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Murine Leukemia Virus R Peptide Inhibits Influenza Virus Hemagglutinin-Induced Membrane Fusion
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     Molecular Surgeon Research Center, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030

    Department of Microbiology and Immunology, Emory University, School of Medicine, Atlanta, Georgia 30322

    ABSTRACT

    The cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein is known to play an important role in regulating viral fusion activity. Upon removal of the C-terminal 16 amino acids, designated as the R peptide, the fusion activity of the Env protein is activated. To extend our understanding of the inhibitory effect of the R peptide and investigate the specificity of inhibition, we constructed chimeric influenza virus-MuLV hemagglutinin (HA) genes. The influenza virus HA protein is the best-studied membrane fusion model, and we investigated the fusion activities of the chimeric HA proteins. We compared constructs in which the coding sequence for the cytoplasmic tail of the influenza virus HA protein was replaced by that of the wild-type or mutant MuLV Env protein or in which the cytoplasmic tail sequence of the MuLV Env protein was added to the HA cytoplasmic domain. Enzyme-linked immunosorbent assays and Western blot analysis showed that all chimeric HA proteins were effectively expressed on the cell surface and cleaved by trypsin. In BHK21 cells, the wild-type HA protein had a significant ability after trypsin cleavage to induce syncytium formation at pH 5.1; however, neither the chimeric HA protein with the full-length cytoplasmic tail of MuLV Env nor the full-length HA protein followed by the R peptide showed any syncytium formation. When the R peptide was truncated or mutated, the fusion activity was partially recovered in the chimeric HA proteins. A low-pH conformational-change assay showed that similar conformational changes occurred for the wild-type and chimeric HA proteins. All chimeric HA proteins were capable of promoting hemifusion and small fusion pore formation, as shown by a dye redistribution assay. These results indicate that the R peptide of the MuLV Env protein has a sequence-dependent inhibitory effect on influenza virus HA protein-induced membrane fusion and that the inhibitory effect occurs at a late stage in fusion pore enlargement.

    INTRODUCTION

    Viral glycoprotein-induced membrane fusion can be categorized into the following two different types: low-pH-dependent and -independent membrane fusion. The influenza virus hemagglutinin (HA) is the prototype for studies of low-pH-induced membrane fusion, which occurs in the acidic endosomes during viral entry (11, 37, 42). In contrast, for many retroviruses, such as murine leukemia virus (MuLV), envelope (Env) protein-induced membrane fusion is pH independent, and virus entry occurs at the plasma membrane at neutral pH (5, 23). Like most other viruses which cause pH-independent membrane fusion, receptor binding is thought to trigger a conformational change of the Env protein and consequent fusion of the viral and cell membranes (14). The retroviral envelope proteins are synthesized as precursor proteins, which are processed by a cellular protease into two subunits, the surface subunit, containing the receptor binding domain, and the transmembrane subunit (TM). The TM subunit contains the following three basic structural elements: an extracellular domain containing the highly hydrophobic N-terminal fusion peptide, which is thought to be directly involved in the fusion process; a membrane-spanning region of 19 to 27 amino acids for anchorage to the cell membrane; and a cytoplasmic tail (8, 17, 20, 39, 45, 46).

    The Env protein of Friend murine leukemia virus has similar structural features to those of other retroviral envelope proteins (9, 41). However, the Env protein of MuLV undergoes an additional processing event during virus assembly that removes its C-terminal 16-amino-acid segment, designated the R peptide (12). It has been shown that the cleavage of the R peptide from the Env protein of MuLV is important in activating the fusion activity of the Env protein and for virus infectivity (12, 17, 30, 31, 39, 45). A point mutation (L627A) in the R peptide significantly reduced the inhibitory effect of the R peptide on MuLV Env-induced membrane fusion (46), indicating that the ability of the R peptide to suppress fusion is sequence dependent. Insertion mutations in the cytoplasmic tail of the MuLV Env protein upstream of the R peptide coding sequence, which may affect a predicted amphipathic helix formed by the connecting region, were also found to reduce the inhibitory effect of the R peptide (20).

