Identification of an 85 kDa phosphoprotein as an immunodominant protein specific for human herpesvirus 7-infected cells.

The reactivity of human cord blood sera was directed most frequently in Western blot assays to a protein with an apparent molecular mass of 85 kDa that belongs to the p85 complex, a family of antigenically related proteins identified previously in our laboratory with the aid of two MAbs. We show that the 85 kDa protein is phosphorylated. As antibodies present in the human sera were directed in part to proteins carrying cross-reactive epitopes between human herpesvirus 6 (HHV-6) and 7 (HHV-7), it is remarkable that reactivity to the 85 kDa phosphoprotein was maintained after preabsorption of the sera with HHV-6 antigen, but abolished after preabsorption with HHV-7 antigen. Therefore, the 85 kDa phosphoprotein may be considered a major determinant of the human immune response to HHV-7, discriminating HHV-6 from HHV-7 infection.

Selection of a monoclonal antibody specific for variant B human herpesvirus 6-infected mononuclear cells.

A monoclonal antibody, designated as MAb 6E2, specific for human herpesvirus 6 variant B (HHV-6B) was derived from the spleen of a mouse immunized with lysates of HHV-6B(Z29) cord blood mononuclear cells. MAb 6E2 reacts by immunofluorescence with all the HIV-6B strains tested (Z29, CV, Hashimoto and SF) and fails to react with variant A prototypes, GS and U1102. The immunofluorescence staining was punctate and localized to the cytoplasm. The protein reacting with MAb 6E2 was identified as protein 48,000 in apparent M(r) value by immunoaffinity chromatography of lysates of HHV-6B-infected mononuclear cells.

Polyvalent and monoclonal antibodies identify major immunogenic proteins specific for human herpesvirus 7-infected cells and have weak cross-reactivity with human herpesvirus 6.

Hyperimmune rabbit and mouse sera raised to human herpesvirus 7 (HHV-7)-infected cells and an immune human serum identified 20[35S]methionine-[35S]cysteine-labelled proteins specific for HHV-7-infected cord blood mononuclear cells, ranging in apparent M(r) from 136K to 30K. The major proteins had apparent M(r) values of 121K, 100K, 87K, 85K, 60K, 51K, 46K, 42K, 40K and 36K. The human serum also identified seven [3H]glucosamine-labelled glycoproteins, with apparent M(r) values of 100K, 89K, 82K, 67K, 63K, 53K and 41K. Four monoclonal antibodies (MAbs) specific for HHV-7-infected cells were derived. Two reacted with a family of five antigenically related polypeptides (87K, 85K, 70K, 61K and 57K in apparent M(r)), designated as the p85 complex. Two reacted with 121K and 51K M(r) proteins designated as p121 and p51, respectively. Human sera react with high frequency with the p85 complex and to a lesser extent with p121; hence these two proteins appear to be immunodominant for both humans and laboratory animals. The hyperimmune mouse serum and some of the MAbs showed some cross-reactivity with HHV-6A(U1102)- and 6B(Z29)-infected cells. The implications of cross-reactivity with respect to the human immune response to HHV-6 and -7 infections and prevalence analyses are discussed.

Human herpesvirus 6 envelope glycoproteins B and H-L complex are undetectable on the plasma membrane of infected lymphocytes.

Membrane immunofluorescence analysis of cells infected with either variant (A or B) of human herpesvirus 6 revealed a typical punctate staining, after labeling with several HHV-6-positive human sera or with two monoclonal antibodies directed to gB and gH. Immunoprecipitation studies showed a sharp difference in glycoprotein content in whole-cell extracts versus on the cell surface, suggesting the occurrence of gB in the extracellular virions juxtaposed to plasma membranes. By immunoelectron microscopy, the extracellular virions still attached to the cell surface appeared consistently and specifically labeled, whereas the plasma membrane was always unlabeled, independent of viral variant, antibody, or target cell used. These findings may reflect an atypical maturation pathway of HHV-6, and could have important implications in the control of cellular immune response to HHV-6-infected lymphocytes.

