a conserved region between the heptad repeats of paramyxovirus fusion proteins is critical for...

A Conserved Region between the Heptad Repeats of Paramyxovirus Fusion ProteinsIs Critical for Proper F Protein Folding†

Amanda E. Gardner, Kimberly L. Martin, and Rebecca E. Dutch*

Department of Molecular and Cellular Biochemistry, UniVersity of Kentucky College of Medicine,Lexington, Kentucky 40536-0509

ReceiVed December 13, 2006; ReVised Manuscript ReceiVed March 9, 2007

ABSTRACT: Paramyxoviruses are a diverse family that utilizes a fusion (F) protein to enter cells via fusionof the viral lipid bilayer with a target cell membrane. Although certain regions of the F protein are knownto play critical roles in membrane fusion, the function of much of the protein remains unclear. Sequencealignment of a set of paramyxovirus F proteins and analysis utilizing Block Maker identified a region ofconserved amino acid sequence in a large domain between the heptad repeats of F1, designated CBF1. Weemployed site-directed mutagenesis to analyze the function of completely conserved residues of CBF1 inboth the simian virus 5 (SV5) and Hendra virus F proteins. The majority of CBF1 point mutants weredeficient in homotrimer formation, proteolytic processing, and transport to the cell surface. For someSV5 F mutants, proteolytic cleavage and surface expression could be restored by expression at 30°C,and varying levels of fusion promotion were observed at this temperature. In addition, the mutant SV5 FV402A displayed a hyperfusogenic phenotype at both 30 and 37°C, indicating that this mutation allowsfor efficient fusion with only an extremely small amount of cleaved, active protein. The recently publishedprefusogenic structure of PIV5/SV5 F (Yin, H. S., et al. (2006)Nature 439, 38-44) indicates that residueswithin and flanking CBF1 interact with the fusion peptide domain. Together, these data suggest that CBF1-fusion peptide interactions are critical for the initial folding of paramyxovirus F proteins from this importantviral family and can also modulate subsequent membrane fusion promotion.

The family ParamyxoViridae comprises many diversemembers, including well-known human pathogens such asmeasles, mumps, and respiratory syncytial virus (RSV) aswell as animal pathogens such as parainfluenza virus 5(PIV5/SV51), and the newly emerged, zoonotic Hendra andNipah viruses. Hendra virus first emerged in 1994 and causedan outbreak of severe respiratory illness near Brisbane,Australia. This resulted in the deaths of 14 horses and 2 outof 3 humans infected, succumbing either to respiratory illnessor to viral meningoencephalitis (1, 2). Nipah virus wasresponsible for an outbreak of viral encephalitis in Malaysiain 1998, which resulted in the deaths of 105 people and thepreventative destruction of over one million swine (3).Hendra and Nipah viruses are classified as NIAID prioritypathogens and DHHS Select Agents, and no antiviraltherapies currently exist for these fatal viruses. Hendra andNipah are grouped into theHenipaVirus genus within thefamily, due in part to the fact that although they possess88% homology to each other, they share less than 30%homology with the rest of the family (4, 5).

Although some enveloped viruses are known to promotevirus-cell or cell-cell membrane fusion via a pH-dependentmechanism within the endosomal pathway (6), the majorityof paramyxoviruses enter cells at the plasma membrane at aneutral pH (7). Recent studies on the paramyxovirus humanmetapneumovirus (HMPV) indicate that the fusion proteinof this virus displays enhanced cell-cell fusion promotionat a slightly acidic pH (8). To enter a cell, paramyxovirusesutilize two surface glycoproteins: a fusion (F) protein, whichpromotes membrane fusion, and an attachment proteindesignated H, HN, or G, which allows for initial bindingbetween the virus and the target cell.

The F protein is a type I integral membrane protein and aclass I viral fusion protein, which exists as an inactive F0

precursor protein when it is initially folded. Within theendoplasmic reticulum, F0 undergoes disulfide bond forma-tion, N-linked glycosylation, and oligomerization into ahomotrimer. At a later stage, F0 is cleaved into two disulfide-linked subunits: F1 and F2 (Figure 1). Furin, a member ofthe proprotein convertase family, is known to be the proteaseresponsible for the cleavage of many paramyxovirus Fproteins, including that of measles virus (9), human parain-fluenza virus 3 (HPIV3) (10), SV5 (11), and RSV (12). Thiscleavage occurs within the trans-Golgi network (TGN) (9).Cathepsin L, an endosomal/lysosomal protease, was recentlyidentified as the protease responsible for the cleavage of bothHendra and Nipah virus F proteins (13, 14). Data indicatethat Hendra and Nipah F are transported from the TGN tothe cell surface and subsequently internalized, cleaved, and

† This work was supported by a grant from the National Institute ofAllergy and Infectious Diseases (AI-51517) to R.E.D. A.E.G. wassupported in part by a predoctoral fellowship from the AmericanHeart Association, Ohio Valley Affiliate (0415223B).

* Corresponding author. Phone: (859) 323-1795. Fax: (859) 323-1037. E-mail: [email protected].

1 Abbreviations: SV5, simian virus 5; PIV5 (SV5), parainfluenzavirus 5; HeV, Hendra virus; F protein, fusion protein; CB, conservedblock; CBF1, conserved block in F1; HRA/HRB, heptad repeat A/B.

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then recycled to the cell surface (15, 16). All of these foldingand processing events, along with a final targeting to thecell surface, must occur in order for the F protein to befusogenically active.

