Protective monoclonal antibodies define maturational and pH-dependent antigenic changes in Sindbis virus E1 glycoprotein

Monoclonal (MC) antibodies specific for either the EI or E2 glycoproteins of Sindbis virus (SIN) were used to probe for differences in the surface topography of SIN epitopes between infected cells and mature virions. Employing an enzyme-linked immunosorbent assay (ELISA) in which binding of individual peroxidase-labeled MC antibodies to immobilized (solid-phase) detergent-disrupted SIN was inhibited specifically by one or more unlabeled antibodies, viral epitopes could be grouped into six spatially distinct antigenic sites--five on E1, designated a through e, and one site on E2. All six sites were represented on the surfaces of SIN-infected cells as shown by the complement (C')-dependent lysis mediated by antibodies of the corresponding epitope specificities. In contrast, virus-neutralizing (NT) activity was restricted to antibodies specific for epitopes on E2 and on site c of E1, irrespective of the presence of added C' and an antiserum against mouse immunoglobulins. That E1 sites a, b, d, and e became inaccessible to antibody binding was shown by a competitive-inhibition ELISA. Whereas all MC antibodies were inhibited from binding to solid-phase SIN when premixed with detergent-treated virions, only those having NT activity could be competitively inhibited by intact virions. Sites E1-d and E1-e could be exposed not only by detergent disruption but also by lowering the virion pH from 7.2 to 6.0. These collective results indicate that a majority of immunologically relevant E1 epitopes present on SIN-infected cell surfaces become cryptic during SIN maturation and, except at low pH, remain undetectable on virion surfaces.     

Sindbis virus mutant ts20 of complementation group E contains a lesion in glycoprotein E2

A technique has been devised to readily obtain the entire structural protein region of Sindbis virus cloned into a plasmid vector. This method uses the fact that the nearest site for restriction enzyme HindIII to the 3' terminal poly(A) occurs at nucleotides 6266-6271 in the genomic RNA. Inserts extending from the poly(A) tract to this HindIII site are 5438 nucleotides long (excluding the poly A tract) and contain the entire 4106-nucleotide structural protein region. Using an oligo(dT)-tailed vector as a primer for first strand cDNA synthesis such clones could be obtained in high yield. We were interested in a precise determination of the mutation responsible for the temperature-sensitive phenotype of ts20, a mutant belonging to complementation group E which has a defect in the function of glycoprotein E2 at the nonpermissive temperature. Using this technique we have cloned and sequenced the structural protein region of ts20 and of several revertants and concluded that the mutation was a change from histidine to leucine at amino acid 291 of E2. Reversion to temperature insensitivity occurred by same site reversion to the parental nucleotide, restoring the original histidine as amino acid 291. Thus, complementation group E of Sindbis virus results from changes in glycoprotein E2 and together with previous results from our laboratory (Arias et al., 1983; Hahn et al., 1985) demonstrates that the three RNA+ complementation groups of Sindbis virus, C, D, and E, result from changes in the three structural proteins of the virus, capsid, glycoprotein E1, and glycoprotein E2, respectively.     

Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.     

应用噬菌体肽文库筛选mAb F3特异性结合肽

目的 应用噬菌体展示肽文库筛选可与汉坦病毒囊膜蛋白中和性单抗（mAb） F3特异性结合的配体肽。方法 以F3mAb为筛选配基，对噬菌体展示和随机12肽文加进行3轮生物亲和淘选；用夹心ELISA和竞争ELISA鉴定筛选克隆的结合特性，并进一步对阳性克隆进行序列测定和分析。结果 通过3轮生物淘选，能被抗体捕获的噬菌体克隆为21／22，ELISA测定显示，筛选到的噬菌体短肽能与F3mAb特异性结合。序列分析表明，7个阳性克隆氨基酸序列相同，均为－MHGPTKNQMWHT；同源性分析显示，该序列与HTNV／SEOV M蛋白G2区第750－759位氨基酸有较高的同源性。结论 本研究为基于表位水平的HFRSV特异性分子多肽疫苗的设计提供了重要的依据。     

Characterization of antibody responses against a neutralizing epitope on the glycoprotein E2 of classical swine fever virus

The sequence TAVSPTTLR is a conserved and linear neutralizing epitope on the glycoprotein E2 of classical swine fever virus. In this study, TAVSPTTLR-directed antibodies, induced either by virions or by an epitope-focused immunogen, were characterized. The results revealed that despite the same epitope specificity, the antibodies induced by different immunogens varied significantly both in the neutralizing test and in binding inhibition assays. This suggests that the protective immunity induced by this epitope is due to more than simply the epitope specificity and that this epitope might need essential contributions from its flanking context to induce functional epitope-specific antibodies.     

