Cellular proteins expressed in herpes simplex virus transformed cells also accumulate on herpes simplex virus infection.

The cell proteins expressed in rat embryo cells transformed by herpes simplex virus (HSV) have been analysed by immunoprecipitation assays to determine those polypeptides which can be identified by immunoprecipitation with the sera of tumour-bearing animals and also with antisera to herpes simplex infected cells. Cell polypeptides commonly recognised by both these sera have been further characterised using a monoclonal antibody directed against a cellular polypeptide which accumulates on HSV-2 lytic infection. This monoclonal antibody recognises in HSV-transformed cells polypeptides of mol. wts. 90 000, 40 000 and 32 000. Further studies show that the accumulation of these polypeptides in HSV-transformed cells is not HSV specific but is a common feature of transformation or of cells which have been immortalised. We suggest that cellular polypeptides accumulating as a result of HSV infection may be of importance in the initiation of transformation by HSV, i.e., at the level of immortalisation of cells.     

Persistent Herpes Simplex Virus Infection In Vitro with Cycles of Cell Destruction and Regrowth.

Hampar, Berge (National Institute of Dental Research, Bethesda, Md.), and Mary Lou Copeland. Persistent herpes simplex virus infection in vitro with cycles of cell destruction and regrowth. J. Bacteriol. 90:205-212. 1965.-The susceptibility of two Chinese hamster cell lines to herpes simplex virus (HSV) was studied from the time of their initiation through successive subcultures. The cells' susceptibility to the cytocidal effects of HSV decreased as the number of cell passages increased. During the early cell passages, the decrease in cell susceptibility to HSV was characterized by an increased time after infection for complete cell destruction to occur, with a concomitant increase in the period when virus could be recovered from supernatant fluids. This was followed by a number of cell passages during which persistent HSV infections were established. The persistent infections were characterized by (i) cycles of virus synthesis and cell destruction followed by regrowth of the cells, (ii) initiation and maintenance under conditions optimal for cell growth in the absence of antibody, (iii) the cells' ability to be passaged while still maintaining their cycling patterns, (iv) a relationship between virus synthesis and cell proliferation, and (v) inability of long-term treatment with antibody to "cure" the persistent infections. The unique characteristics of this HSV infection were compared with other persistent in vitro viral infections.     

Host-Cell Lysosomal Response to Two Strains of Herpes Simplex Virus

A correlation has been made between the host lysosomal responses to and release of infectious virus from HEp-2 cells infected with two strains of herpes simplex virus (HSV). Supravital staining with acridine orange was used for morphological studies of macroplaque and microplaque HSV-infected cells. With the progression of infection, cells infected with either microplaque HSV or macroplaque HSV were observed to undergo different lysosomal and cytopathic changes, which could be correlated with increased accumulation of acid phosphatase and infectious virus in the extracellular fluid.     

Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans

The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.     

Role of T-lymphocyte subsets in recovery from herpes simplex virus infection.

Our investigations probed the nature of different T-lymphocyte subsets effecting clearance of herpes simplex virus after infection of the pinna. Cell populations from animals recently infected subcutaneously or intraperitoneally (acute population) or from animals infected 6 weeks previously (primed population) or the latter cells reimmunized in vitro with virus (memory population) were studied. Viral clearance was a function of the Lyt 1+2- subset in the acute population, but with the memory population both Lyt 1+ and Lyt 2+ cells affected clearance. In primed populations, viral clearance was effected only by the Lyt 2+ subset. The ability of the various cell populations to adoptively transfer delayed-type hypersensitivity was also studied. Only acute population cells from animals infected subcutaneously and memory population cells transferred delayed-type hypersensitivity. In both cases, the cell subtype was Lyt 1+2-. Our results demonstrated that the delayed-type hypersensitivity response does not always correlate with immunity to herpes simplex virus. Multiple subsets of T cells participate in viral clearance, and their respective importances vary according to the stage of the virus-host interaction.     

