). Fusion of SER and rSV5 F551 with both BHK cells and RBC ghosts was temperature-dependent, with both the Fig. 4. Fusion of PIV5 isolate SER and mutant PIV5 virus. (A) Diagram of extent and rate of fusion increasing at elevated temperatures W3A, SER, and F551 F proteins, indicating mutated residues and elongated (Fig. 5). cytoplasmic tails as compared to W3A F. (B) Purified virus preparations (10 μg No fusion with RBC ghosts was observed for rSV5 S443P or total protein) were analyzed by SDS-PAGE on a 10% acrylamide gel followed rSV5 F551-S443P viruses (data not shown). Although the HA by silver staining. (C) Fusion of R18-labeled W3A, SER, and rSV5 F551 with BHK cells. Fusion was analyzed as in Fig. 2A. Fusion with neuraminidase- titers for these viruses were comparable to the others, their treated BHK cells was monitored as a negative control. (D) Fusion of R18- infectivity titers were reduced by ∼1 log. One explanation for labeled W3A, SER, and rSV5 F551 with RBC ghosts. Fusion was analyzed as in the lack of detectable fusion for the rSV5 S443P and rSV5 Fig. 3A. 208 S.A. Connolly, R.A. Lamb / Virology 355 (2006) 203–212

Fig. 5. Temperature dependence of SER and rSV5 F551 fusion with BHK cells. Fusion of R18-labeled SER (A, C) or rSV5 F551 (B, D) with BHK cells (A, B) or RBC ghosts (C, D) was monitored as in Fig. 2 or 3 with the PBS in the cuvette held at the indicated temperatures during fusion. Fusion with neuraminidase-treated RBC ghosts was monitored as a negative control.

F551-S443P viruses may be that their F proteins had become study were more similar to the former, occurring over several partially inactivated due to their temperature lability during the minutes. course of virus growth and thus these virion preparations were Previous studies report widely disparate rates of fusion for fusion inactive. paramyxoviruses. For example, the time required for Sendai virus fusion to approach a maximum varies from approximately Discussion 10 min (Hoekstra and Klappe, 1986) to well over 16 h (Wharton et al., 2000), a time frame that exceeds the replication time of Unlike most low pH-dependent viruses for which fusion is the virus. Nevertheless, no studies show paramyxovirus fusion triggered by acidic pH and a single protein is responsible for both occurring within seconds as has been reported for some low pH- receptor binding and membrane fusion, paramyxovirus fusion triggered viruses (Corver et al., 2000; Smit et al., 1999; Waarts occurs at neutral pH and is triggered by an interaction of HN with et al., 2002). Thus, the slow fusion rates measured for PIV5 in F protein. When influenza virus enters cells by endocytosis, the this study generally agree with previous rates measured for time course of fusion is slow, due to the requirement of Sendai virus (Hoekstra and Klappe, 1986; Ludwig et al., 2003; endocytosis and acidification of endosomes (Lakadamyali et al., Ohki et al., 1998). Both the pyrene and R18 fusion assays used 2003; Nunes-Correia et al., 2002; Stegmann et al., 1987). in this study demonstrate viral fusion within a biologically However, when fusion of influenza virus is triggered by the relevant time frame. The relatively slow fusion kinetics indicate addition of exogenous acid, fusion with target membranes that, even though the virus samples were adsorbed to the target reaches a maximum plateau within 1–5 min (Chu and Whittaker, cells at 4 °C, fusion of the virus population did not proceed 2004; Danieli et al., 1996; Mittal et al., 2002; Ramalho-Santos immediately in a synchronous wave upon heating. The slow and Pedroso De Lima, 1999; Sun and Whittaker, 2003; Takeda et kinetics may be due to a slow conversion of F proteins from the al., 2003). Other studies demonstrate that low pH-triggered pre-fusion to the post-fusion form or to a slow (or stochastic) fusion of viruses such as Sindbis virus, Semliki Forest virus, and triggering of the F proteins by HN. tick-borne encephalitis virus progresses even more rapidly, Just as a reduction in pH increases the rate of fusion of low reaching completion within seconds (Corver et al., 2000; Smit et pH-activated viruses (Mittal et al., 2002), an increase in al., 1999; Waarts et al., 2002). The kinetics of PIV5 fusion in this temperature increased both the rate and extent of fusion for the S.A. Connolly, R.A. Lamb / Virology 355 (2006) 203–212 209