Sindbis virus variants from a persistently infected mosquito cell culture contain an altered E2 glycoprotein

Small plaque temperature-sensitive (SPTS) variants of Sindbis virus were isolated from persistently infected Aedes albopictus cells 73 days after infection with wild-type virus. The viral RNA and protein species synthesized in chick embryo fibroblast (CEF) cells infected by one variant, clone 73-2, were compared with their wild-type virus counterparts. T1 RNase fingerprints of ³²P-labeled intracellular 42 S and 26 S viral RNA species revealed that clone 73-2 virus RNA differed from wild-type virus RNA in four oligonucleotide spots. Three of these were located in the region of the genome coding for structural proteins. Analysis of the virus structural proteins by polyacrylamide gel electrophoresis indicated that clone 73-2 virus glycoprotein E2 had a higher electrophoretic mobility than wild-type virus E2. Analysis of the proteins synthesized in infected cells in the presence and absence of tunicamycin revealed that both the glycosylated and unglycosylated forms of clone 73-2 PE2 migrated slightly faster than their wild-type virus counterparts.     

Biochemical studies of the maturation of the small Sindbis virus glycoprotein E3

A small glycoprotein (E3) was purified from the culture fluid of Sindbis virus-infected primary chick embryo fibroblasts. Tryptic peptide mapping and pulse-chase studies verified that this protein was produced as a by-product of the cleavage of the precursor protein PE2 to produce the envelope glycoprotein E2. A 2600-fold purification was achieved via a procedure which used differential ethanol precipitation, gel filtration, ion-exchange chromatography, and affinity chromatography on a lentil lectin column. Amino acid composition analysis, N-terminal microsequencing, and labeling studies yielded information about the fine structure of E3 and its relationship to E2 and virion maturation. The N-terminal sequence of E3 is identical to that of PE2, including the result that 90% of the molecules appear to be blocked. The first 19 amino acids are uncharged and presumably serve as the signal sequence for the insertion of PE2 into the membrane of the endoplasmic reticulum, but this sequence is unusual in that it is not immediately cleaved from PE2 and is glycosylated at the asparagine at position 14. The two residues at the C-terminus of E3, Lys-Arg, are removed during or shortly after cleavage from PE2. Labeling studies imply that, although the PE2----E2 + E3 cleavage is necessary for virion budding, these two events are not closely coupled. E3 is cleaved and released into the culture fluid under conditions where virions do not bud, and the kinetics of the appearance of E3 in the culture fluid and E2 in virions are quite dissimilar. The maturation of E3 is discussed as it relates to the processing of cellular membrane and secretory glycoproteins.     

Phylogenetic diversity of GB virus C at the antigenic site of E2 protein

Amino acids at position 267–298 in E2 protein of GB virus C (GBV-C) were recognized as the antigenic site, and peptides within the region were previously reported to have inhibitory effect on HIV entry. The effect of sequence variability between different types of GBV-C on the antigenic region of the E2 protein was studied by using phylogenetic analysis. Eighty-one unique sequences encompassing this region derived from all seven GBV-C genotypes were compared to each other in this study. The results showed that GBV-C E2 antigenic nucleotide sites belonging to genotype 3 clustered together regardless of synonymous or nonsynonmous sites in the region, whereas, GBV-C E2 antigenic nucleotide sites belonging to the other 6 genotypes clustered together regardless of genotypes. Despite the fact that GBV-C genotypes might confer different degree of ‘protection’ against HIV, the lack of clustering as a unique group based on the amino acid differences in the antigenic site among the six genotypes suggested some other genomic regions or secondary structure of E2 protein might have played a crucial role in determining the variable protection effect of GBV-C on HIV infection.     