Human herpesvirus-6 glycoprotein H and L homologs are components of the gp100 complex and the gH external domain is the target for neutralizing monoclonal antibodies.

Previous studies have shown that monoclonal antibody (MAb) 2E4 neutralizes infectivity of human herpesvirus-6 (HHV-6) and also inhibits virus-induced T-lymphocyte syncytia formation. Here we characterize two additional MAbs, 1D3 and 5E7, which have similar properties, and identify the glycoprotein targets. The MAbs could immunoprecipitate and immunofluorescence glycoprotein from both A and B variant strain groups of HHV-6. In reactions with infected cells the MAbs immunoprecipitated a complex of glycoproteins, the "gp100" complex, composed of a major glycoprotein species of 100,000 M(r) and minor components of 80,000 M(r) and 32,000 M(r). We show that the 100,000 M(r) product and most likely the 80,000 M(r) correspond to the HHV-6 homologue of herpes simplex virus-1 (HSV-1) glycoprotein H (gH) while the 32,000 M(r) species corresponds to the glycoprotein L (gL) equivalent. All three MAbs could specifically immunoprecipitate either gH expressed on its own in fibroblasts or a complex of gH and gL co-expressed, but could not immunoprecipitate gL expressed on its own. Consistent with these results, the MAbs could recognize gH in an immunofluorescence assay but not gL. Therefore although the MAbs recognized the complex of glycoproteins, they appeared specific for gH. The HHV-6 glycoproteins were produced in a transient expression system induced by T7-vaccinia virus. Immunoprecipitations were carried out in comparisons with an "epitope-tagged" gH, a recombinant glycoprotein designed to contain at the N-terminus the linear epitope for MAb LP14, raised originally against HSV-1 glycoprotein gD. The epitope-tagged gH was also used as a positive control in determining the domain of HHV-6 gH to which MAbs 2E4, 1D3 and 5E7 were directed. Two gH deletions were constructed, one deleting sequences which may serve as a transmembrane and cytoplasmic anchor domains, the second deleting also part of the external domain. MAb LP14 could immunoprecipitate both HHV-6 gH deletions but the gp100 MAbs recognized only the full-length product or the intact external domain minus the transmembrane and cytoplasmic domains. This indicated the epitopes for these MAbs are contained in the external domain of gH, consistent with the MAbs action in neutralization of virion infectivity and inhibition of virus to cell spread by T-lymphocyte fusion.

Monoclonal antibodies to glycoprotein B differentiate human herpesvirus 6 into two clusters, variants A and B.

The distribution of glycoprotein B (gB) among different human herpesvirus 6 (HHV-6) strains was analysed with a panel of three monoclonal antibodies (MAbs) derived from mice immunized with U1102-infected lymphocytes. MAb 2D9 reacted specifically by immunofluorescence and immunoprecipitation with the U1102 and GS isolates, and failed to react with Z29 and the variant B strains Hashimoto and SF. In addition, Z29, Hashimoto and SF gB had a lower M(r) than U1102 and GS gB. MAb 2D9 also failed to react with the exanthem subitum isolate CV, included in this study as an as yet poorly characterized isolate. Consistent with this result, CV failed to react with the variant A-specific MAb to gp82-105 and behaved as a variant B virus even with respect to the diagnostic HindIII endonuclease restriction cleavage site located in a fragment hybridizing to the pZVH14 probe. By contrast with MAb 2D9, MAbs 2B9 and 2D10 reacted with all of the isolates tested, strengthening the argument tha they have common epitopes. Based on the antigenic and M(r) specificities of gB, the HHV-6 isolates tested were arranged into two non-overlapping clusters, which closely parallel the variant A and B strain groups, defined previously by several criteria, including restriction endonuclease polymorphism, antigenic variations, growth in in vitro cultures and sequence analyses.

