Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus

We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA^r) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity. Compared with HSV-1 lytic infection, the recombinant Pol protein exhibited a reduced stability with a half-life of 7 h. Whereas the Pol-associated exonuclease activities, determined from lysates of recombinant VV- and HSV-1-infected cells, were almost identical, the polymerizing activity of recombinant Pol ceased after 10 min of incubation, in correlation with the fact that Pol depends on its cofactor for optimal chain elongation. Kinetics of cellular localization, tracked by a monospecific Pol antibody, revealed that the catalytic subunit initially assembled to a few dot-like nuclear sites, reminiscent of HSV-1 DNA replication compartments. Later during infection, the localization of recombinant Pol matched with that found in lytically HSV-1-infected cells. This study demonstrates that nuclear transport and localization of the Pol subunit is independent of herpesviral functions, and neither requires the presence of herpesviral DNA sequences. Recombinant VV provides a promising alternative to explore protein interactions of the herpesviral replication machinery in their authentic cellular environment.     

Studies on herpes simplex virus type 1 glycoproteins using monoclonal antibodies

Monoclonal antibodies against herpes simplex virus type 1 glycoproteins were isolated and utilized to study the synthesis and processing of glycoproteins B, C, and D (gB, gC, gD, respectively). Monoclonal antibodies against both gB and gD had higher virus-neutralizing activity when compared to that of gC. Differences among these glycoproteins were observed in their time of appearance in the virus-infected cells. The presence of gD was detected at a very early stage of infection when compared to gB and gC. The localization of these glycoproteins during their synthesis and processing was studied.     

Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.     

Fusion activity of lipid-anchored envelope glycoproteins of herpes simplex virus type 1

Expression of the herpes simplex virus type 1 (HSV-1) glycoproteins gB, gD, gH, and gL is necessary and sufficient to cause cell fusion. To identify the requirements for a membrane-spanning domain in HSV-1 glycoprotein-induced cell fusion, we created gB, gD, and gH mutants with transmembrane and cytoplasmic domains replaced by a glycosylphosphatidylinositol (gpi)-addition sequence. The corresponding gBgpi, gDgpi, and gHgpi proteins were expressed with wild-type efficiency at the cell surface and were linked to the plasma membrane via a gpi anchor. The gDgpi mutant promoted cell fusion near wild-type gD levels when co-expressed with gB, gH, and gL in a cell-mixing fusion assay, indicating that the gD transmembrane and cytoplasmic domains were not required for fusion activity. A plasma membrane link was required for fusion because a gD mutant lacking a transmembrane and cytoplasmic domain was nonfunctional for fusion. The gDgpi mutant was also able to cooperate with wild-type gB, gH, and gL to form syncytia, albeit at a size smaller than those formed in the wild-type situation. The gBgpi and gHgpi mutants were unable to promote fusion when expressed with the other wild-type viral glycoproteins, highlighting the requirement of the specific transmembrane and cytoplasmic domains for gB and gH function.     

Neutralizing activity of antibodies against the major herpes simplex virus type 1 glycoproteins

The specificity and neutralizing activity of antibodies against the major herpes simplex virus type 1 (HSV-1) glycoproteins were tested in serum samples of patients with a history of HSV-1 infection. By preabsorption of sera to preparations of native and denatured HSV-1 proteins, followed by immunoblotting and microneutralization, it was shown that the majority of neutralizing antibodies are directed against denaturation-sensitive epitopes. Furthermore, preabsorption of sera to proteins of viral ts and deletion mutants revealed that antibodies specific for gB, gC, and gE had a low neutralizing activity. These results suggest a major role of anti-gD in neutralization of viral infectivity. In addition, it was shown that antibodies directed against the gB monomer were distinct from antibodies against the gB homodimers. The latter, however, did not reveal any measurable neutralizing activity.     

Immunogenicity of subviral herpes simplex virus preparations

Summary Production of neutralizing antibodies was followed in guinea pigs, rabbits hamsters and mice immunized with crude antigen extracts (AM) from human diploid cells infected with herpes simplex virus type 1. The AM induced relatively high levels of neutralizing antibodies in all four species. The antibodies were predominantly complement-requiring and remained so even after administration of repeated AM doses. With the strains used, the antibody response was predominantly type specific and, surprisingly, the type specificity of sera usually increased after administration of repeated doses of AM. Guinea pigs seemed to be the best responsive animal species. They developed the highest levels of antibodies and complement-nonrequiring antibodies were seen in them earlier than in the other animal species. The dose-response experiments carried out in guinea pigs indicated that after a single dose administration the ratio between complement-requiring and complement-nonrequiring antibodies was dependent on the amount of antigen administered. When AM was given without adjuvant less efficient antibody production was observed than after the administration of the same amount of antigen with adjuvant.With 3 Figures     

