Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.

Effect of interferon treatment on expression of gC and gE glycoproteins in herpes simplex virus-infected cells.

The effect of interferon treatment on the herpes simplex virus type 1 (HSV-1)-specific glycoproteins gC and gE in homologous and heterologous cells has been investigated. In human embryonic fibroblastic cells, human leukocyte interferon inhibited virus multiplication and expression of the HSV-1-specific glycoproteins gC and gE on the cell surface in a dose-dependent manner. In heterologous baby hamster kidney cells, the human interferon had no effect on virus multiplication. However, the surface expression of the HSV-1-specific glycoproteins was reduced, as shown by erythrocyte rosette formation, by attachment of monodisperse polystyrene particles coated with antibodies and by immunogold scanning electron microscopy.

Intracellular maturation and sorting of two herpes simplex virus type 1 glycoproteins. Immunogold staining of ultrathin cryosections.

Simultaneous immunocytochemical triple staining of ultrathin cryosections of herpes simplex virus type 1-infected cells was carried out using monoclonal antibodies specific for glycoprotein C, glycoprotein D and alpha + beta tubulin. The viral glycoproteins were identified in the cytoplasm, in the Golgi sacs, on the plasma membrane and on the surface of intra- and extracellular virus particles, but not on the nuclear membrane. The glycoproteins identified in the cytoplasm outside the Golgi region were not always confined to the membranes of vesicles, but were often located in close proximity to the tubulin-labelled structures. The glycoproteins C and D were usually codistributed in the cytoplasm, and both accumulated in the Golgi sacs in the same membrane domains. As the glycoproteins occur in close proximity to the microtubular structures, we speculate that these might be directly involved in the intracellular transport of viral glycoproteins.

Identification and characterization of a novel non-infectious herpes simplex virus-related particle.

During gradient purification of herpes simplex virus type 1 (HSV-1) two bands of particles were observed: a sharp lower band and a more diffuse upper band. The lower band contained almost exclusively HSV-1 virions (H particles) whereas the upper band consisted of membrane-enclosed particles (L particles). These L particles resembled the virions in appearance, but lacked the viral nucleocapsid and were not infectious. Many polypeptides of the viral envelope and the tegument were common to both types of particles. The H particles had polypeptide profiles typical of HSV virions. The L particles contained at least three phosphoproteins (175K, 92K and 55K) and a further two phosphorylated polypeptides not normally observed in virion profiles which comigrated with the 134K and 60K glycoproteins. This clearly indicates that the novel L particles were not merely virions which had formed without the inclusion of a nucleocapsid or virions which had subsequently lost their nucleocapsid during preparative handling. Thus these novel L particles are genuine products of the infectious processes occurring when HSV-1 replicates.

Role of the major capsid protein in herpes simplex virus type-1 capsid assembly.

Two herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutants with defects in the gene for the major capsid protein, ICP5, were examined for their effects on virion capsid assembly. Polyacrylamide gel electrophoresis revealed that both mutants were able to synthesize wild-type (WT) levels of ICP5 at the nonpermissive temperature. However, the 53 kD capsid protein disappeared concomitant with the appearance of a new, 51 kD species. These results, taken together with ultrastructural and immunological analyses indicate that the processing and assembly of capsid proteins, DNA packaging and thermal stability of HSV-1 virions are dependent upon functional ICP5.

Oligomerization of herpes simplex virus glycoprotein B occurs in the endoplasmic reticulum and a 102 amino acid cytosolic domain is dispensable for dimer assembly.

Glycoprotein B (gB) is an essential protein specified by herpes simplex virus and a major envelope component of the virions. It is known to assemble into noncovalently associated dimers. The aim of this study was to investigate the role of topogenic domains of gB in dimer assembly and the intracellular location at which gB dimers are assembled. Therefore, dimer analyses were performed on intact gB and its three COOH-terminus-truncated gB derivatives encoding NH2-terminal 772, 586, and 477 amino acids (aa) of the mature gB, using SDS-polyacrylamide gel electrophoresis and sucrose gradient assays. Dimers were detected in gB and in tgB(772 aa), but were absent from tgB(586 aa) and from tgB(477 aa). These results showed that a 102 aa cytosolic domain (aa 773-874) is not required for the assembly of gB dimers. In addition, using endoglycosidase H treatment and dimer analysis of gB synthesized during 7 min pulse-labeling period, we have demonstrated that ER is the subcellular organelle at which gB monomers are assembled into dimeric forms.

Gel filtration high performance liquid chromatography of envelope polypeptide variants of herpes simplex type 1 strains.