The glycoprotein B homologue of human herpesvirus 6.

The gene for the homologue of herpesvirus glycoprotein B (gB) has been identified in the genome of human herpesvirus 6 (HHV-6), strain U1102, and the nucleotide sequence was determined. The open reading frame encodes a protein of 830 amino acids (93.2K) with the characteristics of a transmembrane glycoprotein and close similarity to the gp58/116 complex of human cytomegalovirus (HCMV). Monoclonal antibodies 2D10 and 2B9 have been shown previously to react with an HHV-6 glycoprotein of apparent M(r) 112K, and its proteolytic cleavage products of M(r) 64K and 58K. We show that both monoclonal antibodies detect prokaryotically expressed carboxy-terminal fragments of the HHV-6 gB homologue. This indicates that the HHV-6 gB homologue is probably processed by proteolytic cleavage similar to its equivalents in HCMV and various other herpesviruses.

Characterization of human herpesvirus-6(U1102) and (GS) gp112 and identification of the Z29-specified homolog.

Monoclonal antibody 2D10 (MAb 2D10) raised toward human herpesvirus-6(U1102) [HHV6(U1102)] immunoprecipitated three glycosylated peptides, M(r) 112,000, 64,000, and 58,000, designated as gp112 from U1102-infected lymphocytes. Pulse-chase experiments suggest that the M(r) 64,000 and 58,000 polypeptides are very likely generated by post-translational cleavage of the M(r) 112,000 polypeptide. MAb 2D10 neutralized virion infectivity in the presence of complement, suggesting that gp112 is located in the virion envelope. MAb 2D10 did not prevent the appearance of HHV6-specific cytopathic effect. MAb 2D10 was reactive with denatured gp112 in immunoblots. HHV6 isolates form two clusters (Schimer, Wyatt, Yamanishi, Rodriguez, and Frenkel, Proc. Natl. Acad. Sci. USA 88, 5922; Ablashi, Balachandran, Josephs, Hung, Krtueger, Kramarsky, Salahuddin, and Gallo, Virology 184, 545). MAb 2D10 reacted by immunofluorescence and immunoprecipitation with the prototypes of each cluster, GS and Z29. Whereas the proteins immunoprecipitated by MAb 2D10 from GS-infected lymphocytes had an electrophoretic pattern very similar to that of U1102 gp112, the homologous glycoprotein immunoprecipitated from Z29-infected lymphocytes consisted of three polypeptides with M(r) 102,000, 59,000, and 50,000. The data suggest a variation among HHV6 isolates as far as this glycoprotein is concerned.

Monoclonal antibodies to gp100 inhibit penetration of human herpesvirus 6 and polykaryocyte formation in susceptible cells.

We report the derivation and properties of a monoclonal antibody (MAb 2E4) which neutralizes human herpesvirus 6 (HHV-6). MAb 2E4 precipitated from lysates of infected cells a glycosylated polypeptide 100,000 in apparent molecular weight and minor components of 80,000, and 32,500. The predominant reactive protein after a pulse was the 100,000-molecular-weight peptide designated as gp100. The smaller polypeptides appeared in the precipitate predominantly after a chase. MAb 2E4 neutralized HHV-6 infectivity in the presence and in the absence of complement, and it inhibited the penetration of virus into the cells. Addition of MAb 2E4 as late as 6 h postinfection inhibited the formation of large polykaryocytes typical of HHV-6-infected cells.

Herpes simplex virus (HSV) glycoprotein H is partially processed in a cell line that expresses the glycoprotein and fully processed in cells infected with deletion or ts mutants in the known HSV glycoproteins.

