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.     

HSV and glycoprotein J inhibit caspase activation and apoptosis induced by granzyme B or Fas

HSV-1 inhibits apoptosis of infected cells, presumably to ensure that the infected cell survives long enough to allow completion of viral replication. Because cytotoxic lymphocytes kill their targets via the induction of apoptosis, protection from apoptosis could constitute a mechanism of immune evasion for HSV. Several HSV genes are involved in the inhibition of apoptosis, including Us5, which encodes glycoprotein J (gJ). Viruses deleted for Us5 showed defects in inhibition of caspase activation after Fas ligation or UV irradiation. Transfected cells expressing the Us5 gene product gJ were protected from Fas- or UV-induced apoptosis, as measured by morphology, caspase activation, membrane permeability changes, or mitochondrial transmembrane potential. In contrast, caspase 3 activation in mitochondria-free cell lysates by granzyme (gr)B was inhibited equivalently by Us5 deletion and rescue viruses, suggesting that gJ is not required for HSV to inhibition this process. However, mitochondria-free lysates from transfected cells expressing Us5/gJ were protected from grB-induced caspase activation, suggesting that Us5/gJ is sufficient to inhibit this process. Transfected cells expressing Us5/gJ were also protected from death induced by incubation with purified grB and perforin. These findings suggest that HSV has a comprehensive set of immune evasion functions that antagonize both Fas ligand- and grB-mediated pathways of CTL-induced apoptosis. The understanding of HSV effects on killing by CTL effector mechanisms may shed light on the incomplete control of HSV infections by the immune system and may allow more rational approaches to the development of immune modulatory treatments for HSV infection.     

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.     

Impaired hepatitis C virus (HCV)-specific effector CD8+ T cells undergo massive apoptosis in the peripheral blood during acute HCV infection and in the liver during the chronic phase of infection

A majority of patients infected with hepatitis C virus (HCV) do not sustain an effective T-cell response, and viremia persists. The mechanism leading to failure of the HCV-specific CD8⁺ T-cell response in patients developing chronic infection is unclear. We investigated apoptosis susceptibility of HCV-specific CD8⁺ T cells during the acute and chronic stages of infection. Although HCV-specific CD8⁺ T cells in the blood during the acute phase of infection and in the liver during the chronic phase were highly activated and expressed an effector phenotype, the majority was undergoing apoptosis. In contrast, peripheral blood HCV-specific CD8⁺ T cells during the chronic phase expressed a resting memory phenotype. Apoptosis susceptibility of HCV-specific CD8⁺ T cells was associated with very high levels of programmed death-1 (PD-1) and low CD127 expression and with significant functional T-cell deficits. Further evaluation of the “death phase” of HCV-specific CD8⁺ T cells during acute HCV infection showed that the majority of cells were dying by a process of cytokine withdrawal, mediated by activated caspase 9. Contraction during the acute phase occurred rapidly via this process despite the persistence of the virus. Remarkably, in the chronic phase of HCV infection, at the site of infection in the liver, a substantial frequency of caspase 9-mediated T-cell death was also present. This study highlights the importance of cytokine deprivation-mediated apoptosis with consequent down-modulation of the immune response to HCV during acute and chronic infections.     

CD4+ Cytotoxic T-Lymphocyte Activity against Macrophages Pulsed with Bovine Herpesvirus 1 Polypeptides

Bovine herpesvirus 1 (BHV-1) induces immune suppression, but the mechanisms for suppression are not well identified. We examined the induction and activity of BHV-1-specific cytolytic CD4⁺ T lymphocytes (CTL) by stimulating peripheral blood mononuclear cells (PBMC) of cattle immunized with attenuated live BHV-1. Cytolytic effector cells were primarily CD4⁺ T lymphocytes and lysed autologous, but not allogeneic, macrophages infected with BHV-1 or pulsed with BHV-1 polypeptides. Apoptosis of BHV-1-expressing target cells was observed in CD4⁺ CTL assays by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. To determine if apoptosis was mediated by a perforin- or Fas-mediated pathway, EGTA, a known selective inhibitor of the perforin pathway, was used. EGTA did not inhibit CD4⁺-T-cell-mediated cytotoxic activity, but it did limit the NK cell cytotoxicity of virus infected cells. These findings support the concept that CD4⁺ CTL lyse macrophages pulsed with BHV-1 polypeptides through a Fas-mediated lytic pathway by inducing apoptosis in the target cells. The prominent cytotoxicity mediated by CD4⁺ CTL suggests a mechanism of selective removal of viral antigen-associated antigen-presenting cells.     

