Association of vaccinia virus fusion regulatory proteins with the multicomponent entry/fusion complex

The proteins encoded by the A56R and K2L genes of vaccinia virus form a heterodimer (A56/K2) and have a fusion regulatory role as deletion or mutation of either causes infected cells to form large syncytia spontaneously. Here, we showed that syncytia formation is dependent on proteins of the recently described entry fusion complex (EFC), which are also required for virus-cell fusion and low-pH-triggered cell-cell fusion. This finding led us to consider that A56/K2 might prevent fusion by direct or indirect interaction with the EFC. To test this hypothesis, we made a panel of recombinant vaccinia viruses that have a tandem affinity purification tag attached to A56, K2, or the A28 EFC protein. Interaction between A56/K2 and the EFC was demonstrated by their copurification from detergent-treated lysates of infected cells and identification by mass spectrometry or Western blotting. In addition, a purified soluble transmembrane-deleted form of A56/K2 was shown to interact with the EFC. Tagged A56 did not interact with the EFC in the absence of K2, nor did tagged K2 interact with the EFC in the absence of A56. The finding that both A56 and K2 are required for efficient binding to the EFC fits well with prior experiments showing that mutation of either A56 or K2 results in spontaneous fusion of infected cells. Because A56 and K2 are located on the surface of infected cells, they are in position to interact with the EFC of released progeny virions and prevent back-fusion and syncytia formation.     

Vaccinia virus H2 protein is an essential component of a complex involved in virus entry and cell-cell fusion

The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.     

Vaccinia virus A56/K2 fusion regulatory protein interacts with the A16 and G9 subunits of the entry fusion complex

Deletion of the A56R or K2L gene of vaccinia virus (VACV) results in the spontaneous fusion of infected cells to form large multinucleated syncytia. A56 and K2 polypeptides bind to one another (A56/K2) and together are required for interaction with the VACV entry fusion complex (EFC); this association has been proposed to prevent the fusion of infected cells. At least eight viral polypeptides comprise the EFC, but no information has been available regarding their interactions either with each other or with A56/K2. Utilizing a panel of recombinant VACVs designed to repress expression of individual EFC subunits, we demonstrated that A56/K2 interacted with two polypeptides: A16 and G9. Both A16 and G9 were required for the efficient binding of each to A56/K2, suggesting that the two polypeptides interact with each other within the EFC. Such an interaction was established by the copurification of A16 and G9 from infected cells under conditions in which a stable EFC complex failed to assemble and from detergent-treated lysates of uninfected cells that coexpressed A16 and G9. A recombinant VACV that expressed G9 modified with an N-terminal epitope tag induced the formation of syncytia, suggesting partial interference with the functional interaction of A56/K2 with the EFC during infection. These data suggest that A16 and G9 are physically associated within the EFC and that their interaction with A56/K2 suppresses spontaneous syncytium formation and possibly "fuse-back" superinfection of cells.     

A conserved region in the F(2) subunit of paramyxovirus fusion proteins is involved in fusion regulation

Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F(1) and F(2). Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F(1) (CBF(1)) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F(2) subunit (CBF(2)). To analyze the functions of CBF(2), alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF(2) mutations resulted in folding and expression defects. However, the CBF(2) mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF(2) Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF(2) I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF(2) in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion.     

The glycine-rich sequence of the beta subunit of Escherichia coli H(+)-ATPase is important for activity

A short sequence motif rich in glycine residues, Gly-X-X-X-X-Gly-Lys-Thr/Ser, has been found in many nucleotide-binding proteins including the beta subunit of Escherichia coli H(+)-ATPase (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residues 149-156). The following mutations were introduced in this region of the cloned E. coli unc operon carried by a plasmid pBWU1: Ala-151----Pro or Val; insertion of a Gly residue between Lys-155 and Thr-156; and replacement of the region by the corresponding sequence of adenylate kinase (Gly-Gly-Pro-Gly-Ser-Gly-Lys-Gly-Thr) or p21 ras protein (ras) (Gly-Ala-Gly-Gly-Val-Gly-Lys-Ser). All F0F1 subunits were synthesized in the deletion strain of the unc operon-dependent on pBWU1 with mutations, and essentially the same amounts of H(+)-ATPase with these mutant beta subunits were found in membranes. The adenylate kinase and Gly insertion mutants showed no oxidative phosphorylation or ATPase activity, whereas the Pro-151 mutants had higher ATPase activity than the wild-type, and the Val-151 and ras mutants had significant activity. It is striking that the enzyme with the ras mutation (differing in three amino acids from the beta sequence) had about half the membrane ATPase activity of the wild-type. These results together with the simulated three-dimensional structures of the wild-type and mutant sequences suggest that in mutant beta subunits with no ATPase activity projection of Thr-156 residues was opposite to that in the wild-type, and that the size and direction of projection of residue 151 are important for the enzyme activity.     