In comparing paramyxovirus F proteins to other class Ifusion proteins such as HIV env/gp160 and influenza virushemagglutinin (HA), similarities and distinctions can bemade. For example, the C-terminal F1 subunit of paramyx-oviruses corresponds to the HIV gp41 subunit and toinfluenza virus HA2. This subunit contains a hydrophobicN-terminal fusion peptide domain, which is responsible forinsertion into the target cell membrane during fusion (17,18), a C-terminal transmembrane anchor, and two heptadrepeats, HRA and HRB, which are known to fold onto eachother into a six-helix bundle to drive the merger of viral andtarget cell membranes (19, 20). However, a major differencebetween the paramyxovirus F1 subunit and HIV gp41 andinfluenza virus HA2 is the length of the peptide sequencebetween the two heptad repeat regions. In HIV gp41 andinfluenza virus HA2, there are only 22 and 8 amino acids,respectively, whereas in F1 there are∼250 amino acids inthis intervening region (21). It is extremely likely that thereare important functions for this large domain within theparamyxovirus F protein, either in F protein folding, process-ing, or fusion.

Although there are certain regions within paramyxovirusfusion proteins with well-defined roles, the function of amajority of regions in the F protein is unknown. To identifyregions of conserved amino acid sequence within theseproteins, sequence alignment of the F proteins from sixparamyxovirus family members, representing four genera,was performed and analyzed for blocks of conservation. Oneconserved block was identified in the fusion peptide/heptadrepeat A region, known to play critical roles in F-mediatedmembrane fusion and to be affected by mutagenesis (22-24). Two other blocks of conservation were identified: onein the F2 subunit (Gardner, A. E., and Dutch, R. E.,unpublished work) and the other in a large intervening regionbetween the heptad repeats of F1, designated as the conservedblock in F1/CBF1 (Figure 1).

We hypothesized that CBF1 was conserved across a diverseviral family because of a critical role in folding or fusion.To address this, point mutations were introduced using site-directed mutagenesis into the completely conserved residueswithin CBF1 for the fusion proteins of the disparate SV5/

PIV5 and Hendra viruses. The amino acids that define CBF1

include residues 374-406 in SV5 F and 380-412 in theHendra virus F protein (HeV F). With one exception, pointmutations to alanine of completely conserved residues withinCBF1 resulted in F proteins that did not properly fold andoligomerize and thus were not significantly proteolyticallyprocessed or expressed on the cell surface. A number of theSV5 F mutants could be induced to fold properly whenexpressed at a reduced temperature, and variations infusogenic activity were observed, suggesting that interactionsin this region can modulate fusion if a sufficient level offolded, activated protein is available on the cell surface. Fourof the 10 conserved cysteines within paramyxovirus Fproteins are located within or just outside of CBF1 andparticipate in intramolecular disulfide bonds (25). Mutationof any of these cysteines resulted in malfolded F proteins.These data indicate a critical role for CBF1 in the initialfolding of paramyxovirus fusion proteins. A recently pub-lished structure of the prefusogenic form of the parainfluenza5 (PIV5/SV5) F protein reveals that CBF1 from one monomerflanks the hydrophobic fusion peptide domain from anothermonomer (26). Our results, therefore, indicate that interac-tions between this conserved region and the fusion peptideare critical for the folding of diverse F proteins.

EXPERIMENTAL PROCEDURES

Cell Lines and Viruses.CV-1, HeLa, Vero, BSR (providedby Karl-Klauss Conzelmann, Pettenkofer Institut), and BHK21F cells were maintained in Dulbecco’s Modified Eagle’sMedium (DMEM; Gibco Invitrogen, Carlsbad, CA) supple-mented with 10% fetal bovine serum (FBS) and 1%penicillin/streptomycin (P/S). CV-1 cells were used topropagate the recombinant vaccinia virus vTF7-3, whichexpresses T7 polymerase (27).

Plasmids. Robert Lamb (Howard Hughes Medical Insti-tute, Northwestern University, Evanston, IL) kindly providedthe pGEM2X-SV5 F and pCAGGS-SV5 F and HN plasmids.A plasmid containing the Hendra virus F gene was kindlyprovided by Lin-Fa Wang (Australian Animal Health Labo-ratory). The F gene was cloned into pGEM4Z and genesequence integrity confirmed as previously described (28).Site-directed mutagenesis was performed on pGEM2X-SV5F and pGEM4Z-HeV F using the QuikChange site-directedmutagenesis system (Stratagene, La Jolla, CA), and mutantF genes were sequenced to verify that no secondary muta-

FIGURE 1: Schematic of a paramyxovirus F protein and identification of CBF1. FP, fusion peptide; HRA/B, heptad repeat A/B; TM,transmembrane domain; residues in black, completely conserved amino acids; residues in dark gray, conserved across most members;residues in light gray, similar amino acids; underlined residues, mutations made to alanine within SV5 F and HeV F.

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tions had occurred. Specific F protein mutants were sub-cloned into the pCAGGS expression plasmid as previouslydescribed (28). Wt HeV F and G genes (also provided byLin-Fa Wang) were ligated into pCAGGS as describedpreviously (29).

Antibodies. SV5 F antipeptide antibodies to amino acids82-96, within the SV5 F2 subunit, were provided by RobertLamb. Antipeptide antibodies (Genemed Custom PeptideAntibody Service, San Francisco, CA) were generated toamino acids 526-539 within the cytoplasmic tail of theHendra virus F protein. The primary monoclonal antibodyF1a, provided by Richard Randall (University of St. An-drews, Fife, Scotland) (30), and the secondary antibodyfluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (Jackson Immunoresearch Labo-ratories, West Grove, PA) were used for flow cytometry.

