Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin.

The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.     

A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains.

When mice were immunized with the A/Okuda/57 (H2N2) strain of influenza virus, a unique monoclonal antibody designated C179 was obtained. Although C179 was confirmed to recognize the hemagglutinin (HA) glycoprotein by immunoprecipitation assays, it did not show hemagglutination inhibition activity to any of the strains of the three subtypes of influenza A virus. However, it neutralized all of the H1 and H2 strains but not the H3 strains. Moreover, it inhibited polykaryon formation induced by the H1 and H2 strains but not by the H3 strains. Two antigenic variants against C179 were obtained, and nucleotide sequence analysis revealed that amino acid sequences, from 318 to 322 of HA1 and from 47 to 58 of HA2, conserved among H1 and H2 strains were responsible for the recognition of C179. Since the two sites were located close to each other at the middle of the stem region of the HA molecule, C179 seemed to recognize these sites conformationally. These data indicated that binding of C179 to the stem region of HA inhibits the fusion activity of HA and thus results in virus neutralization and inhibition of cell-cell fusion. This is the first report which describes the presence of conserved antigenic sites on HA not only in a specific subtype but also in two subtypes of influenza A virus.     

Functions of the stem region of the Semliki Forest virus fusion protein during virus fusion and assembly

Membrane fusion of the alphaviruses is mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 dissociates from its heterodimer interaction with the E2 protein and forms a target membrane-inserted E1 homotrimer. The structure of the homotrimer is that of a trimeric hairpin in which E1 domain III and the stem region fold back toward the target membrane-inserted fusion peptide loop. The E1 stem region has a strictly conserved length and several highly conserved residues, suggesting the possibility of specific stem interactions along the trimer core and an important role in driving membrane fusion. Mutagenesis studies of the alphavirus Semliki Forest virus (SFV) here demonstrated that there was a strong requirement for the E1 stem in virus assembly and budding, probably reflecting its importance in lateral interactions of the envelope proteins. Surprisingly, however, neither the conserved length nor any specific residues of the stem were required for membrane fusion. Although the highest fusion activity was observed with wild-type E1, efficient fusion was mediated by stem mutants containing a variety of substitutions or deletions. A minimal stem length was required but could be conferred by a series of alanine residues. The lack of a specific stem sequence requirement during SFV fusion suggests that the interaction of domain III with the trimer core can provide sufficient driving force to mediate membrane merger.     

Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1 proteins of two subtypes differ in their ability to support assembly

The influenza A virus hemagglutinin (HA) transmembrane domain boundary region and the cytoplasmic tail contain three cysteines (residues 555, 562, and 565 for the H3 HA subtype) that are highly conserved among the 16 HA subtypes and which are each modified by the covalent addition of palmitic acid. Previous analysis of the role of these conserved cysteine residues led to differing data, suggesting either no role for HA palmitoylation or an important role for HA palmitoylation. To reexamine the role of these residues in the influenza virus life cycle, a series of cysteine-to-serine mutations were introduced into the HA gene of influenza virus A/Udorn/72 (Ud) (H3N2) by using a highly efficient reverse genetics system. Mutant viruses containing HA-C562S and HA-C565S mutations had reduced growth and failed to form plaques in MDCK cells but formed wild-type-like plaques in an MDCK cell line expressing wild-type HA. In cell-cell fusion assays, nonpalmitoylated H3 HA, in both cDNA-transfected and virus-infected cells, was fully competent for HA-mediated membrane fusion. When the HA cytoplasmic tail cysteine mutants were examined for lipid raft association, using as the criterion Triton X-100 insolubility, loss of raft association did not show a direct correlation with a reduction in virus replication. However, mutant virus assembly was reduced in parallel with reduced virus replication. Additionally, a reassortant of strain A/WSN/33 (WSN), containing the Ud HA gene with mutations C555S, C562S, and C565S, produced virus that could form plaques on regular MDCK cells and had only moderately decreased replication, suggesting differences in the interactions between Ud and WSN HA and internal viral proteins. Analysis of M1 mutants containing substitutions in the six residues that differ between the Ud and WSN M1 proteins indicated that a constellation of residues are responsible for the difference between the M1 proteins in their ability to support virus assembly with nonpalmitoylated H3 HA.     