Cell lines that constitutively express herpes simplex virus 1 (HSV-1) glycoprotein H (gH-1) failed to synthesize the mature form of gH and accumulated a precursor-like form of the glycoprotein, which was retained intracellularly, most likely in RER. Fine-structure analysis of the oligosaccharides present in recombinant gH revealed oligosaccharides processed by RER enzymes; sialylated complex-type and biantennary oligosaccharides, which are assembled in the trans-Golgi, were absent. A small fraction had the characteristics of oligosaccharides processed by the early mannosidases of the Golgi. These findings suggest that a defect in the transport out of RER to the Golgi may account for the intracellular retention of the immature form of gH in cells that express the glycoprotein constitutively. Upon superinfection of cells expressing gH-1 with HSV-2, recombinant gH-1 underwent maturation, indicating that a viral function is required to attain full processing of gH. The known HSV glycoproteins do not appear to carry out this function, since in cells infected with deletion mutants in gD, gG, gE, and gE-gI, with a spontaneous gC- mutant, or with a temperature-sensitive mutant in gB, maturation of gH occurred independently of the presence or of the maturation of the single glycoproteins tested. The present findings together with previous observations on HSV, human CMV, and the EBV homologue of gH suggest that inability of gH to undergo full processing in the absence of viral protein(s) is a property of gH.

Glycoprotein D of herpes simplex virus encodes a domain which precludes penetration of cells expressing the glycoprotein by superinfecting herpes simplex virus.

Earlier studies have shown that herpes simplex viruses adsorb to but do not penetrate permissive baby hamster kidney clonal cell lines designated the BJ series and constitutively expressing the herpes simplex virus 1 (HSV-1) glycoprotein D (gD). To investigate the mechanism of the restriction, the following steps were done. First, wild-type HSV-1 strain F [HSV-1(F)] virus was passaged blindly serially on clonal line BJ-1 and mutant viruses [HSV-1(F)U] capable of penetration were selected. The DNA fragment capable of transferring the capacity to infect BJ cells by marker transfer contains the gD gene. The mutant gD, designated gDU, differed from wild-type gD only in the substitution of Leu-25 by proline. gDU reacted with monoclonal antibodies which neutralize virus and whose epitopes encompass known functional domains involved in virus entry into cells. It did not react with the monoclonal antibody AP7 previously shown to react with an epitope which includes Leu-25. Second, cell lines expressing gDU constitutively were constructed and cloned. Unlike the clonal cell lines constitutively expressing gD (e.g., the BJ cell line), those expressing gDU were infectable by both HSV-1(F) and HSV-1(F)U. Lastly, exposure of BJ cells to monoclonal antibody AP7 rendered the cells capable of being infected with HSV-1(F). The results indicate that (i) gD expresses a specific function, determined by sequences at or around Leu-25, which blocks entry of virus into cells synthesizing gD, (ii) the gD which blocks penetration by superinfecting virus is located in the plasma membrane, (iii) the target of the restriction to penetration is the identical domain of the gD molecule contained in the envelope of the superinfecting virus, and (iv) the molecular basis of the restriction does not involve competition for a host protein involved in entry, as was previously thought.

Glycoprotein C-dependent attachment of herpes simplex virus to susceptible cells leading to productive infection.

