Identification of amino acids recognized by syncytium-inhibiting and neutralizing monoclonal antibodies to the human parainfluenza type 3 virus fusion protein.

Neutralizing monoclonal antibodies specific for the fusion (F) glycoprotein of human parainfluenza type 3 virus (PIV3) were used to select neutralization-resistant antigenic variants. Sequence analysis of the F genes of the variants indicated that their resistance to antibody binding, antibody-mediated neutralization or to both was a result of specific amino acid substitutions within the neutralization epitopes of the F1 and F2 subunits. Comparison of the locations of PIV3 neutralization epitopes with those of Newcastle disease and Sendai viruses indicated that the antigenic organization of the fusion proteins of paramyxoviruses is similar. Furthermore, some of the PIV3 epitopes recognized by syncytium-inhibiting monoclonal antibodies are located in an F1 cysteine cluster region which corresponds to an area of the measles virus F protein involved in fusion activity.     

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.     

Expression of measles virus antigens in Streptococcus gordonii

The measles virus proteins haemagglutinin (HA) and fusion protein (F), which together mediate attachment and penetration of the virus in the host cell and can elicit production of neutralising antibodies in the course of natural infection were expressed in the vaccine vector Streptococcus gordonii, a Gram-positive bacterium normally present in the human oral cavity. HA and F were expressed as fusion proteins attached to the bacterial surface, and were both found to be immunogenic when the recombinant S. gordonii were inoculated subcutaneously in mice.     

麻疹病毒融合蛋白(F)和血凝素(HA)在痘苗病毒中的表达

将麻疹病毒F和HA基因插入到痘苗病毒中,分别处于痘苗启动子P7.5与P11控制下,获得重组病毒vLmF和vCmH。用抗F多肽抗体和HA单抗进行ELISA检测,结果表明,两株重组病毒均能表达相应的麻疹蛋白。蛋白印迹显示重组病毒表达产物在分子大小,蛋白切割和糖化方面与麻疹病毒糖蛋白一致。两株重组病毒分别免疫家兔都能产生较高滴度的麻疹抗体,这些抗体具有中和作用和血溶抑制作用。此外,vCmH产生的抗体还具有血凝抑制作用。     

Participation of two fusion peptides in measles virus-induced membrane fusion: emerging similarity with other paramyxoviruses

Paramyxoviruses penetrate into their host cells by fusing their membranes with the plasma membrane. The hydrophobic N terminus of their F1 protein, termed the 'fusion peptide', is thought to be responsible for this process. Recently, an additional internal fusion peptide, homologous in sequence to the N-terminal fusion peptide of HIV-1, was identified in the Sendai virus F1 protein. Here, we investigated whether the presence of an additional internal fusion peptide is a general feature of paramyxoviridae. To this end, we synthesized and structurally and functionally characterized three peptides: (i) MV-197, which corresponds to an internal segment of the F1 protein of the measles virus (amino acids 197-225), homologous in location but not in sequence to the internal fusion peptide of the Sendai virus, (ii) Mu-MV-197, a randomized version of MV-197, and (iii) the 33 amino acid N-terminal fusion peptide of the measles virus. Remarkably, only MV-197 was highly fusogenic toward large unilamellar vesicles composed of either zwitterionic (phosphatidylcholine or phosphatidylcholine/sphingomyelin/cholesterol, a composition similar to that of human cell membranes) or negatively charged phospholipids. Binding experiments, circular dichroism spectroscopy in phospholipid membranes, and homo energy-transfer studies with fluorescently labeled peptides revealed that MV-197 adopts a predominant alpha-helical structure and shares properties similar to those reported for known fusion peptides. These results suggest that the presence of two fusion peptides in the F1 protein is a general feature of paramyxoviruses.     

Antigenic modulation induced by monoclonal antibodies: antibodies to measles virus hemagglutinin alters expression of other viral polypeptides in infected cells

Polyclonal antibody to measles virus can have profound effects on external (outer plasma membrane) as well as internal (cytoplasmic) viral polypeptides expressed in infected cells. The process, termed "antibody-induced antigenic modulation," was further investigated by using monoclonal antibody to several viral polypeptides. Four monoclonal antibodies against the viral hemagglutinin had the ability to decrease the expression of the phosphoprotein, fusion, and membrane protein. A monoclonal antibody to the nucleocapsid protein did not cause these changes. The observed decreases were not due to preferential degradation of viral polypeptides as determined by pulse-chase experiments. Our results indicate that a specific signal to an epitope on the plasma membrane (monoclonal antibody measles virus hemagglutinin) can alter the expression of measles virus phosphoprotein and membrane protein, both polypeptides present in the cytoplasm of infected cells.     