Cross-Presentation of a Spread-Defective MCMV Is Sufficient to Prime the Majority of Virus-Specific CD8+ T Cells

CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.     

HSV-2 Regulates Monocyte Inflammatory Response via the Fas/FasL Pathway

Monocytic cells represent important cellular elements of the innate and adaptive immune responses in viral infections. We assessed the role of Fas/FasL in promoting monocyte apoptosis during HSV-2 infection by using an in vitro model based on the murine RAW 264.7 monocytic cell line and an in vivo murine model of HSV-2 infection applied to C57BL6, MRL-Fas^lpr/J (Fas−/−) and C3-Fasl^gld/J (FasL−/−) mice. HSV-2 infection of the monocytic cell line led to early induction of apoptosis, with no protective expression of anti-apoptotic Bcl-2. HSV-2 infected monocytes up-regulated Fas and FasL expression early during in vitro infection but were susceptible to Fas induced apoptosis. The vaginal monocytes in the HSV-2 murine model of infection up-regulated FasL expression and were susceptible to Fas induced apoptosis. HSV-2 infection of Fas and FasL- deficient mice led to decreased apoptosis of monocytes and impaired recruitment of NK, CD4+ and CD8+ T cells within the infection sites. The vaginal lavages of HSV-2 infected Fas and FasL- deficient showed decreased production of CXCL9, CXCL10 and TNF-α in comparison to HSV-2 infected wild-type mice strain. The decreased recruitment of immune competent cells was accompanied by delayed virus clearance from the infected tissue. Triggering of the Fas receptor on HSV-2 infected monocytes in vitro up-regulated the expression of CXCL9 chemokines and the cytokine TNF-α. Our study provides novel insights on the role of Fas/FasL pathway not only in apoptosis of monocytes but also in regulating local immune response by monocytes during HSV-2 infection.     

Poly-(ADP-ribose) polymerase partially contributes to target cell death triggered by cytolytic T lymphocytes.

We hypothesized that CTL-induced target cell (TC) death is partially due to processes that follow the DNA damage in target cells and include the activation of poly-ADP-ribose transferase (PADPRT) by DNA strand breaks. According to this model, the activated PADPRT is expected to deplete NAD, ATP, and to contribute to the TC death. We used inhibitors of PADPRT and a PADPRT-deficient cell mutant, as well as other nucleated TC and SRBC to test the role of PADPRT in CTL-induced cytotoxicity. It is found that inhibitors of PADPRT (3-aminobenzamide, benzamide (aromatic amides)) and nicotinamide all inhibit the CTL-mediated lysis of both Ag-specific TC and of Ag-nonbearing TC. The effect of PADPRT inhibitors was not due to inhibition of the lethal hit delivery by CTL, because in parallel control experiments, the same inhibitors did not interfere with CTL-induced lysis of SRBC, cells that are devoid of nuclei and PADPRT. Moreover, the effect of inhibitors of PADPRT did not affect earlier stages of lethal hit delivery because 3-aminobenzamide and benzamide did not interfere with CTL-induced DNA fragmentation in TC at concentration which protected TC lysis. Importantly, a PADPRT-deficient cell line was also much more resistant to CTL-induced lysis as tested in retargeting (4 and 8 h) assays; this was expected if activation of PADPRT is indeed involved in TC death. Control experiments reveal that the relative resistance of the PADPRT-deficient cell mutant to CTL-induced lysis was not related to its impaired ability to form conjugates and to trigger CTL (as tested in granule exocytosis assay). In addition, PADPRT-deficient cells were as susceptible to CTL-induced DNA fragmentation as were the control cells; yet, they were resistant to CTL-induced 51Cr-release. Control cells and PADPRT-deficient mutant were equally susceptible to antibody+C'-mediated lysis. Our data support the view that the activation of PADPRT can contribute to the CTL-induced cytolysis of some TC, but is not involved in lysis of other TC, as evidenced by the ability of CTL to efficiently lyse SRBC. These data suggest that there could be multiple molecular pathways of TC death in CTL-mediated cytotoxicity and the relative contribution of PADPRT and/or other enzymes will reflect the individual make-up of a particular TC.     

