Herpes simplex virus 1 infection alters the mRNA translation processing in L-02 cells

HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off. Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.     

Herpes simplex virus 2 causes apoptotic infection in monocytoid cells

Increasing evidence indicates that apoptosis can be associated with several viral infections. Here we demonstrate, that infection of monocytoid cells by Herpes simplex virus 2 (HSV-2) resulted, in time- and dose-dependent induction of apoptosis as an exclusive cytopathic effect. The phenomenon was confirmed using four different techniques. Conversely, apoptosis was not observed in the Vero cell line. Virus yield in monocytoid cells was delayed and reduced, although well detectable, in comparison with that observed in Vero cells. Nevertheless, released virions exhibited full infecting capability. Apoptosis induced by HSV-2 was not inhibited by cycloheximide and only partially by an UV-treatment which completely abrogated infectivity. Virus-induced apoptosis was partly inhibited by indomethacin and was associated with a down-regulation of Bcl-2. A similar, but less pronounced, apoptosis-inducing effect in monocytoid cells was also observed with HSV-1 infection. Depending on the target cells, therefore, HSV could complete a cycle of infection which is characterized by apoptosis of infected cells.     

Herpes simplex virus glycoprotein K promotes egress of virus particles.

Herpes simplex virus (HSV) glycoprotein K (gK) is thought to be intimately involved in the process by which infected cells fuse because HSV syncytial mutations frequently alter the gK (UL53) gene. Previously, we characterized gK produced in cells infected with wild-type HSV or syncytial HSV mutants and found that the glycoprotein was localized to nuclear and endoplasmic reticulum membranes and did not reach the cell surface (L. Hutchinson, C. Roop, and D. C. Johnson, J. Virol. 69:4556-4563, 1995). In this study, we have characterized a mutant HSV type 1, denoted F-gK beta, in which a lacZ gene cassette was inserted into the gK coding sequences. Since gK was found to be essential for virus replication, F-gK beta was propagated on complementing cells which can express gK. F-gK beta produced normal plaques bounded by nonfused cells when plated on complementing cells, although syncytia were observed when the cells produced smaller amounts of gK. In contrast, F-gK beta produced only microscopic plaques on Vero cells and normal human fibroblasts (which do not express gK) and these plaques were reduced by 10(2) to 10(6) in number. Further, large numbers of nonenveloped capsids accumulated in the cytoplasm of F-gK beta-infected Vero cells, virus particles did not reach the cell surface, and the few enveloped particles that were produced exhibited a reduced capacity to enter cells and initiate an infection of complementing cells. Overexpression of gK in HSV-infected cells also caused defects in virus egress, although particles accumulated in the perinuclear space and large multilamellar membranous structures juxtaposed with the nuclear envelope were observed. Together, these results demonstrate that gK regulates or facilitates egress of HSV from cells. How this property is connected to cell fusion is not clear. In this regard, gK may alter cell surface transport of viral particles or other viral components directly involved in the fusion process.     

Herpes simplex virus 2 infection impacts stress granule accumulation

Interference with stress granule (SG) accumulation is gaining increased appreciation as a common strategy used by diverse viruses to facilitate their replication and to cope with translational arrest. Here, we examined the impact of infection by herpes simplex virus 2 (HSV-2) on SG accumulation by monitoring the localization of the SG components T cell internal antigen 1 (TIA-1), Ras-GTPase-activating SH3-domain-binding protein (G3BP), and poly(A)-binding protein (PABP). Our results indicate that SGs do not accumulate in HSV-2-infected cells and that HSV-2 can interfere with arsenite-induced SG accumulation early after infection. Surprisingly, SG accumulation was inhibited despite increased phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), implying that HSV-2 encodes previously unrecognized activities designed to maintain translation initiation downstream of eIF2α. SG accumulation was not inhibited in HSV-2-infected cells treated with pateamine A, an inducer that works independently of eIF2α phosphorylation. The SGs that accumulated following pateamine A treatment of infected cells contained G3BP and PABP but were largely devoid of TIA-1. We also identified novel nuclear structures containing TIA-1 that form late in infection. These structures contain the RNA binding protein 68-kDa Src-associated in mitosis (Sam68) and were noticeably absent in infected cells treated with inhibitors of viral DNA replication, suggesting that they arise as a result of late events in the virus replicative cycle.     

