Stoichiometry of murine leukemia virus envelope protein-mediated fusion and its neutralization

Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.     

Modulation of lentivirus replication by antibodies: Fc portion of immunoglobulin molecule is essential for enhancement of binding, internalization, and neutralization of visna virus in macrophages.

Antibodies to visna virus neutralized the virus in fibroblasts and macrophages but specifically enhanced the binding, penetration, and uncoating of the virus in the latter cells. F(ab')2 fragments of the immune antibody neutralized the virus in fibroblasts but did not enhance the early stages of the virus life cycle in macrophages. Furthermore, these fragments did not neutralize infectivity in macrophages but delayed the appearance of infectious virus in cells after the inoculation of preincubated virus-F(ab')2 complexes.     

Rubella virus antigens: localization of epitopes involved in hemagglutination and neutralization by using monoclonal antibodies.

Monoclonal antibodies (MAbs) against the rubella virion were used to locate epitopes involved in hemagglutination and neutralization. The MAbs exhibiting high-level hemagglutination-inhibiting activity were shown by Western blot analysis to be specific for the E1 polypeptide; this is consistent with the presence of the hemagglutinin on the E1 polypeptide. Some of the E1-specific MAbs also neutralized viral infectivity. However, hemagglutination-inhibiting activity and neutralizing activity did not always correlate. Three distinct functional epitopes were identified on the E1 polypeptide by competition analyses: one which reacted with MAbs with high-level hemagglutination-inhibiting activity and with neutralizing activity, one which reacted with MAbs with low-level hemagglutination-inhibiting activity and with neutralizing activity, and one which reacted with MAbs with only hemagglutination-inhibiting activity. A MAb specific for the E2 polypeptide exhibited neutralizing activity. This E2-specific MAb and two E1-specific MAbs with neutralizing activity were capable of precipitating intact virus which indicates that at least three epitopes involved in neutralization are accessible on the surface of the virion.     

Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.     

Antibody-selected variation and reversion in Sindbis virus neutralization epitopes.

Sindbis virus variants evidencing a complex and bidirectional tendency toward spontaneous antigenic change were isolated and characterized. Variants were selected on the basis of their escape from neutralization by individual monoclonal antibodies to either of the two envelope glycoproteins, E2 and E1. Multisite variants, including one altered in three neutralization sites, were obtained by selecting mutants consecutively in the presence of different neutralizing monoclonal antibodies. Two phenotypic revertants, each of which reacquired prototype antigenicity, were back-selected on the basis of their reactivity with a neutralizing monoclonal antibody. An incidental oligonucleotide marker distinguished these and the variant from which they arose from parental Sindbis virus and other mutants, thereby confirming that the revertants were true progeny of the antigenic variant. Prototype Sindbis virus and variants derived from it were compared on the basis of their reactivities with each of a panel of monoclonal antibodies; patterns revealed a minimum of five independently mutable Sindbis virus neutralization epitopes, segregating as three antigenic sites (two E2 and one E1).     

Relative contributions of measles virus hemagglutinin- and fusion protein-specific serum antibodies to virus neutralization

The relative contribution of measles virus hemagglutinin (H)- or fusion protein (F)-specific antibodies to virus neutralization (VN) has not been demonstrated. We have depleted these specific antibodies from sera collected from young adults, who had been vaccinated during childhood, by prolonged incubation with viable transfected human melanoma cells expressing H or F. Simultaneous depletion of antibodies of both specificities completely abrogated VN activity. Depletion of F-specific antibodies only had a minimal effect, whereas removal of H-specific antibodies resulted in almost complete reduction of VN activity. These results demonstrate that measles virus neutralizing antibodies are mainly directed to the H protein.     

Variation in the biological function of envelope protein epitopes of yellow fever vaccine viruses detected with monoclonal antibodies.

Three different virus strains (17D-204, 17DD and the French neurotropic vaccine) have been used as live attenuated yellow fever (YF) vaccines and are manufactured in different centres around the world. The envelope proteins of these vaccine viruses were examined and compared using mouse monoclonal antibodies (MAbs) in haemagglutination inhibition (HAI) and neutralization (N) tests. The epitopes eliciting HAI and/or N were found to vary depending on the virus examined. Such variation was also found between vaccine viruses of the same strain manufactured in different centres. These data were confirmed by the use of mouse polyclonal antisera. On the basis of the MAb results in HAI tests a dendrogram of the similarity coefficients between the viruses was constructed and showed that the viruses could be placed into three major groups. Thus, it is concluded that YF vaccines manufactured in different centres are antigenically distinct as recognized by the mouse immune system.     

Carbohydrate epitopes are responsible for antibody cross-reactivity in Trypanosoma cruzi-infected mice.