    Although several studies have analyzed the inhibitory effect of the R peptide on membrane fusion (20, 28, 31, 46), the molecular mechanism of inhibition remains unclear. One of the limitations of studies on membrane fusion by the MuLV Env protein is the lack of specific antibodies for detecting conformational changes in the external domain. Studies with the influenza virus HA protein have shown that it undergoes a conformational change at low pHs, which exposes the buried fusion peptide and expels it to the distal tip of the protein for insertion into the target membrane, where it induces fusion between viral and cell membranes (7, 34, 43, 44). Many studies have been done to analyze the low-pH-induced conformational change of the HA external domain, and a panel of monoclonal antibodies against the HA protein's external domain have been used for such studies (4, 38). To investigate the specificity of the inhibitory effect of the MuLV R peptide on the fusion activities of viral glycoproteins and to extend studies on the role of the influenza virus HA cytoplasmic tail in the fusion process, we constructed chimeric influenza virus-MuLV HA genes in which the full-length or R peptide-truncated cytoplasmic domain of the MuLV Env protein was attached to the end of the transmembrane domain or the end of the cytoplasmic tail of the influenza virus HA protein. We also determined the effect of the L627A mutant R peptide sequence on HA proteins. We determined the expression levels of the chimeric HA proteins by enzyme-linked immunosorbent assays (ELISAs) and Western blotting, analyzed the fusion activities of these proteins by syncytium formation and dye redistribution assays, and investigated the conformational changes of the chimeric HA proteins in response to different pHs.

    MATERIALS AND METHODS

    Cells and viruses. BHK21, HeLa, MDCK, 293T, XC, and CV-1 cells were obtained from the American Type Culture Collection, Rockville, MD. They were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (GIBCO BRL). Construction of recombinant vaccinia viruses expressing the wild-type (WT) or chimeric HA proteins was carried out as described by Blasco and Moss (3), and the titers of the viruses were determined on CV-1 cells.

    Plasmid construction and site-directed mutagenesis. The Pfu polymerase and DpnI for PCR were purchased from Stratagene (La Jolla, Calif.), and other restriction endonucleases and DNA modification enzymes used for plasmid construction were purchased from Roche (Indianapolis, Ind.). The wild-type H3 influenza virus (A/Aichi/2/68) HA gene was cloned into plasmid pRB21 as described by Blasco and Moss (3).

    A gene encoding a chimeric HA protein containing the full-length cytoplasmic tail of the MuLV Env protein (HAM) was constructed by using PCR amplification. Two primers were synthesized, with the forward primer spanning the start codon of the wild-type HA gene and the reverse primer containing the coding sequence for the full-length cytoplasmic tail of MuLV Env following the end of the HA transmembrane domain. PCR was performed with the pGEM4-HA plasmid, in which the wild-type H3 HA gene was cloned into the pGEM4 plasmid, as the template, and the PCR product was purified and cloned into the pRB21 plasmid. The construction of the R peptide-truncated (HAM-13 or HAM-16) or L627A mutant (HAMRA) chimeric HA fusion gene was carried out by using a Stratagene Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Two pairs of primers were synthesized which contained the desired mutation, with 10 to 15 bases of the complementary sequence on both sides, based on the cytoplasmic tail sequence of the wild-type Friend MuLV Env protein (18). The primers for the HAM-16 construct were as follows: forward primer, 5'-GGATCTCAGTAGTCTAGGCTTTAGTCGCG-3'; and reverse primer, 5'-CGCGACTAAAGCCTAGACTACTGAGATCC-3'. The primers for HAMRA were as follows: forward primer, 5'-CCAGGCTTTAGTCGCGACTCAACAATACCACCAGC-3'; and reverse primer, 5'-GCTGGTGGTATTGTTGAGTCGCGACTAAAGCCTGG-3'. PCR amplification was carried out by using the pRB21-HAM plasmid, which contains the full-length cytoplasmic tail coding sequence of the MuLV Env protein in the pRB21 vector, as the template, with 20 cycles of 94°C for 1 min, 55°C for 1 min, and 68°C for 10 min. The PCR products were incubated with DpnI at 37°C for 1 h to digest the methylated parental supercoiled double-stranded DNA and then transformed into Escherichia coli DH5 competent cells to repair the nicks in the mutated plasmids.

