Deoxycytidine kinase from rabbit kidney cells infected with herpes simplex virus type 1 or 2.

When rabbit kidney cells were infected with herpes simplex virus type 1 (strain Seibert) or herpes simplex virus type 2 (strain 316D), deoxycytidine kinase (CdR kinase) activity, assayed at 38 degrees, increased 5- to 15-fold relative to controls. The CdR kinase activity induced by type 2 virus was more thermolabile than the enzyme activity induced by type 1 virus. When CdR kinase activity was assayed at various temperatures between 0.5 and 38 degrees, maximum activity for type 1 enzyme was obtained at 16 degrees while maximum activities for host and type 2 enzymes were obtained at 38 degrees. Both type 1 and type 2 induced CdR kinase activities eluted at the same positions as deoxythymidine kinase activities on a Sephadex G-100 column. The estimated mol wt for HSV-1 (Seibert) and HSV-2 (316D) induced CdR kinases are 67,000 and 60,000, respectively.     

Increased adherence of human granulocytes to herpes simplex virus type 1 infected endothelial cells

Summary We studied the interaction of human polymorphonuclear leukocytes (PMNs) with umbilical vein endothelial cells infected with herpes simplex virus (HSV) type 1. PMNs labeled with⁵¹Cr were added to endothelial monolayers at varying times after infection and their adherence assessed 1 h later. Granulocyte adherence (GA) to uninfected cells averaged 26.5±1.9%. Increased adherence began 6 h postinfection and rose to a maximum at 20 to 24 h. HSV-1 glycoproteins seemed to mediate the increase in GA: tunicamycin treatment of infected monolayers for 18 h abolished the increased GA as did incubation of infected cells with F(ab')₂ fragments prepared from human antiserum containing HSV-1 antibody. Supported by grants R01-AA-06029 and T32-AA07233 from the National Institute of Alcohol Abuse and Alcoholism, and R01-HL-28220 from the National Heart, Lung, and Blood Institute.     

Immature monocyte-derived dendritic cells are productively infected with herpes simplex virus type 1

Herpes simplex viruses (HSV) have developed several immunoevasive strategies. Here we demonstrate a novel mechanism by which HSV type 1 may interfere with the immune response through infection of immature dendritic cells (DC) and selective downmodulation of costimulatory molecules. In our study we show productive infection of immature monocyte-derived DC, which closely resemble sessile Langerhans cells, by sequential expression of immediate-early, early, and late viral proteins and of glycoprotein D mRNA, as well as production of infectious virus of moderate titers. Infection was cytopathic, with the progressive loss of 20 to 45% of cells from 24 to 48 h after infection, with no more than 80% of DC found to be infected. These results are in contrast to those of previous findings of nonpermissive or abortive infection of monocytes and mature monocyte-derived DC. Infection of immature DC also led to selective and asynchronous downregulation of CD1a, CD40, CD54 (ICAM-1) (12 h postinfection), CD80 (24 h postinfection), and CD86 (48 h postinfection) but not of CD11c or major histocompatibility complex class I and II molecules when compared to DC exposed to UV-inactivated virus. Thus, we propose that productive infection of epidermal Langerhans cells in vivo may lead to delayed activation of T cells, allowing more time for replication of HSV type 1 in epidermal cells.     

Polyphosphoinositide metabolism in baby-hamster kidney cells infected with herpes simplex virus type 1.

The incorporation of [32P]Pi and [3H]inositol into the inositol lipids of baby-hamster kidney cells was studied in herpes-simplex-virus-type-1(HSV-1)-infected and mock-infected cells. The infection was conducted during incorporation of, as well as after prelabelling with, the precursors. These methods were used in order to study both synthesis de novo of, and steady-state changes in, the phosphoinositides. Both with infection during labelling, and after prelabelling, we found increased [32P]- and [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) and decreased [32P]- and [3H]-phosphatidylinositol 4-monophosphate in infected as compared with mock-infected cells, whereas no effect was observed on phosphatidylinositol. This altered inositol-lipid metabolism was (at least in the case of PIP2) not present until 3-6 h after infection and remained stable, or increased slightly, throughout the infection period. Polyphosphoinositide metabolism constitutes an important step in signal processing in many forms of cellular stimulation, and the results obtained suggest that HSV-1 infection may induce such events in our cell system.     

Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells

How viral infections affect host cell mitochondrial functions is largely unknown. In this study, uptake of radiolabeled precursors was used to assess how a herpes simplex virus type 1 (HSV 1) infection influences synthesis of macromolecules comprising Vero cell mitochondria. Total macromolecular synthesis in infected cells was determined for comparative purposes. Mitochondrial and total cellular DNA syntheses were approximately halved at 1-2.5 h postinfection (PI). Mitochondrial DNA synthesis in infected cells then rose to 3.5-fold that in control cells at 3-4.5 h PI. Total DNA synthesis in infected cells also rose, but more slowly, reaching threefold that for control cells at 5-6.5 h PI. Mitochondrial and total RNA synthesis in infected cells were both decreased by approximately 40% at 1-3 h PI. Over the next 4 h, total RNA synthesis in infected cells slowly continued to decrease, while that in mitochondria recovered to control levels. Synthesis of mitochondrial proteins in infected cells decreased progressively, dropping to about 60% of control levels by 5-6.5 h PI. With the metabolic inhibitors ethidium bromide and cycloheximide, it was determined that nuclear DNA and mitochondrial DNA and mitochondrial DNA directed synthesis of mitochondrial proteins were each partially inhibited in infected cells. Total cellular protein synthesis was decreased by 30% at 1-2.5 h PI and then recovered to control levels by 5-6.5 h PI. Finally, phospholipid synthesis in mitochondria from infected cells was elevated 2.3-fold at 1-5 h PI, but dropped to 14% below control levels during 4-8 h PI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early induction of autophagy in human fibroblasts after infection with human cytomegalovirus or herpes simplex virus 1

The infection of human fetal foreskin fibroblasts (HFFF2) with human cytomegalovirus (HCMV) resulted in the induction of autophagy. This was demonstrated by the increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, and by the visualization of characteristic vesicles within infected cells. The response was detected first at 2 h postinfection and persisted for at least 3 days. De novo protein synthesis was not required for the effect, since HCMV that was irradiated with UV light also elicited the response, and furthermore the continuous presence of cycloheximide did not prevent induction. Infection with herpes simplex virus type 1 (HSV-1) under conditions that inhibited viral gene expression provoked autophagy, whereas UV-irradiated respiratory syncytial virus did not. The induction of autophagy occurred when cells were infected with HCMV or HSV-1 that was gradient purified, but HCMV dense bodies and HSV-1 light particles, each of which lack nucleocapsids and genomes, were inactive. The depletion of regulatory proteins Atg5 and Atg7, which are required for autophagy, reduced LC3 modification in response to infection but did not result in any detectable difference in viral or cellular gene expression at early times after infection. The electroporation of DNA into HFFF2 cultures induced the lipidation of LC3 but double-stranded RNA did not, even though both agents stimulated an innate immune response. The results show a novel, early cellular response to the presence of the incoming virion and additionally demonstrate that autophagy can be induced by the presence of foreign DNA within cells.     

Release of a virus coded glycoprotein from herpes simplex virus type 1 infected cells

HEp-2 cells, which were infected with HSV-1, excrete besides other proteins a soluble glycoprotein (Mr 125000–130000) related to the virus protein gC. The excretion of the glycoprotein and the production of extracellular virus particles is reduced to a similar extent when the cells were treated with monensin. Possible consequences of the excretion of soluble viral proteins to a modulation of the immune response are discussed.Abbreviations HSV-1 Herpes simplex virus type 1 - PAGE Polyacrylamide gel electrophoresis - SDS Sodium dodecylsulfate     

Fate of microfilaments in vero cells infected with measles virus and herpes simplex virus type 1.