Herpes simplex viruses encode several glycoproteins dispensable for infection and replication in cell culture. Evidence is presented that there exist at least two pathways for viral attachment to cells, i.e., one mediated by the dispensable glycoprotein C (gC) and one independent of that glycoprotein. Thus, whereas the polycations neomycin and polylysine inhibit attachment but not entry of already attached herpes simplex virus 1 (HSV-1) into baby hamster kidney (BHK) cell line, they have no effect on HSV-2 attachment to the same cells (N. Langeland, H. Holmsen, G.R. Lilehaug, and L. Haarr, 1987, J. Virol. 61, 3388-3393; N. Langeland, L.J. Moore, H. Holmsen, and L. Haarr, 1988, J. Gen. Virol. 69, 1137-1145). We report that (i) analyses of intertypic HSV-1 X HSV-2 recombinants indicated that the HSV-2 locus which confers ability to infect BHK cells in the presence of neomycin or polylysine comaps with the gene specifying gC but not with or near the genes specifying the other viral glycoproteins (gB, gD, gE, and gG, and gI), (ii) the smallest HSV-2 DNA fragment capable of transferring this function to HSV-1 was a 2880-bp Sa/l fragment encoding the entire gC (UL44 open reading frame) gene, 515 bp of coding sequences from the UL43 open reading frame and 393 bp of coding sequences from the UL45 open reading frame, but analyses of the recombinant virus DNA excluded UL43 and most of the UL45 sequences, and (iii) definitive evidence that HSV-2 gC confers upon HSV the capacity to infect BHK cells in the presence neomycin or polylysine emerged from studies showing that site-specific mutagenesis which inactivated the gene yielded a recombinant whose attachment to BHK cells was blocked by the polycations. We conclude that in BHK cells there exists in addition to the pathway blocked by neomycina and polylysine a pathway which is parallel and HSV-2 gC dependent.

Inhibition of herpes simplex virus type 1 (HSV-1) plaque enlargement by neutralizing sera.

The inhibitory activity of neutralizing sera on Herpes simplex virus type 1 (HSV-1) plaque enlargement (PE) is easily detected using the S variant of low passage clinical isolates (Mannini-Palenzona et al., 1985b; Costanzo et al., 1986). The same sera show little or no activity on PE of the product of the S variant rapid in vitro conversion, the L variant, and laboratory strains HSV-1 (F) and (MP). No significant difference was found in the inhibitory activity of sera from healthy individuals with no history of recurrence and patients with recurrences.

Individual herpes simplex virus 1 glycoproteins display characteristic rates of maturation from precursor to mature form both in infected cells and in cells that constitutively express the glycoproteins.

Pulse-chase experiments in conjunction with quantitative immunoprecipitation have been used to study the time-course of conversion from precursor to mature form of herpes simplex virus 1 glycoproteins C, D and B (gC, gD, and gB). The experimental systems employed were two infected cell lines and cells that constitutively express gD or gB. The relative rates of conversion among the glycoproteins did not vary in the systems used; the rate of maturation of gC was about two-fold higher than that of gD which, in turn, was about one and a half-fold higher than that of gB. Treatment with phosphonoacetate which inhibits viral DNA synthesis and hence virion morphogenesis induced a striking increase in the time course of conversion of immature gC, gD, and gB to fully glycosylated forms when measured late in the infection. The model of HSV glycoproteins maturation as integral components of the virion envelope is discussed.

A herpes simplex virus type 1 mutant resistant to benzhydrazone, an inhibitor of glycoprotein synthesis in herpesvirus-infected cells. Preliminary mapping of benzhydrazone-resistance and of a novel syncytial mutation.

Benzhydrazone (BH) is an inhibitor of glycoprotein biosynthesis. It acts selectively in Herpes simplex virus (HSV)-infected cells and does not significantly affect glycoprotein synthesis in uninfected cells and in cells infected with other viruses. Previously, we reported on a syncytial (syn) mutant, designated HSV-1(13)S11, resistant to BH, and showed that BH-resistance is encoded in the mutant virus DNA and therefore can be transferred into the genome of wild type HSVs. The present paper reports on a preliminary mapping in HSV-1(13)S11 genome of the loci which confer resistance to BH and of three distinct syn mutations present simultaneously in this mutant. Two of them were mapped in previously described syn loci localized in BamHI fragment L (map units 0.707-0.745) (locus syn 1) and BamHI fragment Q (map units 0.296-0.317) (locus syn 5). A third mutation not described before and mapping in BamHI fragment SP (c.a. map units 0.81-0.85) conferred the syn phenotype to both HEp-2 and Vero cells. This novel mutation has been designated herein locus syn 6. Transfer of BH-resistance could be achieved in cotransfection experiments involving two HSV-1(13)S11 fragments, BamHIL and BamHISP.