Herpes Simplex Virus Inhibits Apoptosis through the Action of Two Genes, Us5 and Us3

Apoptosis of virus-infected cells occurs either as a direct response to viral infection or upon recognition of infection by the host immune response. Apoptosis reduces production of new virus from these cells, and therefore viruses have evolved inhibitory mechanisms. We previously showed that laboratory strains of herpes simplex virus type 1 (HSV-1) protect infected cells from apoptosis induced by cytotoxic T lymphocytes or ethanol. We have now evaluated the ability of HSV-1 and HSV-2 laboratory and clinical isolates to inhibit apoptosis induced by anti-Fas antibody or UV irradiation and explored the genetic basis for this inhibition. HSV-1 isolates inhibited apoptosis induced by UV or anti-Fas antibody. In contrast, HSV-2 clinical isolates failed to inhibit apoptosis induced by either stimulus, although the HSV-2 laboratory strain 333 had a partial inhibitory effect on UV-induced apoptosis. Inhibition of apoptosis by HSV was accompanied by marked reduction of caspase-3 and caspase-8 activity. Deletion of the HSV-1 Us3 gene markedly reduced inhibition of UV-induced apoptosis and partially abrogated inhibition of Fas-mediated apoptosis. Conversely, deletion of the HSV-1 Us5 gene markedly reduced protection from Fas-mediated apoptosis and partially abrogated protection from UV. The Us11 and Us12 genes were not necessary for protection from apoptosis induced by either stimulus. The differences between HSV-1 and HSV-2 in the ability to inhibit apoptosis may be factors in the immunobiology of HSV infections.     

Asymptomatic human CD4+ cytotoxic T-cell epitopes identified from herpes simplex virus glycoprotein B

The identification of “asymptomatic” (i.e., protective) epitopes recognized by T cells from herpes simplex virus (HSV)-seropositive healthy individuals is a prerequisite for an effective vaccine. Using the PepScan epitope mapping strategy, a library of 179 potential peptide epitopes (15-mers overlapping by 10 amino acids) was identified from HSV type 1 (HSV-1) glycoprotein B (gB), an antigen that induces protective immunity in both animal models and humans. Eighteen groups (G1 to G18) of 10 adjacent peptides each were first screened for T-cell antigenicity in 38 HSV-1-seropositive but HSV-2-seronegative individuals. Individual peptides within the two immunodominant groups (i.e., G4 and G14) were further screened with T cells from HLA-DR-genotyped and clinically defined symptomatic (n = 10) and asymptomatic (n = 10) HSV-1-seropositive healthy individuals. Peptides gB_161-175 and gB_166-180 within G4 and gB_661-675 within G14 recalled the strongest HLA-DR-dependent CD4⁺ T-cell proliferation and gamma interferon production. gB_166-180, gB_661-675, and gB_666-680 elicited ex vivo CD4⁺ cytotoxic T cells (CTLs) that lysed autologous HSV-1- and vaccinia virus (expressing gB)-infected lymphoblastoid cell lines. Interestingly, gB_166-180 and gB_666-680 peptide epitopes were strongly recognized by CD4⁺ T cells from 10 of 10 asymptomatic patients but not by CD4⁺ T cells from 10 of 10 symptomatic patients (P < 0.0001; analysis of variance posttest). Inversely, CD4⁺ T cells from symptomatic patients preferentially recognized gB_661-675 (P < 0.0001). Thus, we identified three previously unrecognized CD4⁺ CTL peptide epitopes in HSV-1 gB. Among these, gB_166-180 and gB_666-680 appear to be “asymptomatic” peptide epitopes and therefore should be considered in the design of future herpes vaccines.     

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.     