PIV5 strains studied (Figs. 1, 2, 3, and 5). Prior to fusion, F membrane (Yin et al., 2005, 2006). These two residues flank protein exists in a metastable form and increasing the HRA helix 2, with residue 132 at a bend that connects helix 1 temperature reduces the energy threshold required to trigger F and helix 2 and residue 149 within a loop that connects helix 2 into a fusogenic form. The temperature dependence of PIV5 to beta strand 1 (Yin et al., 2006). Mutation of these residues fusion has been shown in cell–cell fusion assays, and specific may affect the rate at which the HRA refolds into a single long mutations in F can increase or decrease the activation energy helix. Previous work demonstrated that a mutation at residue threshold (Paterson et al., 2000; Russell et al., 2004; Russell et 132 confers HN independence to cell–cell fusion of another al., 2003). The temperature dependence of virus fusion has also PIV5 mutant (Ito et al., 2000). Residues 438 and 443 lie within been demonstrated for other viruses, including both pH- the HRB linker region at the base of the head of the pre-fusion dependent and pH-independent viruses such as influenza molecule. Mutations at these residues may affect the efficiency virus, Rous sarcoma virus, and Newcastle disease virus (Carr at which HRB refolds upon fusion to form a 6-helix bundle with et al., 1997; Gilbert et al., 1990; San Roman et al., 1999). HRA. In this regard, previous work has shown that the S443P Correspondingly, exposing PIV5 to high temperature (65 °C) in mutation in a W3A background results in a hyperfusogenic the absence of target membrane inactivated virus fusion (Fig. phenotype in cell–cell fusion assays (Paterson et al., 2000; 1E) and heat treatment was observed to inhibit fusion more Russell et al., 2003). effectively than F1a antibody or C1 peptide. Most likely, the Analysis of SER fusion in this study demonstrates a high temperature treatment prevents fusion by triggering F into a difference between cell–cell fusion and virus–cell fusion. post-fusion conformation prior to contact with the target cells. Although BHK cells infected with SER do not form syncytia Alternatively, elevated temperature may alter the virus particles and cells transfected with SER F and HN have greatly reduced in another manner to prevent fusion. syncytium formation compared to W3A (Bissonnette et al., The overall molecular mechanism of F protein-mediated 2006; Seth et al., 2003; Tong et al., 2002), SER virus fusion with fusion is thought to be the same for virus–cell fusion and cell– BHK cells or RBC ghosts occurred as rapidly as that of W3A cell fusion. Both fusion events are temperature-dependent, (Figs. 4C, D). This may explain why SER and W3A grow to inhibited by C1 peptide, and blocked by the neutralizing MAb equivalent titers (Bissonnette et al., 2006). Thus, while the F1a (Figs. 1B, C, D) (Paterson et al., 2000; Randall et al., 1987; extended cytoplasmic tail of SER F inhibits cell–cell fusion, Russell et al., 2001). The level of C1 peptide required to inhibit virus–cell fusion for SER appears to be unaffected by the long virus–cell fusion exceeded the IC50 for C1 inhibition of cell–cell tail. fusion (35 nM; Russell et al., 2001), even after taking into Cell–cell fusion differs from virus–cell fusion in that the account the fact that the peptide concentration was reduced 30- potential region of contact between membranes is larger, fold upon injection into the cuvette. This may reflect differences cytoskeletal rearrangements on both sides of the fusion reaction in the concentration of F protein or the accessibility of the C1 may affect the rate and extent of fusion, the densities of F and HN binding site in the context of a virion compared to a transfected in the membrane may be different, and the lipid composition of cell. the membranes may differ from the virion membrane. PIV5 We expected that both the SER and rSV5 F551 virus strains fusion most likely requires the cooperation of multiple trimers of would have slower kinetics of fusion than W3A as these viruses F protein (Dutch et al., 1998), and thus the concentration of F in demonstrate reduced fusion in cell–cell fusion assays (Bissonn- the membrane may affect the extent of fusion. Monitoring the ette et al., 2006; Seth et al., 2003; Tong and Compans, 1999; fusion of PIV5 viruses with cells in real time revealed both Waning et al., 2004). However, it was observed that, whereas similarities