Induction of uracil-DNA glycosylase and dUTP nucleotidohydrolase activity in herpes simplex virus-infected human cells

HeLa BU cells infected with either the type 1 or the type 2 forms of herpes simplex virus show an increase in the activities of uracil-DNA glycosylase and dUTP nucleotidohydrolase. Under optimal conditions, uracil-DNA glycosylase activity increases approximately 40-fold in HSV type 2-infected cells. In herpes simplex virus (HSV) type 1-infected cells, uracil-DNA glycosylase activity increases only 6-fold. At a KCl concentration of 100 mM, uracil-DNA glycosylase derived from HSV type 2-infected cells is activated 2-fold, while the glycosylase extracted from mock infected HeLa BU cells is inhibited almost 90% at 100 mM KCl. dUTP nucleotidohydrolase activity increases 4-fold and 3-fold, respectively, in HSV type 1- and HSV type 2-infected HeLa BU cells. Nondenaturing polyacrylamide gel electrophoresis of extracts derived from the type 1- and type 2-infected cells indicates distinct electrophoretic mobilities from the host cell enzyme. dUTP nucleotidohydrolase RF values for the mock infected cells, HSV type 1, and HSV type 2 are 0.5, 0.25, and 0.33, respectively. Serum from rabbits immunized against cells infected with herpes simplex virus type 1 or type 2 specifically neutralizes the dUTPase and uracil-DNA glycosylase activities extracted from herpes simplex virus-infected cells. This serum does not neutralize dUTPase or uracil-DNA glycosylase activity derived from mock infected cells.     

Deoxyribonucleic Acid Synthesis in Synchronized Mammalian KB Cells Infected with Herpes Simplex Virus

We examined the patterns of host cell and virus deoxyribonucleic acid (DNA) synthesis in synchronized cultures of KB cells infected at different stages of the cell cycle with herpes simplex virus (HSV). We found that the initiation of HSV DNA synthesis, we well as the production of new infectious virus, is independent of the S, G1, and G2 phases of the mitotic cycle of the host cell. This is in contrast to data previously found with equine abortion virus. Because HSV replicates independently of the cell cycle, we were able to establish conditions that would permit the study of rates of HSV DNA synthesized in logarithmically growing cells in the virtual absence of cellular DNA synthesis. This eliminates the need for separation of viral and cellular DNA by isopycnic centrifugation in CsCl. We found that HSV DNA synthesis was initiated between 2 to 3 hr after infection. The rate of DNA synthesis increased rapidly, reaching a maximum 4 hr after infection, and decreased to 50% of maximum by 8 hr. Evidence is also presented which suggests that HSV infection can inhibit both the ongoing synthesis of host DNA as well as the initiation of the S phase.     

Cellular localization of nectin-1 and glycoprotein D during herpes simplex virus infection

During viral entry, herpes simplex virus (HSV) glycoprotein D (gD) interacts with a specific cellular receptor such as nectin-1 (PRR1/HveC/CD111) or the herpesvirus entry mediator A (HVEM/HveA). Nectin-1 is involved in cell-to-cell adhesion. It is located at adherens junctions, where it bridges cells through homophilic or heterophilic interactions with other nectins. Binding of HSV gD prevents nectin-1-mediated cell aggregation. Since HSV gD affects the natural function of nectin-1, we further investigated the effects of gD expression on nectin-1 during HSV infection or in transfected cells. We also studied the importance of the interaction between nectin-1 and the cytoplasmic protein afadin for HSV entry and spread as well as the effects of infection on this interaction. In these investigations, we used a panel of cells expressing nectin-1 or nectin-1-green fluorescent protein fusions as the only mediators of HSV entry. During HSV infection, nectin-1 localization at adherens junction was dramatically altered in a manner dependent on gD expression. Nectin-1 and gD colocalized at cell contact areas between infected and noninfected cells and at the edges of plaques. This specific accumulation of gD at junctions was driven by expression of nectin-1 in trans on the surface of adjacent cells. Reciprocally, nectin-1 was maintained at junctions by the trans expression of gD in the absence of a cellular natural ligand. Our observations indicate that newly synthesized gD substitutes for nectin-1 of infected cells at junctions with noninfected cells. We propose that gD attracts and maintains the receptor at junctions where it can be used for virus spread.     