Alterations to influenza virus hemagglutinin cytoplasmic tail modulate virus infectivity.

The influenza virus hemagglutinin (HA) contains a cytoplasmic domain that consists of 10 to 11 amino acids, of which five residues have sequence identity for 10 of 13 HA subtypes. To investigate properties of these conserved residues, oligonucleotide-directed mutagenesis was performed, using an HA cDNA of influenza virus A/Udorn/72 (H3N2) to substitute the conserved cysteine residues with other residues, to delete the three C-terminal conserved residues, or to remove the entire cytoplasmic domain. The altered HAs were expressed in eukaryotic cells, and the rates of intracellular transport were examined. It was found that substitution of either conserved cysteine residue within the cytoplasmic domain did not affect the rate of intracellular transport, whereas deletion of residues within the C-terminal domain resulted in delayed cell surface expression. All the altered HAs were biologically active in hemadsorption and fusion assays. To investigate whether the wild-type HA and HAs with altered cytoplasmic tails could complement the influenza virus temperature-sensitive transport-defective HA mutant A/WSN/33 ts61S, the HA cDNAs were expressed by using a transient expression system and released virus was assayed by plaque analysis. The wild-type HA expression resulted in a release of approximately 10(3) PFU of virus per ml. Antibody neutralization of complemented virus indicated that the infectivity was due to incorporation of wild-type H3 HA into ts61S virions. Sucrose density gradient analysis of released virions showed that each of the HA cytoplasmic domain mutants was incorporated into virus particles. Virions containing HAs with substitution of the cysteine residues in the cytoplasmic domain were found to be infectious. However, no infectivity could be detected from virions containing HAs that had deletions in their cytoplasmic domains. Possible roles of the HA cytoplasmic domain in forming protein-protein interactions in virions and their involvement in the initiation of the infection process in cells are discussed.     

Aspartic acid-66 is the only essential negatively charged residue in the putative hydrophilic loop region of the metal-tetracycline/H+ antiporter encoded by transposon Tn10 of Escherichia coli.

Of the 16 acidic amino acid residues located in the hydrophilic region of the metal-tetracycline/H+ antiporter of transposon Tn10, five glutamic acids and three aspartic acids are conserved among the tetracycline/H+ antiporters of Gram-negative bacteria. When these conserved acidic residues were each replaced by a neutral polar residue, glutamine or asparagine, only the Asp66 substitution mutants completely lost their transport activity. The substitution of Glu274, Asp120, Glu181, or Asp38 caused significant reduction of the transport activity, whereas the substitution of the other three residues had no detectable effect on the activity. These findings led to the conclusion that only Asp66 is essential for the transport function.     

Mutational analysis of the leucine zipper motif in the Newcastle disease virus fusion protein.

The paramyxovirus fusion proteins have a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit (R. Buckland and F. Wild, Nature [London] 338:547, 1989). To determine the role of the conserved leucines in the oligomeric structure and biological activity of the Newcastle disease virus (NDV) fusion protein, the heptadic leucines at amino acids 481, 488, and 495 were changed individually and in combination to an alanine residue. While single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein, although cell surface expression of the mutants was higher than that of the wild-type protein. Substitution of all three leucine residues with alanine did not alter the size of the fusion protein oligomer as determined by sedimentation in sucrose gradients. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain, resulted in secretion of an oligomeric polypeptide. These results indicate that the conserved leucines are not necessary for oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative changes of serine to alanine (position 473), glutamic acid to lysine or alanine (position 482), asparagine to lysine (position 485), or aspartic acid to alanine (position 489), the fusogenic ability of the protein was not significantly disrupted. In addition, a double mutant (E482A,D489A) which removed negative charges along one side of the helix had negligible effects on fusion activity.     

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.     

Human influenza virus hemagglutinin with high sensitivity to proteolytic activation.