    Constructs encoding the chimeric HA proteins containing the R peptide (HA+R) or the L627A mutant R peptide (HA+RA) attached at the end of the transmembrane domain of the HA protein were constructed by using PCR amplification. Two primers were synthesized, with the forward primer spanning the start codon of the wild-type HA gene and the reverse primer containing the coding sequence for the R peptide or L627A mutant R peptide sequence following the end of the HA transmembrane domain. PCRs were performed by using the pGEM4-HA plasmid as the template, and the PCR products were purified and cloned into the pRB21 plasmid. Similarly, to construct the chimeric HA protein retaining the full-length cytoplasmic tail of HA followed by the R peptide, the L627A mutant R peptide, or the upstream region of the R peptide in the MuLV Env cytoplasmic tail, pairs of primers were designed, with the forward primer spanning the start codon of the wild-type HA gene and the reverse primer containing the sequence encoding the R peptide, L627A mutant R peptide, or the upstream region of the R peptide sequence following the end of the HA cytoplasmic domain. PCRs were performed by using the pGEM4-HA plasmid as the template, and the PCR products were purified and cloned into the pRB21 plasmid as described previously. All constructs were sequenced to confirm the presence of the desired mutations and the absence of additional mutations.

    Expression and cleavage of cell surface HA proteins. Cell surface HA proteins were detected by ELISA. Briefly, HeLa cells were infected with recombinant vaccinia viruses expressing chimeric HA proteins at a multiplicity of infection (MOI) of 5 at 37°C. At 12 to 18 h postinfection, cells were incubated with different monoclonal antibodies against the HA protein, and binding to the antibodies was detected by ELISA as described previously (4). The cleavage of HA proteins was detected by Western blotting. Briefly, CV-1 cells were infected with the recombinant vaccinia viruses expressing the chimeric HA proteins. At 12 to 18 h postinfection, cells were washed and treated with tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK) trypsin (5 μg/ml) for 5 min at 37°C and lysed with lysis buffer, and proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis using a rabbit anti-HA polyclonal antibody.

    Conformational-change assay and fusion assay of wild-type and chimeric HA proteins. The assay of HA conformational changes by ELISA was described previously (4, 38). Briefly, HeLa cells cultured in a 96-well plate were infected with a recombinant vaccinia virus expressing either WT or chimeric HA at an MOI of 5. At 12 to 18 h postinfection, the cell monolayers were treated with TPCK trypsin (5 μg/ml) for 5 min at 37°C. The cells were washed once and incubated with serial low-pH buffer (20 mM HEPES, 2 mM CaCl2, 0.15 N NaCl, 20 mM citric acid buffer) at 37°C for 1 min, followed by washing with neutral buffer and fixation in 0.05% glutaraldehyde-phosphate-buffered saline (PBS). Monoclonal antibodies HC3 (1:1,000) and HC68 (1:500) in 2% bovine serum albumin-PBS were added and incubated at 37°C for 1 h, followed by incubation with horseradish peroxidase-conjugated protein A (1:1,000) in 2% bovine serum albumin-PBS at 37°C for 1 h. After the cell monolayers were washed with PBS five times, 50 μl of substrate (0.015% 3,3',5,5' tetramethylbenzidine, 0.03% H2O2 in 20 mM citric acid buffer, pH 4.5) was added, and the reaction was stopped with 50 μl of 0.1 N H2SO4. The absorbance was detected at a wavelength of 450 nm by an ELISA reader. The fusion activities of the chimeric HA-MuLV Env proteins were determined by the following procedure. BHK21 cells were infected with recombinant vaccinia viruses expressing a wild-type or mutant HA gene at an MOI of 5. Cells were treated by trypsin cleavage at 37°C for 5 min, followed by exposure of the cells to low pH for 1 min at 37°C. Syncytium formation was observed under a phase-contrast microscope. Syncytia were defined as giant cells with more than four nuclei within one single membrane. Ten fields from each sample were randomly selected, and the fusion activity was calculated based on the average value for the ratio of nuclei in syncytia to total nuclei in the same field.

    Fluorescent dye redistribution assay. We also determined fusion activity based on the redistribution of fluorescent dyes between chicken red blood cells (RBCs) or XC cells and target HeLa cells upon fusion (13), using the following procedures.

