Human cytomegalovirus TRS1 and IRS1 gene products block the double-stranded-RNA-activated host protein shutoff response induced by herpes simplex virus type 1 infection

Human cytomegalovirus (HCMV) attachment and entry stimulates the expression of cellular interferon-inducible genes, many of which target important cellular functions necessary for viral replication. Double-stranded RNA-dependent host protein kinase (PKR) is an interferon-inducible gene product that limits viral replication by inhibiting protein translation in the infected cell. It was anticipated that HCMV encodes gene products that facilitate the evasion of this PKR-mediated antiviral response. Using a deltagamma1 34.5 herpes simplex virus type 1 (HSV-1) recombinant that triggers PKR-mediated protein synthesis shutoff, experiments identified an HCMV gene product expressed in the initial hours of infection that allows continued protein synthesis in the infected cell. Recombinant HSV-1 viruses expressing either the HCMV TRS1 or IRS1 protein demonstrate that either of these HCMV gene products allows the deltagamma1 34.5 recombinant viruses to evade PKR-mediated protein shutoff and maintain late viral protein synthesis.     

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.     

Polyamine metabolism in MRC5 cells infected with different herpesviruses.

Both methyglyoxal bis(guanylhydrazone), an inhibitor of S-adenosyl-L-methionine decarboxylase (EC.4.1.1.50) and DL-α-methylornithine, an inhibitor of ornithine decarboxylase (EC.4.1.1.17), are shown to be potent inhibitors of the replication of human cytomegalovirus (HCMV) in MRC-5 cells. These compounds, both inhibitors of polyamine biosynthesis, do not affect the replication of either herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). This difference in antiviral effect is shown to be related to the stimulation of spermidine and spermine synthesis in host cells following HCMV infection and the inhibition of polyamine metabolism in HSV-1 or HSV-2-infected cells. Inhibition of HCMV replication by the inhibitors of polyamine biosynthesis is accompanied by a marked decrease in the formation of intranuclear, DNA-containing inclusions characteristic of HCMV infection. These results suggest significant differences in the mechanisms of replication of different herpesviruses.     

Cellular elongation factor 1delta is modified in cells infected with representative alpha-, beta-, or gammaherpesviruses

Earlier reports (Y. Kawaguchi, R. Bruni, and B. Roizman, J. Virol. 71:1019-1024, 1997; Y. Kawaguchi, C. Van Sant, and B. Roizman, J. Virol. 72:1731-1736, 1998) showed that herpes simplex virus 1 (HSV-1) infection causes the hyperphosphorylation of translation elongation factor 1delta (EF-1delta) and that the modification of EF-1delta is the consequence of direct phosphorylation by a viral protein kinase encoded by the UL13 gene of HSV-1. The UL13 gene is conserved in members of all herpesvirus subfamilies. Here we report the following. (i) In various mammalian cells, accumulation of the hyperphosphorylated form of EF-1delta is observed after infection with alpha-, beta-, and gammaherpesviruses, including HSV-2, feline herpesvirus 1, pseudorabiesvirus, bovine herpesvirus 1, human cytomegalovirus (HCMV), and equine herpesvirus 2. (ii) In human lung fibroblast cells infected with recombinant HSV-1 lacking the UL13 gene, the hypophosphorylated form of EF-1delta is a minor species, whereas the amount of the hyperphosphorylated form of EF-1delta significantly increases in cells infected with the recombinant HSV-1 in which UL13 had been replaced by HCMV UL97, a homologue of UL13. These results indicate that the posttranslational modification of EF-1delta is conserved herpesvirus function and the UL13 homologues may be responsible for the universal modification of the translation factor.     

Induction of interleukin-8 gene expression is associated with herpes simplex virus infection of human corneal keratocytes but not human corneal epithelial cells.