To examine the prerequisites for cleavage activation of the hemagglutinin of human influenza viruses, a cDNA clone obtained from strain A/Port Chalmers/1/73 (serotype H3) was subjected to site-directed mutagenesis and expressed in CV-1 cells by using a simian virus 40 vector. The number of basic residues at the cleavage site, which consists of a single arginine with wild-type hemagglutinin, was increased by inserting two, three, or four additional arginines. Like wild-type hemagglutinin, mutants with up to three additional arginines were not cleaved in CV-1 cells, but insertion of four arginines resulted in activation. When the oligosaccharide at asparagine 22 of the HA1 subunit of the hemagglutinin was removed by site-directed mutagenesis of the respective glycosylation site, only three inserted arginines were required to obtain cleavage. Mutants containing a series of four basic residues were also generated by substituting arginine for uncharged amino acids immediately preceding the cleavage site. The observation that these mutants were not cleaved, even when the carbohydrate at asparagine 22 of HA1 was absent, underscores the fact that the basic peptide had to be generated by insertion to obtain cleavage. The data show that the hemagglutinin of a human influenza virus can acquire high cleavability, a property known to be an important determinant for the pathogenicity of avian influenza viruses. Factors important for cleavability are the number of basic residues at the cleavage site, the oligosaccharide at asparagine 22, and the length of the carboxy terminus of HA1.     

Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics

Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.     

Variant influenza virus hemagglutinin that induces fusion at elevated pH.

The hemagglutinin (HA) glycoprotein of influenza virus performs two critical roles during infection: it binds virus to cell surface sialic acids, and under mildly acidic conditions it induces fusion of the virion with intracellular membranes, liberating the genome into the cytoplasm. The pH dependence of fusion varies for different influenza virus strains. Here we report the isolation and characterization of a naturally occurring variant of the X31 strain that fuses at a pH 0.2 units higher than the parent strain does and that is less sensitive to the effects of ammonium chloride, a compound known to elevate endosomal pH. The bromelain-solubilized ectodomain of the variant HA displayed a corresponding shift in the pH at which it changed conformation and bound to liposomes. Cloning and sequencing of the variant HA gene revealed amino acid substitutions at three positions in the polypeptide. Two substitutions were in antigenic determinants in the globular region of HA1, and the third occurred in HA2 near the base of the molecule. By using chimeric HA molecules expressed in CV-1 cells from simian virus 40-based vectors, we demonstrated that the change in HA2 was solely responsible for the altered fusion phenotype. This substitution, asparagine for aspartic acid at position 132, disrupted a highly conserved interchain salt bridge between adjacent HA2 subunits. The apparent role of this residue in stabilizing the HA trimer is consistent with the idea that the trimer dissociates at low pH. Furthermore, the results demonstrate that influenza virus populations contain fusion variants, raising the possibility that such variants may play a role in the evolution of the virus.     

Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety.

The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.     

Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes.     

Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.     

An Induced Pocket for the Binding of Potent Fusion Inhibitor CL-385319 with H5N1 Influenza Virus Hemagglutinin

The influenza glycoprotein hemagglutinin (HA) plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA) calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.     

Functional interactions of the influenza virus glycoproteins

Hemagglutinin (HA) and neuraminidase (NA) are functionally related coat glycoproteins of the influenza virus (Flu). HA interacts with terminal sialyl residues of oligosaccharides and ensures the binding of the virus particle to the cell surface. NA is a receptor-destroying enzyme that removes sialyl residues from oligosaccharides contained in cell and virus components and thereby prevents aggregation of virus particles. Analysis of reasortants combining low-functional NA of human Flu with HA of avian Flu showed that sialyl residues are not completely removed in some cases. With high HA affinity for sialyl substrates, such virus particles aggregate, aggregates accumulate on the cell surface, and virus yield decreases. Serial passaging of low-yield aggregating reassortants may lead to selection of high-yield variants, which do not aggregate. A loss of aggregation is due to a decrease in HA affinity for high-molecular-weight sialyl substrates. On evidence of sequencing of the HA gene in original reassortants and their nonaggregating variants, HA affinity is reduced and aggregation lost owing to a mechanism common for different HA antigenic subtypes (H2, H3, H4, and H13). This is an increase in the negative charge as a result of an amino acid substitution in the vicinity of the receptor-binding pocket of HA. Taken together, these findings suggest a way of postreassortment adaptation which improves the functional match of HA and NA. The experimental system employed provides a model of natural processes associated with generation of Flu variants having a pandemic potential.     

