Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1

Cultured human epithelial cells infected with an ICP27 deletion strain of herpes simplex virus type 1 (HSV-1) show characteristic features of apoptotic cells including cell shrinkage, nuclear condensation, and DNA fragmentation. These cells do not show such apoptotic features when infected with a wild-type virus unless the infections are performed in the presence of a protein synthesis inhibitor. Thus, both types of virus induce apoptosis, but the ICP27-null virus is unable to prevent this process from killing the cells. In this report, we show that this ICP27-deficient virus induced apoptosis in human HEp-2 cells through a pathway which involved the activation of caspase-3 and the processing of the death substrates DNA fragmentation factor and poly(ADP-ribose) polymerase. The induction of apoptosis by wild-type HSV-1 occurred prior to 6 h postinfection (hpi), and de novo viral protein synthesis was not required to induce the process. The ability of the virus to inhibit apoptosis was shown to be effective between 3 to 6 hpi. Wild-type HSV-1 infection was also able to block the apoptosis induced in cells by the addition of cycloheximide, staurosporine, and sorbitol. While U(S)3- and ICP22-deficient viruses showed a partial prevention of apoptosis, deletion of either the U(L)13 or vhs gene products did not affect the ability of HSV-1 to prevent apoptosis in infected cells. Finally, we demonstrate that in UV-inactivated viruses, viral binding and entry were not sufficient to induce apoptosis. Taken together, these results suggest that either gene expression or another RNA metabolic event likely plays a role in the induction of apoptosis in HSV-1-infected human cells.     

Accumulation of herpes simplex virus type 1 early and leaky-late proteins correlates with apoptosis prevention in infected human HEp-2 cells

We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803-2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (gamma(2)) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (beta) and leaky-late (gamma(1)) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.     

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.     

Phenotype of a herpes simplex virus type 1 mutant that fails to express immediate-early regulatory protein ICP0

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein ICP0 is required for efficient progression of infected cells into productive lytic infection, especially in low-multiplicity infections of limited-passage human fibroblasts. We have used single-cell-based assays that allow detailed analysis of the ICP0-null phenotype in low-multiplicity infections of restrictive cell types. The major conclusions are as follows: (i) there is a threshold input multiplicity above which the mutant virus replicates normally; (ii) individual cells infected below the threshold multiplicity have a high probability of establishing a nonproductive infection; (iii) such nonproductively infected cells have a high probability of expressing IE products at 6 h postinfection; (iv) even at 24 h postinfection, IE protein-positive nonproductively infected human fibroblast cells exceed the number of cells that lead to plaque formation by up to 2 orders of magnitude; (v) expression of individual IE proteins in a proportion of the nonproductively infected cells is incompletely coordinated; (vi) the nonproductive cells can also express early gene products at low frequencies and in a stochastic manner; and (vii) significant numbers of human fibroblast cells infected at low multiplicity by an ICP0-deficient virus are lost through cell death. We propose that in the absence of ICP0 expression, HSV-1 infected human fibroblasts can undergo a great variety of fates, including quiescence, stalled infection at a variety of different stages, cell death, and, for a minor population, initiation of formation of a plaque.     

The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.     

Human neuron-committed teratocarcinoma NT2 cell line has abnormal ND10 structures and is poorly infected by herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 stimulates the initiation of lytic infection and reactivation from quiescence in human fibroblast cells. These functions correlate with its ability to localize to and disrupt centromeres and specific subnuclear structures known as ND10, PML nuclear bodies, or promyelocytic oncogenic domains. Since the natural site of herpesvirus latency is in neurons, we investigated the status of ND10 and centromeres in uninfected and infected human cells with neuronal characteristics. We found that NT2 cells, a neuronally committed human teratocarcinoma cell line, have abnormal ND10 characterized by low expression of the major ND10 component PML and no detectable expression of another major ND10 antigen, Sp100. In addition, PML is less extensively modified by the ubiquitin-like protein SUMO-1 in NT2 cells compared to fibroblasts. After treatment with retinoic acid, NT2 cells differentiate into neuron-like hNT cells which express very high levels of both PML and Sp100. Infection of both NT2 and hNT cells by HSV-1 was poor compared to human fibroblasts, and after low-multiplicity infection yields of virus were reduced by 2 to 3 orders of magnitude. ICP0-deficient mutants were also disabled in the neuron-related cell lines, and cells quiescently infected with an ICP0-null virus could be established. These results correlated with less-efficient disruption of ND10 and centromeres induced by ICP0 in NT2 and hNT cells. Furthermore, the ability of ICP0 to activate gene expression in transfection assays in NT2 cells was poor compared to Vero cells. These results suggest that a contributory factor in the reduced HSV-1 replication in the neuron-related cells is inefficient ICP0 function; it is possible that this is pertinent to the establishment of latent infection in neurons in vivo.     