Expression and characterization of baculovirus expressed herpes simplex virus type 1 glycoprotein L

Summary We have constructed a recombinant baculovirus expressing high levels of the herpes simplex virus type 1 (HSV-1) glycoprotein L (gL) in Sf9 cells. Sf9 cells infected with this recombinant virus synthesized three polypeptides of 26–27 kDa 28 kDa, and 31 kDa. The 28 and 31 kDa species were sensitive to tunicamycin and N-glycosidase F (PNGase F) treatment, suggesting that they were glycosylated. As shown by both indirect immunofluorescence and Western blot analysis, using polyclonal antibodies to synthetic gL peptides indicated that the baculovirus expressed gL was abundant on the surface of baculovirus gL infected Sf9 cells. A small fraction of the 31 kDa polypeptide was secreted into the extracellular medium as judged by Western blot analysis. The secreted form of gL was completely resistant to Endoglycosidase H (Endo-H), while the membrane associated form of gL was only partially resistant to Endo-H treatment, suggesting that the secreted gL represented a subpopulation of the membrane bound gL. Mice vaccinated with baculovirus expressed gL produced serum antibodies that reacted with authentic HSV-1 gL. However, these mice produced no HSV-1 neutralizing antibody (titer <1: 10) and they were not protected from lethal intraperitoneal or lethal ocular challenge with HSV-1. Thus, when used as a vaccine in the mouse model, gL, similar to our findings with HSV-1 gH, but unlike our results with the other 6 HSV-1 glycoproteins that we have expressed in this baculovirus system, did not provide any protection against HSV-1 challenge.     

Monoclonal antibodies to herpes simplex virus type 1 glycoproteins show that epitope location influences virus neutralization

Three monoclonal antibodies, G8D1 , C2D2 , and TI57 , reacting with herpes simplex virus type 1 glycoproteins have been characterised according to the location of their epitope and ability to neutralize infective virus. Immune electron microscopy and a blocking radioimmunoassay were used to locate the epitopes. The results indicate that the epitope recognised by G8D1 is located on the surface of the glycoprotein fringe, whereas those recognized by C2D2 and TI57 are interior with respect to this. Only G8D1 has neutralizing activity alone, whereas C2D2 can neutralize when antiglobulin is added. Thus, epitope location and density determine the neutralizing capacity of individual antibody molecules.     

Hemagglutination by herpes simplex virus type 1

Summary The McIntyre and HSZP strains as well as clinical isolate of herpes simplex virus type 1 were found to agglutinate C57B1/10su and CBA mouse red blood cells. The hemagglutinating activity was inhibited by antisera that neutralized the infectivity of the virus.     

Some properties of recombinants between type 1 and type 2 herpes simplex viruses.

Four intertypic recombinants of herpes simplex virus have been shown to possess genetic information for functions characteristic of each of the two parental types. The functions were identified by (a) polyacrylamide gel electrophoresis of purified virus particles and of polypeptides synthesized in cells infected with the recombinants and (b) analysis of antigenic sites interacting with type specific neutralizing antibody. The analysis shows that each recombinant possess a different combination of these type specific markers. Finally we have been unable to detect recombination between herpes simplex type 1 and pseudorabies viruses.     

Cell-mediated immune responses to influenza virus antigens expressed by vaccinia virus recombinants

Recombinant vaccinia viruses enable studies of immune recognition of antigens expressed from single viral genes. We have constructed recombinants expressing the haemagglutinin (HA) and nucleoprotein (NP) genes of the influenza virus A/PR/8/34 (H1N1). These recombinant viruses together with a recombinant expressing the HA from influenza virus A/JAP/305/57 (H2N2) have been used to examine the cytotoxic T lymphocyte (CTL) response to these influenza virus antigens. Both antigens are recognised by murine CTL and recognition of HA is influenza virus subtype-specific, whereas recognition of NP is crossreactive. In limiting dilution studies approximately 10% of the influenza CTL response is HA-specific, while approximately 30% of the response is NP-specific. Despite the ability of NP to stimulate a significant CTL response, mice immunised with the NP-vaccinia recombinant are not as well protected from subsequent lethal challenge with influenza virus, as mice immunised with the HA vaccinia recombinant. These studies demonstrate that viral antigens expressed from vaccine recombinants can provide protective immunity and that the influenza-poxvirus recombinants can provide data on protective immunity generated by individual viral proteins.     

