D. Falke

Herpes simplex virus binds to human serum lipoprotein.

Binding of herpes simplex virus (HSV) type 1 to the various subclasses of human serum lipoproteins was investigated. Studies were performed with human serum lipoproteins purified by differential ultracentrifugation and artificial proteoliposomes containing only one type of apolipoprotein (A1, E) by using an enzyme-linked immunosorbent assay technique, column chromatography, and electron microscopy. All tested lipoprotein subclasses (very low, low-, high-density lipoproteins; VLDL, LDL, HDL, HDL1) showed significant binding of purified HSV type 1. Furthermore, HSV bound to all different synthetic proteoliposomes. Adsorption of envelope proteins isolated from purified HSV to Sepharose-bound lipoproteins revealed binding of HSV glycoprotein B. Based on these results we reached the conclusion that in HSV-lipoprotein complex formation the lipid component in the lipoproteins and the glycoprotein B in HSV are the preferential reaction partners.

Intracellular distribution of the La antigen in CV-1 cells after herpes simplex virus type 1 infection compared with the localization of U small nuclear ribonucleoprotein particles

The La antigen is known to associate, at least transiently, with a series of small nuclear and cytoplasmic ribonucleoprotein particles (snRNPs and scRNPs), e.g. U1 and U6 snRNPs. In CV-1 cells a monoclonal antibody (MAb), directed against the La protein (La1B5), immunostained intranuclear speckles. These speckles were found to co-localize with speckles that were stained by MAbs directed against either all U snRNPs or only against U1 snRNPs. Two h after infection of CV-1 cells with herpes simplex virus type 1 (HSV-1) (strain HFEM) the staining of nuclear speckles with the anti-La MAb disappeared and the La protein was found quantitatively in the cytoplasm. In contrast nuclear speckles remained stained with the MAbs against the U snRNPs. Similar results were obtained using HSV-1 strains Lenette or 17 syn+ or temperature-sensitive (ts) mutants defective either in DNA synthesis (tsS) or in the immediate early protein (Mr 175 K) (tsK). Later in infection the La protein returned to the nucleus. Six h after infection most of the nuclear La protein was found to localize within patchy regions. These areas seem to be related to heterogeneous nuclear RNA transcription and/or processing sites, but not to DNA replication sites.

9-β-d-Arabinofuranosyladenine as a tool to study herpes simplex virus DNA replication in vitro

Abstract 9-β- d -Arabinofuranosyladenine (araAdo) strongly suppresses herpes simplex virus (HSV) DNA synthesis in intact cell systems. After incubation with araAdo, two HSV-DNA fractions can be isolated by neutral isopycnic CsCl density gradient centrifugation, a light fraction with a buoyant density of 1.726 g/cm 3 , and a heavy fraction with a density of 1.738 g/cm 3 . After recentrifugation in neutral CsCl gradients, the light and heavy fractions are detected at a density of 1.729 and 1.741, respectively. Analysis of the two HSV-DNA fractions by hydroxylapatite chromatography and by digestion with nuclease S 1 revealed that the light fraction consists predominantly of native DNA and the heavy fraction of denatured DNA. AraAdo is incorporated into HSV-DNA and has been shown to be present in terminal positions at the 3′-hydroxyl position. The HSV-DNA pieces, containing incorporated araAdo, are of low molecular weight (less than 2.6 × 10 6 ) and are not assembled to higher molecular weight aggregates.

Fusion from Without Induced by Herpes Simplex Virus Type 1

Strains ANG and ANG path of herpes simplex virus type 1 (HSV1) produced fusion from without (FFWO) of cells in culture. FFWO required 45 min to become complete. In contrast, fusion from within (FFWI) was not detected until 3-4 h after infection, depending on the cell type. FFWO was temperature dependent: at 0 degrees no fusion could be observed, but increase of temperature increased the degree of fusion. The pH optimum for FFWO was 7.8-8.5. The FFWO activity of the virus was found to be slightly more heat stable at 46 degrees than was infectivity. FFWO was produced in Vero, CV-1 and BSC1 cells, but not in BHK clone 13 or in primary or secondary rabbit kidney cells. FFWO was linked to the presence of virus particles and perhaps to other sedimentable, infected-cell material but not to soluble factors. Actinomycin D, cycloheximide, and UV irradiation did not block this activity, indicating no direct activity of the HSV1 genome for FFWO.

Thymidine-, uridine- and choline-kinase in rabbit kidney cells infected with herpesvirus hominis, type I and II

Data are presented about the activity of the thymidine-, uridine- and choline-kinase after infection with 21 strains ofherpesvirus hominis of serotype I or II in rabbit kidney cells. Type I strains increase the activity of the thymidine-kinase 15–20 fold over the controls, whereas the type II strains demonstrate a moderate activity, the level of the enzyme is increased 2–5 fold. One giant cell forming strain exhibits unusual properties, the TK activity decreases in correlation to the controls. The uridine- and choline-kinases induce the respective enzymes in different manner. The strains tested are divided into 5 groups depending upon the activity of the enzymatic activity. The implications of the results are discussed.