STAT-1- and IRF-3-dependent pathways are not essential for repression of ICP0-null mutant herpes simplex virus type 1 in human fibroblasts

Efficient herpes simplex virus type 1 (HSV-1) infection of human fibroblasts (HFs) is highly dependent on the viral immediate-early regulatory protein ICP0 unless the infection is conducted at a high multiplicity. ICP0-null mutant HSV-1 exhibits a plaque-forming defect of up to 3 orders of magnitude in HFs, whereas in many other cell types, this defect varies between 10- and 30-fold. The reasons for the high ICP0 requirement for HSV-1 infection in HFs have not been established definitively. Previous studies using other cell types suggested that ICP0-null mutant HSV-1 is hypersensitive to interferon and that this sensitivity is dependent on the cellular promyelocytic leukemia (PML) protein. To investigate the roles of two important aspects of interferon signaling in the phenotype of ICP0-null mutant HSV-1, we isolated HFs depleted of STAT-1 or interferon regulatory factor 3 (IRF-3). Surprisingly, plaque formation by the mutant virus was not improved in either cell type. We found that the sensitivity to interferon pretreatment of both ICP0-null mutant and wild-type (wt) HSV-1 was highly dependent on the multiplicity of infection. At a low multiplicity in virus yield experiments, both viruses were extremely susceptible to interferon pretreatment of HFs, but the sensitivity of the wild type but not the mutant could be overcome at higher multiplicities. We found that both wt and ICP0-null mutant HSV-1 remained sensitive to interferon in PML-depleted HFs albeit to an apparently lesser extent than in control cells. The data imply that the substantial reduction in ICP0-null HSV-1 infectivity at a low multiplicity in HFs does not occur through the activities of STAT-1- and IRF-3-dependent pathways and cannot be explained solely by enhanced sensitivity to interferon. We suggest that antiviral activities induced by interferon may be separable from and additive to those resulting from PML-related intrinsic resistance mechanisms.     

The potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1

Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more serious diseases. It can establish a latent stage in sensory ganglia after primary epithelial infections, and reactivate in response to stress or sunlight. Previous studies have demonstrated that viral immediate-early protein ICP0 plays a key role in regulating the balance between lytic and latent infection. Recently, It has been determined that promyelocytic leukemia (PML) nuclear bodies (NBs), small nuclear sub-structures, contribute to the repression of HSV-1 infection in the absence of functional ICP0. In this review, we discuss the fundamentals of the interaction between ICP0 and PML NBs, suggesting a potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1.     

The ribonucleotide reductase R1 subunits of herpes simplex virus 1 and 2 protect cells against poly(I · C)-induced apoptosis

We recently provided evidence that the ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 (HSV-1 and -2) protect cells against tumor necrosis factor alpha- and Fas ligand-induced apoptosis by interacting with caspase 8. Double-stranded RNA (dsRNA) is a viral intermediate known to initiate innate antiviral responses. Poly(I · C), a synthetic analogue of viral dsRNA, rapidly triggers caspase 8 activation and apoptosis in HeLa cells. Here, we report that HeLa cells after HSV-1 and HSV-2 infection were quickly protected from apoptosis caused by either extracellular poly(I · C) combined with cycloheximide or transfected poly(I · C). Cells infected with the HSV-1 R1 deletion mutant ICP6Δ were killed by poly(I · C), indicating that HSV-1 R1 plays a key role in antiapoptotic responses to poly(I · C). Individually expressed HSV R1s counteracted caspase 8 activation by poly(I · C). In addition to their binding to caspase 8, HSV R1s also interacted constitutively with receptor-interacting protein 1 (RIP1) when expressed either individually or with other viral proteins during HSV infection. R1(1-834)-green fluorescent protein (GFP), an HSV-2 R1 deletion mutant protein devoid of antiapoptotic activity, did not interact with caspase 8 and RIP1, suggesting that these interactions are required for protection against poly(I · C). HSV-2 R1 inhibited the interaction between the Toll/interleukin-1 receptor domain-containing adaptor-inducing beta interferon (IFN-β) (TRIF) and RIP1, an interaction that is essential for apoptosis triggered by extracellular poly(I · C) plus cycloheximide or TRIF overexpression. TRIF silencing reduced poly(I · C)-triggered caspase 8 activation in mock- and ICP6Δ-infected cells, confirming that TRIF is involved in poly(I · C)-induced apoptosis. Thus, by interacting with caspase 8 and RIP1, HSV R1s impair the apoptotic host defense mechanism prompted by dsRNA.     

Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.     

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.     

Infection and inhibition of human cytotoxic T lymphocytes by herpes simplex virus.