Further characterization of virus obtained from herpes simplex virus type 1 recurrences and primary infections. Influence of the temperature of incubation upon glycoprotein synthesis and virus release. Brief report.

The virus contained in clinical isolates of herpes simplex virus type 1 (HSV-1) which have not undergone previous in vitro passages (new isolates) differs from HSV-1 prototype strains with respect to infected cell glycoprotein pattern, and, most probably efficiency of virus egress at 37 degrees C. The differences can be abolished by lowering the temperature of incubation to 33 degrees C. A few tissue culture passages cause the conversion of the original virus to a virus undistinguishable from HSV-1 prototype strains with respect to the parameters mentioned above.

Establishment and characterization of a persistent infection of MDBK cells with herpes simplex virus.

A persistent, dynamic-state infection of a variant of herpes simplex virus type 1 strain MP [HSV-1 (MP)] in MDBK cells was established without supportive measures and maintained for over three years by routine passaging of the cells at 7-9 day intervals. The infection was characterized by a cyclic pattern of monolayer damage and reconstitution, correlated with virus production, which was most evident when the interval between subcultures was intentionally prolonged and the cells were left undisturbed. Occasional periods of cell crisis, with increased virus replication and extensive cytopathology, occurred. Passaging of the cells at higher density avoided eventual loss of the culture during the most severe crises. Presence of specific antibodies did not alter the course of infection. Interferon was constantly found during periods of cell crisis; it appeared on the second day after subculture, reached a maximum in correspondance of the first peak of cytopathology, and disappeared well before the onset of the second wave of cytopathology. Addition of exogenous interferon cured the cells of infection. Defective interfering particles could not be found. Virus isolated during persistence differed from parental virus regarding plaque morphology, temperature sensitivity of growth, and electrophoretic pattern of infected cells glycoproteins. A discussion on the possible mechanisms of persistence is provided.

Study of herpes simplex virus type 1 populations obtained from recurrences and primary infections.

The analysis of 23 clinical isolates of herpes simplex virus type 1 (HSV-1) showed that 15 of 15 isolates that had undergone a few passages in tissue culture (fresh isolates) and two of eight isolates that had never been passaged (new isolates) were composed of a mixed population with respect to plaque morphology in Vero cells. Cloning and characterization of 10 large plaque viruses (L variants) and nine small plaque viruses (S variants), obtained from seven different isolates, showed the following. BamHI DNA restriction patterns of the L and the S variants from a single isolate differed only with respect to the electrophoretic mobility of the fragments that contain reiteration of specific sequences; they did not differ regarding the presence or the absence of restriction endonuclease cleavage sites. The L and S variants differed with respect to the electrophoretic profiles of infected cell glycoproteins, thermosensitivity of growth and plaquing efficiency at 39 degrees C, and, at least in the case of the two couples of variants that we tested, pathogenicity for the mouse. The hypothesis that the L variants might arise from the S variant during in vivo replication is discussed.

Effect of ribosome-inactivating proteins on virus-infected cells. Inhibition of virus multiplication and of protein synthesis.

HEp-2 cells were infected with herpes simplex virus-1 (HSV-1) or with polio-virus I in the presence of plant proteins which inactivate ribosomes in cell-free systems, while exerting scarce effect on whole cells. Ribosome-inactivating proteins used were gelonin, from seeds of Gelonium multiflorum, an inhibitor from the seeds of Momordica charantia, dianthin 32, from the leaves of Dianthus caryophyllus (carnation), and PAP-S, from the seeds of Phytolacca americana (pokeweed). All proteins tested had the following effects: 1. They reduced viral yield; 2. They decreased HSV-1 plaque-forming efficiency; 3. They inhibited protein synthesis more in infected than in uninfected cells. These results strongly suggest that ribosome-inactivating proteins impair viral replication by inhibiting protein synthesis in virus-infected cells, in which presumably they enter more easily than in uninfected cells.