Second-site mutation outside of the U(S)10-12 domain of Deltagamma(1)34.5 herpes simplex virus 1 recombinant blocks the shutoff of protein synthesis induced by activated protein kinase R and partially restores neurovirulence.

Earlier studies have shown that herpes simplex virus type 1 (HSV-1) activated protein kinase R (PKR) but that the product of the product of the gamma(1)34.5 gene binds and redirects the host phosphatase 1 to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In consequence, the gamma(1)34.5 gene product averts the threatened shutoff of protein synthesis caused by activated PKR. Serial passages of Deltagamma(1)34.5 mutants in human cells led to isolation of two classes of second-site, compensatory mutants. The first, reported earlier, resulted from the juxtaposition of the alpha promoter of the U(S)12 gene to the coding sequence of the U(S)11 gene. The mutant blocks the phosphorylation of eIF-2alpha but does not restore the virulence phenotype of the wild-type virus. We report another class of second-site, compensatory mutants that do not map to the U(S)10-12 domain of the HSV-1 genome. All mutants in this series exhibit sustained late protein synthesis, higher yields in human cells, and reduced phosphorylation of PKR that appears to be phosphatase dependent. Specific dephosphorylation of eIF-2alpha was not demonstrable. At least one mutant in this series exhibited a partial restoration of the virulence phenotype characteristic of the wild-type virus phenotype. The results suggest that the second-site mutations reflect activation of fossilized functions designed to block the interferon response pathways in cells infected with the progenitor of present HSV.     

Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.     

Cells lacking NF-kappaB or in which NF-kappaB is not activated vary with respect to ability to sustain herpes simplex virus 1 replication and are not susceptible to apoptosis induced by a replication-incompetent mutant virus

Earlier we reported that NF-kappaB is activated by protein kinase R (PKR) in herpes simplex virus 1-infected cells. Here we report that in PKR(-/-) cells the yields of wild-type virus are 10-fold higher than in PKR(+/+) cells. In cells lacking NF-kappaB p50 (nfkb1), p65 (relA), or both p50 and p65, the yields of virus were reduced 10-fold. Neither wild-type nor mutant cells undergo apoptosis following infection with wild-type virus. Whereas PKR(+/+) and NF-kappaB(+/+) control cell lines undergo apoptosis induced by the d120 (Deltaalpha4) mutant of HSV-1, the mutant PKR(-/-) and NF-kappaB(-/-) cell lines were resistant. The evidence suggests that the stress-induced apoptosis resulting from d120 infection requires activation of NF-kappaB and that this proapoptotic pathway is blocked in cells in which NF-kappaB is not activated or absent. Activation of NF-kappaB in the course of viral infection may have dual roles of attempting to curtain viral replication by rendering the cell susceptible to apoptosis induced by the virus and by inducing the synthesis of proteins that enhance viral replication.     

Virus-cell interactions regulating induction of tumor necrosis factor alpha production in macrophages infected with herpes simplex virus

Macrophages respond to virus infections by rapidly secreting proinflammatory cytokines, which play an important role in the first line of defense. Tumor necrosis factor alpha (TNF-alpha) is one of the major macrophage-produced cytokines. In this study we have investigated the virus-cell interactions responsible for induction of TNF-alpha expression in herpes simplex virus (HSV)-infected macrophages. Both HSV type 1 (HSV-1) and HSV-2 induced TNF-alpha expression in macrophages activated with gamma interferon (IFN-gamma). This induction was to some extent sensitive to UV treatment of the virus. Virus particles unable to enter the cells displayed reduced capacity to stimulate TNF-alpha expression but retained a significant portion which was abolished by HSV-specific antibodies. Recombinant HSV-1 glycoprotein D was able to trigger TNF-alpha secretion in concert with IFN-gamma. Sugar moieties of HSV glycoproteins have been reported to be involved in induction of IFN-alpha but did not contribute to TNF-alpha expression in macrophages. Moreover, the entry-dependent portion of the TNF-alpha induction was investigated with HSV-1 mutants and found to be independent of the tegument proteins VP16 and UL13 and partly dependent on nuclear translocation of the viral DNA. Finally, we found that macrophages expressing an inactive mutant of the double-stranded RNA (dsRNA)-activated protein kinase (PKR) produced less TNF-alpha in response to infectious HSV infection than the empty-vector control cell line but displayed the same responsiveness to UV-inactivated virus. These results indicate that HSV induces TNF-alpha expression in macrophages through mechanisms involving (i) viral glycoproteins, (ii) early postentry events occurring prior to nuclear translocation of viral DNA, and (iii) viral dsRNA-PKR.     