O-glycosidic carbohydrate-peptide linkages of herpes simplex virus glycoproteins

Summary Electrophoretically purified HSV-specified glycoproteins with radiolabelled carbohydrates were subjected to mild alkaline borohydride treatment (0.5m NaOH and 0.5m NaBH₄). The treatment liberated significant amounts of the labelled oligosaccharides. The latter demonstrated molecular weights of about 3,000 as determined by gel filtration. The glycoproteins involved probably belong to the gA/gB complex or gC. The results suggest that HSV specified glycoproteins contain oligosaccharides linked with an O-glycosidic bond to a threonine or serine residue of the polypeptide.With 3 Figures     

Replication of animal viruses in differentiating muscle cells: vaccinia and herpes simplex virus type 1.

Cells cultured from the breast muscles of 11 to 12-day-old chick embryos were infected in the undifferentiated mitotic myoblast stage or in the terminally differentiated non-mitotic myotube stage with one of two DNA viruses, vaccinia and herpes simplex virus type 1 (HSV-1). DNA synthesis was measured and production ov virus-specific DNA detected in cells infected as myoblasts or myotubes by isotope labelling, autoradiographic and buoyant density centrifugation techniques. Furthermore, fully fused myotubes resemble myoblasts in their ability to support productive infection by these DNA viruses although DNA replication and nuclear division have ceased in myotubes and only minimum levels of host-cell DNA polymerase activity are present.     

Typing of herpes simplex virus types 1 and 2 by immunoblotting analysis using polyclonal antisera to herpes simplex virus glycoproteins

Herpes simplex virus (HSV) isolates were differentiated by immunoblotting analysis using a mixture of polyclonal antisera directed against HSV type 1 (HSV-1) and HSV type 2 (HSV-2) glycoprotein fractions (gB/gC of HSV-1 and gC/gE/84-kDa protein of HSV-2), since the mixed antisera recognized viral polypeptides with different molecular weights in HSV-1- and HSV-2-infected cells. Results of typing by immunoblotting analysis were consistent with those obtained by restriction endonuclease analysis of DNAs extracted from 10 HSV isolates. These results suggest that the immunoblotting technique will be applicable to reliable typing of HSV isolates using polyclonal antisera showing the difference in reaction patterns between HSV-1- and HSV-2-infected cells.     

Immunogenicity of purified glycoprotein gB of herpes simplex virus

Summary The efficacy of a herpes simplex virus (HSV) component vaccine consisting of viral glycoprotein gB was examined in a mouse system. Immunization of mice with HSV type 1 (HSV-1) gB emulsified in Freund's complete adjuvant or with HSV-1 gB adsorbed to aluminum gel was fully protective against subsequent challenge with HSV-1 or HSV type 2. Latent infection in the trigeminal ganglion was also prevented by immunization with gB.With 6 Figures     

Apical expression of herpes simplex virus type 2 glycoproteins in human neuroblastoma cells.

The expression of herpes simplex virus type 2 (HSV-2) glycoproteins on the surface of human neuroblastoma cells has been investigated using Millipore Millicell culture plate inserts. Utilizing a modified radioimmunoassay, we learned that glycoproteins B, C, D, E, and I were expressed predominantly on the apical membrane domain of the infected neuroblastoma cells. The unidirectional transport of HSV-2 glycoproteins was substantiated by the analysis of extracellular glycoproteins released from neuroblastoma cells. The results suggest that the evaluated HSV-2 glycoproteins were transported primarily to the apical plasma membrane domain of human neuroblastoma cells.     

5-Methoxymethyldeoxyuridine-resistant mutants of herpes simplex virus type 1

5-Methoxymethyldeoxyuridine (MMdU)-resistant (MMdU^r) mutants of herpes simplex virus type 1 (HSV-1) were isolated by a single passage of the virus in MMdU at 100 μg/ml (3.7 × 10^?4M). The isolates were cloned and passed in the presence of MMdU at 5 μg/ml (0.18 × 10^?4M). All MMdU^r mutants showed considerable cross-resistance to the nucleoside analogs acycloguanosine (ACG), (E)-5-bromovinyldeoxyuridine (BVdU), and arabinosylthymine (araT), but were as sensitive as the parental strain to phosphonoacetate (PAA). One mutant, MMdU^r-20, induced significant deoxythymidine kinase (dTK) activity. Because it was only 30 times more resistant to BVdU than the parental wild-type virus, while mutants MMdU^r-2 and -12-3 were about 10,000 times more resistant, it was suspected that the mutation was in the dTK locus. All three mutants, however, showed a similar pattern of resistance to the other nucleoside analogs ACG, MMdU, and araT. These results suggest differences in the active sites for PAA and nucleoside analogs (with respect to the viral DNA polymerase), and also among the nucleoside analogs (with respect to the viral dTK).