Membrane cell fusion activity of the vaccinia virus A17-A27 protein complex

Vaccinia virus enters cells by endocytosis and via a membrane fusion mechanism mediated by viral envelope protein complexes. While several proteins have been implicated in the entry/fusion event, there is no direct proof for fusogenic activity of any viral protein in heterologous systems. Transient coexpression of A17 and A27 in mammalian cells led to syncytia formation in a pH-dependent manner, as ascertained by confocal fluorescent immunomicroscopy. The pH-dependent fusion activity was identified to reside in A17 amino-terminal ectodomain after overexpression in insect cells using recombinant baculoviruses. Through the use of A17 ectodomain deletion mutants, it was found that the domain important for fusion spanned between residues 18 and 34. To further characterize A17–A27 fusion activity in mammalian cells, 293T cell lines stably expressing A17, A27 or coexpressing both proteins were generated using lentivectors. A27 was exposed on the cell surface only when A17 was coexpressed. In addition, pH-dependent fusion activity was functionally demonstrated in mammalian cells by cytoplasmic transfer of fluorescent proteins, only when A17 and A27 were coexpressed. Bioinformatic tools were used to compare the putative A17–A27 protein complex with well-characterized fusion proteins. Finally, all experimental evidence was integrated into a working model for A17–A27-induced pH-dependent cell-to-cell fusion.     

The membrane fusion step of vaccinia virus entry is cooperatively mediated by multiple viral proteins and host cell components

For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.     

The vaccinia virus 14-kilodalton fusion protein forms a stable complex with the processed protein encoded by the vaccinia virus A17L gene.

The mechanism by which the 14-kDa fusion protein of vaccinia virus (VV) is anchored in the envelope of intracellular naked virions (INV) is not understood. In this investigation, we demonstrate that the 14-kDa protein interacts with another virus protein with an apparent molecular mass of 21 kDa. Microsequence analysis of the N terminus of the 21-kDa protein revealed that this protein is encoded by the VV A17L gene. The 21-kDa protein is processed from a 23-kDa precursor, by cleavage at amino acid position 16, at the consensus motif Ala-Gly-Ala, previously identified as a cleavage site for several VV structural proteins. The 21-kDa protein contains two large internal hydrophobic domains characteristic of membrane proteins. Pulse-chase analysis showed that within 1 h after synthesis, the 14-kDa protein forms a stable complex with the 21-kDa protein. Formation of the complex was not inhibited by rifampin, indicating that the interaction between these two proteins occurs prior to virion morphogenesis. Immunoprecipitation analysis of disrupted virions showed the presence of the 21-kDa protein in the viral particle. Release of the 14-kDa-21-kDa protein complex from INV required treatment with the nonionic detergent Nonidet P-40 and a reducing agent. The protein complex consisted of 14-kDa trimers and of 21-kDa dimers. Since the 14-kDa fusion protein lacks a signal sequence and a large hydrophobic domain characteristic of membrane proteins, our findings suggest that the 21-kDa protein serves to anchor the 14-kDa protein to the envelope of INV.     

Four conserved cytoplasmic sequence motifs are important for transport function of the Leishmania inositol/H(+) symporter

The protozoan Leishmania donovani has a myo-inositol/proton symporter (MIT) that is a member of a large sugar transporter superfamily. Active transport by MIT is driven by the proton electrochemical gradient across the parasite membrane, and MIT is a prototype for understanding the function of an active transporter in lower eukaryotes. MIT contains two duplicated 6- or 7-amino acid motifs within cytoplasmic loops, which are highly conserved among 50 members of the sugar transporter superfamily and are designated A(1), A(2) ((V)(D/E)(R/K)PhiGR(R/K)), and B(1) (PESPRPhiL), B(2) (VPETKG). In particular, the three acidic residues within these motifs, Glu(187)(B(1)), Asp(300)(A(2)), and Glu(429)(B(2)) in MIT, are highly conserved with 96, 78, and 96% amino acid identity within the analyzed members of this transporter superfamily ranging from bacteria, archaea, and fungi to plants and the animal kingdom. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopus oocytes and overexpression in Leishmania transfectants to investigate the significance of these three acidic residues in the B(1), A(2), and B(2) motifs. Alteration to the uncharged amides greatly reduced MIT transport function to 23% (E187Q), 1.4% (D300N), and 3% (E429Q) of wild-type activity, respectively, by affecting V(max) but not substrate affinity. Conservative mutations that retained the charge revealed a less pronounced effect on inositol transport with 39% (E187D), 16% (D300E) and 20% (E429D) remaining transport activity. Immunofluorescence microscopy of oocyte cryosections confirmed that MIT mutants were expressed on the oocyte surface in similar quantity to MIT wild type. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy) phenylhydrazone and dinitrophenol inhibited inositol transport by 50-70% in the wild type as well as in E187Q, D300N, and E429Q, despite their reduced transport activities, suggesting that transport in these mutants is still proton-coupled. Furthermore, temperature-dependent uptake studies showed an increased Arrhenius activation energy for the B(1)-E187Q and the B(2)-E429Q mutants, which supports the idea of an impaired transporter cycle in these mutants. We conclude that the conserved acidic residues Glu(187), Asp(300), and Glu(429) are critical for transport function of MIT.     