Expression of wt F and F Protein Mutants, MetabolicLabeling, and Immunoprecipitation. Subconfluent monolay-ers of HeLa T4 or CV-1 cells were first infected with vTF7-3recombinant vaccinia virus (1× 107 pfu/cell in DMEM +1% bovine serum albumin (BSA)) for 60 min to allow forthe expression of T7 polymerase (27). Cells were washedwith phosphate-buffered saline (PBS) and transfected with2 µg of pGEM2X-SV5 F or pGEM4Z-HeV F wt or mutantconstructs, using Lipofectamine Plus reagents (InvitrogenLife Technologies, Carlsbad, CA) per the manufacturer’sprotocol (HeLa) or cationic liposomes (CV-1) prepared asdescribed previously (31) in Opti-MEM (Gibco Invitrogen,Carlsbad, CA) according to established laboratory protocols.Following a 3 hincubation at 37°C, cells were washed withPBS and either starved in cysteine- and methionine-deficientDMEM for 30 min and metabolically labeled for 30 min to3 h with Tran[35S] (100 µCi/mL; MP Biomedicals, Inc.,Irvine, CA) in cysteine- and methionine-deficient DMEMor labeled overnight with Tran[35S] (50µCi/mL). Cells werethen washed with PBS and either lysed in radioimmunopre-cipitation assay (RIPA) buffer supplemented with proteaseinhibitors (32) and 25 mM iodoacetamide or incubated incold (non-radioactive) DMEM for 2 to 3 h at 37°C prior tolysis. Immunoprecipitations were performed as describedpreviously (33) using SV5 F or HeV F antipeptide antibodiesand protein A-conjugated sepharose beads (Amersham/GEHealthcare Bio-Sciences, Piscataway, NJ) to pull downantibody-bound proteins. Samples were separated on a 15%polyacrylamide gel in the presence of the reducing agentdithiothreitol (DTT, Bio-Rad, Hercules, CA) to allowvisualization of both the F1 and F2 subunits and analyzed bystorage phosphorautoradiography using a Storm or Typhoonimaging system (Amersham/GE Healthcare Bio-Sciences,Piscataway, NJ).

Flow Cytometry to Detect Surface Populations. SV5 F wtor mutant proteins were expressed using the recombinantvTF7-3/pGEM expression system in HeLa T4 cells usingcationic liposomes as described above. After the 3 hincubation at 37°C, cells were shifted to 33°C overnight.Cells were prepared for flow cytometry as described previ-ously (22) with the primary monoclonal antibody F1a andthe secondary antibody fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G. The fluo-rescence intensity of 10,000 cells was measured by flowcytometry (Flow Cytometry Core Facility, University of

Kentucky and FACSCalibur, Becton Dickinson, MountainView, CA).

Biotinylation of Cell Surface Proteins. Following thetransient transfection of BSR cells with wt or mutant HeVF proteins using pGEM vector expression, cells weremetabolically labeled overnight and chased for 2 h. Cellswere placed on ice and washed three times with ice-coldPBS deficient in calcium and magnesium chloride (PBS-)at pH 8. Cells were then incubated in 1 mL of thecell-impermeable biotin analogue EZ-Link sulfo-N-hydrox-ysuccinimide-biotin (sulfo-NHS-biotin, 1 mg/mL; Pierce,Rockford, IL) in PBS- (pH 8), first rocking gently at 4°Cfor 10 min and then resting at 4°C for 25 min. Cells werethen washed six times with ice-cold PBS- (pH 8) and lysedin RIPA buffer and immunoprecipitated as described for themetabolic labeling experiments. One hundred microliters of10% SDS was added to the protein A-sepharose beads andthe samples boiled for 10 min and beads pelleted. Thesupernatant was removed and saved. Fifty microliters of 10%SDS was added to the protein A-sepharose beads and samplesboiled for another 10 min and beads pelleted. The supernatantwas removed and added to the previous supernatant. Thirtymicroliters (one-fifth) of the combined supernatant was savedfor an analysis of the total F protein in the lysed cells. Tothe remaining 120µL, 400µL of biotinylation dilution buffer(20 mM Tris (pH 8), 150 mM NaCl, 5 mM EDTA, 1%Triton X-100, and 0.2% bovine serum albumin) and 30µLof immobilized streptavidin beads (Pierce) were added, andthe samples were rocked at 4°C for 1 h. Samples werewashed with RIPA buffer as described previously. Both totaland surface portions were analyzed on 15% polyacrylamidegels under reducing conditions and visualized by using theStorm or Typhoon imaging system.

Chemical Cross-Linking. SV5 F and HeV F wt and mutantproteins were expressed in HeLa T4 cells from pGEMconstructs using vTF7-3 infection and transfection withLipofectamine Plus reagents. Following overnight incubation,cells were starved and labeled for 30 min with Tran[35S] (100µCi/mL) and then chased in cold DMEM for 1 h. Cells wereremoved from culture dishes using PBS- and 50 mM EDTA.Chemical cross-linking using the reagent 3,3′-dithiobi-(sulfosuccinimidyl propionate) (DTSSP, 1 mM; Pierce Bio-technology Inc., Rockford, IL) was performed in the presenceof 0.2% NP-40 as described previously (34). Samples werelysed and immunoprecipitated as described above, resolvedunder nonreducing conditions on a 3.5% acrylamide gel, andvisualized with the Storm system.

Syncytia Assay for Fusion. Wt SV5 F, HeV F, or F proteinmutants, in the presence or absence of the respectiveattachment proteins, were expressed via the pCAGGS systemin BHK 21F cells using the Lipofectamine Plus reagent asdescribed above. Syncytia were examined at 100× magni-fication 24-48 h post-transfection using a Nikon TS100inverted phase-contrast microscope (Nikon Inc., Garden City,NY), and pictures were taken using a Nikon Coolpix995digital camera.

