Preliminary characterization of an epitope involved in neutralization and cell attachment that is located on the major bovine rotavirus glycoprotein.

The 38,200-molecular weight (unreduced)/41,900-molecular-weight (reduced) glycoprotein of bovine rotavirus, isolate C486, was identified as the major neutralizing antigen. This glycoprotein as well as the corresponding glycoprotein of another bovine rotavirus serotype also specifically attached to cell monolayers under normal conditions for virus adsorption in vitro. Further support for this glycoprotein being directly responsible for virus attachment to cells was that (i) infectious virus of both serotypes could compete with the C486 glycoprotein for cell surface receptors, and (ii) neutralizing monospecific antiserum and neutralizing monoclonal antibodies directed toward the glycoprotein could block this virus-cell interaction. Preliminary epitope mapping of the glycoprotein with monoclonal antibodies further localized the neutralization-adsorption domain to a peptide with an approximate molecular weight of 14,000. The effect of two protein modifications, glycosylation and disulfide bridging, on the reactivity of this peptide with antibodies and cell surface receptors was investigated. It was demonstrated that, whereas glycosylation did not appear to affect these reactivities, disulfide bridging seemed to be essential.     

Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein.

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.     

Rotavirus neutralizing protein VP7: antigenic determinants investigated by sequence analysis and peptide synthesis.

The rotavirus neutralizing antigen, VP7, is a 37,000-molecular-weight glycoprotein which is a major component of the outer shell of the virion. The amino acid sequence of VP7 for strain S2 (human serotype 2) and Nebraska calf diarrhea virus (bovine serotype) has been inferred from the nucleic acid sequence of cloned copies of genomic segment nine. Comparison of the amino acid sequences of these two VP7 proteins with those already determined for other rotavirus strains reveals extensive sequence conservation between serotypes with clusters of amino acid differences sited predominantly in hydrophilic domains of the protein. Six peptides have been synthesized that span the hydrophilic regions of the molecule. Antisera to these peptides both recognize the respective homologous peptides in a solid-phase radioimmunoassay and bind to denatured VP7 in a Western blot. However, none of the antisera either recognize virus or exhibit significant neutralizing activity, indicating that these peptide sequences are not available on the surface of the virus.     

Cell-free translation of bovine viral diarrhea virus RNA.

Bovine viral diarrhea virus RNA was translated in a reticulocyte cell-free protein synthesizing system. The purified, 8.2-kilobase, virus-specific RNA species was unable to serve an an efficient message unless it was denatured immediately before translation. In this case, several polypeptides, ranging in molecular weight from 50,000 to 150,000 and most of which were immunoprecipitated by bovine viral diarrhea virus-specific antiserum, were synthesized in vitro. When polyribosomes were used to program cell-free synthesis, mature viral 80,000- and 115,000-molecular-weight proteins were detected; no precursor to the viral 55,000-molecular-weight glycoprotein was noted. The implications of these results with respect to virus-specific protein synthesis are discussed.     

Resistance to neutralization by broadly reactive antibodies to the human immunodeficiency virus type 1 gp120 glycoprotein conferred by a gp41 amino acid change.

A neutralization-resistant variant of human immunodeficiency virus type 1 (HIV-1) that emerged during in vitro propagation of the virus in the presence of neutralizing serum from an infected individual has been described. A threonine-for-alanine substitution at position 582 in the gp41 transmembrane envelope glycoprotein of the variant virus was responsible for the neutralization-resistant phenotype (M.S. Reitz, Jr., C. Wilson, C. Naugle, R. C. Gallo, and M. Robert-Guroff, Cell 54:57-63, 1988). The mutant virus also exhibited reduced sensitivity to neutralization by 30% of HIV-1-positive sera that neutralized the parental virus, suggesting that a significant fraction of the neutralizing activity within these sera can be affected by the amino acid change in gp41 (C. Wilson, M. S. Reitz, Jr., K. Aldrich, P. J. Klasse, J. Blomberg, R. C. Gallo, and M. Robert-Guroff, J. Virol. 64:3240-3248, 1990). It is shown here that the change of alanine 582 to threonine specifically confers resistance to neutralizing by antibodies directed against both groups of discontinuous, conserved epitopes related to the CD4 binding site on the gp120 exterior envelope glycoprotein. Only minor differences in binding of these antibodies to wild-type and mutant envelope glycoproteins were observed. Thus, the antigenic structure of gp120 can be subtly affected by an amino acid change in gp41, with important consequences for sensitivity to neutralization.     