Functional Interactions of the Influenza Virus Glycoproteins

Hemagglutinin (HA) and neuraminidase (NA) are functionally related envelope glycoproteins of the influenza virus (Flu). HA interacts with terminal sialyl residues of oligosaccharides and ensures the binding of the virus particle to the cell surface. NA is a receptor-destroying enzyme that removes sialyl residues from oligosaccharides contained in cell and virus components and thereby prevents aggregation of virus particles. Analysis of reassortants combining low-functional NA of human Flu with HA of avian Flu showed that sialyl residues are not completely removed in some cases. With high HA affinity for sialyl substrates, such virus particles aggregate, aggregates accumulate on the cell surface, and virus yield decreases. Serial passaging of low-yield aggregating reassortants may lead to selection of high-yield variants, which do not aggregate. The loss of aggregation is due to a decrease in HA affinity for high-molecular-weight sialyl substrates. On evidence of sequencing of the HA gene in original reassortants and their nonaggregating variants, for different HA antigenic subtypes (H2, H3, H4, and H13) the affinity is reduced and aggregation lost through a common mechanism: an increase in the negative charge as a result of an amino acid substitution in the vicinity of the receptor-binding pocket of HA. Taken together, these findings suggest a way of postreassortment adaptation, which improves the functional match of HA and NA. The experimental system employed provides a model of natural processes associated with emergence of Flu variants having a pandemic potential.     

Computational studies of pandemic 1918 and 2009 H1N1 hemagglutinins bound to avian and human receptor analogs

The purpose of this work was to study the binding properties of two pandemic influenza A virus 1918 H1N1 (SC1918) and 2009 H1N1 (CA09) hemagglutinin (HA) with avian and human receptors. The quantum chemical calculations have been performed to analyze the interactions of 130 loop, 190 helix, 220 loop region, and conserved residues 95,145,153–155, of pandemic viruses’ HA with sialo-trisaccharide receptor of avian and human using density functional theory. The HA’s residues Tyr 95, Ala 138, Gln 191, Arg 220, and Asp 225 from the above regions have stronger interaction with avian receptor. The residues Thr 136, Trp 153, His 183, and Asp 190 of HA are important and play a significant role to bind with human receptor. The residues Tyr 95, Ala 138, Lys 145, Trp 153, Gln 192, and Gln 226 of HA of CA09 virus have found more interaction energies with human than avian receptors. Due to mutations in the active residues of HA of CA09 virus comparing with SC1918, the binding capabilities of HA with human have been increased. The molecular dynamics simulation was made to understand the different dynamical properties of HA and molecular interactions between HA of these two viruses with sialo-trisaccharide receptors of avian and human receptors. The interaction energy of HA of CA09 virus with human receptor decreases due to the human receptor far away from conserved residue region of HA protein. This reveals that the conserved residues particularly Lys 145 play major contribution to interaction with human receptor in HA of CA09 virus.     

Characterization of the putative fusogenic domain in vesicular stomatitis virus glycoprotein G.

The envelope glycoprotein G of vesicular stomatitis virus induces membrane fusion at low pH. Site-directed mutagenesis of specific amino acids within a segment spanning amino acids 123 to 137 of G protein, which is highly conserved in vesiculoviruses and was previously shown by us to be involved in fusogenic activity (Y. Li, C. Drone, E. Sat, and H. P. Ghosh, J. Virol. 67:4070-4077, 1993), was used to determine the role of this region in low-pH-induced membrane fusion. The mutant glycoproteins expressed in COS cells were assayed for acid-pH-induced cell-cell fusion. Substitution of the variant Pro-123 with Leu had no effect on the fusogenic activity, while substitution of conserved Phe-125 and Asp-137 with Tyr and Asn, respectively, shifted the pH optimum of membrane fusion to a more acidic pH value and decreased the fusion efficiency. The deletion of amino acid residues 124 to 127, 131 to 137, or 124 to 137 produced mutants defective in transport. Mutation of the conserved residues Gly-124 and Pro-127 to Ala and to Gly or Leu, respectively, inhibited cell-cell fusion activity by about 90% without affecting transport of the mutant proteins to the cell surface, suggesting that these two residues may be present within the fusion peptide and thus may be directly involved in fusion. This highly conserved domain containing neutral amino acids of G protein may therefore represent the putative fusion domain of vesicular stomatitis virus G protein.     

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.