DNA Fragmentation Induced by Cytotoxic T Lymphocytes Can Result in Target Cell Death

Cytotoxic T lymphocyte (CTL)-mediated lysis is accompanied by fragmentation of target cell DNA into an oligonucleosome ladder, a hallmark of apoptosis. Is this a fortuitous coincidence, or could CTL be inducing lysis by activation of the suicide signal? In this report we demonstrate that CTL-mediated target cell death can be blocked with the drug aurintricarboxylic acid (ATA). The abrogation of death correlates with the inhibition of DNA fragmentation. While ATA prevented DNA fragmentation, it failed to significantly alter protein, RNA, or DNA synthesis in the cell lines over the dose range used. In addition, there was no inhibition of cell-cell interaction or granule exocytosis during CTL-mediated killing. ATA also significantly inhibited the cytolysis and DNA fragmentation mediated by isolated cytolytic granules, as well as the granular protein fragmentin. We developed an assay in which target cells could be separated from CTL after binding and programming for lysis. Once they had received the "kiss of death," target cells could be rescued from lysis (as indicated by inhibition of DNA fragmentation and increased target cell viability) by treatment with ATA. These results suggest that ATA blocks target cell death by inhibition of DNA fragmentation, and further, that chromatin degradation is a cause rather than a result of cell death in CTL-mediated lysis.     

Early passage neonatal and adult keratinocytes are sensitive to apoptosis induced by infection with an ICP27-null mutant of herpes simplex virus 1

Herpes simplex virus 1 (HSV-1) is a enveloped, double stranded DNA virus that is the causative agent of various diseases including cold sores, encephalitis, and ocular keratitis. Previous research has determined that HSV-1 modulates cellular apoptotic pathways. Apoptosis is triggered in infected cells early in infection; however, later in the infection the apoptotic response is suppressed due to the expression of several viral apoptotic antagonists. This sets us a delicate balance between pro- and anti-apoptotic processes during the lytic phase of infection. Several studies have demonstrated that the apoptotic balance can be shifted during infection of certain cell types, leading to apoptosis of the infected cells (HSV-1-dependent apoptosis). For example, HEp-2 cells infected with an ICP27-null recombinant HSV-1 virus undergo HSV-1-dependent apoptosis. Differences in the sensitivity to HSV-1-dependent apoptosis have been revealed. Although many tumor cells have been found to be highly sensitive to this apoptotic response, with the exception hematological cells, all primary human cells tested prior to this study have been shown to be resistant to HSV-1-dependent apoptosis. Here, we demonstrate that early passage neonatal and adult human keratinocytes, which are usually the first cells to encounter HSV-1 in human infection and support the lytic stage of the life cycle, display membrane blebbing and ballooning, chromatin condensation, caspase activation, and cleavage of cellular caspase substrates when infected with an ICP27-null recombinant of HSV-1. Furthermore, caspase activation is needed for the efficient apoptotic response. These results suggest that apoptotic machinery may be a target for modulating HSV-disease in patients.     

An ��Escape Clock�� for Estimating the Turnover of SIV DNA in Resting CD4+ T Cells

Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an “escape clock” approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT) SIV DNA present in early infection by CTL escape mutant (EM) strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication.     

Differential CD4+ T-lymphocyte apoptosis and bystander T-cell activation in rhesus macaques and sooty mangabeys during acute simian immunodeficiency virus infection

In contrast to pathogenic lentiviral infections, chronic simian immunodeficiency virus (SIV) infection in its natural host is characterized by a lack of increased immune activation and apoptosis. To determine whether these differences are species specific and predicted by the early host response to SIV in primary infection, we longitudinally examined T-lymphocyte apoptosis, immune activation, and the SIV-specific cellular immune response in experimentally infected rhesus macaques (RM) and sooty mangabeys (SM) with controlled or uncontrolled SIV infection. SIVsmE041, a primary SIVsm isolate, reproduced set-point viremia levels of natural SIV infection in SM but was controlled in RM, while SIVmac239 replicated to high levels in RM. Following SIV infection, increased CD8⁺ T-lymphocyte apoptosis, temporally coinciding with onset of SIV-specific cellular immunity, and elevated plasma Th1 cytokine and gamma interferon-induced chemokine levels were common to both SM and RM. Different from SM, SIV-infected RM showed a significantly higher frequency of peripheral blood activated CD8⁺ T lymphocytes despite comparable magnitude of the SIV-specific gamma interferon enzyme-linked immunospot response. Furthermore, an increase in CD4⁺ and CD4⁻CD8⁻ T-lymphocyte apoptosis and plasma tumor necrosis factor-related apoptosis-inducing ligand were observed only in RM and occurred in both controlled SIVsmE041 and uncontrolled SIVmac239 infection. These data suggest that the “excess” activated T lymphocytes in RM soon after SIV infection are predominantly of non-virus-specific bystander origin. Thus, species-specific differences in the early innate immune response appear to be an important factor contributing to differential immune activation in natural and nonnatural hosts of SIV infection.     