and differences between the cell–cell and virus–cell rSV5 F551 had a slower rate and extent of fusion, SER and W3A fusion reactions. The fluorescence-based virus–cell fusion fused at similar levels (Figs. 4C, D). In cell–cell fusion assays, assays developed in this study complement the various cell– the extended cytoplasmic tails of the SER and F551 F proteins cell fusion assays currently employed to examine the mechanism inhibit fusion, possibly by forming a cytoplasmic structure that of PIV5 fusion. slows the conformational changes required for fusion (Seth et al., 2003; Waning et al., 2004). In the context of virus–cell fusion, this inhibition remains for rSV5 F551, but not for SER. Materials and methods Presumably, the inhibition is overcome by eight residue changes within the SER F protein (Fig. 4A). Antibodies and C1 peptide The location of the residue changes between W3A and SER in the pre-fusion structure of the PIV5 F protein (Yin et al., Monoclonal antibody (MAb) F1a (Randall et al., 1987) 2006) suggests a scenario by which the SER mutations specific for PIV5 F and MAb 14C2 (Zebedee and Lamb, 1988) overcome the inhibition imposed by an extended cytoplasmic specific for influenza virus M2 protein were used, and their tail. With the exception of M370, the individual mutation of any IgGs were purified using AffinityPak Protein A columns one of these residues to the corresponding W3A residue was (Pierce). The concentration of IgG preparations was determined reported to restore syncytium formation in transfected cells by OD280. Polyclonal antibodies (PAb) vacF and vacHN (Seth et al., 2004). Residues 132 and 149 lie within HRA, a (Paterson et al., 1987) were raised against vaccinia-expressed region that undergoes a drastic conformational change during PIV5 F and PIV5 HN respectively. The C1 peptide contains the fusion to reposition the fusion peptide toward the target amino acid residues of the W3A F protein HRB and extended 210 S.A. Connolly, R.A. Lamb / Virology 355 (2006) 203–212 chain region and was expressed in E. coli and purified as To determine HA titer, virus preparations were serially described previously (Joshi et al., 1998). diluted in 96 well U-bottom plates with PBS+ (100 μL/well). After the addition of 100 μL/well 0.5% hematocrit chicken Cells and viruses RBC, plates were incubated at 4 °C for 2 h. The HA titer was recorded as the reciprocal of the highest dilution at which Madin-Darby bovine kidney (MDBK), CV-1, and baby agglutination occurred. hamster kidney-21F (BHK) cells were maintained as described previously (Paterson and Lamb, 1993; Russell et al., 2003). Pyrene virus–cell fusion assay Human red blood cells (RBC) were obtained with the approval of the Northwestern University Institutional Review Board. To BHK cells were released from plates using 530 μM EDTA in prepare human erythrocyte ghosts, RBCs were washed extens- PBS. Cells were pelleted and resuspended in PBS at 3×107 ively with PBS (50 mM phosphate buffer, pH 7.0, 150 mM cells/mL. Purified pyrene-labeled W3A was added to BHK cells NaCl) followed by PBS+ (PBS supplemented with 500 μM in 100 μL PBS at 3 pfu/cell for 30 min on ice. The virus–cell Mg2Cl and 900 μM CaCl2) at pH 8. To release hemoglobin, mixtures were then injected into cuvettes holding 3 mL PBS+ RBCs were washed 3 times with 5 mM Na2HPO4 at pH 8, pre-warmed to 37 °C. Fluorescence emission from 351 to pelleted at 17,000×g for 10 min, resuspended in PBS+, pelleted 510 nm was monitored using a spectrofluorimeter (AB2) with again, and resuspended in PBS+. After 30 min at 37 °C, RBC continuous scanning at 5 nm/s and 343 nm excitation. To ghosts were diluted to 2.5 mg/mL protein and stored at 4 °C. For measure fusion kinetics, pyrene monomer fluorescence was neuraminidase (NA)-treated BHK cells or RBC ghosts, NA monitored continuously with 1 s time resolution at 343 and (Sigma Aldrich) was added at 100 mU/mL for 1 h at 37 °C. 396 nm of excitation and emission, respectively. Temperature PIV5 isolates W3A and SER (kindly provided by Dr. Richard was controlled using a waterbath circulation system and W. Compans, Emory University, Atlanta, GA) and PIV5 W3A constant stirring. To determine the maximum level of monomer with an F protein containing mutations (rSV5 F551, rSV5 fluorescence (Ft), Triton X-100 was injected at 1000 s at a final S443P, and rSV5 F551-S443P) (Waning et al., 2004) were concentration of 1%. To