Antibody to Viruses Affecting Cattle in Commercial Tissue Culture Grade Fetal Calf Serum

Commercial fetal calf serum (FCS) for tissue culture use was tested for neutralizing activity against several viruses which affect cattle. Certain lots of FCS contained no neutralizing activity, whereas other lots contained neutralizing activity to several viruses. It was concluded that the neutralizing activity found in certain lots of sera was due to specific antibody and that its presence could be most easily explained by the contamination of the FCS with serum from postcolostral bovine serum. A nonantibody inhibitor to vesicular stomatitis virus was also found at low levels in most lots of serum. Because those sera which had antibody had antibody to several viruses, it was suggested that the use of the micro-serum neutralization test with a few bovine viruses which are widespread in the bovine population should be satisfactory to detect FCS which was contaminated with postcolostral bovine serum.     

A predominant group-specific neutralizing epitope of human immunodeficiency virus type 1 maps to residues 342 to 511 of the envelope glycoprotein gp120.

Recombinant native human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp160 and gp120 (residues 1 to 511) expressed in insect cells quantitatively adsorbed the group-specific neutralizing antibodies found in human sera. However, these antibodies were not adsorbed by envelope fragment 1 to 471 or 472 to 857 or by both fragments sequentially, even though together they add up to the full-length gp160 sequence. A hybrid envelope glycoprotein was constructed with residues 342 to 511 of the HIV-1 sequence and residues 1 to 399 of the simian immunodeficiency virus type 1 sequence to vary the HIV-1 sequence while preserving its conformation. This hybrid glycoprotein quantitatively adsorbed human neutralizing antibodies, while native simian immunodeficiency virus type 1 envelope glycoprotein did not. These results identify a new neutralizing epitope that depends on conformation and maps to residues 342 to 511 of gp120. It overlaps the extended CD4-binding site but is distinct from the V3 loop described previously (K. Javaherian et al., Proc. Natl. Acad. Sci. USA 86:6768-6772, 1989; J. R. Rusche et al., Proc. Natl. Acad. Sci. USA 85:3198-3202). Since it is conserved among diverse HIV-1 isolates, this new epitope may be a suitable target for future vaccine development.     

The transfer of J blood-group activity from J-containing bovine serum to J-negative erythrocytes.

The J blood-group activity of bovine serum is contained both in a lipid and in a nonlipid fraction. This is also true for calf serum. It demonstrated that the J determinant is transferred from a serum protein onto the erythrocyte membrane by incubation in vitro. Even though the donor of J activity is a lipid-free serum protein (probably a glycoprotein), the transferred J activity is detectable only in the lipid fraction of erythrocytes. Thus, the J determinant (probably a carbohydrate unit) must have been detached from a serum glycoprotein and transferred to a lipidic receptor (probably a glycosphingolipid) at the erythrocyte membrane. It is suggested than an enzyme system located in or at the erythrocyte membrane is responsible for the transfer of J substance. The transfer of J substance is inhibited by a polar lipid present in bovine serum.     

Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F.     

Plaque size phenotype as a selectable marker to generate vaccinia virus recombinants.

In this report, we provide a new method for selection of vaccinia virus recombinants expressing foreign genes. The method is based on the use of the gene encoding the viral 14,000-molecular-weight envelope protein that rescues the small-plaque-size phenotype of a vaccinia virus variant to large-plaque-size virus. Selection of recombinants is easily obtained after visual inspection of large viral plaques.     

Structural analysis of precursor and product forms of type-common envelope glycoprotein D (CP-1 antigen) of herpes simplex virus type 1.