Anti-Trypanosoma cruzi antibodies can be eluted from western blots of T. cruzi antigens and thereby are fractionated on the basis of the electrophoretic mobility of the antigens to which they bind. Antibodies fractionated by these methods can bind antigens with electrophoretic mobility different from those antigens from which they are eluted. Such antibodies thus are considered cross-reactive. Studies in which the target antigens are reacted with sodium periodate to destroy carbohydrate epitopes prior to exposure to the eluted antibodies revealed that antibodies are produced that bind to both carbohydrate and noncarbohydrate epitopes on western blots, but that most of the cross-reactive antibodies are directed toward carbohydrate moieties.     

Antiviral antibodies are necessary for control of simian immunodeficiency virus replication

To better define the role of B cells in the control of pathogenic simian immunodeficiency virus (SIV) replication, six rhesus monkeys were depleted of B cells by intravenous infusion of rituximab (anti-CD20) 28 days and 7 days before intravaginal SIVmac239 inoculation and every 21 days thereafter until AIDS developed. Although the blood and tissues were similarly depleted of B cells, anti-SIV immunoglobulin G (IgG) antibody responses were completely blocked in only three of the six animals. In all six animals, levels of viral RNA (vRNA) in plasma peaked at 2 weeks and declined by 4 weeks postinoculation (PI). However, the three animals prevented from making an anti-SIV antibody response had significantly higher plasma vRNA levels through 12 weeks PI (P = 0.012). The remaining three B-cell-depleted animals made moderate anti-SIV IgG antibody responses, maintained moderate plasma SIV loads, and showed an expected rate of disease progression, surviving to 24 weeks PI without developing AIDS. In contrast, all three of the B-cell-depleted animals prevented from making anti-SIV IgG responses developed AIDS by 16 weeks PI (P = 0.0001). These observations indicate that antiviral antibody responses are critical in maintaining effective control of SIV replication at early time points postinfection.     

Evidence for similar recognition of the conserved neutralization epitopes of human immunodeficiency virus type 1 envelope gp120 in humans and macaques

We compared the immune responses to the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins in humans and macaques with the use of clade A and clade B isogenic V3 loop glycan-possessing and -deficient viruses. We found that the presence or absence of the V3 loop glycan affects to similar extents immune recognition by a panel of anti-HIV human and anti-simian/human immunodeficiency virus (anti-SHIV) macaque sera. All sera tested neutralized the glycan-deficient viruses, in which the conserved CD4BS and CD4i epitopes are more exposed, better than the glycan-containing viruses. The titer of broadly neutralizing antibodies appears to be higher in the sera of macaques infected with glycan-deficient viruses. Collectively, our data add legitimacy to the use of SHIV-macaque models for testing the efficacy of HIV-1 Env-based immunogens. Furthermore, they suggest that antibodies to the CD4BS and CD4i sites of gp120 are prevalent in human and macaque sera and that the use of immunogens in which these conserved neutralizing epitopes are more exposed is likely to increase their immunogenicity.     

The amphotropic and ecotropic murine leukemia virus envelope TM subunits are equivalent mediators of direct membrane fusion: implications for the role of the ecotropic envelope and receptor in syncytium formation and viral entry.

The murine leukemia virus (MuLV) envelope protein was examined to determine which sequences are responsible for the differences in direct membrane fusion observed with the ecotropic and amphotropic MuLV subtypes. These determinants were studied by utilizing amphotropic-ecotropic chimeric envelope proteins that have switched their host range but retain their original fusion domain (TM subunit). Fusion was tested both in rodent cells and in 293 cells bearing the human homolog of the ecotropic MuLV receptor. The results demonstrate that the amphotropic TM is able to mediate cell-to-cell fusion to an extent equivalent to that mediated by the ecotropic TM, indicating that their fusion domains are equivalent. The "murinized" human homolog of the ecotropic receptor supports syncytium formation as well as the native murine receptor. These findings suggest that interactions between the ecotropic envelope protein and conserved sequences in the ecotropic receptor are the principal determinants of syncytium formation. The relationship of the fusion phenotype to pH-dependent infection and the route of viral entry was examined by studying virions bearing the chimeric envelope proteins. Such virions appear to enter cells via a pathway that is directed by the host range-determining region of their envelope rather than by sequences that confer pH dependence. Therefore, the pH dependence of infection may not reflect the initial steps in viral entry. Thus, it appears that both the syncytium phenotype and the route of viral entry are properties of the viral receptor, the amino-terminal half of the ecotropic envelope protein, or the interaction between the two.     

Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.     

Neutralizing antibodies to visna lentivirus: mechanism of action and possible role in virus persistence.