High performance liquid chromatography (HPLC) with a TSK-4000SW gel filtration column was used to compare envelope polypeptides from four strains of herpes simplex virus type 1 (HSV-1). The chromatographic profiles demonstrated polypeptide variability among three clinical strains and the wild-type F strain. Radioimmunoprecipitation of the HPLC fractions with polyclonal anti-HSV-1 followed by SDS-polyacrylamide gel electrophoresis (PAGE) of the immunoprecipitates revealed molecular weight differences of various polypeptides in fractions from the area containing major peaks. This HPLC method could prove useful for the analysis of polypeptide polymorphism in clinical isolates of HSV-1, as well as in other viruses.

Comparative studies of HSV-1 antigens solubilised from infected cells by using non-ionic or zwitterionic detergents.

HSV-1 antigen preparations solubilised from Vero cells by using either the non-ionic detergent Nonidet P40 or the zwitterionic detergent Empigen BB, and purified on sucrose density gradients or over a sucrose cushion, were tested by ELISA with anti-HSV-1 glycoprotein monoclonal antibodies and by radioimmunoprecipitation (RIP) with polyclonal HSV-1 antiserum. Amongst several proteins detected in these preparations, the four major HSV-1 glycoproteins, gB, gC, gD, and gE, were found to be present. Differences between NP40 or Empigen-solubilised HSV-1 antigen preparations with respect to two of these glycoproteins, gB and gE, were detected by using a small panel of monoclonal antibodies. Comparative studies in mice showed the Empigen-solubilised HSV-1 antigen preparations elicited greater antibody responses and greater protection against lethal HSV-1 challenge infection than the NP40-solubilised preparation.

Estudio inmunoelectromicroscópico con oro coloidal en muestras de cerebro de dos gemelos monocigóticos de madre esquizofrénica / Immunoelectromicroscopic study with colloidal gold in brain samples from two monocigotic twins from a schizophrenic mother

Se hace un estudio mediante técnicas inmunoelectromicroscópicas con oro coloidal en muestras de cerebro de dos fetos gemelos monocigóticos de una madre esquizofrénica paranoide. La presencia de antígeno del virus herpes simplex tipo I (HSV-1) en las muestras de cerebro de los dos gemelos estudiados, confirman los resultados de estudios anteriores mediante técnicas de inmuno-peroxidasa en cerebros de adultos esquizofrénicos fallecidos y en fetos de madres esquizofrénicas, lo que plantea una posible relación de la enfermedad con este tipo de virus y la posibilidad de la adquisición de la misma durante las primeras etapas del desarrollo intrauterino. Estos resultados a su vez se analizan a la luz de observaciones clínicas y epidemiológicas que apoyan esta hipótesis

Estudio inmunoelectromicroscópico con oro coloidal en muestras de cerebro de dos gemelos monocigóticos de madre esquizofrénica / Immunoelectromicroscopic study with colloidal gold in brain samples from two monocigotic twins from a schizophrenic mother

Se hace un estudio mediante técnicas inmunoelectromicroscópicas con oro coloidal en muestras de cerebro de dos fetos gemelos monocigóticos de una madre esquizofrénica paranoide. La presencia de antígeno del virus herpes simplex tipo I (HSV-1) en las muestras de cerebro de los dos gemelos estudiados, confirman los resultados de estudios anteriores mediante técnicas de inmuno-peroxidasa en cerebros de adultos esquizofrénicos fallecidos y en fetos de madres esquizofrénicas, lo que plantea una posible relación de la enfermedad con este tipo de virus y la posibilidad de la adquisición de la misma durante las primeras etapas del desarrollo intrauterino. Estos resultados a su vez se analizan a la luz de observaciones clínicas y epidemiológicas que apoyan esta hipótesis

Purification of the structural proteins of herpes simplex virus type 1 by reverse-phase high performance liquid chromatography.

Herpes simplex virus type-1 structural proteins were solubilized in formic acid and purified by reverse-phase high performance liquid chromatography. Purified proteins have been used to prepare monospecific polyclonal antibodies which neutralized virus infectivity in vitro.

Nucleocapsid, nuclear association, and genome location of the herpes simplex virus type 2 38-kD DNA-binding protein.

A previously-described herpes simplex virus type 2 DNA-binding protein with a molecular size of 38 kD has been further characterized. Using purified nucleocapsids, a DNase release assay, and intertypic recombinants, this protein was found to be a component of the nucleocapsid, intimately associated with the nuclear matrix, and encoded between 0.605 and 0.720 on the herpes simplex virus type 2 genome.

Oral cavity herpes simplex virus--a risk factor to dental personnel and patients. An overview.

Herpes virus antigens were found in the sulcular epithelium of approximately 60% of patients with clinically healthy gingiva. In addition, specific antigens for herpes simplex virus (HSV) were found in the sulcular epithelial cells of patients undergoing periodontal treatment. Specific antibodies were also detected in 70-80% of the gingival fluids collected. On the basis of these data we hypothesized that the oral cavity may act as a preferential site for latent HSV. Thus, stressful events such as traumatic dental treatment and tissue damage may elicit herpetic episodes, risking dental personnel. Measures of precaution are indicated for routine dental treatment.