Enhancement by TNF-α of reactivation and replication of latent herpes simplex virus from trigeminal ganglia of mice

SummaryThe influence of tumor-necrosis-factor-α (TNF-α), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukine-1 (IL-1) and IL-3 on the in vitro reactivation frequency and replication rate of trigeminal ganglia of mice latently infected with herpes simplex virus (HSV) strain KOS was studied. It could be demonstrated that TNF-α and possibility GM-CSF, but not IL-1 and IL-3, enhanced the reactivation frequency and replication of HSV. Interferon α/β (IFNα/β) prevented reactivation and replication.

Involvement of actin-containing microfilaments in HSV-induced cytopathology and the influence of inhibitors of glycosylation.

SummaryTwo and a half hours after infection with a high dose of different strains of HSV-1 which induce rounding of cells, breakdown of actin containing microfilaments can be observed. At the periphery of the cell, actin containing knob-like protuberances were visible. Later on, actin seems to be located exclusively on the surface of cells. Observations were done by immunofluorescence microscopy, scanning electron-microscopy and immunoperoxidase staining of ultrathin sections. The envelope of HSV appears to be stained by anti-actin. Strain IES produces rounding of cells at a high dose of infection before fusion proceeds at 37°C. Similar alterations were not observed with the fusing strains MP and HFEM. Incubation of infected cells at 39°C revealed strain dependent differences of the fusion activity. At 41°C no “fusion from within” of cells but only rounding was detectable. Application of tunicamycin resulted in complete inhibition of fusion by all strains. The fusion activity of some strains of HSV-1 (ANG, HFEM, and MP) was not inhibited by addition of 2-deoxy-D-glucose and 2-fluoro-deoxy-D-glucose. A variant from strain MP could be isolated, which is sensitive to the effects of 2-deoxy-D-glucose. Inhibitors of processing of glycoproteins did not affect fusion of cells.

Infections of susceptible and resistant mouse strains with herpes simplex virus type 1 and 2

SummaryThe spread of HSV of type 1 and 2 was investigated after intraperitoneal, intraplantar and intracerebral infections of resistant (C57/bl) and susceptible (NMRI) mice. The virus spreads after i.p. infection to the spleen and the liver to the same extent in both strains of mice. However, virus is eliminated earlier in resistant mice. Intracerebral infections revealed a peculiar type of resistance of C57/bl mice especially for type 2 of HSV. HSV multiplies in the thymus at the early stage of infection and can be detected in this organ in sick mice of NMRI strain. HSV-1 and 2 can be detected in the spinal cord of C57/bl mice without sickness or death of these animals.

Replication of HSV-1 in murine peritoneal macrophages: Comparison of various virus strains with different properties

SummaryThein vitro replication of eleven different strains of herpes simplex virus type 1 was studied in resident or thioglycollate-stimulated mouse macrophages. The strains of herpes simplex virus differed in the type of cytopathic effect, induction capacity for herpes simplex virus coded thymidine kinase and pathogenicity in the mouse. Herpes simplex virus replicated better in thioglycollate-stimulated macrophages than in resident macrophages.In vitro ageing of macrophages increased their replicative potency. Herpes simplex virus replicated better in macrophages from homozygous bg/bg C57/BL6J mice than in macrophages from their heterozygous littermates. Separation of macrophages on discontinous Percoll-gradients revealed 4 fractions with identical potency for replication. The ability of herpesvirus to replicate in macrophages varied from strain to strain of virus i.e. Wal > Len, clone 4 of Len, >L3-2s, JES, Ang−, Ang+path, clone 2 of Len and >MDK clones. The ability to cause cytopathology also varied. Only strains Ang− and Ang+path showed limited or late cytopathology in macrophages. The cell-fusing property of herpes simplex virus appeared to be more closely correlated with lower replication rates than production cell rounding. Thymidine kinase− viruses replicated less well than thymidine kinase+ or thymidine kinase(+) strains. Strains of herpes simplex virus with high or low pathogenicity for mice replicated in macrophages to the same degree. The phagocytic activity of macrophages for IgM-coated sheep red blood cells was inhibited earlier by strains of herpes simplex virus of type 2 than by strains of herpes simplex virus of type 1.

Variation of DNA polymerase and RNA polymerase activities in cells infected with herpes simplex virus type 1

Abstract Infection of rabbit kidney cells with herpes simplex virus (HSV) leads to pronounced alterations of the different species of DNA-dependent DNA and DNA-dependent RNA polymerases. The activity of distinct polymerase species was determined; it was ruled out first that deoxyribonucleases or ribonucleases had an influence on these determinations and second that the activities of isoenzymes modified the evaluation of the activity of a distinct enzyme species. The three different DNA-dependent DNA polymerases (form a and β and HSV-induced enzyme) were separated by velocity sedimentation through sucrose density gradients and were asscyayed using activated DNA as template. DNA polymerase a and HSV polymerase were resolved by different assay conditions. The cellular polymerase a decreases by 80% 10 hr after infection with HSV, while the cellular polymerase β shows a slight increase during this period. The activity of HSV-induced DNA polymerase at the beginning of infection is virtually zero; it increases to a maximum 8 hr postinfection. The activity of DNA-dependent RNA polymerase I declines dramatically, by 68%, after infection with HSV while the alterations of the activities of RNA polymerases II and III are less pronounced.