normalize the data, readings were purified from supernatants of MDBK cells infected at 0.2 pfu/ expressed as a percentage of this maximum with initial readings cell for 4 days. To incorporate pyrene biosynthetically into subtracted, i.e. 100 (F−F0)/(Ft −F0), where F0 and F are the virions, subconfluent MDBK monolayers were infected with fluorescence at time zero and at a given time point. When noted, W3A at 4 pfu/cell. After 1.5 h at 37 °C, 1-pyrenehexadecanoic MAb or peptide was added to the virus on ice for 30 min prior to acid (15 μg/mL) (Invitrogen) was added to the medium. binding cells. Supernatants were harvested after 48 h at 37 °C. Influenza strain A/Udorn/72 was purified from the allantoic fluid of R18 virus–cell fusion assays infected embryonated chicken eggs. For purification, infected cell supernatants were clarified at 1600×g for 15 min at 4 °C. Purified virus (20 μg per sample) was diluted to 240 μg/mL Virus was pelleted at 118,000×g for 1 h, resuspended in NTE in PBS+ and mixed with the lipid probe octadecyl rhodamine (100 mM NaCl, 10 mM Tris hydrochloride pH 7.4, 1 mM B (R18; Molecular Probes) at 4 μg/mL. After 30 min at room EDTA), and banded through 20–60% sucrose gradients for 75 temperature, virus was pelleted through 20% sucrose at min at 77,000×g. Virus bands were collected, pelleted at 66,000×g for 1 h and resuspended in PBS+ (40 μL per 118,000×g for 1 h, and resuspended in TE (10 mM Tris sample). For each sample, 40 μL of virus was added to 80 μL hydrochloride pH 7.4, 1 mM EDTA). Protein concentrations of of BHK cells (∼3×107 cells/mL) or 40 μL of RBC ghosts on the purified virus stocks and RBC ghosts were determined using ice for 30 min. The virus–cell mixtures were injected into BCA Protein Assay Reagent (Pierce). cuvettes holding 3 mL PBS+ pre-warmed to 37 °C. Fluore- scence dequenching was monitored continuously as above at Immunostain plaque assay and HA titer 560 and 590 nm of excitation and emission, respectively. A 570 nm filter was placed in the emission pathway to reduce Confluent CV-1 cell monolayers were infected with serial scattering. Temperature was controlled as above. To determine dilutions of virus preparations in DMEM supplemented with the maximum level of dequenching, Triton X-100 was 1% bovine serum albumin (BSA). After 1.5 h at 37 °C, infected injected at 1000 or 200 s at a final concentration of 1%. To cells were overlaid with DMEM containing 1% agarose and 2% normalize the data, readings were expressed as a percentage FCS. Cells were incubated for 4 days at 37 °C and fixed with of this maximum. F0 was not subtracted from samples with 1% glutaraldehyde in PBS+ for 2 h. Blocking solution (3% egg RBC ghosts due to spikes in the readings. When noted, MAb albumin in PBS) was added to the cells for 30 min followed by was added to the virus on ice for 30 min prior to binding PAb sera vacF and vacHN for 1 h at a 1:100 dilution in blocking cells. solution. Monolayers were washed 4 times with PBS, and HRP- conjugated goat-anti-rabbit secondary Ab (Jackson) was added SDS-PAGE and silver stain for 1 h at a 1:500 dilution in blocking solution. Cells were washed again, and plaques were visualized by adding DAB Ten micrograms of each purified virus sample was diluted in substrate (Pierce). loading buffer (0.5 mM Tris pH 6.8, 5% SDS, 7.5% DTT, 50% S.A. Connolly, R.A. Lamb / Virology 355 (2006) 203–212 211 glycerol) and boiled for 5 min. Proteins were analyzed by SDS- Ludwig, K., Baljinnyam, B., Herrmann, A., Bottcher, C., 2003. The 3D structure PAGE using a 10% acrylamide gel. Polypeptides were of the fusion primed Sendai F-protein determined by electron cryomicro- scopy. EMBO J. 22, 3761–3771. visualized by silver staining. Mittal, A., Shangguan, T., Bentz, J., 2002. Measuring pKa of activation and pKi of inactivation for influenza hemagglutinin from kinetics of membrane Acknowledgments fusion of virions and of HA expressing cells. Biophys. J. 83, 2652–2666. Nunes-Correia, I., Eulalio, A., Nir, S., Duzgunes, N., Ramalho-Santos, J., We thank R.W. Compans for providing SER virus and R.G. Pedroso de Lima, M.C., 2002. Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability. Biochim. Biophys. Paterson,M.L.Z.Bissonnette,B.J.Chen,G.P.Leser,Y. Acta 1561, 65–75. Ohigashi, J. Panickaveetil, J. Rausch, O. Rozhok, and C.J. Ohki, S., Flanagan, T.D., Hoekstra, D., 1998. 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