Interleukin-8 (IL-8) is a proinflammatory cytokine released at sites of tissue damage by various cell types. One important function of IL-8 is to recruit neutrophils into sites of inflammation and to activate their biological activity. Stromal keratitis induced by herpes simplex virus type 1 (HSV-1) is characterized by an initial infiltration of neutrophils. This study was carried out to determine whether cells resident in the cornea synthesize IL-8 after virus infection. Pure cultures of epithelial cells and keratocytes established from human corneas were infected with HSV-1, and the medium overlying the cells was subsequently assayed for IL-8 by an enzyme-linked immunosorbent assay. Cytokine mRNA levels in cell lysates were monitored by Northern (RNA) blot analysis. It was found that virus infection of keratocyte cultures led to the synthesis of IL-8-specific mRNA with more than 30 ng of IL-8 made per 10(6) cells. Neither UV-inactivated virus nor virus-free filtrates collected from HSV-1-infected keratocytes could induce IL-8 protein or mRNA, suggesting that viral gene expression was needed for induction of IL-8 gene expression. Unlike keratocytes, HSV-1-infected epithelial cells failed to synthesize IL-8 protein or mRNA. However, these cells readily produced both molecules following tumor necrosis factor alpha stimulation. HSV-1 had similar titers in both cell types. Thus, the failure to induce IL-8 synthesis was not due to an inability of the virus to replicate in epithelial cells. The capacity of HSV-1-infected corneal keratocytes to synthesize IL-8 suggests that these cells can contribute to the induction of the acute inflammatory response seen in herpes stromal keratitis.     

A novel strategy for converting recombinant viral protein into high immunogenic antigen.

Interleukin 2 (IL-2) is a lymphokine promoting immune response and therefore has been investigated as an immunological adjuvant. In order to enhance the immunogenicity of recombinant viral protein, herpes simplex virus type 1 (HSV-1) glycoprotein D (gD), we genetically created a fusion protein consisting of gD and human IL-2. The fusion protein, without any other adjuvants, induced high antibody responses and cell-mediated immunity to HSV-1 in mice. Mice immunized with the fusion protein were protected against HSV-1 infection. The results indicate that IL-2-fusing can provide a means for converting a weak immunogenic protein into a high immunogenic antigen, and the strategy would be widely applicable to the other antigens for pathogens.     

Inhibition of calmodulin-dependent kinase kinase blocks human cytomegalovirus-induced glycolytic activation and severely attenuates production of viral progeny

Viruses depend on the host cell to provide the energy and biomolecular subunits necessary for production of viral progeny. We have previously reported that human cytomegalovirus (HCMV) infection induces dramatic changes to central carbon metabolism, including glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and nucleotide biosynthesis. Here, we explore the mechanisms involved in HCMV-mediated glycolytic activation. We find that HCMV virion binding and tegument protein delivery are insufficient for HCMV-mediated activation of glycolysis. Viral DNA replication and late-gene expression, however, are not required. To narrow down the list of cellular pathways important for HCMV-medicated activation of glycolysis, we utilized pharmaceutical inhibitors to block pathways reported to be both involved in metabolic control and activated by HCMV infection. We find that inhibition of calmodulin-dependent kinase kinase (CaMKK), but not calmodulin-dependent kinase II (CaMKII) or protein kinase A (PKA), blocks HCMV-mediated activation of glycolysis. HCMV infection was also found to target calmodulin-dependent kinase kinase 1 (CaMKK1) expression, increasing the levels of CaMKK1 mRNA and protein. Our results indicate that inhibition of CaMKK has a negligible impact on immediate-early-protein accumulation yet severely attenuates production of HCMV viral progeny, reduces expression of at least one early gene, and blocks viral DNA replication. Inhibition of CaMKK did not affect the glycolytic activation induced by another herpes virus, herpes simplex virus type 1 (HSV-1). Furthermore, inhibition of CaMKK had a much smaller impact on HSV-1 replication than on that of HCMV. These data suggest that the role of CaMKK during the viral life cycle is, in this regard, HCMV specific. Taken together, our results suggest that CaMKK is an important factor for HCMV replication and HCMV-mediated glycolytic activation.     

Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism

Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV.     

Genetically Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for Treatment of Malignant Disease

We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.     

Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis.

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.     

A human cytomegalovirus function inhibits replication of herpes simplex virus.

Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 micrograms/ml) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5 degrees C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.     

Monocyte-derived inhibitor of interleukin 1 induced by human cytomegalovirus.

It has previously been shown that human cytomegalovirus (HCMV) can exert immunosuppressive effects, and it has been suggested that these may be mediated by monocytes, although the mechanism is unclear. We showed that infection of human monocytes with the AD169 strain of HCMV abrogates their production of interleukin 1 (IL-1) activity. This was associated with the release from infected monocytes of an inhibitor of IL-1 activity which was also released after HCMV infection of the U937 macrophage-like cell line. The inhibitor of IL-1 activity is a protein with an apparent molecular weight of ca. 95,000. This action of HCMV strain AD169 was virus specific and required infectious virus but occurred without virus replication or detectable expression of viral proteins. This effect may account, at least in part, for the previously observed immunosuppressive properties of HCMV.     