Characterization of a Natural Mutation in an Antigenic Site on the Fusion Protein of Measles Virus That Is Involved in Neutralization

Although measles virus is an antigenically monotypic virus, nucleotide sequence analysis of the hemagglutinin and nucleoprotein genes has permitted the differentiation of a number of genotypes. In contrast, the fusion (F) protein is highly conserved; only three amino acid changes have been reported over a 40-year period. We have isolated a measles virus strain which did not react with an anti-F monoclonal antibody (MAb) which we had previously shown to be directed against a dominant antigenic site. This virus strain, Lys-1, had seven amino acid changes compared with the Edmonston strain. We have shown that a single amino acid at position 73 is responsible for its nonreactivity with the anti-F MAb. With the same MAb, antibody-resistant mutants were prepared from the vaccine strain. A single amino acid change at position 73 (R→W) was observed. The possibility of selecting measles virus variants in vaccinated populations is discussed.     

Measles virus fusion: role of the cysteine-rich region of the fusion glycoprotein.

Measles virus (MV) fusion requires the participation of both the fusion (F) and hemagglutinin (H) glycoproteins. The canine distemper virus fusion protein (CDVF) cannot substitute for the measles virus fusion protein (MVF) in this process. Introduction of restriction enzyme sites into the cDNAs of CDVF and MVF by site-directed mutagenesis facilitated the production of chimeric F proteins which were tested for their capacity to give fusion when coexpressed with MVH. Fusion resulted when the amino-terminal half of the MVF cysteine-rich region was transferred to CDVF.     

Development of antibody to measles virus polypeptides during complicated and uncomplicated measles virus infections.

Immune precipitation of 181 sera from 152 patients with natural measles was studied to determine the temporal course and frequency of antibody responses to nucleocapsid, fusion, hemagglutinin, and matrix proteins of measles virus. Large amounts of antibody to nucleocapsid protein developed in all patients by day one of the rash. Antibody to hemagglutinin and fusion proteins developed in all patients over the next 3 weeks, the former to high levels and the latter to low levels. Antibody to matrix protein developed to very low levels and was detectable in only 41% of the patients; this poor response to matrix protein was not correlated with the age of the patient or the acute neurological complications of measles.     

The paramyxovirus fusion protein C-terminal region: mutagenesis indicates an indivisible protein unit

Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Fusion is mediated by the viral fusion (F) protein, and it undergoes large irreversible conformational changes to cause membrane merger. The C terminus of PIV5 F contains a membrane-proximal 7-residue external region (MPER), followed by the transmembrane (TM) domain and a 20-residue cytoplasmic tail. To study the sequence requirements of the F protein C terminus for fusion, we constructed chimeras containing the ectodomain of parainfluenza virus 5 F (PIV5 F) and either the MPER, the TM domain, or the cytoplasmic tail of the F proteins of the paramyxoviruses measles virus, mumps virus, Newcastle disease virus, human parainfluenza virus 3, and Nipah virus. The chimeras were expressed, and their ability to cause cell fusion was analyzed. The chimeric proteins were variably expressed at the cell surface. We found that chimeras containing the ectodomain of PIV5 F with the C terminus of other paramyxoviruses were unable to cause cell fusion. Fusion could be restored by decreasing the activation energy of refolding through introduction of a destabilizing mutation (S443P). Replacing individual regions, singly or doubly, in the chimeras with native PIV5 F sequences restored fusion to various degrees, but it did not have an additive effect in restoring activity. Thus, the F protein C terminus may be a specific structure that only functions with its cognate ectodomain. Alanine scanning mutagenesis of MPER indicates that it has a regulatory role in fusion since both hyperfusogenic and hypofusogenic mutations were found.     

Production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody.

The severe disease atypical measles occurred when individuals immunized with a poorly protective inactivated vaccine contracted measles, and was postulated to be due to a lack of fusion-inhibiting antibodies. Here, rhesus macaques immunized with formalin-inactivated measles vaccine developed transient neutralizing and fusion-inhibiting antibodies, but no cytotoxic T-cell response. Subsequent infection with measles virus caused an atypical rash and pneumonitis, accompanied by immune complex deposition and an increase in eosinophils. Fusion-inhibiting antibody appeared earlier in these monkeys than in non-immunized monkeys. These data indicate that atypical measles results from previous priming for a nonprotective type 2 CD4 T-cell response rather than from lack of functional antibody against the fusion protein.     

Contribution of measles virus fusion protein in protective immunity: anti-F monoclonal antibodies neutralize virus infectivity and protect mice against challenge.

To study the contribution of the measles virus fusion (F) protein in the immune response, anti-F monoclonal antibodies were prepared by using a vaccinia-measles virus F recombinant. In contrast to previously described anti-F monoclonal antibodies, these antibodies not only neutralized virus infectivity and inhibited fusion but also passively protected mice. Since these monoclonal antibodies recognize a configurational epitope, presentation of the antigen during infection may play an important role in the immune response. These factors are discussed in relation to vaccination.     