Expression of Proteins at a Reduced Temperature. Wt SV5F, HeV F, or the CBF1 mutants were transfected using theLipofectamine 2000 reagent (Invitrogen Life Technologies,Carlsbad, CA) per the manufacturer’s protocol in duplicateinto BSR cells, a variant of BHK cells that stably expressesT7 polymerase and thus does not require vaccinia virus

5096 Biochemistry, Vol. 46, No. 17, 2007 Gardner et al.

infection to drive the transcription of genes using pGEMexpression (35). For the SV5 F-expressing cells, after a30 min starve and 30 min pulse with Tran[35S] (100 µCi/mL) at 37 °C, one population was shifted to 30°C for a3 h chase period, while the other cell population remainedat 37°C. The HeV F-expressing cells were also divided into30 and 37°C populations, and labeled overnight with Tran-[35S] (50µCi/mL) followed by a 2 hchase. Immunoprecipi-tation and analysis were performed as described above.

Mutants that were proteolytically processed at 30°C weresubcloned into the pCAGGS expression vector. After trans-fection of Vero cells using Lipofectamine Plus or Lipo-fectamine 2000 reagents, overnight metabolic labeling(without a chase period) and surface biotinylation wereperformed as described above.

RESULTS

Identification and Mutagenesis of CBF1. Regions withinthe F1 subunit of paramyxovirus fusion proteins with well-defined functions include the fusion peptide, transmembranedomain, and heptad repeat regions (Figure 1). However, nodefined functions have been ascribed to the 250 amino acidregion between heptad repeat A and B (HRA and HRB). Toidentify regions of amino acid conservation across theparamyxovirus family and to shed light upon domains ofunknown function in the F protein, sequences from the Fproteins of paramyxovirus family members, representing fourgenera, were aligned and analyzed. F protein amino acidsequences from the Rubulaviruses mumps virus (36) and SV5(37), the Avulavirus Newcastle Disease virus (NDV) (38),the Respirovirus Sendai virus (39), and the HenipavirusesNipah virus (4) and Hendra virus (40), were analyzed byBlock Maker, utilizing both the GIBBs and MOTIF programs(41). Three regions were identified when either the MOTIFprogram or the GIBBs sampler was used in the analysis. Thefirst identified block was in the fusion peptide/heptad repeatA region, known to play critical roles in F-mediatedmembrane fusion. The other two identified blocks ofconservation were in the undefined F2 subunit (Gardner, A.E., and Dutch, R. E., unpublished work) and the largeintervening region between the heptad repeats of F1, whichwe designated the conserved block in F1 (CBF1) (Figure 1).A protein BLAST search identified no matches to the CBF1

region outside of paramyxovirus fusion proteins, indicatingthat this conserved stretch of amino acids is unique to Fproteins. A small number of mutations in the large, undefinedregion between the heptad repeats have been shown to inducefolding defects (42, 43). However, none of these residuesare located in CBF1. Because sequence conservation wasidentified in both the fusion peptide and HRA, and both haveimportant functions in F-mediated membrane fusion, wehypothesized that CBF1 may also be conserved because ofa critical role in the initial folding or biological activity ofparamyxovirus F proteins.

Site-directed mutagenesis was utilized to mutate to alanineseven conserved residues within CBF1 in the fusion proteinsof the disparate paramyxoviruses SV5 (G374A, N379A,I394A, Q396A, V402A, I405A, and D406A) and Hendravirus (G380A, N385A, I400A, Q402A, L408A, I411A, andD412A) in order to determine the function for this conservedregion in paramyxovirus F proteins (Figure 1). The only

conserved residue that differs between the two F proteins isV402A in SV5 F and the corresponding L408A in HendraF. However, these two residues are considered highly similar,and one of these is present at this position in all paramyx-ovirus F proteins aligned.

Proteolytic Processing of the CBF1 Mutants. We firstexamined the expression and proteolytic processing of thewt and mutant F proteins to assess proper folding andintracellular transport. The SV5 F protein undergoes pro-teolytic processing by the cellular protease furin aftertransport from the ER through to the TGN, whereas HendraF protein processing is mediated by the endosomal proteasecathepsin L after endocytosis of the uncleaved precursor fromthe cell surface (13, 16). Thus, proteolytic processing of bothSV5 F and Hendra F requires exit from the ER followingprotein folding, with subsequent proper intracellular traf-ficking. Transient expression was performed using therecombinant vaccinia-T7 polymerase (vTF7-3) system. Mocktransfected cells (empty plasmid vector) were used as anegative control. Overnight labeling with Tran[35S], immu-noprecipitation, and visualization via reducing SDS-PAGEand the STORM phosphoimaging system revealed that themutant F0 precursor proteins of the SV5 F (Figure 2A) andHendra virus F mutants (Figure 2B) were only minimallycleaved into F1 and F2, compared to a much higher level ofprocessing for wild-type F, suggesting a defect in initialprotein folding and/or transport. The only exception to thisis the HeV F G380A mutant, which was processed in amanner similar to that used for wt HeV F (Figure 2B). It

FIGURE 2: Processing of the CBF1 mutants. Wt SV5 F (A) andHeV F (B) and the CBF1 mutants were expressed in HeLa T4 orCV-1 cells, respectively, by using the vTF7-3 system. Three hourspost-transfection, cells were labeled with Tran[35S] overnight andchased for 2 h. The samples were immunoprecipitated, separatedon a 15% SDS-polyacrylamide gel in the presence of a reducingagent, and analyzed using the Storm or Typhoon system.



should be noted that there is an additional glycine at aminoacid 379 in HeV F, absent in SV5 F, which could compensatefor the loss in flexibility at G380A.