    Labeling with rhodamine R-18. Fresh chicken RBCs were incubated with 15 μl of octadecyl rhodamine B (R-18) (1 mg/ml; Molecular Probes) in 10 ml PBS for 30 min at room temperature in the dark (19, 22). Unbound probe was absorbed by adding 30 ml of DMEM with 10% serum and shaking for 20 min at room temperature. RBCs were then washed five times in PBS and resuspended in PBS.

    Labeling with the cytoplasmic probe calcein-AM. XC cells were selected for labeling with calcein-AM because of their size advantage and better fluorescence emission. XC cells were incubated with 5 μM of calcein-AM (Molecular Probes, Eugene, Oreg.) (21, 27) in 10 ml of DMEM for 45 min at 37°C in the dark, washed once, and then incubated in fresh complete medium containing 10% fetal bovine serum for another 30 min at 37°C. XC cells were washed three times with PBS and resuspended in DMEM.

    Dye redistribution assay. Labeled RBCs or XC cells were used to overlay neuraminidase (NA; 30 mU)- and TPCK trypsin-treated HeLa cells expressing a wild-type or chimeric influenza virus HA protein and then incubated at 37°C for 15 min. Cells were then treated with low-pH or neutral buffer and incubated at 37°C to allow fusion to occur. Dye redistribution was monitored for 2 min after the cells were transferred to 37°C by using a Nikon fluorescence microscope. Fluorescein isothiocyanate and rhodamine optical filter cubes were used for observing green and red fluorescent dye transfer, respectively.

    Reverse genetics. The chimeric HA-MuLV genes were introduced into the RNA expression plasmid pHH21, which contains the noncoding regions of both ends of the HA gene. Influenza viruses were rescued from plasmid cDNA essentially as described by Neumann et al. (26). Briefly, human 293T cells were transfected with 17 protein and RNA expression plasmids (kindly provided by Y. Kawaoka), using Mirus (Panvera) transfection reagent following the manufacturer's instructions. At 3 days posttransfection, cell supernatants were titrated on MDCK cells.

    RESULTS

    Construction and expression of chimeric HA-MuLV Env protein. Recent studies have shown that the C-terminal R peptide of the MuLV Env protein has a potent inhibitory effect on viral membrane fusion and that this sequence could also inhibit fusion activity when it was attached to the C terminus of a distantly related simian immunodeficiency virus (SIV) Env protein (45). To extend our understanding of the mechanism of the inhibitory effect of the R peptide, we determined whether it could affect the activity of a viral fusion protein of a distinct virus family. We constructed three groups of genes which encode chimeric influenza virus HA-MuLV Env proteins. In the first group, the cytoplasmic tail of the influenza virus HA protein was replaced by the full-length (HAM) or R peptide-deleted (HAM-13) cytoplasmic tail of the MuLV Env protein. We also constructed a gene which encodes a chimeric HA protein (HAMRA) containing an L627A point mutation in the R peptide coding sequence of the MuLV Env cytoplasmic tail, with which the inhibitory effect of the R peptide on MuLV-induced fusion is no longer observed (46). In the second group, the R peptide sequence (HA+R), the R peptide with the L627A point mutation (HA+RA), or the cytoplasmic tail region upstream of the R peptide in the MuLV Env protein (HAM-16) was attached directly at the end of the HA protein transmembrane domain. In the third group, we used the intact full-length HA protein, and the R peptide sequence (HACT+R), the R peptide with the L627A point mutation (HACT+RA), or the upstream region of the R peptide in the MuLV Env protein (HACT+16) was attached directly at the end of the HA protein cytoplasmic tail. The amino acid sequences of the cytoplasmic tails of the chimeric HA proteins are shown in Fig. 1, and all sequences of the chimeric HA genes were confirmed to determine that there was no additional mutation during PCR amplification or plasmid construction.

    To compare the expression of the wild-type and chimeric HA proteins, we used the recombinant vaccinia virus system (3). The cell surface expression and conformation of the chimeric HA proteins were assessed by ELISA, using conformation-specific monoclonal antibodies which recognize different antigenic sites of HA proteins. As shown in Table 1, most antibodies bound to chimeric HA proteins at similar levels to those for the wild-type HA protein; however, we found that HAM-16 showed less reactivity to the HC31 and HC68 monoclonal antibodies, and HA+RA showed less reactivity to the HC3 monoclonal antibody. These results indicate that all of the chimeric HA proteins are efficiently expressed on the cell surface and that the expression of antigenic loops is not affected by the cytoplasmic tail of the MuLV Env protein. To further investigate the sensitivities of the chimeric HA proteins to trypsin cleavage, we treated CV-1 cells expressing chimeric HA proteins with trypsin before analysis by Western blotting. As shown in Fig. 2, all of the mutant HA proteins were effectively cleaved into HA1 and HA2 subunits at comparable levels to those of the wild-type HA protein. These results indicate that the chimeric HA proteins were not affected with respect to cell surface expression, conformation, or susceptibility to cleavage by trypsin.