Vaccinia virus A21 virion membrane protein is required for cell entry and fusion

We provide the initial characterization of the product of the vaccinia virus A21L (VACWR140) gene and demonstrate that it is required for cell entry and low pH-triggered membrane fusion. The A21L open reading frame, which is conserved in all sequenced members of the poxvirus family, encodes a protein of 117 amino acids with an N-terminal hydrophobic domain and four invariant cysteines. Expression of the A21 protein occurred at late times of infection and was dependent on viral DNA replication. The A21 protein contained two intramolecular disulfide bonds, the formation of which required the vaccinia virus-encoded cytoplasmic redox pathway, and was localized on the surface of the lipoprotein membrane of intracellular mature virions. A conditional lethal mutant, in which A21L gene expression was regulated by isopropyl-beta-d-thiogalactopyranoside, was constructed. In the absence of inducer, cell-to-cell spread of virus did not occur, despite the formation of morphologically normal intracellular virions and extracellular virions with actin tails. Purified virions lacking A21 were able to bind to cells, but cores did not penetrate into the cytoplasm and synthesize viral RNA. In addition, virions lacking A21 were unable to mediate low pH-triggered cell-cell fusion. The A21 protein, like the A28 and H2 proteins, is an essential component of the poxvirus entry/fusion apparatus for both intracellular and extracellular virus particles.     

Transmembrane domain of IFITM3 is responsible for its interaction with influenza virus HA2 subunit

Interferon-inducible transmembrane protein 3 (IFITM3) inhibits influenza virus infection by blocking viral membrane fusion, but the exact mechanism remains elusive. Here, we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin (HA). The restriction of IFITM3 on HA-mediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses. Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA₂ subunit but not HA₁ subunit of H5 and H7 virus. Truncated analyses showed that the transmembrane domain of the IFITM3 and HA₂ subunit might play an important role in their interaction. Finally, this interaction of IFITM3 was also verified with HA₂ subunits from other subtypes of influenza A virus and influenza B virus. Overall, our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain, providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.     

c-Myb is an evolutionary conserved miR-150 target and miR-150/c-Myb interaction is important for embryonic development

Human c-Myb proto-oncogene is highly expressed in hematopoietic progenitors as well as leukemia and certain solid tumor. However, the regulatory mechanisms of its expression and biological functions remain largely unclear. Recently, c-Myb has been shown to be targeted by microRNA-150 (miR-150) which thereby controls B cell differentiation in mice. In this study, we demonstrated that c-Myb is an evolutionary conserved target of miR-150 in human and zebrafish, using reporter assays. Ectopic expression of miR-150 in breast cancer and leukemic cells repressed endogenous c-Myb at both messenger RNA (mRNA) and protein levels. Among several leukemia cell lines, primary leukemia cells, and normal lymphocytes, expression levels of miR-150 inversely correlated with c-Myb. The miR-150 overexpression or c-Myb silencing in zebrafish zygotes led to similar and serious phenotypic defects in zebrafish, and the phenotypic aberrations induced by miR-150 could be reversed by coinjection of c-Myb mRNA. Our findings suggest that c-Myb is an evolutionally conserved target of miR-150 and miR-150/c-Myb interaction is important for embryonic development and possibly oncogenesis.     

A large region within the Rous sarcoma virus matrix protein is dispensable for budding and infectivity.

All retroviruses have a layer of matrix protein (MA) situated directly beneath the lipid of their envelope. This protein is initially expressed as the amino-terminal sequence of the Gag polyprotein, where it plays an important role in binding Gag to the plasma membrane during the early steps of the budding process. Others have suggested that MA may provide additional functions during virion assembly, including the selective incorporation of viral glycoproteins and the RNA genome into the emerging virion. To further study the role of the Rous sarcoma virus MA sequence in the viral replication cycle, we have pursued an extensive deletion analysis. Surprisingly, the entire second half of MA (residues 87 to 155) and part of the neighboring p2 sequence were found to be dispensable not only for budding but also for infectivity in avian cells. Thus, all of the functions associated with the Rous sarcoma virus MA sequence must be contained within its first half.     

Highly conserved eight amino acid sequence in SH2 is important for recognition of phosphotyrosine site.