DNA-binding proteins induced by herpes simplex virus type 2 in HEp-2 cells.

Affinity chromatography on single-stranded and double-stranded DNA-cellulose indicates that 12 proteins previously identified from herpes simplex virus type 2-infected cells, ranging in molecular weight from 28 X 10(3) to 186 X 10(3), bind to DNA-cellulose. The DNA-binding proteins found in infected cells differed in relative binding strengths for denatured DNA-cellulose. The virus specificity of these DNA-binding proteins was further studied by comparison with DNA-binding proteins isolated from mock-infected cells, and by immunoprecipitation of infected-cell DNA-binding proteins with antisera specific for viral antigens. The promise this technique holds for the purification and study of polypeptides involved in virus DNA replication, recombination, or repair is discussed.     

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.     

Establishment of a persistent measles virus infection in HEp-2 cells.

A productive measles virus persistent infection has been established in HEp-2 cells. Greater than 90% of the persistently infected HEp-2 cells (H2MV) exhibited measles specific immunofluorescence and haemadsorption. Although most of the H2MV cells contained measles specific antigens, only a small percentage (less than 1%) actually produced infectious measles virus as determined by infectious centre assays. The measles virus produced by H2MV cells exhibited properties different from the initiating parent Edmonston strain virus, being reduced in virulence and also temperature sensitive for replication at 39 degrees C. The role of these altered virus properties in the establishment of persistence is considered.     

Bcl-2 blocks a caspase-dependent pathway of apoptosis activated by herpes simplex virus 1 infection in HEp-2 cells

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type virus blocks the execution of the cell death program triggered by expression of viral genes, by the Fas and tumor necrosis factor pathways, or by nonspecific stress agents. In particular, an earlier report from this laboratory showed that the mutant virus d120 lacking the genes encoding infected cell protein 4 (ICP4), the major regulatory protein of the virus, induces a caspase-3-independent pathway of apoptosis in human SK-N-SH cells. Here we report that the pathway of apoptosis induced by the d120 mutant in human HEp-2 cells is caspase dependent. Specifically, in HEp-2 cells infected with d120, (i) a broad-range inhibitor of caspase activity, z-vad-FMK, efficiently blocked DNA fragmentation, (ii) cytochrome c was released into the cytoplasm, (iii) caspase-3 was activated inasmuch as poly(ADP-ribose) polymerase was cleaved, and (iv) chromatin condensation and fragmentation of cellular DNA were observed. In parallel studies, HEp-2 cells were transfected with a plasmid encoding human Bcl-2 and a clone (VAX-3) expressing high levels of Bcl-2 was selected. This report shows that Bcl-2 blocked all of the manifestations associated with programmed cell death caused by infection with the d120 mutant. Consistent with their resistance to programmed cell death, VAX-3 cells overproduced infected cell protein 0 (ICP0). An unexpected observation was that ICP0 encoded by the d120 mutant accumulated late in infection in small, quasi-uniform vesicle-like structures in all cell lines tested. Immunofluorescence-based colocalization studies indicated that these structures were not mitochondria or components of the endoplasmic reticulum or the late endosomal compartment. These studies affirm the conclusion that HSV can induce programmed cell death at multiple steps in the course of its replication, that the d120 mutant can induce both caspase-dependent and -independent pathways of programmed cell death, and that virus-induced stimuli of programmed cell death may differ with respect to the pathway that they activate.     

Herpes simplex virus type 1 glycoprotein E is not indispensable for viral infectivity.