    The R peptide inhibits the syncytium formation activity of the HA protein. To investigate if the R peptide inhibits the fusion activity of the HA protein, we examined the syncytium formation activities of the chimeric HA proteins 12 to 18 h after infection of BHK21 cells (Fig. 3). The quantitative measurements of the cell fusion activities are summarized in Fig. 4. The cells expressing the wild-type HA protein showed significant syncytium formation after exposure to low-pH (5.1) fusion buffer, while no syncytia were observed at neutral pH. Of the first group of chimeric HA proteins, the HAM protein, in which the cytoplasmic tail of the HA protein was replaced by the full-length cytoplasmic tail of the MuLV Env protein, did not cause any detectable syncytium formation at low pH. However, the HAMRA protein, which contains an L627A point mutation in the R peptide sequence, caused syncytium formation at about 40% the level of wild-type HA at low pH. Similarly, the HAM-13 protein, in which the R peptide was deleted from the C terminus, also caused syncytium formation at similar levels to those of HAMRA. These results indicate that the R peptide specifically inhibits HA-induced syncytium formation in the context of the full-length cytoplasmic tail of the MuLV Env protein, while the L627A point mutation or R peptide deletion in the cytoplasmic tail of the MuLV Env resulted in partial recovery of syncytium formation by the chimeric HA proteins.

    For the second group of constructs, where the R peptide, the R peptide with the L627A point mutation, or the R peptide-deleted cytoplasmic tail of MuLV Env was directly attached to the end of the transmembrane domain of the HA protein, all three chimeric HA proteins, HAM-16, HA+R, and HA+RA, caused syncytium formation at a low pH, ranging from 10 to 40% of that of the wild-type HA protein. Thus, the R peptide did not inhibit syncytium formation by the HA protein in molecules lacking the cytoplasmic tail of the HA protein.

    For the third group, where the full-length cytoplasmic tail of the HA protein was intact, we found that the HACT+R protein, in which the R peptide sequence was attached directly at the end of the full-length HA protein, did not cause any syncytium formation at a low pH. The HACT+16 protein, in which the R peptide-deleted cytoplasmic domain was attached to the full-length HA protein, caused syncytium formation at a level of 5 to 10% that of the wild-type HA protein. HACT+RA, which contains the L627A point mutation, caused 60% syncytium formation compared to the wild-type HA protein. These results indicate that the R peptide specifically inhibits HA-induced cell fusion when attached directly at the end of the full-length HA protein, and the chimeric HA proteins containing the R peptide-deleted cytoplasmic tail of the MuLV Env protein or an L627A point mutation in the R peptide showed partial recovery of syncytium formation activity.

    Conformational changes of chimeric HA proteins. To further investigate if the chimeric HA proteins undergo the conformational changes in the external domain which are required for membrane fusion, we examined the conformational changes of all chimeric HA proteins at different pHs, using the monoclonal antibodies HC3 and HC68. HC3 recognizes the HA protein at both acidic and neutral pHs, while HC68 only recognizes the HA protein at neutral pH. As shown in Table 2 and Fig. 5, all chimeric HA proteins showed conformational changes at low pH values similar to that of the wild-type HA protein, although the precise pH at which the conformational changes of the chimeric HA proteins occurred was slightly different from that for the wild-type HA protein. As shown in Table 1, most antibodies bound to chimeric HA proteins at similar levels to those for the wild-type HA protein; however, we found that HAM-16 showed less reactivity to the HC31 and HC68 monoclonal antibodies and that HA+RA showed less reactivity to the HC3 monoclonal antibody. Thus, the ratio of HC68/HC3 signals for HAM-16 was lower than those observed with the rest of the chimeric HA proteins and the ratio for HA+RA was higher than those for the rest of the chimeric HA proteins.