Surface Expression of F Proteins. Because the majorityof the CBF1 mutations produced F proteins that were notproperly proteolytically processed, we next examined thesurface expression of these mutants to determine if they wereindeed defective in transport to the cell surface. SV5 Fproteins were transiently expressed using the vTF7-3 systemand examined by flow cytometry using a monoclonalantibody to F1 and a FITC-conjugated secondary antibody.An examination of the point mutations of the sevenconserved residues in CBF1 from SV5 F indicates that thesemutations lead to a defect in transport to the cell surfacebecause the surface expression of the mutant F proteins wascomparable to that of the negative controls (Table 1). Thisconfirms that mutations in SV5 F lead to a transport defectconsistent with defects in initial F protein folding.

Flow cytometry was not performed on Hendra virus Fproteins because of the lack of an appropriate antibody.Instead, HeV F protein surface populations were analyzedby biotinylation (Figure 3). F proteins were expressed in BSRcells. Cells were labeled overnight with Tran[35S], chasedfor 2 h in cold media, and then biotinylated as described in

Experimental Procedures. Similar to expression of the SV5CBF1 mutants, the HeV F CBF1 mutants were not expressedon the cell surface, confirming a defect in protein foldingand transport (Figure 3). As expected, the only exception tothis was the HeV F G380A mutant, which had been shownto be properly proteolytically processed (Figure 2). HeV FG380A displayed surface expression of the cleaved F1 proteinat ∼60% of wild-type F1 (representative of several experi-ments). However, the amount of F0 was also reduced,suggesting that the lowered expression on the cell surface isdue to a slight folding defect (i.e., less protein made andtransported to the surface) rather than due to inefficientendosomal trafficking, proteolytic processing by cathepsinL, and recycling to the cell surface.

Fusion Promotion by HeV F G380A. The fusogenicallyactive form of the F protein exists as a proteolytically cleavedtrimer on the cell surface. Because the majority of CBF1

mutations resulted in misfolded F proteins, which wereneither proteolytically processed nor expressed on the cellsurface, these mutants were not assayed for fusion promotionactivity. However, HeV F mutant G380A was expressed onthe cell surface in a cleaved form at levels comparable tothat of the wt protein, and this mutant was, therefore,examined for its ability to induce the formation of giantmultinucleated cells termed syncytia. To examine well-defined syncytia in the absence of vaccinia virus infection,HeV F G380A was expressed using the pCAGGS plasmid,which allows for high-level expression from a chicken actinpromoter (44). Expression assays confirmed that HeV FG380A was proteolytically processed after expression in thissystem (data not shown). Membrane fusion promoted by theHendra virus F protein requires expression of the attachmentprotein G (45). Therefore, the expression of HeV F alone,without the coexpression of HeV G, was used as a negativecontrol for syncytium formation. HeV F G380A displayedthe formation of syncytia in transfected BHK 21F cells,although they were slightly smaller and in fewer numbersthan those seen for wt HeV F (Figure 4). The reduced fusionseen for G380A was not due to a reduced surface expressionof HeV F G380A because this mutant was shown to beexpressed on the cell surface at levels slightly higher thanwild-type HeV F levels in the pCAGGS system (data notshown), suggesting a slight fusion defect for this mutant.

Oligomerization of CBF1 Mutants. Surface expression andprocessing studies suggested that mutations in CBF1 resultin a defect in protein folding. Because the oligomerizationof F0 into a homotrimer is known to occur in the endoplasmicreticulum prior to cleavage and transport to the cell surface(46), studies on the oligomerization of wild-type SV5 F and

Table 1: Surface Expression of the SV5 CBF1 Mutantsa

mutation % positiveb M.F.I.c

control (2°Ab only) 0.495 12.5mock 0.862 17.7wild-type SV5 F 100 100G374A 7.53 17.4N379A 0.977 19.1I394A 4.93 21.1Q396A 16.1 21.8V402A 16.3 23.4I405A 2.60 14.4D406A 3.27 20.2

a The results are representative of three separate experiments andare normalized to percent wild-type SV5 F set at 100%.b Percent cellspositive for F protein expression, determined by flow cytometry asdescribed in Experimental Procedures.c M.F.I., mean fluorescentintensity determined by flow cytometry.

FIGURE 3: Surface expression of biotinylated HeV F proteins. WtHeV F and the CBF1 mutants were expressed in BSR cells. Cellswere labeled overnight with Tran[35S] and chased for 2 h. Thesamples were biotinylated and immunoprecipitated as total andsurface populations as described in Experimental Procedures. Thesamples were separated on a 15% SDS-polyacrylamide gel underreducing conditions and analyzed using the Storm or Typhoonsystem.

FIGURE 4: Syncytia assay for cell-cell fusion. Wt HeV F alone, F+ G and G380A+ G were expressed via the pCAGGS vector inBHK 21 F cells. Twenty-nine hours post-transfection, pictures weretaken at 100× magnification and cells examined for the formationof giant multinucleated cells or syncytia (indicated with arrows).




Hendra virus F and mutant F proteins were performed. HeLaT4 cells transiently expressed the F proteins via the vTF7-3system and were briefly labeled with Tran[35S]. Nonidet P40(NP40) was used to permeabolize the cell membrane, andDTSSP was used to chemically cross-link F with its bindingpartners. In the absence of the cross-linker, the wt and mutantF proteins were primarily monomeric, with some dimer,trimer, and higher molecular weight forms detected. Wild-type SV5 and HeV F proteins formed discrete trimers in thepresence of the cross-linker, with SV5 F more efficientlycross-linked to a trimer than HeV F (Figure 5A and B). Theaddition of the cross-linker stabilizes interactions with Fprotein monomers within the length of the spacer arm, whichresults in a trimeric form for wild-type paramyxovirus Fproteins. However, no corresponding trimer was seen for themutant proteins except the processed, fusogenically activeHeV F G380A (Figure 5B). Instead, cross-linking of themajority of the CBF1 mutants resulted in a smear at the topof the gels, suggesting that rather than stabilizing a discretetrimeric form of F, the addition of the cross-linker resultedin the stabilization of high molecular weight aggregates.These data indicate that CBF1 is critical for proper foldingand oligomerization of the paramyxovirus fusion proteins.