The type-common CP-1 antigen of herpes simplex virus type 1 (HSV-1) is associated in the infected cell with two components, a 52,000-molecular-weight glycoprotein (gp52 or pD) and a 59,000-molecular-weight glycoprotein (gp59 or D). The larger form (D) is also found in the virion envelope. It was postulated that pD is a precursor of D. We found that pD shared methionine and arginine tryptic peptides with D isolated from infected cell extracts. D isolated from infected extracts had the same trypric methionine peptide profile as D isolated from the virion envelope. Thus, processing of pD to D does not involve any major alterations in polypeptide structure. Furthermore, D did not share tryptic methionine peptides with the other major glycoproteins of HSV-1. Using [2-3H]mannose as a specific glycoprotein label, we found that pD, which is a basic protein (isoelectric point = 8.0) contained a 1,800-molecular-weight oligomannosyl core moiety and was processed by further glycosylation and sialyation to a more acidic and heterogeneous molecule D, which as a molecular weight of at least 59,000.     

Analysis of foot-and-mouth disease virus-neutralizing idiotypes from immune bovine and swine with anti-murine idiotype antibody probes

Rabbit anti-idiotypic antibodies (a-IdAb) induced by foot-and-mouth disease virus (FMDV) neutralizing mAb were used as probes to identify anti-FMDV Id in immune serum from bovine and swine. In a competitive RIA, at least two of the a-IdAb exhibited a dose-dependent capacity to compete with labeled virus for anti-FMDV antibodies from a convalescent bovine serum. These a-IdAb were immobilized on activated Sepharose and used to isolate anti-viral Id from bovine, swine, and murine FMDV immune sera. Both the bovine and swine antibodies recovered from the a-IdAb/Sepharose columns reacted with virus, and to a lesser extent with corresponding mAb-resistant virus variants. The binding of affinity isolated bovine and swine antibodies to virus was specifically inhibited by the homologous a-IdAb, and in addition, both were capable of neutralizing FMDV in suckling mouse protection and plaque reduction neutralization assays. Therefore, by means of a-IdAb probes generated against FMDV murine Id, two neutralizing Id were identified in bovine and swine. These results suggest that FMDV-neutralizing epitopes recognized by murine systems play a role in the overall immunity of foot-and-mouth disease-susceptible animals.     

Neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the amino-terminal half of the spike glycoprotein.

Neuroattenuated variants of mouse hepatitis virus type 4 (MHV-4) selected for resistance to neutralizing monoclonal antibodies (R.G. Dalziel, P.W. Lampert, P. J. Talbot, and M. J. Buchmeier, J. Virol. 59:463-471, 1986) were found to harbor large deletions in both mRNA 3 and its protein product, the 180-kilodalton viron spike (S) glycoprotein. By using antipeptide antibodies directed against selected portions of the chain, deletions were mapped to the middle of the amino-terminal S1 fragment, one of the two posttranslational cleavage products of S, and involved omission of 15 kilodaltons of protein. Deletion mutants could be selected only after multiple passage of virus through cultured cell lines; minimally passaged MHV-4 stocks contained putative point mutants selectable by neutralizing monoclonal antibodies but no deletions. Enhanced growth of deletion mutants relative to wild-type virus was observed in four cell lines used for virus propagation and was attributed to delayed and diminished cytopathic effects that allowed cultures to support virus production for prolonged periods. This hypothesis was reinforced by the finding that no selective advantage for the deletion mutants was observed in two cell lines resistant to virus-induced cytopathic effects. These results indicate that the passaging of MHV-4 in culture generates heterogeneity in S structure and eventually selects for rare neutralization-resistant deletion mutants with decreased virulence properties.     

Herpes simplex virus type 2 glycoprotein gF and type 1 glycoprotein gC have related antigenic determinants.

The 104-S monoclonal antibody immunoprecipitated from herpes simplex virus type 2 (HSV-2)-infected cell extracts the 75,000-molecular-weight glycoprotein gF and its 65,000-molecular-weight precursor (pgF). The precursor pgF was sensitive to endoglycosidase H digestion, indicating the presence of high mannose-type oligosaccharides, whereas the stable gF product was sensitive to neuraminidase digestion, indicating the presence of sialic acid residues. The 104-S antibody also weakly precipitated the 130,000-molecular-weight herpes simplex virus type 1 (HSV-1) glycoprotein gC from both infected cell extracts and purified preparations obtained through the use of monoclonal antibody-containing immunoadsorbent columns. Immunofluorescence tests demonstrated that the 104-S antibody reacted with antigen present in cells infected with HSV-2 strain 333 and HSV-1 strain 14012 but not with antigen present in cells infected with HSV-1 strain MP, a strain deficient in HSV-1 gC production. These findings indicate that HSV-1 gC and HSV-2 gF have antigenic determinants that are related.     