    The R peptide does not affect the hemifusion activity of chimeric HA proteins. To further determine at which stage the R peptide inhibits chimeric HA protein-induced membrane fusion, we used a lipid mixing assay. HeLa cells expressing a wild-type or chimeric HA protein were treated with trypsin and overlaid with R-18-labeled chicken RBCs, and lipid mixing was monitored under a fluorescence microscope. As shown in Fig. 6, at a low pH all the cells expressing the chimeric HA proteins were able to induce lipid mixing at a level similar to that of the wild-type HA protein, as shown by fluorescent dye redistribution, indicating that the R peptide does not affect the hemifusion activity of the HA protein. No dye transfer was observed in cells treated with neutral pH fusion buffer (Fig. 6). We also included controls in which the cells expressing chimeric HA proteins were not cleaved by trypsin but underwent the low-pH treatment, and no dye transfer was observed in those controls either (data not shown), indicating that the dye transfers at low pH shown in Fig. 6 were induced specifically by the chimeric HA proteins.

    The R peptide does not affect small fusion pore formation. To further investigate if the R peptide inhibits small fusion pore formation, we labeled XC cells with a small aqueous fluorescent dye, calcein-AM. Once calcein-AM enters cells, it is cleaved into calcein, a smaller molecule which is fluorescent and can be transferred into the target cells when small pore formation occurs. After incubation of the XC cells with trypsin-treated HeLa cells expressing wild-type or chimeric HA protein at 37°C for 15 min, cells were incubated with low-pH or neutral-pH buffer and transferred to 37°C for 2 min. As shown in Fig. 7, at a low pH all the chimeric HA proteins were able to induce aqueous dye transfer at a level similar to that for the wild-type HA protein, indicating that the R peptide did not have a significant effect on small pore formation during the membrane fusion process. It therefore seems most likely that the R peptide is involved in the regulation of a late stage of membrane fusion induced by the influenza virus HA protein.

    We also compared the time courses of dye transfer mediated by the wild-type and chimeric HA proteins, and we did not find any significant difference between the wild-type and chimeric HA proteins, suggesting that the R peptide does not retard hemifusion and small pore formation.

    Reverse genetics. We generated recombinant influenza viruses containing the WT Aichi/2/68 HA gene and other gene segments derived from the WSN/1/33 virus (H3N1/WSN) at a titer of 103 to 104 PFU/ml by using the methods described by Neumann et al. (26). In order to further analyze the function of the chimeric HA-MuLV proteins, we attempted to rescue influenza viruses containing the chimeric HA-MuLV genes. However, we were not able to rescue the influenza viruses. Fujii et al. (10) have shown that the package signals of gene segments are located not only in the noncoding region but also near both ends of the coding region. Our results are consistent with their observations.

    DISCUSSION

    Although the cytoplasmic tails of viral envelope proteins are not directly involved in membrane fusion, they play important roles in regulating the membrane fusion process. In the current study, we have investigated the inhibitory effects of the MuLV Env cytoplasmic tail and the fusion-suppressive R peptide on HA-induced membrane fusion. We modified the cytoplasmic tail region of the wild-type HA protein by replacing the cytoplasmic tail of HA with the full-length cytoplasmic tail of MuLV Env or the MuLV Env cytoplasmic tail with the R peptide truncated or modified by an L627A point mutation or by replacing the cytoplasmic tail of HA with the R peptide or L627A mutant R peptide sequence only. We also added the R peptide or L627A mutant R peptide sequence directly at the end of the full-length HA protein. We found that all of the chimeric HA proteins were effectively expressed, folded, and cleaved by trypsin. The R peptide specifically inhibited HA-induced membrane fusion in the context of the full-length cytoplasmic tail of MuLV Env or when added directly at the end of the full-length HA protein. A dye redistribution assay showed that the R peptide did not affect hemifusion or small pore formation induced by the chimeric HA proteins.