Cytochemical analysis of DNA organization during encapsidation in herpes simplex virus.

The organization of intranuclear Herpes simplex virus DNA in rabbit fibroblast cells infected for 7 hr with HSV type 1 was examined before and during encapsidation by electron microscopic cytochemistry. Most non-encapsidated viral deoxyribonucleoprotein fibers exhibited a non-nucleosomal configuration. Empty capsids within the virus-specific regions of infected nuclei were wrapped with portions of the viral genome which adhered tightly to their surfaces even under conditions that loosened and spread apart other nucleoprotein fibers. During encapsidation, the internal surface of the capsid shell also appeared to bind a part of the viral genome, specifically the outer cage portion, which is detectable in methanol-dehydrated cells. Variations in the amount of DNA within the capsids indicated that the insertion of HSV genome into the capsid is a progressive process. The cage and core cylinder portions of the viral nucleoid appear to form and develop simultaneously. We propose that there may be binding sites on both the external and internal surfaces of the capsid shells which might play a role in the encapsidation process.

In situ detection of DNA-binding proteins in herpes simplex virus type 1-infected cells.

An in situ assay for detecting DNA-binding proteins in herpes simplex virus type 1 (HSV-1)-infected cells is described. Seventeen HSV-induced DNA-binding species were visible with nicked, double-stranded DNA as a substrate, while fourteen virus-induced DNA-binding fractions were present in gels containing nuclease-treated, single-stranded DNA. The effects of HSV on cellular DNA-binding protein expression could also be seen. The resolution of DNA-binding fractions was dependent upon the type of DNA substrate utilized, high salt extraction of DNA-binding components and their physical separation from infected cell DNAs, dialysis of the high salt and the length of DNase treatment of gels following electrophoresis.

Mapping of epitopes on the 65k DNA-binding protein of herpes simplex virus type 1.

Previously we have described the isolation of seven monoclonal antibodies (MAbs) and two polyvalent rabbit sera directed against the product of herpes simplex virus type 1 (HSV-1) gene UL42, a 65K DNA-binding protein (65KDBP) which is essential for HSV DNA replication and virus growth. We now report the synthesis of all 483 overlapping hexapeptides of this 488 amino acid protein and describe their use for the identification of epitopes recognized by these MAbs and polyvalent sera. MAb 6898, derived from one fusion, recognizes the peptides EDLDGA and DLDGAA which correspond to amino acids 363 to 369 of 65KDBP. MAbs Z4D4, Z6F3, Z1A8, Z10Cl, Z3H12 and Z1F11, derived from a second fusion, all recognize the peptides GDPEDL and DPEDLD which correspond to amino acids 360 to 366. As expected both polyvalent sera recognize several different epitopes.

A major transcriptional regulatory protein (ICP4) of herpes simplex virus type 1 is associated with purified virions.

Herpes simplex virus type 1 was purified by density gradient centrifugation, and the virion-associated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By Western blot (immunoblot) analysis with an anti-ICP4 monospecific serum, the results indicated that ICP4, one of the five immediate-early proteins of herpes simplex virus type 1, was associated with the purified virions. To define the location of ICP4 within the virion, trypsin digestion experiments were performed. Purified virions were treated with trypsin in the presence or absence of detergent. The virus envelope appeared to protect ICP4 from the trypsin, since virus-associated ICP4 was sensitive to digestion only after detergent treatment. In addition, ICP4 remained associated with the virus particle when the virion-specific glycoproteins were removed after detergent treatment. Finally, ICP4 was not detected in purified preparations of type A and B capsids isolated from the nuclear fraction of virus-infected cells. The above-mentioned data suggest that detectable amounts of ICP4 are present within the tegument region of the virion.

Detection of viral antigens in patients with IgA nephropathy.

Immunofluorescent analysis of viral antigens in the cultured fibroblasts and renal tissues in patients with IgA nephropathy was described. Freeze and thawed extracts of pharyngeal cells obtained from patients with IgA nephropathy, chronic proliferative glomerulonephritis without IgA deposition (PGN) and healthy adults were cultured with human fibroblasts, i.e. Hel cells, with or without addition of 5-iodine 2'-deoxy-uridine (IUDR) at 37 degrees C for 2 weeks. These fibroblasts and renal sections were stained with several kinds of FITC-labeled antiviral antibodies. Deposition of adeno, herpes simplex, varicella zoster or parainfluenza 3 was observed not only in the renal sections but also in the nuclear regions and/or cytoplasm of Hel cells after incubation of extracts of pharyngeal cells with or without IUDR from patients with IgA nephropathy. It is indicated that antigenic stimulation in the upper respiratory tracts may be due to several different types of DNA and/or RNA viruses in patients with IgA nephropathy. It appears that these antigenic substances show some heterogeneity among these patients.