Requirements for the induction of interleukin-6 by herpes simplex virus-infected leukocytes

Cytokines play important roles in the clearance of herpes simplex virus (HSV) infections and in virus-induced immunopathology. One cytokine known to contribute to resistance against HSV is interleukin-6 (IL-6). Here we have investigated virus-cell interactions responsible for IL-6 induction by HSV in leukocytes. Both HSV type 1 and type 2 are potent inducers of IL-6, and this phenomenon is augmented in the presence of gamma interferon. The ability to induce IL-6 is dependent on de novo protein synthesis and is sensitive to UV irradiation of the virus. Virus mutants lacking the virion-transactivating protein VP16 or any of the immediate-early proteins ICP0, ICP4, or ICP27 displayed unaltered capacities to induce IL-6. However, wild-type virus was unable to induce IL-6 in a macrophage cell line overexpressing a mutant of double-stranded RNA-activated protein kinase (PKR). This suggests a role for PKR in HSV-induced IL-6 expression. HSV infection led to enhanced binding to the kappaB, CRE, and AP-1 sites of the IL-6 promoter, and inhibitors against NF-kappaB and the p38 kinase strongly reduced accumulation of IL-6 mRNA in infected cells. Moreover, macrophage cell lines expressing dominant negative mutants of IkappaBalpha and p38 responded to HSV-1 infection with reduced IL-6 expression compared to the control-vector-transfected cell line. The results show that induction of IL-6 by HSV in leukocytes is dependent on PKR and cellular signaling through NF-kappaB and a p38-dependent pathway.     

Suppression of proinflammatory cytokine expression by herpes simplex virus type 1

Viral immune evasion strategies are important for establishment and maintenance of infections. Many viruses are in possession of mechanisms to counteract the antiviral response raised by the infected host. Here we show that a herpes simplex virus type 1 (HSV-1) mutant lacking functional viral protein 16 (VP16)-a tegument protein promoting viral gene expression-induced significantly higher levels of proinflammatory cytokines than wild-type HSV-1. This was observed in several cell lines and primary murine macrophages, as well as in peritoneal cells harvested from mice infected in vivo. The enhanced ability to stimulate cytokine expression in the absence of VP16 was not mediated directly by VP16 but was dependent on the viral immediate-early genes for infected cell protein 4 (ICP4) and ICP27, which are expressed in a VP16-dependent manner during primary HSV infection. The virus appeared to target cellular factors other than interferon-induced double-stranded RNA-activated protein kinase R (PKR), since the virus mutants remained stronger inducers of cytokines in cells stably expressing a dominant-negative mutant form of PKR. Finally, mRNA stability assay revealed a significantly longer half-life for interleukin-6 mRNA after infection with the VP16 mutant than after infection with the wild-type virus. Thus, HSV is able to suppress expression of proinflammatory cytokines by decreasing the stability of mRNAs, thereby potentially impeding the antiviral host response to infection.     

Herpes simplex virus with highly reduced gD levels can efficiently enter and spread between human keratinocytes

The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.     

An oncogenic role of sphingosine kinase

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.     

Herpes simplex virus infection and propagation in a mouse L cell mutant lacking heparan sulfate proteoglycans.

We have isolated a variant line of mouse L cells, termed gro2C, which is partially resistant to infection by herpes simplex virus type 1 (HSV-1). Characterization of the genetic defect in gro2C cells revealed that this cell line harbors a specific defect in the heparan sulfate synthesis pathway. Specifically, anion-exchange high-performance liquid chromatography of metabolically radiolabeled glycosaminoglycans indicated that chondroitin sulfate moieties were synthesized normally in the mutant cells, whereas heparin-like chains were absent. Because of these properties, we have used these cells to investigate the role of heparan sulfate proteoglycans in the HSV-1 life cycle. In this report, we demonstrate that the partial block to HSV-1 infection in gro2C cells occurs in the virus entry pathway. Virus adsorption assays using radiolabeled HSV-1 (KOS) revealed that the gro2C cell surface is a relatively poor target for HSV-1 in that virus attachment was 85% lower in the mutant cells than in the parental L cell controls. A portion of the 15% residual virus adsorption was functional, however, insofar as gro2C cells were susceptible to HSV-1 infection in plaque assays and in single-step growth experiments. Moreover, although the number of HSV-1 plaques that formed in gro2C monolayers was reduced by 85%, the plaque morphology was normal, and the virus released from the mutant cells was infectious. Taken together, these results provide strong genetic evidence that heparan sulfate proteoglycans enhance the efficiency of HSV attachment to the cell surface but are otherwise not essential at any stage of the lytic cycle in culture. Moreover, in the absence of heparan sulfate, other cell surface molecules appear to confer susceptibility to HSV, leading to a productive viral infection.     

Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [35S]sulfate-labeled or [6-3H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.     

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.     

Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells

We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.     

The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) blocks apoptosis in hippocampal neurons, involving activation of the MEK/MAPK survival pathway.