Kinetics of herpes simplex virus photoinhibition by haematoporphyrin in both diploid and heteroploid cells

The kinetics of inhibition of herpes simplex virus type 1 (HSV 1) on both diploid (CEF) and heteroploid cells (HEp2) by light-irradiated haematoporphyrin (HP) was studied. The inactivation of HSV1 by HP was drug-dose dependent and light-irradiation dependent; the viruses grown in heteroploid cells being in all cases more sensitive to inhibition than viruses grown in diploid cells. Cell toxicity by HP was markedly more evident on HEp2 cells than on CEF. The highest viral sensitivity to photodynamic inactivation by HP was found to be between the 4th and the 5th hour after cell infection, when the viral DNA synthesis is at its peak and before it is incorporated into complete virions. Microfluorometric and spectrofluorometric assays revealed that virus infected cells always take up more HP than uninfected cells, and heteroploid cells incorporated more HP than diploid cells. The possibility that an increased uptake of HP and modifications of the cell micro-environment in virus infected cells could account for the viral-inhibiting properties of HP, is discussed.     

Role of NK cells in protection of mice against herpes simplex virus-1 infection

Natural killer (NK) cells have been implicated in the recognition and killing of a variety of virus infected target cells in vitro, yet their role in vivo remains uncertain. In these experiments, the role of NK cells in the regulation of resistance to herpes simplex virus-1 (HSV-1) was studied. Adult C57BL/6 mice are resistant to HSV-1 (HFEM strain), but are rendered highly susceptible by treatment with cyclophosphamide 24 hr prior to infection. In this model, passive transfer of 10(8) normal spleen cells or 10(7) poly I:C-treated spleen cells provided protection for 72% of the recipients. Spleen cells from NK cell-deficient beige mice similarly treated failed to engender passive protection. The phenotype of the cells responsible for transferring protection was NK1.1+, and asialo GM1+. Transfer of NK cells resulted in marked reduction of HSV titers in the livers and brains of recipients. These experiments provide direct evidence for a role for NK cells in protection against development of fatal HSV infection in mice.     

Cell surface expression of H2 antigens on primary sensory neurons in response to acute but not latent herpes simplex virus infection in vivo.

CD8(+) T lymphocytes and class I major histocompatibility complex (MHC-I) molecules profoundly influence the severity of neuronal herpes simplex virus (HSV) infection in experimentally infected mice. Paradoxically, neurons are classically regarded as MHC-I deficient. However, it is shown here that H2-encoded heavy chains (alphaCs) and their associated light chain, beta2 microglobulin, are present on the surfaces of primary sensory neurons recovered from sensory ganglia within 1 to 2 weeks of HSV infection. During this time, some neurons are found to be tightly associated with T cells in vivo. Prior data showed that termination of productive HSV infection in the peripheral nervous system is not dependent on cell-mediated lysis of infected neurons. Consistent with these data, immunogold electron microscopy showed that the density of cell surface H2 on neurons is an order of magnitude lower than on satellite glia, which is predicted to favor a noncytolytic CD8 cell response.     

Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection.

We showed that the expression of a single protein, glycoprotein D (gD-1), specified by herpes simplex virus type 1 (HSV-1) renders cells resistant to infection by HSV but not to infection by other viruses. Mouse (LMtk-) and human (HEp-2) cell lines containing the gene for gD-1 under control of the human metallothionein promoter II expressed various levels of gD-1 constitutively and could be induced to express higher levels with heavy metal ions. Radiolabeled viruses bound equally well to gD-1-expressing and control cell lines. Adsorbed viruses were unable to penetrate cells expressing sufficient levels of gD-1, based on lack of any cytopathic effects of the challenge virus and on failure to detect either the induction of viral protein synthesis or the shutoff of host protein synthesis normally mediated by a virion-associated factor. The resistance to HSV infection conferred by gD-1 expression was not absolute and depended on several variables, including the amount of gD-1 expressed, the dosage of the challenge virus, the serotype of the challenge virus, and the properties of the cells themselves. The interference activity of gD-1 is discussed in relation to the role of gD-1 in virion infectivity and its possible role in permitting escape of progeny HSV from infected cells.     