A mutant of the herpes simplex virus type 1 Angelotti was isolated in which 87% of the coding region of glycoprotein E (gE) was deleted and replaced by a functional neomycin resistance gene of the Tn5 transposon. The mutant was characterized by restriction enzyme analyses and Southern blotting. Western blotting of proteins and immunofluorescence assays revealed that gE was completely absent and that the Fc receptor was not expressed in cells infected with the mutant. The fact that this mutant was viable and that it replicated to a slightly lower titer than did the wild-type virus suggests that the presence of gE is not a prerequisite of viral infectivity in tissue culture.     

Localization of highly phosphorylated proteins in cells altered by Herpes simplex virus infection

Highly phosphorylated proteins detectable by their ability to bind bismuth ions were localized in rabbit fibroblasts before and during infection with Herpes simplex viruses type 1 and type 2. The bismuth tartrate procedure of Locke and Huie applied to glutaraldehyde-fixed cells revealed a low level of bismuth binding in a restricted portion of the normal nucleolus in non-infected cells. From 2.5-17 hr post-infection during virus development and maturation, the phosphorylated proteins were more widespread and the intensity of reaction was augmented. Bismuth deposits were then associated with virus-modified pre-existing structures including all of the nucleolar fibrils, the more abundant interchromatin granules, reduplications of some areas of the inner nuclear membrane and the Golgi apparatus. Virus-induced structures which were stained included nuclear dense bodies, the teguments of enveloped virions and the contents of extranuclear enveloped structures devoid of capsids. Following detergent-induced destruction of membranes, staining was lost from the nuclear envelope and cytoplasmic virions, which demonstrated that the highly phosphorylated proteins were tightly bound to nuclear and viral membranes. Bismuth staining of nitrocellulose sheets containing proteins extracted from whole cells revealed no reaction in normal cells but three positive bands were found in infected cells.     

Deoxyribonucleotide metabolism in Herpes simplex virus infected HeLa cells.

The effect of Rolly No. 11 strain herpes simplex virus infection of HeLa cells in culture on deoxynucleotide metabolism and the level of various enzymes concerned with the biosynthesis of DNA has been investigated. Of 18 enzyme activities studied, thymidine kinase, DNA polymerase and deoxyribonuclease were markedly augmented, a finding in agreement with previous reports. Deoxycytidine kinase, ribonucleotide reductase, thymidylate kinase and deoxycytidylate deaminase activities, in contrast with previous reports, did not increase; the activities of the other enzymes studied, also did not increase. Whereas most of the radioactivity derived from [14-C] thymidine in the acid-soluble fraction of the uninfected cells was present as deoxythymidine triphosphate, that present in the infected cells was primarily in the form of deoxythymidine monophosphate. Thus, in the infected cell deoxythymidylate kinase is a rate-limiting enzyme in the biosynthesis of deoxythymidine triphosphate. A marked increase in the pools of the four naturally occurring deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP) was found. The rate of formation of the virus-induced enzymes was determined, as were the various nucleoside triphosphate pools and the other phosphorylated derivatives of thymidine; a maximum was reached for all these csmponents between 6 to 8 h post infection. Although an apparent greater synthesis of DNA occurred in the uninefected cells, when the specific activity of the radioactive deoxythymidine triphosphate was taken into account, there was actually a greater rate of DNA synthesis in the infected cells, with the peak at 8 h post infection.     

Herpes simplex virus infection of human dendritic cells induces apoptosis and allows cross-presentation via uninfected dendritic cells

HSV efficiently infects dendritic cells (DCs) in their immature state and induces down-regulation of costimulatory and adhesion molecules. As in mice, HSV infection of human DCs also leads to their rapid and progressive apoptosis, and we show that both early and late viral proteins contribute to its induction. Because topical HSV infection is confined to the epidermis, Langerhans cells are expected to be the major APCs in draining lymph nodes. However, recent observations in murine models show T cell activation to be mediated by nonepidermal DC subsets, suggesting cross-presentation of viral Ag. In this study we provide an explanation for this phenomenon, demonstrating that HSV-infected apoptotic DCs are readily phagocytosed by uninfected bystander DCs, which, in turn, stimulate virus-specific CD8+ T cell clones.