Importance of CBF1 in Disulfide Bond Formation. Iwataet al. (25) demonstrated that the F protein of the paramyx-ovirus Sendai virus possesses 10 conserved cysteine residues,with 2 participating in an intermolecular disulfide bondbetween F1 and F2 and the other 8 in intramolecular disulfide

bonds within F1. Three of these eight cysteines are locatedwithin CBF1, with a fourth just C-terminal to this conservedblock. The proposed disulfide bonds between these fourcysteines were confirmed by the recently published prefu-sogenic structure of PIV5/SV5 F (26). We tested whetherthe presence of individual or all four cysteines is critical forthe proper folding of paramyxovirus F proteins. SV5 FC380S, C385S, C387S and C410S, and the quadruple mutant4CysS were constructed and expressed as described above.Removal of any of these individual cysteines disrupted animportant intramolecular disulfide bond and left an unpairedcysteine, resulting in multiple sizes of the precursor F0 on anonreducing gel. A more compact form is seen for wild-type SV5 F0 (Figure 6A). All mutations resulted in F proteinsthat did not oligomerize into homotrimers (Figure 6B) andthus were not properly processed (Figure 6C). These fourcysteines located within or near CBF1 likely participate incritical intramolecular disulfide bond formation (25, 26),important for the proper folding of paramyxovirus F proteins.

Expression of CBF1 Mutants at a Reduced Temperature.Some mutations that result in misfolded proteins at nonper-missive temperatures can permit properly folded proteinswhen expressed at reduced temperatures. For example, thetemperature-sensitive vesicular stomatitis virus (VSV) Gmutant ts045 folds improperly at a nonpermissive 39°C,yet this folding defect can be rescued when the mutant isexpressed at 32°C (47). Similarly, the E1 envelope proteinof the Sindbis virusts23 mutant undergoes proper foldingand maturation when expressed at 28°C (48). Presumably,a lower temperature slows the folding process and allowsfor proper associations with ER folding chaperones. Wetherefore assessed the proteolytic processing of the CBF1

mutants upon expression at a lowered temperature. Inaddition, to ensure that expression utilizing the vaccinia virusvTF7-3 expression system had not affected the results, thewt F and mutant proteins were transiently expressed in BSRcells, which stably express the T7 polymerase, and thus driveexpression from the T7 promoter in the absence of the virus(35). The expression of CBF1 mutants at the physiological37 °C gave extremely low to undetectable levels of pro-teolytic processing for SV5 (Figure 7A) and Hendra virus(data not shown), indicating that the defects in folding and/or trafficking to allow processing were not due to theexpression system utilized. The expression of wt SV5 F at30 °C allowed for proper proteolytic processing, though ata slightly reduced rate (Figure 7B), suggesting slowedmovement and maturation through the secretory pathway ofa properly folded protein at the reduced temperature.Expression at 30°C resulted in greatly increased processingof a number of the SV5 CBF1 mutant proteins (Figure 7B),with percent cleaved F (F2/(F2 + F0) × 100) ranging from34 wt % SV5 F cleaved for G374A up to 54 wt % F cleavedfor V402A. The numbers represent the averages from threeseparate experiments. However, while the wt HeV F proteinwas efficiently expressed and processed at the loweredtemperature, the HeV CBF1 mutants (except G380A) werestill processing defective (data not shown).

To assess the ability of the SV5 F mutants to promotefusion, mutant F proteins which were proteolytically pro-cessed at 30°C using pGEM expression (G374A, I394A,Q396A, and V402A), were expressed using the pCAGGSplasmid. To confirm the pGEM results, wt SV5 F and the

FIGURE 5: Oligomerization of the CBF1 mutants. Wt SV5 F (A)or HeV F (B) and the CBF1 mutants were expressed in HeLa T4cells by using the vTF7-3 system. Three hours post-transfection,cells were starved for 30 min, pulsed with Tran[35S] for 30 min,and chased for 1 h. Chemical cross-linking was performed on cellsuspensions in the presence of 0.2% NP-40 and 1 mM DTSSP.The samples were immunoprecipitated, analyzed on a 3.5%polyacrylamide gel in the absence of a reducing agent, andvisualized by using the Storm or Typhoon system.



CBF1 mutants were expressed in Vero cells and assayed forproteolytic processing as well as surface expression usingbiotinylation. After expression and overnight metaboliclabeling at both 37 and 30°C, cell surface expression wasexamined. All of the CBF1 mutants were expressed in thecleaved form on the cell surface at 30°C, with the amountof cleaved F protein at 30°C versus that at 37°Cincreasing∼150% for Q396A and almost 400% for V402A(Figure 7C and D).

FIGURE 6: Expression of SV5 F cysteine mutants. (A) Wt SV5 Fand the Cys mutants were expressed in HeLa T4 cells by using thevTF7-3 system. Three hours post-transfection, cells were starvedand labeled with Tran[35S] for 30 min followed by a 3 h chase.The samples were immunoprecipitated, separated on a 7.5% SDS-polyacrylamide gel in the absence of a reducing agent and analyzedas described previously. (B) Wt SV5 F and the Cys mutants wereexpressed in HeLa T4 cells by using the vTF7-3 system. Threehours post-transfection, cells were starved for 30 min, pulsed withTran[35S] for 30 min, and chased for 1 h. Chemical cross-linkingwas performed on cell suspensions in the presence of 0.2% NP-40and 1 mM DTSSP. The samples were immunoprecipitated andanalyzed on a 3.5% polyacrylamide gel in the absence of a reducingagent. (C) Wt SV5 F and the Cys mutants were expressed in CV-1cells by using the vTF7-3 system. Three hours post-transfection,cells were labeled overnight followed by a 2 hchase. The sampleswere immunoprecipitated, separated on a 15% SDS-polyacryla-mide gel in the presence reducing agent, and analyzed as describedpreviously.