Trigeminal ganglion infection by thymidine kinase-negative mutants of herpes simplex virus after in vivo complementation.

Infection of trigeminal ganglion by herpes simplex virus (HSV) thymidine kinase-negative (TK-) mutants was investigated in mixed infection studies in mice. Mice were corneally inoculated with TK- HSV alone or with mixtures of TK- HSV-TK+ HSV. When inoculated alone, an arabinosylthymine-selected HSV type 1 TK- mutant and a HSV type 2 TK- deletion mutant infected mouse ocular tissues but rarely infected ganglion tissues. However, both TK- mutants readily infected ganglion tissues when they were inoculated in mixtures with TK+ HSV. By means of mixed infection studies, it was demonstrated that TK- HSV could readily establish acute and latent ganglion infections. It was thought that the frequent infection of trigeminal ganglion tissue by both TK- mutants after mixed TK(-)-TK+ HSV infection was the result of in vivo complementation. After mixed TK(-)-TK+ HSV infection and subsequent cultivation of ganglion explants in arabinosylthymine, results supported the conclusion that when TK- was present in ganglia it was in the same neurons that contained TK+ HSV.     

Modulation of apoptosis during herpes simplex virus infection in human cells

Human herpes simplex virus (HSV) is cytolytic and has profound impacts on its host cells. Consequences of HSV infection include the induction of apoptosis and the concomitant synthesis of proteins which act to block this process. We review recent evidence showing how this important human pathogen modulates the fundamental cell death process.     

Alterations in the protein synthetic apparatus of Friend erythroleukemia cells infected with vesicular stomatitis virus or herpes simplex virus.

We have compared the effects of infection with herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) on the protein synthetic apparatus of Friend erythroleukemia cells. Previous studies demonstrated that infection with HSV rapidly shuts off the synthesis of globin and other cellular polypeptides (Y. Nischioka and S. Silverstein, 1977, Proc. Natl. Acad. Sci. U.S.A. 74: 2370-2374). In contrast to these findings, globin synthesis persists in Friend erythroleukemia cells infected with VSV. Physical measurements of the size of bulk-infected cell mRNA, using hybridization with polyuridylic acid, demonstrated that there was no detectable change in the size of mRNA's after infection with VSV. A comparison of the kinetics of hybridization of cytoplasmic RNA extracted from cells infected with either HSV or VSV with globin complementary DNA revealed that by 4 h postinfection with HSV only about 15% of the globin mRNA sequences remained, whereas there was no discernible change in the sequence abundance of globin mRNA in VSV-infected cells.     

Persistent cyclic herpes simplex virus infection in vitro. IV. Changes in the severity of the infections in the presence of antibody

Hampar, Berge (National Institute of Dental Research, Bethesda, Md.). Persistent cyclic herpes simplex virus infection in vitro. IV. Changes in the severity of the infections in the presence of antibody. J. Bacteriol. 92:1741-1747. 1966.-The severity of persistent herpes simplex virus (HSV) infections maintained in the presence of viral antibody (AB) changed with time. These changes could be divided into three distinct phases during which the severity of the infections were maintained at low (phase 1), high (phase 2), and intermediate (phase 3) levels. Paralleling these changes was the appearance in the cultures of a new HSV variant which induced the formation of giant cells. The changes in the severity of the infections were attributable to three factors. The first was the presence in the cultures of virus-resistant cells. The second was the suppressive effects due to virus neutralization by the AB. The third was the inherent properties of each HSV variant which determined the amount of virus remaining in an infectious form in the presence of AB. Finally, the period of "exacerbation" in vitro (phase 2) was compared with recurrent episodes in human herpes infection.     