Lentiviruses are nononcogenic retroviruses that cause persistent infections and slowly progressive diseases. Visna virus, a lentivirus of sheep, persists in cells of the macrophage lineage despite the presence of neutralizing antibodies in the animal. These antibodies are measured by prevention of virus replication in sheep fibroblast cell cultures. In this study we have compared the antiviral properties of the antibodies in sheep fibroblast and macrophage cell cultures, the latter being more relevant to infection in the animal. Using infectivity assays, binding of radiolabeled virus to cell membranes, cellular processing of labeled virus into acid-precipitable and acid-soluble components, and in situ hybridization of viral nucleic acid, we show that the antibodies prevented virus replication in both fibroblasts and macrophages. However, the site of neutralization differed between the two cell types. In fibroblasts, the site of virus neutralization was at the cell membrane, when the antibodies prevented virus attachment. In macrophages, virus incubated with the antibodies was phagocytized rapidly, followed by uncoating of the virions. However, virus RNA was not transcribed. Despite this ability of the antibodies to abort virus replication in macrophages, the kinetics of binding of the antibodies to the virus was much slower than the binding of virus to the macrophages. Therefore, persistent virus replication in immune sheep may be the result of virus spreading from macrophage to macrophage before the agent can be neutralized by antibodies in the plasma.     

Cryptic nature of envelope V3 region epitopes protects primary monocytotropic human immunodeficiency virus type 1 from antibody neutralization.

Characterization of biological and immunological properties of human immunodeficiency virus type 1 (HIV-1) is critical to developing effective therapies and vaccines for AIDS. With the use of a novel CD4+ T-cell line (PM-1) permissive to infection by both monocytotropic (MT) and T-cell-tropic virus types, we present a comparative analysis of the immunological properties of a prototypic primary MT isolate of HIV-1 strain JR-CSF (MT-CSF) with those of a T-cell-tropic variant (T-CSF) of the same virus, which emerged spontaneously in vitro. The parental MT-CSF infected only PM-1 cells and was markedly resistant to neutralization by sera from HIV-1-infected individuals, rabbit antiserum to recombinant MT-CSF gp120, and anti-V3 monoclonal antibodies. The T-CSF variant infected a variety of CD4+ T-cell lines, contained positively charged amino acid substitutions in the gp120 V3 region, and was highly sensitive to antibody neutralization. Neutralization and antibody staining of T-CSF-expressing cells were significantly inhibited by HIV-1 V3 peptides; in contrast, the MT strain showed only weak V3-specific binding of polyclonal and monoclonal antibodies. Exposure of PM-1 cells to a mixture of both viruses in the presence of human anti-HIV-1 neutralizing antiserum resulted in infection with only MT-CSF. These results demonstrate that although the V3 region of MT viruses is immunogenic, the target epitopes in the V3 principal neutralizing domain on the membrane form of the MT envelope appear to be cryptic or hidden from blocking antibodies.     

The visna virus genome: evidence for a hypervariable site in the env gene and sequence homology among lentivirus envelope proteins.

The complete nucleotide sequence of the visna virus 1514 genome was determined. Our sequence confirms the relationship of visna virus and other lentiviruses to human immunodeficiency virus (HIV) both at the level of sequence homology and of genomic organization. Sequence homology is shown to extend to the transmembrane proteins of lentivirus env genes; this homology is strongest in the extracellular domain, suggesting that close structural and functional similarities may also exist among these envelope proteins. Comparison of our data with the sequence of visna virus LV1-1, an antigenic variant derived from strain 1514, demonstrates that the rate of divergence has been about 1.7 x 10(-3) substitutions per nucleotide per year in vivo. This rate is orders of magnitude higher than that for most DNA genomes, but agrees well with estimates of the rate for HIV. A statistically significant cluster of mutations in the env gene appears to represent a hypervariable site and may correspond to the epitope responsible for the antigenic differences between 1514 and LV1-1. Analysis of the potential RNA folding pattern of the visna virus env gene shows that this hypervariable site falls within a region with little potential for intramolecular base pairing. This correlation of hypervariability with lack of RNA secondary structure is strengthened by the fact that it also holds for a hypervariable site in the env gene of HIV.     

The noncoding and surface envelope coding sequences of myeloblastosis-associated virus are respectively responsible for nephroblastoma development and renal hyperplasia.

The use of chimeric viruses allowed us to establish that myeloblastosis-associated virus long terminal repeat sequences are necessary and sufficient for induction of nephroblastoma in chickens and that the blastemal hyperplasia induced by env SU is not a prerequisite for tumor development but rather constitutes a predisposing stage.     

Discontinuous, conserved neutralization epitopes overlapping the CD4-binding region of human immunodeficiency virus type 1 gp120 envelope glycoprotein.