Production of monoclonal antibody to bovine leukemia virus glycoprotein gp51 by in vitro immunization

The monoclonal antibody against glycoprotein gp51 of bovine leukemia virus (BLV) envelope antigen was produced by in vitro immunization. This monoclonal antibody reacted with viral antigen was observed at the 69 kilodalton (kDa) glycoprotein. However, this monoclonal antibody was not involved in neutralizing. It was shown that in comparison with in vivo immunization, in vitro immunization has some advantages, namely a short immunization period and a small antigen quantity.     

Map location of the gene for a 130,000-dalton glycoprotein of bovine herpesvirus 1.

A bovine herpesvirus 1 variant (mar6) containing a mutation in a viral glycoprotein with a molecular weight of 130,000 (g130) was isolated by selecting for resistance to a neutralizing monoclonal antibody (130-6) directed against g130. Mar6 was completely resistant to neutralization by monoclonal antibody 130-6 in the presence and absence of complement, but was neutralized by polyvalent immune sera. The mar6 mutant synthesized and processed g130, but produced plaques which failed to react with monoclonal antibody 130-6 in an in situ immunoassay (black plaque). However, monoclonal antibody 130-6 was capable of binding and immunoprecipitating g130 from infected-cell extracts produced by lysis of mar6-infected cells with nonionic detergents. The mutation in mar6 was mapped by marker rescue with cloned bovine herpesvirus 1 restriction enzyme fragments to a 3.8-kilobase fragment at approximate map units 0.405 to 0.432. In addition, it was found that a DNA probe containing the glycoprotein B gene of herpes simplex type 1 hybridized uniquely to the same 3.8-kilobase fragment which was shown by marker rescue to contain the mutation site in the gene for bovine herpesvirus 1 g130.     

In vitro generation of an HTLV-III variant by neutralizing antibody

Transmission and culture of "parental" virus (HTLV-III) from H9 cells transfected with the cloned isolate (lambda HXB-2D) in human serum possessing HTLV-III neutralizing antibody selected for a "variant" that was not neutralized by the selecting serum but was neutralized by another antibody-positive serum "Control" virus, selected in serum lacking neutralizing antibody, and the variant showed highly similar tryptic peptide maps of the major envelope glycoprotein, and no changes in restriction enzyme patterns of viral DNA. These findings show that HTLV-III type-specific neutralizing antibodies occur, can influence the propagation of variant viruses that may arise, and presumably result from minor changes in the eliciting antigen. The extent to which such type-specific neutralizing antibodies influence immune surveillance against HTLV-III infection in vivo, a question with relevance to future vaccination attempts, remains to be determined. Nucleotide sequencing of the control and variant envelope genes may elucidate a region important for virus neutralization and vaccine development.     

Effect of glycosylation inhibitors on the release of enveloped vaccinia virus.

Addition of 1 to 10 mM 2-deoxy-D-glucose (2-dg) or glucosamine (gln) to the growth medium of vaccinia virus-infected cells inhibited the release of extracellular enveloped vaccinia virus (EEV) without affecting the production of intracellular naked vaccinia virus (INV) particles. In contrast, INV infectivity (particles per PFU) was decreased sevenfold by 50 mM 2-dg. Treatment with 2-dg reduced but did not eliminate glycosylation of the INV 37,000-molecular-weight glycoprotein. The kinetics of sensitivity to inhibitor addition experiments and inhibitor reversal experiments indicated that EEV release was dependent on glycosylation before 8 h postinfection. This was supported by polyacrylamide gel electrophoretic analysis of the synthesis kinetics for cell membrane-associated vaccinia glycoproteins in 2-dg-inhibited infected cells. The dependence of vaccinia protein glycosylation before 8 h postinfection for efficient EEV release was observed in spite of the fact that the period of greatest glycoprotein synthesis was 8 to 12 h postinfection. The presence of 2-dg resulted in an incompletely glycosylated 89,000-molecular-weight glycoprotein, as indicated by a reduction in the apparent glycoprotein molecular weight. The morphological event affected by the inhibitors was the acquisition by INV of a double-membrane structure from the Golgi apparatus. This morphological intermediate is necessary for release of EEV.