Antigenic and genetic characterization of Sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein E2

The Sindbis virus mutant SB-RL, in contrast to its parent, Sindbis strain AR339 (SB), is attenuated in neonatal mice, has an increased rate of penetration in tissue culture cells, and is more sensitive to neutralization by E2-specific monoclonal antibodies (MCAbs) R6 and R13. These phenotypic differences are controlled by substitution of an arginine for serine at amino acid 114 of the E2 glycoprotein. To explore these relationships further, MCAb R6 and R13 neutralization escape mutants of both SB and SB-RL were isolated and characterized. All mutants bound both MCAb R6 and R13 significantly less effectively in ELISA, and were more resistant to complement-mediated neutralization than their respective parental strains. Single coding changes in the E2 glycoprotein gene of each 11 mutants were identified. SB/R6, SB/R13, and SB-RL/R13 mutants contained a mutation at either E2 codon 96 or 159. SB-RL/R6 mutants contained changes at E2 codon 62, 96, or 159. These coding changes included two intragenic suppressor mutations. Mutation of E2 codon 159 from lysine to glutamate or codon 62 from asparagine to aspartate suppressed the attenuated phenotype conferred by E2 arginine 114 in SB-RL. However, only the change at E2 codon 62 significantly suppressed the rapid penetration phenotype of SB-RL. Mutation in E2 codon 96 of SB, replacing tyrosine with histidine, reduced the virulence of SB for neonatal mice but had no effect on penetration of cultured cells. Therefore, mutation in E2 codons 62, 96, 114, or 159 affected both virulence in animals and the binding or biological activity of these E2c-specific MCAbs. These results suggest that an E2 antigenic site (E2c), defined by MCAbs R6 and R13, is conformational in nature and may constitute a surface domain on Sindbis virions important for virulence in neonatal mice.     

Single and multiple deletions in the transmembrane domain of the Sindbis virus E2 glycoprotein identify a region critical for normal virus growth

Sindbis virus is composed of two nested T = 4 icosahedral protein shells containing 240 copies each of three structural proteins: E1, E2, and Capsid in a 1:1:1 stoichiometric ratio. E2 is a 423 amino acid glycoprotein with a membrane spanning domain 26 amino acids in length and a 33 amino acid cytoplasmic endodomain. The interaction of the endodomain with the nucleocapsid is an essential step in virus maturation and directs the formation of the outer protein shell as envelopment occurs. A previous study had determined that deletions in the transmembrane domain could affect virus assembly and infectivity (Hernandez et al., 2003. J. Virol. 77 (23), 12710-12719). Unexpectedly, a single deletion mutant (from 26 to 25 amino acids) resulted in a 1000-fold decrease in infectious virus production while another deletion of eight amino acids had no affect on infectious virus production. To further investigate the importance of these mutants, other single deletion mutants and another eight amino acid deletion mutant were constructed. We found that deletions located closer to the cytoplasmic (inner leaflet) of the membrane bilayer had a more detrimental effect on virus assembly and infectivity than those located closer to the luminal (outer leaflet) of the membrane bilayer. We also found that selective pressure can restore single amino acid deletions in the transmembrane domain but not necessarily to the wild type sequence. The partial restoration of an eight amino acid deletion (from 18 to 22 amino acids) also partially restored infectious virus production. The amount of infectious virus produced by this revertant was equivalent to that produced for the four amino acid deletion produced by site directed mutagenesis. These results suggest that the position of the deletion and the length of the C terminal region of the E2 transmembrane domain is vital for normal virus production. Deletion mutants resulting in decreased infectivity produce particles that appear to be processed and transported correctly suggesting a role involved in virus entry.     

Characterization of neutralizing domains on varicella-zoster virus glycoprotein E defined by monoclonal antibodies

Summary. The genome of varicella-zoster virus (VZV), encodes at least six glycoproteins and they elicit the formation of complement-independent, complement-dependent, and non-neutralizing antibody responses. We have used our library of MAbs to VZV glycoprotein E (gE) to determine the neutralizing epitopes of gE, and shown that gE has 3 distinct neutralizing domains. In this report we have used the baculovirus expression system to identify the antigenic domains of gE. We have generated 3 recombinant baculoviruses, expressing the full-length gE and two overlapping truncated forms (the amino-terminal and the carboxy-terminal) of gE. By immuno-fluorescence and immunoblotting we have explored the physical interactions of Mabs to gE on these constructs. Our panel of MAbs revealed 3 district antigenic domains on gE. All MAbs reacted with the full-length gE; MAbs with high titered complement-dependent neutralizing activities reacted with the N-terminal truncated gE; MAbs with low titered or non-neutralizing activities reacted with the C-terminal truncated gE; MAbs with complement-enhanced neutralizing activities reacted with both truncated constructs. However, although the antibody binding in immunofluorescence and immunoblotting was carried out under denatured conditions, whereas the neutralization is under non-denatured conditions, still the antigenic mapping was similar in both conditions. Received April 24, 1996 Accepted July 29, 1996     