FIGURE 7: Expression of the SV5 CBF1 mutants at a reducedtemperature. Wt SV5 F and the CBF1 mutants were expressed inBSR cells. After a 30 min starve and 30 min pulse period, onepopulation of SV5 F mutants remained at 37°C for a 3 hchaseperiod (A), while the other population was shifted to 30°C (B).The samples were immunoprecipitated and analyzed as describedpreviously. Mutants that were processed at 30°C were expressedin Vero cells via the pCAGGS vector and metabolically labeledovernight at either 37°C (C) or 30°C (D), and surface biotinylationand analysis were performed as described previously.




Cell-cell fusion promotion at both 37 and 30°C wasexamined using the syncytia assay. The strain of SV5 usedin these studies has been shown to promote cell-cell fusion,albeit at reduced efficiency, after expression of the F proteinin the absence of the normally required attachment proteinHN (49-53). Wt SV5 F promoted syncytium formation atboth temperatures (Figure 8A and B), although syncytia weremuch smaller, and expression of HN was required at 30°Cfor the detection of significant syncytia formation over thebackground. G374A could promote membrane fusion whencoexpressed with the attachment protein at the loweredtemperature (Figure 8B), but no syncytia formation abovethe background was observed at 37°C (data not shown),demonstrating that this mutation does not impair fusion iftransport and proteolytic processing are rescued. Both I394A

and Q396A were incapable of inducing syncytium formationat either temperature, indicating that some mutations in thisregion have a negative impact on both folding and transport,and on subsequent fusogenic activity. Interestingly, themutant SV5 F V402A was found to be hyperfusogenic atboth temperatures, forming extremely large syncytia thatincluded a vast majority of the cells in the plate, in thepresence or absence of HN (Figure 8A and B). Proteolyticprocessing for this mutant was barely detectable at 37°C(Figure 7A and 7C), and surface expression was significantlyreduced compared to that of the wt protein (Table 1),indicating that this high level of fusion is promoted by onlyan extremely small level of surface-expressed, activatedprotein. These data suggest that the overall structure of SV5F around this region may be slightly more stable and tolerant

FIGURE 8: Syncytium formation at a reduced temperature. Wt SV5 F and the CBF1 mutants were expressed alone or in the presence of theattachment protein HN in BHK 21 F cells via the pCAGGS vector. Forty-seven hours post-transfection, pictures were taken at 100×magnification and cells examined for the formation of syncytia.



to changes than HeV F, thus allowing F proteins with foldingdefects to be properly folded, processed, and trafficked tothe cell surface, particularly after expression at a reducedtemperature. These results also indicate that residues in CBF1

can modulate membrane fusion.

DISCUSSION

SV5 (PIV5) and Hendra virus are two disparate membersof the paramyxovirus family. We have shown that CBF1, aregion conserved across the family, is critical for the properfolding and oligomerization of both the SV5 and Hendravirus F protein. The conservation of amino acids throughoutthe family suggests that the critical role in protein folding isa conserved function of this region. The recently publishedprefusogenic structure of PIV5/SV5 F (26) indicates thatCBF1 interacts with the fusion peptide. As seen in Figure9A (adapted from Yin et al. (26)), CBF1 and flanking residues(purple) from one monomer of the F protein lie adjacent tothe fusion peptide (orange) from another monomer, showingpositioning that is consistent with our findings that thisdomain is critical for forming stable oligomers. The fusionpeptide region is well defined as the domain that inserts intoa target cell membrane during the initiation of fusion. Fusionpeptide domains are extremely hydrophobic (7), and theyinitially remain shielded from the solvent within the F protein(Figure 9C, hydrophobic residues in green), whereas theadjacent cleavage site remains accessible for proteolyticprocessing, as seen in the prefusogenic PIV5/SV5 F structure(26). Paramyxovirus F proteins are cleaved just N-terminalto the fusion peptide, thus making it the new N-terminus ofthe F1 ectodomain. We hypothesize that the conservation ofCBF1 is due to a critical role in burying the fusion peptidein the precursor F0 protein by its position on the exterior ofthe F protein globular head (Figure 9B). Thus, point mutationof completely conserved residues within this block resultsin misfolding of the F protein, likely due to both exposureof this extremely hydrophobic region and failure to formproper monomer-monomer contacts. The conservationacross the family indicates a similar function of this regionfor all F proteins.

CBF1 may also properly stabilize trimers to assist in theproper folding of heptad repeat B (HRB), a region involvedin membrane fusion promotion. CBF1 is immediately N-terminal to HRB (Figure 9B, purple residues and stalkregion), separated only by an HRB linker region. During theprocess of cotranslational protein folding, CBF1 would beat least partially folded during HRB translation and insertioninto the endoplasmic reticulum. If proper interactionsbetween residues within CBF1 are perturbed, interactionsbetween C-terminal residues may also be affected. In theprefusogenic structure of SV5 F, HRB monomers associateinto a trimeric coiled-coil. However, it has been shown thatHRB peptides alone in solution do not form coiled-coilstructures (54-56), suggesting that they must be expressedalong with the entire F protein ectodomain in order to obtainthe correct homotrimeric interaction. Also, mutations madeto the HRB region result in fusion rather than folding defects(57, 58), consistent with a role for HRB in fusion promotionand for CBF1 in F protein folding and oligomerization.