Monoclonal antibodies have been isolated from human immunodeficiency virus type 1 (HIV-1)-infected patients that recognize discontinuous epitopes on the gp120 envelope glycoprotein, that block gp120 interaction with the CD4 receptor, and that neutralize a variety of HIV-1 isolates. Using a panel of HIV-1 gp120 mutants, we identified amino acids important for precipitation of the gp120 glycoprotein by three different monoclonal antibodies with these properties. These amino acids are located within seven discontinuous, conserved regions of the gp120 glycoprotein, four of which overlap those regions previously shown to be important for CD4 recognition. The pattern of sensitivity to amino acid change in these seven regions differed for each antibody and also differed from that of the CD4 glycoprotein. These results indicate that the CD4 receptor and this group of broadly neutralizing antibodies recognize distinct but overlapping gp120 determinants.     

A single point mutation in the envelope gene is responsible for replication and XC fusion deficiency of the endogenous ecotropic C3H/He murine leukemia virus and for its repair in culture.

The molecular basis has been determined for differences in infectivity and XC phenotype of endogenous ecotropic murine leukemia virus of the low-leukemia mouse strain C3H/He, its relative in the high-leukemia mouse strain AKR, and highly infectious, XC-positive C3H virus variants selected in vitro. Endogenous ecotropic type C virus induced by iododeoxyuridine from the nontransformed C3H/10T1/2 cell line is XC negative and replication deficient. In contrast, viruses produced late after iododeoxyuridine induction in chemically transformed C3H/10T1/2 cells (MCA5) are XC positive and infectious. XC-negative viruses can be converted to XC-positive viruses by being grown in certain transformed cell lines. We have cloned the endogenous ecotropic provirus of C3H/He from MCA5 cells, which is XC negative and replication deficient, as well as two XC-positive C3H proviruses derived by in vitro conversion. Fragment exchange between the XC-negative molecular clone p110 and the XC-positive AKR virus clone p623 revealed that the defect in p110 lies 3' of the SalI site located in the pol region. Nucleotide sequencing established that the C3H p110 provirus was integrated within the R region of an endogenous VL30 long terminal repeat (LTR) in reverse orientation and that the virus differed from the infectious AKR p623 provirus by a point mutation, substituting Lys for Arg at the potential precursor cleavage site for gp70 and p15E. In vitro-converted XC-positive C3H proviral clones 3211 and 4211 are identical to XC-negative C3H p110, except that they have Arg at this site and the normal cleavage site is thus regenerated in these clones. The XC-negative C3H p110 was blocked in processing of Pr85env, whereas clones 3211 and 4211 had normal cleavage of the env precursor into gp70. Both the XC-negative C3H provirus and the in vitro-converted XC-positive C3H proviruses had a single copy of a 99-base-pair enhancer element in the LTR, whereas two copies of this sequence are present in the AKR proviral LTR. Substitution of Arg for Lys at the envelope precursor processing site of C3H p110 by site-directed mutagenesis is sufficient by itself to convert the virus to the XC-positive replication-competent phenotype. Thus, we have established that a single point mutation at the processing site of the envelope precursor protein Pr85 is responsible for the difference in the infectivity and XC phenotype of endogenous ecotropic murine leukemia virus from C3H/He and AKR mice and that the basis for in vitro conversion is a mutation at this site.     

B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins.

In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines.     

Residues of the human metapneumovirus fusion (F) protein critical for its strain-related fusion phenotype: implications for the virus replication cycle

The paramyxovirus F protein promotes fusion of the viral and cell membranes for virus entry, as well as cell-cell fusion for syncytium formation. Most paramyxovirus F proteins are triggered at neutral pH to initiate membrane fusion. Previous studies, however, demonstrated that human metapneumovirus (hMPV) F proteins are triggered at neutral or acidic pH in transfected cells, depending on the strain origin of the F sequences (S. Herfst et al., J. Virol. 82:8891-8895, 2008). We now report an extensive mutational analysis which identifies four variable residues (294, 296, 396, and 404) as the main determinants of the different syncytial phenotypes found among hMPV F proteins. These residues lie near two conserved histidines (H368 and H435) in a three-dimensional (3D) model of the pretriggered hMPV F trimer. Mutagenesis of H368 and H435 indicates that protonation of these histidines (particularly His435) is a key event to destabilize the hMPV F proteins that require low pH for cell-cell fusion. The syncytial phenotypes were reproduced in cells infected with the corresponding hMPV strains. However, the low-pH dependency for syncytium formation could not be related with a virus entry pathway dependent on an acidic environment. It is postulated that low pH may be acting for some hMPV strains as certain destabilizing mutations found in unusual strains of other paramyxoviruses. In any case, the results presented here and those reported by Schowalter et al. (J. Virol. 83:1511-1522, 2009) highlight the relevance of certain residues in the linker region and domain II of the pretriggered hMPV F protein for the process of membrane fusion.