In herpes simplex virus type 1-infected Vero cells, reorganization of microfilaments was observed approximately 4 h postinfection. Conversion of F (filamentous) actin to G (globular) actin, as assessed by a DNase I inhibition assay, was continuous over the next 12 to 16 h, at which time a level of G actin of about twice that observed in uninfected cells was measured. Fluorescent localization of F actin, using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, demonstrated that microfilament fibers began to diminish at about 16 to 18 h postinfection, roughly corresponding to the time that G actin levels peaked and virus-induced cytopathology was first observable. In measles virus-infected cells, no such disassembly of microfilaments occurred. Rather, there was a modest decrease in G actin levels. Fluorescent localization of F actin showed that measles virus-infected Vero cells maintained a complex microfilament network characterized by fibers which spanned the entire length of the newly formed giant cells. Disruption of microfilaments with cytochalasin B, which inhibits measles virus-specific cytopathology, was not inhibitory to measles virus production at high multiplicities of infection (MOI) but was progressively inhibitory as the MOI was lowered. The carbobenzoxy tripeptide SV-4814, which inhibits the ability of Vero cells to fuse after measles virus infection, like cytochalasin B, inhibited measles virus production at low MOI but not at high MOI. Thus, it appears that agents which affect the ability of Vero cells to fuse after measles virus infection may be inhibitory to virus production and that the actin network is essential to this process.     

Viral DNA synthesis in cells infected with temperature-sensitive mutants of herpes simplex virus type 1.

Temperature-sensitive mutants of herpes simplex virus type 1 representing eight DNA-negative complementation groups were grouped into the following three categories based on the viral DNA synthesis patterns after shift-up from the permissive to the nonpermissive temperature and after shift-down from the nonpermissive to the permissive temperature in the presence and absence of inhibitors of RNA and protein synthesis. (i) Viral DNA synthesis was inhibited after shift-up in cells infected with tsB, tsH, and tsJ. After shift-down, tsB- and tsH-infected cells synthesized viral DNA in the absence of de novo RNA and protein synthesis whereas tsJ-infected cells synthesized no viral DNA in the absence of protein synthesis. The B, H, and J proteins appear to be continuously required for the synthesis of viral DNA. (ii) Viral DNA synthesis continued after shift-up in cells infected with tsD and tsK whereas no viral DNA was synthesized after shift-down in the absence of RNA and protein synthesis. Mutants tsD and tsK appear to be defective in early regulatory functions. (iii) Cells infected with tsL, tsS, and tsU synthesized viral DNA after shift-up and after shift-down in the absence of RNA and protein synthesis. The functions of the L, S, and U proteins cannot yet be determined.     

Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity

Mature dendritic cells (DC) are the most potent antigen-presenting cells within the entire immune system. Interference with the function of these cells therefore constitutes a very powerful mechanism for viruses to escape immune responses. Several members of the Herpesviridae family have provided examples of such escape strategies, including interference with antigen presentation and production of homologous cytokines. In this study we investigated the infection of mature DC with herpes simplex virus type 1 (HSV-1) and the way in which infection alters the phenotype and function of mature DC. Interestingly, the T-cell-stimulatory capacity of these DC was strongly impaired. Furthermore, we demonstrated that HSV-1 leads to the specific degradation of CD83, a cell surface molecule which is specifically upregulated during DC maturation. These data indicate that HSV-1 has developed yet another novel mechanism to escape immune responses.     

Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.     

Dynamic 1H NMR-based extracellular metabonomic analysis of oligodendroglia cells infected with herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) is a large, neurotropic, double-stranded DNA virus that establishes a lifelong latent infection in neurons and glial cells. Previous studies reveal that several metabolic perturbations are associated with HSV-1 infection. However, the extracellular metabolic alterations associated with HSV-1 infection have not been systematically profiled in human cells. Here, a proton nuclear magnetic resonance-based metabonomic approach was applied to differentiate the extracellular metabonomic profiles of HSV-1 infected human oligodendroglia cells (n = 18) and matched control cells (n = 18) at three time points (12, 24, and 36 h post-infection). Resulting spectra were analyzed by chemometric and statistical methods. Metabonomic profiling revealed perturbations in 21 extracellular metabolites. Partial least squares discriminant analysis demonstrated that the whole metabolic patterns enabled statistical discrimination between HSV-1 infected human oligodendroglia cells and control cells. Eight extracellular metabolites, seven of which were amino acids, were primarily responsible for score plot discrimination between HSV-1 infected human oligodendroglia cells and control cells at 36 h post-infection: alanine, glycine, isoleucine, leucine, glutamate, glutamine, histidine, and lactate. HSV-1 infection alters amino acid metabolism in human oligodendroglia cells cultured in vitro. HSV-1 infection may disturb these host cellular pathways to support viral replication. Through elucidating the extracellular metabolic changes incident to HSV-1 infection, this study also provides future directions for investigation into the pathogenic mechanism of HSV-1.