Src homology region 2(SH2) has been demonstrated to recognize phosphotyrosine site. To clarify the precise mechanism of the recognition, we developed in vitro binding assay system using EGF receptor and SH2/SH3 region of phospholipase C(PLC) gamma 1. Phosphorylated EGF receptor bound to immobilized SH2/SH3 of PLC gamma 1 in Sepharose beads, while nonphosphorylated EGF receptor did not bind. In SH2 domain of PLC gamma 1, there are several highly conserved amino acid sequences that are common in a variety of SH2-containing proteins. Especially the eight amino acid sequence, G(S/T)FLVR(E/D)S is highly conserved in these proteins. We synthesized several peptides related to these sequences and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC gamma 1. P1, GSFLVRES was the most effective inhibitor to suppress the binding. P2, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine is still effective. But a peptide, P3, SFLVRE in which two amino acids are deleted from P1 did not inhibit markedly. Moreover, P1 peptide immobilized in Sepharose beads also bound phosphorylated EGF receptor. These data suggest that highly conserved amino acid sequence GSFLVRES is the minimum essential unit to recognize tyrosine phosphorylated site.     

Histone H2A.Z has a conserved function that is distinct from that of the major H2A sequence variants

Saccharomyces cerevisiae contains three genes that encode members of the histone H2A gene family. The last of these to be discovered, HTZ1 (also known as HTA3), encodes a member of the highly conserved H2A.Z class of histones. Little is known about how its in vivo function compares with that of the better studied genes (HTA1 and HTA2) encoding the two major H2As. We show here that, while the HTZ1 gene encoding H2A.Z is not essential in budding yeast, its disruption results in slow growth and formamide sensitivity. Using plasmid shuffle experiments, we show that the major H2A genes cannot provide the function of HTZ1 and the HTZ1 gene cannot provide the essential function of the genes encoding the major H2As. We also demonstrate for the first time that H2A.Z genes are functionally conserved by showing that the gene encoding the H2A.Z variant of the ciliated protozoan TETRAHYMENA: thermophila is able to rescue the phenotypes associated with disruption of the yeast HTZ1 gene. Thus, the functions of H2A.Z are distinct from those of the major H2As and are highly conserved.     

The capsid-binding nucleolar helicase DDX56 is important for infectivity of West Nile virus

Recent findings suggest that in addition to its role in packaging genomic RNA, the West Nile virus (WNV) capsid protein is an important pathogenic determinant, a scenario that requires interaction of this viral protein with host cell proteins. We performed an extensive multitissue yeast two-hybrid screen to identify capsid-binding proteins in human cells. Here we describe the interaction between WNV capsid and the nucleolar RNA helicase DDX56/NOH61. Coimmunoprecipitation confirmed that capsid protein binds to DDX56 in infected cells and that this interaction is not dependent upon intact RNA. Interestingly, WNV infection induced the relocalization of DDX56 from the nucleolus to a compartment in the cytoplasm that also contained capsid protein. This phenomenon was apparently specific for WNV, as DDX56 remained in the nucleoli of cells infected with rubella and dengue 2 viruses. Further analyses showed that DDX56 is not required for replication of WNV; however, virions secreted from DDX56-depleted cells contained less viral RNA and were 100 times less infectious. Together, these data suggest that DDX56 is required for assembly of infectious WNV particles.     

Acylation-mediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity

Attachment of palmitic acid to cysteine residues is a common modification of viral glycoproteins. The influenza virus hemagglutinin (HA) has three conserved cysteine residues at its C terminus serving as acylation sites. To analyze the structural and functional roles of acylation, we have generated by reverse genetics a series of mutants (Ac1, Ac2, and Ac3) of fowl plague virus (FPV) containing HA in which the acylation sites at positions 551, 559, and 562, respectively, have been abolished. When virus growth in CV1 and MDCK cells was analyzed, similar amounts of virus particles were observed with the mutants and the wild type. Protein patterns and lipid compositions, characterized by high cholesterol and glycolipid contents, were also indistinguishable. However, compared to wild-type virus, Ac2 and Ac3 virions were 10 and almost 1,000 times less infectious, respectively. Fluorescence transfer experiments revealed that loss of acyl chains impeded formation of fusion pores, whereas hemifusion was not affected. When the affinity to detergent-insoluble glycolipid (DIG) domains was analyzed by Triton X-100 treatment of infected cells and virions, solubilization of Ac2 and Ac3 HAs was markedly facilitated. These observations show that acylation of the cytoplasmic tail, while not necessary for targeting to DIG domains, promotes the firm anchoring and retention of FPV HA in these domains. They also indicate that tight DIG association of FPV HA is essential for formation of fusion pores and thus probably for infectivity.     

The membrane-proximal region of vesicular stomatitis virus glycoprotein G ectodomain is critical for fusion and virus infectivity

The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.