E1 glycoprotein of rubella virus carries an epitope that binds a neutralizing antibody

We identified by immunoprecipitation and Western blot analysis, using a monoclonal antibody that neutralizes rubella virus, that E1 glycoprotein carries an epitope linked with neutralization. Glycosidase treatment of virus does not prevent blotting of this monoclonal antibody with the E1 glycoprotein, dissociating this epitope from the hemagglutination epitope which is linked with the oligosaccharide side chains. We also investigated by Western blot analysis human serum reactivity toward E1 glycoprotein and the two other structural proteins of rubella virus, E2 and C: all positive sera detected E1 and C, irrespective of their titers, indicating the importance of glycoprotein E1 in immunity. Frequent lack of reactivity against E2 might suggest that this glycoprotein is either less exposed or less immunogenic.     

Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus.

Current serological assays using whole rubella virus (RV) as a target antigen for detecting RV-specific antibodies fail to define specific RV proteins and antigenic determinants such as hemagglutinin (HA) and virus-neutralizing (VN) epitopes of rubella virus. A panel of E1 deletion mutants and a subset of E1-specific monoclonal antibodies (MAb) were used for the initial analysis of HA and VN epitopes of E1 glycoprotein. A peptide region (E1(193) to E1(269)) was found to contain HA and VN epitopes. Using both overlapping synthetic peptides and truncated fusion proteins within this region, the HA epitope defined by MAb 3D9F mapped to amino acid residues E1(214) to E1(240), while two VN epitopes defined by MAb 21B9H and MAb 16A10E mapped to amino acid residues E1(214) to E1(233) and E1(219) to E1(233), respectively. The epitopes defined in this study are recognized by antibody whether or not the epitopes are glycosylated.     

Heterogeneity of the antigenic site responsible for the induction of neutralizing antibodies in infectious bursal disease virus

Summary Using neutralizing monoclonal antibodies, three categories of escape mutants were selected from a stock of wild-type infectious bursal disease virus (IBDV). Additional mutants were found, where alterations coexisted in two or three of these epitopes. Although each group of mutants had a distinct reaction pattern with neutralizing monoclonal antibodies, all types of mutants were neutralized by convalescent chicken sera to the same extent. In spite of the lack of homogeneity in these antigenic sites located on IBDV structural polypeptide VP2, all neutralizing monoclonal antibodies reacted with epitopes in extracts prepared from the bursa of Fabricius from animals that had died during recent outbreaks of infectious bursal disease in the F.R.G. and Africa. Since binding to VP2 of the escape mutants, demonstrable by immunoprecipitation, correlated with the neutralizing capacity of these antibodies, a combined immunoprecipitation-immunoblotting technique was established as equivalent for a neutralization assay. The results of our experiments indicate that IBDV did not undergo a major antigenic variation in these two areas of Europe and Africa. The significance of protein conformation for the interaction of VP2 with neutralizing antibodies was underlined by the finding that renatured VP2 was capable of binding neutralizing antibodies; the antibodies induced in animals by immunization with this protein, however, were not neutralizing.     

Epitopes of the G1 glycoprotein of La Crosse virus form overlapping clusters within a single antigenic site

Antigenic sites on the G1 glycoprotein of La Crosse bunyavirus were defined by constructing a panel of neutralizing and nonneutralizing monoclonal antibodies (F. Gonzalez-Scarano, R. E. Shope, C. H. Calisher, and N. Nathanson (1982), Virology 120, 42-53). To analyze the relationship between the individual epitopes delineated by monoclonal antibodies, 11 neutralizing antibodies were used to select variant viruses. These variant viruses were tested against the panel of anti-G1 protein monoclonal antibodies by neutralization and by ELISA. The neutralization tests assigned the 11 epitopes to five groups, consisting of 6, 2, 1, 1, and 1 epitopes. ELISA tests gave a similar pattern, but also demonstrated interrelationships between four of the five epitope groups, suggesting that there may be a single immunodominant antigenic site on the G1 protein. When eight nonneutralizing anti-G1 monoclonal antibodies were tested in ELISA, they fell into three of the five epitope groups defined by neutralization; there was no evidence of a separate noneutralizing antigenic site on the G1 protein.     