Do cells treated with human interferon survive virus infection?

Abstract HeLa cells pretreated with human lympho-blastoid interferon (Hu IFN-α (Ly)), at concentrations up to 100 IU / ml and infected with moderate multiplicities of encephalomyocarditis (EMC) virus (10 PFU / cell) died 1 or 2 days after infection. However, if cells were repeatedly treated with high doses of IFN (800 IU / ml) they survived infection by EMC virus for at least a month. Cells survived Semliki Forest virus (SFV) infection when even lower IFN concentrations were used. By contrast infection of IFN-treated HeLa cells with other RNA-containing viruses, such as poliovirus, vesicular stomatitis virus (VSV), Newcastle disease virus (NDV) and reovirus type 3 resulted in cell death. Similarly, infection with a number of DNA-containing viruses such as adenovirus type 5, Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and vaccinia virus killed cells. The results are discussed in the light of different models for the molecular mechanism of action of interferon.     

Cytomegalovirus infection stimulates expression of monocyte-associated mediator genes

Monocytes and tissue macrophages play important roles in host defense against virus infections and, in the case of human cytomegalovirus (HCMV) and HIV, may also be the reservoir for latent disease. Because these cells can also rapidly respond to most infections by secretion of inflammatory mediators, we were interested in determining if HCMV infection could have a direct activating effect on macrophage cytokine production. To do this, we primarily investigated the influence of HCMV infection on IL-1 beta-mRNA expression in peripheral blood monocytes and the promyelocytic cell line, ML-3 as well as the inflammatory response genes TNF-alpha, MAD-9, MAD-6, and MAD-2 in the promyelocytic ML-3 cell line. Exposure of ML-3 cells to the virus prior to induction of differentiation had little influence on mediator gene expression. However, induction of the macrophage phenotype by pretreatment of ML-3 cells with the phorbol ester, PMA, followed by HCMV challenge, resulted in a greatly extended period of expression of IL-1 beta, TNF-alpha, MAD-9, and CSF-1 but not MAD-6 and MAD-2. Constitutively expressed genes such as lysozyme and actin were not similarly modulated. Both RNA dot-blot and in situ hybridization studies demonstrated that infection of human peripheral blood monocytes with HCMV leads to sustained expression of IL-1 beta mRNA for up to 96 h, which contrasted markedly with mock-infected or LPS-stimulated monocytes. Flow cytometric analysis of the intracellular levels of IL-1 beta protein in ML-3 cells indicated that not only was there more protein produced in infected cells, but that the majority of the cells had responded. Enhanced levels of the intracellular form of IL-1 beta in monocytes was confirmed by Western blot analysis. Cotransfection experiments were performed using IL-1 beta-CAT chimeric plasmids together with plasmids encoding HCMV-immediate-early gene region products. Transactivation of the IL-1 beta gene by region 2 of the immediate-early gene was observed in ML-3 cells that had been induced to differentiate prior to transfection. No stimulation of IL-1 beta promoter activity was observed in ML-3 cells that were undifferentiated prior to transfection. In summary, HCMV infection, although not leading to productive infection, nonetheless may contribute to the pathology of the infection through enhancement of monocyte inflammatory mediator gene expression with subsequent stimulation of protein synthesis.     

The UL97 protein kinase of human cytomegalovirus and homologues in other herpesviruses: impact on virus and host

The human herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV-6A), HHV-6B, HHV-7 and HHV-8, establish persistent infections with possible recurrence during immunosuppression. HCMV replication is inhibited by the nucleoside analogue ganciclovir (GCV), the compound of choice for the treatment of HCMV diseases and preemptive treatment of infections. The viral UL97 protein (pUL97) which shares homologies with protein kinases and bacterial phosphotransferases is able to monophosphorylate GCV. Homologues of pUL97 are found in HSV (UL13), VZV (ORF47), EBV (BGLF4), HHV-6 (U69), HHV-8 (ORF36) as well as in murine CMV (M97) or rat CMV (R97). Several indolocarbazoles have been reported to be specific inhibitors of pUL97. The protein is important for efficient replication of the virus. Autophosphorylation of pUL97 was observed using different experimental systems. Most recently, it has been shown that pUL97 interacts with the DNA polymerase processivity factor pUL44. Indolocarbazole protein kinase inhibitors are promising lead compounds for the development of more specific inhibitors of HCMV.