The effect of herpes simplex virus type 1 (HSV-1) infection on human cytotoxic T-lymphocyte (CTL) lytic function was assessed. All HSV-infected CTL populations tested were significantly inhibited in lysing target cells. The inhibition of CTL lytic function by infection with HSV-1 was independent of T-cell receptor-mediated antigen recognition and did not involve virus-induced shutoff of host protein synthesis, the expression of the HSV-1 transactivation protein, ICP4, or replicating virus. Understanding the functional impairment of CTL following infection with HSV may have important implications for HSV-induced immunosuppression and the mechanism of HSV persistence in immunocompetent hosts.     

ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency

Relative to wild-type herpes simplex virus type 1 (HSV-1), ICP0-null mutant viruses reactivate inefficiently from explanted, latently infected mouse trigeminal ganglia (TG), indicating that ICP0 is not essential for reactivation but plays a central role in enhancing the efficiency of reactivation. The validity of these findings has been questioned, however, because the replication of ICP0-null mutants is impaired in animal models during the establishment of latency, such that fewer mutant genomes than wild-type genomes are present in latently infected mouse TG. Therefore, the reduced number of mutant viral genomes available to reactivate, rather than mutations in the ICP0 gene per se, may be responsible for the reduced reactivation efficiency of ICP0-null mutants. We have recently demonstrated that optimization of the size of the ICP0 mutant virus inoculum and transient immunosuppression of mutant-infected mice with cyclophosphamide can be used to establish wild-type levels of ICP0-null mutant genomes in latently infected TG (W. P. Halford and P. A. Schaffer, J. Virol. 74:5957-5967, 2000). Using this procedure to equalize mutant and wild-type genome numbers, the goal of the present study was to determine if, relative to wild-type virus, the absence of ICP0 function in two ICP0-null mutants, n212 and 7134, affects reactivation efficiency from (i) explants of latently infected TG and (ii) primary cultures of latently infected TG cells. Although equivalent numbers of viral genomes were present in TG of mice latently infected with either wild-type or mutant viruses, reactivation of n212 and 7134 from heat-stressed TG explants was inefficient (31 and 37% reactivation, respectively) relative to reactivation of wild-type virus (KOS) (95%). Similarly, n212 and 7134 reactivated inefficiently from primary cultures of dissociated TG cells plated directly after removal from the mouse (7 and 4% reactivation, respectively), relative to KOS (60% reactivation). The efficiency and kinetics of reactivation of KOS, n212, and 7134 from cultured TG cells (treated with acyclovir to facilitate the establishment of latency) in response to heat stress or superinfection with a nonreplicating HSV-1 ICP4(-) mutant, n12, were compared. Whereas heat stress induced reactivation of KOS from 69% of latently infected TG cell cultures, reactivation of n212 and 7134 was detected in only 1 and 7% of cultures, respectively. In contrast, superinfection with the ICP4(-) virus, which expresses high levels of ICP0, resulted in the production of infectious virus in nearly 100% of cultures latently infected with KOS, n212, or 7134 within 72 h. Thus, although latent mutant viral genome loads were equivalent to that of wild-type virus, in the absence of ICP0, n212 and 7134 reactivated inefficiently from latently infected TG cells during culture establishment and following heat stress. Collectively, these findings demonstrate that ICP0 is required to induce efficient reactivation of HSV-1 from neuronal latency.     

一种1型单纯庖疹病毒载体系统的建立

1型单纯疱疹病毒（HSV-1）作为溶瘤病毒和病毒载体的研究已有很长的历史. 本研究利用细菌人工染色体技术建立了一种HSV-1载体系统. 首先,将HSV-1内部反向重复序列（internal inverted repeat sequences, IR）两侧的片段克隆入pKO5获得穿梭质粒pKO5/BN,其电转含pHSV-BAC的大肠杆菌后筛选获得删除IR区重组DNA的 pHSVΔIR-BAC. pHSVΔIR-BAC转染Vero细胞获得删除IR区的重组病毒HSVΔIR（MH1001）.上述pKO5/BN和含pHSVΔIR BAC的大肠杆菌构成了HSV-1载体系统. 利用该系统获得了表达绿色荧光蛋白EGFP的重组病毒HSVΔIR/EGFP（MH1002）.MH1001和MH1002在感染的Vero细胞中增殖水平略低于野生型HSV-1,但无显著差异|Western印迹检测表明,重组病毒早期蛋白质ICP0、ICP4、ICP8、ICP22、ICP27在感染细胞中的表达水平下降|免疫荧光及激光共聚焦检测表明,重组病毒与野生型病毒均存在于细胞质中.以上结果表明,删除IR区的重组HSV-1保留了复制能力,能够携载并表达外源基因,建立的HSV-1载体系统可用于构建携载外源基因的复制型重组HSV-1.