We have shown that the mutation of completely conservedresidues within CBF1, including four cysteines known to

participate in intramolecular disulfide bonds, results in Fproteins that do not form homotrimers. The formation ofintramolecular disulfide bonds is known to occur early duringcotranslational folding within the endoplasmic reticulum andis essential for the subsequent proper folding of manyglycoproteins (46, 59). The mutation of correspondingcysteine residues in Sendai virus F (59) and RSV F (60)resulted in F proteins that did not transit through the secretorypathway and reach the cell surface, and thus were defectivefor fusion. These results indicate that disulfide bond forma-tion plays an integral part in the critical folding function ofCBF1 and that this function is conserved across the paramyx-ovirus family.

The observed folding defect for four of the SV5 CBF1

mutants can be partially rescued by expressing these mutants

FIGURE 9: CBF1 and adjacent residues modeled within theprefusogenic form of PIV5/SV5 F. (A) Close-up view of CBF1 andinteracting residues. Purple, CBF1 and adjacent residues; orange,fusion peptide; green, F103, the first residue of the fusion peptide;yellow, disulfide bond; red, completely conserved residues in CBF1.(B) CBF1 (purple) in the context of the entire F ectodomain. (C)Potential hydrophobic interactions between CBF1 and flankingresidues (purple) and the fusion peptide domain (orange); green,hydrophobic residues. (Figure 7 was adapted from ref 26 (26) usingPyMOL 0.99 and color coded for clarification.)



at a reduced temperature, but no rescue of the HeV F mutantswas observed, suggesting that this region is more stable inSV5 F than in Hendra F. A difference in stability could benecessary because of the different mechanisms of proteolyticprocessing that SV5 F and Hendra F undergo. Furin cleavesSV5 F in the TGN and recognizes multiple basic amino acids.Cathepsin L cleaves Hendra F in the endosomal pathway,yet it has no specific consensus sequence. It is possible thata more structurally accessible cleavage site and a less rigidfold surrounding the fusion peptide domain allows for thecleavage of Hendra F by cathepsin L.

For the four SV5 F mutants whose folding could bepartially rescued at 30°C, differences in the fusogenicactivity of the cleaved, surface-expressed molecules wereobserved. SV5 F I394A and SV5 F Q396A were unable topromote syncytia formation above the background at 30°C(Figure 8B), even though cleaved protein was present onthe cell surface (Figure 7D). In contrast, fusion promotioncapability was restored at 30°C for mutant G374A, indicat-ing that this mutation affects folding but not subsequentmembrane fusion. Finally, mutation V402A produced an Fprotein that exhibited an extreme hyperfusogenic phenotypeat both 30 and 37°C, in the presence or absence of the SV5HN attachment protein (Figure 8). This mutant was mini-mally processed at 37°C, indicating that only a smallpercentage of the properly folded and processed V402Amutant is required to promote significantly high levels ofcell-cell fusion. Several other SV5 F protein mutants havebeen described with large decreases in surface expressionand processing but high levels of fusion, including themutants SV5 F L447F or L447W and SV5 F I449F andI449W within the HRB linker region (61) and SV5 F mutantI49F, which is located within the conserved block in the F2

subunit (Gardner, A. E., and Dutch, R. E., unpublished work).We hypothesize that mutations such as V402A destabilizethe F protein, leading to malfolding of much of the producedprotein but increased fusion promotion for the small percent-age that is initially folded, processed, and transported to thecell surface. V402 is located at one of the bends in the middleof the immunoglobulin-like, seven-strandedâ-sheet thatcomprises CBF1 and flanking residues. A change in side-chain volume and hydrophobic interactions (Figure 9C) maysignificantly contribute to this phenotype because the sub-stitution of valine for alanine decreases the relative hydro-phobicity of residue 402 by over 50%.

Our results demonstrate that the mutation of residueswithin CBF1 results in defects in F protein folding andtrimerization, which can be reversed for some mutants ifconditions are altered to allow folding and transport to occurmore slowly. In comparing the location of CBF1 in both theprefusogenic structure of SV5 F (26) and the knownpostfusogenic structures of NDV F (62) and HPIV3 F (63),the secondary and tertiary structures of CBF1 alone do notchange. CBF1 remains in an immunoglobulin-like shape inthe postfusogenic structure, consisting of sevenâ-strandswith disulfide bonds intact. The region as a whole isdisplaced during the transition from the metastable to stablepostfusion structure, exposing the fusion peptide to initiatemembrane fusion and allowing for the heptad repeat B linkerto swing HRB around to form the stable six-helix bundlewith HRA. This stable fold of CBF1 is consistent with itsrole in initial protein folding and suggests that observed

changes in fusion result from CBF1 interactions with thefusion peptide region, rather than the direct participation ofCBF1 in the fusion process. Those mutations that displayedincreased proteolytic processing at a reduced temperature butno cell-cell fusion may have increased the stability ofCBF1-fusion peptide interactions. In contrast, SV5 F V402Amay significantly destabilize hydrophobic interactions withinthis region and with the fusion peptide such that the fusionpeptide is triggered to undergo the transition from the pre topostfusion form much more readily than in wt SV5 F.

ACKNOWLEDGMENT

We thank Dr. Robert Lamb (HHMI, Northwestern Uni-versity) for the pGEM2X-SV5 F, pCAGGS-SV5 F, andpCAGGS-SV5 HN plasmids and SV5 F-specific antibodies,Dr. Lin-Fa Wang (Australian Animal Health Laboratory) forthe Hendra virus F and G plasmids, and Dr. Richard Randall(University of St. Andrews, Fife, Scotland) for the F1amonoclonal antibody. We are grateful to Dr. David Rodgers(University of Kentucky) for his assistance with figureconstruction. We also thank Dr. Trevor Creamer (Universityof Kentucky) and members of the Dutch lab for criticallyreviewing this manuscript.

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BI6025648


a conserved region between the heptad repeats of paramyxovirus fusion proteins is critical for...

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