Inhibition of phosphorylation of cellular dUTP nucleotidohydrolase as a consequence of herpes simplex virus infection

During an infection with herpes simplex virus, activity of cellular dUTPase decreases as a function of time, post-infection, while virus-encoded dUTPase activity increases. Prelabeling of cells with 35S-methionine and immunoprecipitation analysis, using monoclonal antibodies, indicates that cellular dUTPase protein levels remain the same (with respect to levels in uninfected cells) throughout the infection period. New synthesis of cellular dUTPase does not occur in infected cells as determined by 35S-methionine labeling during infection. Further characterization of the cellular dUTPase, in uninfected cells, reveals that the protein is post-translationally phosphorylated at serine residues. Pulse labeling of virus-infected cells with 32P-orthophosphate reveals that the phosphorylation rate of the cellular dUTPase protein decreases significantly as a function of time post-infection. In an effort to establish that phosphate turnover was occurring on the cellular dUTPase protein, cells were prelabeled with 32P-orthophosphate and then infected with HSV in the absence of label. Evidence from this experiment indicates that the phosphate moiety is removed from the cellular dUTPase protein during the infection. A series of viable virus mutants was generated by insertional inactivation of the HSV dUTPase gene. These mutants do not express viral dUTPase activity and HSV dUTPase protein is not detected by western blot analysis. However, in contrast to the wild-type situation, these mutant virus retain significant cellular dUTPase activity throughout infection. Interestingly, phosphorylation of cellular dUTPase protein is now readily detectable in each of the mutant virus-infected cells. These studies indicate that cellular dUTPase activity is diminished in wild-type HSV-infected cells by a process of dephosphorylation. It also appears that in mutant HSV, lacking the virus dUTPase, the mechanism of dephosphorylation and thus inactivation of cellular dUTPase is not functional. The end result is that the mutant virus can now rely on the cellular activity for its survival.     

Appearanc of cytotoxic cells within the bronchus after local infection with herpes simplex virus.

Cells from rabbit spleens, bronchial washings (BW) and bronchus-associated lymphoid tissues (BALT) were examined for their ability to lyse cells infected with herpes simplex virus (HSV). Specific lysis of HSV-infected cells was mediated by BW cells as early as 4 days after intratracheal infection of the rabbits with the virus whereas lysis by spleen cells and BALT cells was not detected until 7 or more days after infection. Lysis by spleen cells was initially detected 7 days after intraperitoneal injection of the virus but lysis by BW and BALT cells was not observed until 14 days after infection. Although spleen, BW, and BALT cells could lyse antibody-coated target cells, antibodies detectable by antibody-dependent cellular cytotoxicity could not be detected in bronchial washings until 7 or more days after infection. The data suggest that cells capable of direct cytotoxicity of virus-infected cells appear within the bronchus after local infection by the virus.     

Partial purification and characterization of the ribonucleotide reductase induced by herpes simplex virus infection of mammalian cells.

In this report we confirm and further characterize the induction of a novel ribonucleotide reductase after herpes simplex virus infection of mammalian cells. Induction of the enzyme was observed at a multiplicity of infection of 1 PFU/cell or greater and was found to be maximal (three- to sixfold the activity in mock-infected controls at 6 to 8 h postinfection at a multiplicity of infection of 10 PFU/cell. Partial purification and subsequent characterization of the reductase activity from infected cells demonstrated the existence of two enzymes which could be separated by precipitation with ammonium sulfate. One of the activities precipitated at between 35 and 55% salt saturation, as did the enzyme from control cells, whereas the novel activity precipitated at 0 to 35% saturation. This latter enzyme was similar to the herpes simplex virus-induced reductase described by others in its lack of requirement for Mg2 and its resistance to inhibition by dTTP and dATP; in addition, we found that it was inhibited by ATP, whereas the enzyme from control cells displayed an absolute requirement for the nucleotide. Both enzymes were equally inhibited by pyridoxal phosphate and showed similar cold and heat stability. The enzyme induced by herpes simplex virus infection, however, was much more labile than the control enzyme upon purification.