Herpes simplex virus type 1 (HSV-1) and HSV-2 trigger or counteract apoptosis by a cell-specific mechanism. Our studies are based on previous findings that the protein kinase (PK) domain of the large subunit of HSV-2 ribonucleotide reductase (ICP10) activates the Ras/MEK/MAPK pathway (Smith et al., J. Virol. 74:10417, 2000). Because survival pathways can modulate apoptosis, we used cells that are stably or transiently transfected with ICP10 PK, an HSV-2 mutant deleted in ICP10 PK (ICP10DeltaPK) and the MEK-specific inhibitor U0126 to examine the role of ICP10 PK in apoptosis. Apoptosis was induced by staurosporine or D-mannitol in human (HEK293) cells or HEK293 cells stably transfected with the ICP10 PK-negative mutant p139 (JHL15), as determined by morphology, DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage. HEK293 cells stably transfected with ICP10 (JHLa1) were protected from apoptosis. ICP10 but not p139 protected neuronally differentiated PC12 cells from death due to nerve growth factor withdrawal, and apoptosis (determined by TUNEL) and caspase-3 activation were seen in primary hippocampal cultures infected with ICP10DeltaPK but not with HSV-2 or a revertant virus [HSV-2(R)]. The data indicate that ICP10 has antiapoptotic activity under both paradigms and that it requires a functional PK activity. The apoptotic cells in primary hippocampal cultures were neurons, as determined by double immunofluorescence with fluorescein-labeled dUTP (TUNEL) and phycoerythrin-labeled antibodies specific for neuronal proteins (TuJ1 and NF-160). Protection from apoptosis was associated with MEK/MAPK activation, as evidenced by (i) increased levels of activated (phosphorylated) MAPK in HSV-2- but not ICP10DeltaPK-infected cultures and (ii) inhibition of MAPK activation by the MEK-specific inhibitor U0126. MEK and MAPK were activated by infection with UV-inactivated but not antibody-neutralized HSV-2, suggesting that activation requires cellular penetration but is independent of de novo viral protein synthesis.     

Protection from herpes simplex virus type 1 lethal and latent infections by secreted recombinant glycoprotein B constitutively expressed in human cells with a BK virus episomal vector.

The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.     

The Herpes Simplex Virus 1 Us11 Protein Inhibits Autophagy through Its Interaction with the Protein Kinase PKR

Autophagy is now known to be an essential component of host innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral host defense. Herpes simplex virus 1 (HSV-1) blocks autophagy in fibroblasts and in neurons, and the ICP34.5 protein is important for the resistance of HSV-1 to autophagy because of its interaction with the autophagy machinery protein Beclin 1. ICP34.5 also counteracts the shutoff of protein synthesis mediated by the double-stranded RNA (dsRNA)-dependent protein kinase PKR by inhibiting phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) in the PKR/eIF2α signaling pathway. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. In the present study, we unveil a previously uncharacterized function of Us11 by demonstrating its antiautophagic activity. We show that the expression of Us11 is able to block autophagy and autophagosome formation in both HeLa cells and fibroblasts. Furthermore, immediate-early expression of Us11 by an ICP34.5 deletion mutant virus is sufficient to render the cells resistant to PKR-induced and virus-induced autophagy. PKR expression and the PKR binding domain of Us11 are required for the antiautophagic activity of Us11. However, unlike ICP34.5, Us11 did not interact with Beclin 1. We suggest that the inhibition of autophagy observed in cells infected with HSV-1 results from the activity of not only ICP34.5 on Beclin 1 but also Us11 by direct interaction with PKR.     

Establishment of mutant murine mammary carcinoma FM3A cell strains transformed with the herpes simplex virus type 2 thymidine kinase gene

To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. IV. Recognition and activation by cloned glycoproteins gB and gD

Results of studies in mice and clinical observations in man indicate that T cell-mediated immunity is important in resistance to herpes simplex virus (HSV) infections. This study was undertaken to elucidate the viral antigen specificity of human HSV-immune T cells. Purified HSV-1 glycoproteins gB-1 and gD-1, cloned and expressed in mammalian cells, were found to stimulate proliferation of, and interleukin 2 (IL 2) production by, peripheral blood lymphocytes (PBL) of HSV seropositive individuals, indicating the presence of memory T cells to gB-1 and gD-1 in individuals with serologic evidence of immunity to HSV. Second, T cell clones, generated by stimulation of PBL with HSV-1, were found to recognize gB-1 or gD-1, as evidenced by the ability of the clones to proliferate in response to stimulation with gB-1 or gD-1 in the absence of exogenous IL 2. Third, HSV-specific T cell clones, lytic for HSV-1 or both HSV-1- and HSV-2-infected autologous target cells, were generated after stimulation of PBL with purified cloned gB-1 or gD-1. Our findings, that human HSV-specific T cells can recognize and be activated by HSV subunit antigens gB-1 or gD-1, imply that these glycoproteins play a role in human T cell-mediated immunity to HSV and support the contention that a gB-1 or gD-1 subunit vaccine may be protective in man.