Immunoglobulin g antibody-mediated enhancement of measles virus infection can bypass the protective antiviral immune response

Antibodies to viral surface glycoproteins play a crucial role in immunity to measles by blocking both virus attachment and subsequent fusion with the host cell membrane. Here, we demonstrate that certain immunoglobulin G (IgG) antibodies can also enhance the entry of measles virus (MV) into monocytes and macrophages. Antibody-dependent enhancement of infectivity was observed in mouse and human macrophages using virions opsonized by a murine monoclonal antibody against the MV hemagglutinin (H) glycoprotein, polyclonal mouse anti-MV IgG, or diluted measles-immune human sera. Neither H-specific Fab fragments nor H-specific IgM could enhance MV entry in monocytes or macrophages, indicating involvement of a Fc γ receptor (FcγR)-mediated mechanism. Preincubation with an anti-fusion protein (anti-F) monoclonal antibody or a fusion-inhibitory peptide blocked infection, indicating that a functional F protein was required for viral internalization. Classical complement pathway activation did not promote infection through complement receptors and inhibited anti-H IgG-mediated enhancement. In vivo, antibody-enhanced infection allowed MV to overcome a highly protective systemic immune response in preimmunized IfnarKo-Ge46 transgenic mice. These data demonstrate a previously unidentified mechanism that may contribute to morbillivirus pathogenesis where H-specific IgG antibodies promote the spread of MV infection among FcγR-expressing host cells. The findings point to a new model for the pathogenesis of atypical MV infection observed after immunization with formalin-inactivated MV vaccine and underscore the importance of the anti-F response after vaccination.     

C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein

The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus, and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the virus-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in the extent of cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F protein fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in the extent of fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of addition of the HA tag varied with other fusion proteins, as parainfluenza virus 5 F-HA showed a decreased level of surface expression and no stimulation of fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope-tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in the modulation of the membrane fusion reaction promoted by these viral glycoproteins.     

Effect of epitope position on neutralization by anti-human immunodeficiency virus monoclonal antibody 2F5

The membrane-proximal region of the human immunodeficiency virus type 1 (HIV-1) transmembrane protein (TM) is critical for envelope (Env)-mediated membrane fusion and contains the target for broadly reactive neutralizing antibody 2F5. It has been proposed that 2F5 neutralization might involve interaction of its long, hydrophobic, complementarity-determining region (CDR) H3, with adjacent viral membrane. Using Moloney murine leukemia virus (MLV) as a tool, we examined the effect of epitope position on 2F5 neutralization. When the 2F5 epitope was inserted in the proline-rich region of MLV Env surface protein (SU), 2F5 blocked cell fusion and virus infection, whereas MLV with a hemagglutinin (HA) epitope at the same position was not neutralized by anti-HA, even though the antibodies bound their respective Envs on the surface of infected cells and viruses equally well. When the 2F5 epitope was inserted in the MLV Env TM at a position comparable to its natural position in HIV-1 TM, 2F5 antibody blocked Env-mediated cell fusion. Epitope position had subtle effects on neutralization by 2F5: the antibody concentration for 50% inhibition of cell fusion was more than 10-fold lower when the 2F5 epitope was in SU than in TM, and inhibition was less complete at high concentrations of antibody; we discuss possible explanations for these effects of epitope position. Since membrane proximity was not required for neutralization by 2F5 antibody, we speculate that the CDR H3 of 2F5 contributes to neutralization by destabilizing an adjacent protein rather than by inserting into an adjacent membrane.     

Measles Virus Fusion Machinery Activated by Sialic Acid Binding Globular Domain

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.     

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.     

Engineered serine protease inhibitor prevents furin-catalyzed activation of the fusion glycoprotein and production of infectious measles virus.

We have identified the major cellular endoprotease that activates the fusion (F) glycoprotein of measles virus (MV) and have engineered a serine protease inhibitor (serpin) to target the endoprotease and inhibit the production of infectious MV. The F-protein precursor of MV was not cleaved efficiently into the mature F protein in human colon carcinoma cells lacking functional furin, indicating that furin is the major enzyme responsible for activation of the MV F protein. A human serpin alpha 1-antitrypsin variant was engineered to specifically inhibit furin. When expressed from a recombinant vaccinia virus in primate cells infected by MV, the engineered serpin (alpha 1-PDX) specifically inhibited furin-catalyzed cleavage of the F-protein precursor without affecting synthesis of other MV proteins. We generated human glioma cells stably expressing alpha 1-PDX. MV infection in these cells did not result in syncytia. The infected cells produced all the MV proteins, but the F-protein precursor remained largely uncleaved. This did not prevent virus assembly. However, the released virions contained inactive F-protein precursor rather than mature F protein, and infectious-virus titers were reduced by 3 to 4 orders of magnitude. These results show that a mature F protein is not required for the assembly of MV but is crucial for virus infectivity. The engineered serpin may offer a novel molecular antiviral approach against MV.