Antigenic mimicking with cysteine-based cyclized peptides reveals a previously unknown antigenic determinant on E2 glycoprotein of classical swine fever virus

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. The conformational epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779 (Chang et al., 2010a). To mimic the conformational epitopes, a set of synthetic cyclized peptides spanning the B/C domains of E2 were used to react with monoclonal antibodies (mAbs) against E2 and with swine anti-CSFV polyclonal sera. All antibodies recognized a highest common element, ⁷⁵³RYLASLHKKALPTSV⁷⁶⁷, on the double-looped peptides. This epitope region has not been revealed previously in the literature. Both substitution-scanning of residues ⁷⁵³RYLASLHKKALPTSV⁷⁶⁷ on a double-looped peptide and site-directed mutagenesis of expressed E2 demonstrated that residues ⁷⁶¹K, ⁷⁶³L and ⁷⁶⁴P were critical for the reactivity with mAbs. In addition, the up- and downstream residues ⁷⁵³R, ⁷⁵⁴Y, ⁷⁵⁵L and ⁷⁶⁵T were also crucial. Alignment showed that this stretch of amino acids was relatively conserved among various CSFVs. Thus, we identified a motif ⁷⁵³RYLASLHKKALPT⁷⁶⁵, which may be part of group-specific antigen and important for the structural integrity of conformational epitope recognition.     

Monoclonal antibodies identify a strain-specific epitope on the HN glycoprotein of Newcastle disease virus strain Australia-Victoria

A panel of monoclonal antibodies raised against the hemagglutinin-neuraminidase glycoprotein (HN) of the Australia-Victoria strain of Newcastle disease virus has been used to compare that strain and eight other strains of the virus. The ability of the antibodies to neutralize infectivity, inhibit hemagglutination and neuraminidase, and bind to purified virions in solid-phase radioimmunoassays was determined for each strain. Of the four antigenic sites delineated by these antibodies on the HN of the homologous strain, site 1 (that with the greatest neutralizing susceptibility), is apparently conserved in all the strains tested as revealed by neutralization assays. The least neutralizing site, number 4, is also conserved in most of the strains tested. Site 2, which lies at or near the neuraminidase site, appears to be conserved in the avirulent strains but not in the virulent strains. An antibody to site 3 is unable to bind to a significant extent to any of the heterologous strains tested, and thus recognizes a strain-specific epitope. Inhibition of hemagglutination and neuraminidase by antibodies to each site were also examined and the results suggest that antibodies to sites 1 and 2 may distinguish virulent and avirulent strains at least with respect to these functions.     

The effect of ribonuclease on the replicative forms of Sindbis virus RNA

Summary Three species of double-stranded RNA, designated RF I, RF II, and RF III in order of decreasing size (25), are produced by ribonuclease treatment of extracts of chicken embryo cells infected for 6 hours with Sindbis virus. Only one class of replicative form RNA is present in extracts not treated with ribonuclease; this class contains some molecules which can be enzymatically cleaved to produce the other two replicative forms. At a low level of enzyme (0.001 µg/ml) the major species obtained was RF I, the replicative form of the genome. When the enzyme concentration was increased 10-, 100-, and 1000-fold, there was a progressive increase in the proportions of RF's II and III and a concomitant decrease in the proportion of RF I. The generation of RF's II and III by nuclease resulted in the ratio expected for these two species if they are produced by cleavage of RF I-like molecules. In preparations of isolated double-stranded RNA, only RF I and replicative intermediate RNA were present. Mild nuclease treatment of these preparations converted the replicative intermediates primarily to RF I. Higher enzyme levels generated greater proportions of RF II and RF III, but RF I-like molecules were the major source for these increased proportions. Treatment of the isolated naturally occurring replicative form with 0.01 µg of ribonuclease per ml cleaved some molecules migrating as RF I during gel electrophoresis into molecules which migrated as RF II and RF III.With 5 Figures     

The antigenic structure of glycoprotein E2 in the Venezuelan equine encephalomyelitis virus studied using rat monoclonal antibodies]

A collection of 21 rat hybridomas secreting high-affinity monoclonal antibodies to Venezuelan equine encephalomyelitis (VEE) virus was generated. Using a panel of 15 monoclonal antibodies to glycoprotein E2, the antigenic structure of this protein of VEE strains TC-83 and 230 was studied. A competitive radioimmunoassay suggested a new map of the antigenic structure of glycoprotein E2 in which 5 sites including 11 epitopes of monoclonal antibody binding were distinguished. Antibody to E2-2 site neutralized virus infectivity and blocked hemagglutination test and antibody to E2-3 site could only block hemagglutination. Antibodies to other E2 protein sites lacked any biological activity.