    Cytoplasmic tails of viral glycoproteins play an important role in regulating viral membrane fusion. Modification of the cytoplasmic tail has different consequences in different viral families. The long cytoplasmic tails of the retroviruses human immunodeficiency virus and SIV are thought to be involved in regulating virus infectivity and fusion activity, and deletion of the cytoplasmic tail has been shown to increase the fusion activity of the viral envelope protein (15, 25, 29, 32, 35, 36). Several studies have shown that the C-terminal R peptide of the MuLV Env protein's cytoplasmic tail exerts a potent inhibitory effect on MuLV-induced cell fusion (16, 24, 39). Truncation of the cytoplasmic domain of the human paramyxovirus type 2 F protein did not affect its fusion activity (47). In contrast, removal of the cytoplasmic tail of the human paramyxovirus type 3 F protein or the simian virus 5 (SV5) F protein debilitated the fusion activity (1, 47). For influenza virus, a previous study showed that elongation of the cytoplasmic tail of the HA protein by as little as one amino acid reduced fusion activity significantly, whereas the addition of five amino acids abolished fusion activity completely (27). Although replacing the cytoplasmic tail of the HA protein with the corresponding region of the Sendai virus F protein did not affect the fusion activity, replacement of the cytoplasmic tail with that of the CD4 molecule impaired fusion pore enlargement significantly (19). Our recent studies on SER virus, a paramyxovirus closely related to SV5, indicated that the long cytoplasmic tail of the SER F protein plays an important role in F protein-induced syncytium formation and that truncation or mutations of the cytoplasmic tail result in enhanced syncytium formation (33, 40). These studies indicate that the cytoplasmic tails of many viral envelope proteins play an important role in modulating viral membrane fusion. In our current study, we extended our understanding of the inhibitory effect of the MuLV R peptide by replacing the cytoplasmic tail of the influenza virus HA protein with that of the MuLV Env protein. We found that the R peptide specifically inhibited the fusion activity of the HA protein; however, when the R peptide was removed or mutated such that the chimeric HA protein retained the same length of cytoplasmic tail to that of the chimeric HA protein containing the R peptide, the resulting chimeric HA protein was fusogenic. These results indicate that the MuLV R peptide has a potent inhibitory role in the fusion activity of an unrelated viral glycoprotein, the HA protein, and that this inhibition is sequence specific and not length specific.

    With constructs in which we directly attached the R peptide or L627A mutant R peptide to the end of the transmembrane domain of HA, we did not find specific inhibition of membrane fusion by the R peptide. One of the possible explanations is that the inhibitory effect of the R peptide involves sequences from the cytoplasmic tail region upstream of the R peptide in the MuLV Env protein. The sequence of the MuLV Env cytoplasmic tail indicates that the MuLV Env is predicted to form helical structures in the membrane-proximal region of the cytoplasmic tail, which plays important roles in connecting the cytoplasmic tail to the external domain and regulating the membrane fusion activity of MuLV Env. Without this connecting region, the R peptide may not form a stable helix structure and therefore may not be able to modulate the membrane fusion caused by the HA protein.

    When exposed to a low pH after trypsin cleavage, the external domain of the HA protein undergoes a conformational change, which is required for fusion pore formation, leading to complete membrane fusion (7, 34). We found that all of the chimeric HA proteins that we constructed undergo conformational changes similar to those of the wild-type HA protein, although the pH values at which this occurs are slightly different from that for the wild-type HA protein. We also found that the R peptide did not affect hemifusion and small pore formation, which are early stages of the membrane fusion process, by the chimeric HA proteins. It is possible that a late stage in HA-induced membrane fusion might be altered by the MuLV R peptide, resulting in impaired fusion activity of the HA protein. According to current fusion models (2, 6, 7, 8, 34), after conformational changes, class I viral fusion proteins form rod-like structures with a central coiled coil and antiparallel polypeptide chains that pack against it. This generates a trimer-of-hairpins structure that brings the two membranes together at the same end of the helical rods to initiate the fusion process. It will be interesting to determine whether the R peptide has an impact on the proper formation of the low-pH structure of the HA external domain in the chimeric proteins, thus affecting a later phase of the fusion process.

    ACKNOWLEDGMENTS

    This study was supported by grants AI054337, AI066870, and DE015543 from the National Institutes of Health.

    We thank Kim M. Gernert (Emory Molecular Modeling Center) for helpful discussions concerning molecular modeling of the MuLV Env protein and the HA protein. We thank Y. Kawaoka for providing the plasmids for reverse genetics and Tanya Cassingham for assistance in preparing the manuscript.

    M.L. and Z.-N.L. contributed equally to this work.

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