Effector CD4+ T-cell involvement in clearance of infectious herpes simplex virus type 1 from sensory ganglia and spinal cords

In primary infection, CD8⁺ T cells are important for clearance of infectious herpes simplex virus (HSV) from sensory ganglia. In this study, evidence of CD4⁺ T-cell-mediated clearance of infectious HSV type 1 (HSV-1) from neural tissues was also detected. In immunocompetent mice, HSV-specific CD4⁺ T cells were present in sensory ganglia and spinal cords coincident with HSV-1 clearance from these sites and remained detectable at least 8 months postinfection. Neural CD4⁺ T cells isolated at the peak of neural infection secreted gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), or IL-4 after stimulation with HSV antigen. HSV-1 titers in neural tissues were greatly reduced over time in CD8⁺ T-cell-deficient and CD8⁺ T-cell-depleted mice, suggesting that CD4⁺ T cells could mediate clearance of HSV-1 from neural tissue. To examine possible mechanisms by which CD4⁺ T cells resolved neural infection, CD8⁺ T cells were depleted from perforin-deficient or FasL-defective mice. Clearance of infectious virus from neural tissues was not significantly different in perforin-deficient or FasL-defective mice compared to wild-type mice. Further, in spinal cords and brains after vaginal HSV-1 challenge of chimeric mice expressing both perforin and Fas or neither perforin nor Fas, virus titers were significantly lower than in control mice. Thus, perforin and Fas were not required for clearance of infectious virus from neural tissues. These results suggest that HSV-specific CD4⁺ T cells are one component of a long-term immune cell presence in neural tissues following genital HSV-1 infection and play a role in clearance of infectious HSV-1 at neural sites, possibly via a nonlytic mechanism.     

Intracellular hepatitis B virus nucleocapsids survive cytotoxic T-lymphocyte-induced apoptosis

Following antigen recognition, hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) induce a necroinflammatory liver disease in HBV-transgenic mice. An early event in this process is CTL-dependent activation of apoptosis in a small fraction of HBV-positive hepatocytes. Here we show that cytoplasmic HBV nucleocapsids and their cargo of replicative DNA intermediates survive CTL-induced apoptosis of hepatocytes in vitro. These results suggest that destruction of infected cells per se is not sufficient to destroy the replicating HBV genome in infected tissue and that other events in addition to this process are required for viral clearance to occur.     

Loss of CD4+ T Cells in Human Immunodeficiency Virus Type 1-Infected Chimpanzees Is Associated with Increased Lymphocyte Apoptosis

Supportive evidence that apoptosis contributes to loss of CD4⁺ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected humans comes from an apparent lack of abnormal apoptosis in apathogenic lentivirus infections of nonhuman primates, including HIV-1 infection of chimpanzees. Two female chimpanzees were inoculated, one cervically and the other intravenously, with HIV-1 derived from the LAI/LAV-1b strain, which was isolated from a chimpanzee infected with the virus for 8 years. Within 6 weeks of infection, both recipient chimpanzees developed a progressive loss of CD4⁺ T cells which correlated with persistently high viral burdens and increased levels of CD4⁺ T-cell apoptosis both in vitro and in vivo. Lymph nodes from both animals also revealed evidence of immune hyperactivation. Intermediate levels of T-cell apoptosis in both peripheral blood and lymph nodes were seen in a third chimpanzee that had been infected with the LAI/LAV-1b strain for 9 years; this animal has maintained depressed CD4/CD8 T-cell ratios for the last 3 years. Similar analyses of cells from 4 uninfected animals and 10 other HIV-1-infected chimpanzees without loss of CD4⁺ cells revealed no difference in levels of apoptosis in these two control groups. These results demonstrate a correlation between immune hyperactivation, T-cell apoptosis, and chronic loss of CD4⁺ T cells in HIV-1-infected chimpanzees, providing additional evidence that apoptosis is an important factor in T-cell loss in AIDS. Furthermore, the results show that some HIV-1 strains are pathogenic for chimpanzees and that this species is not inherently resistant to HIV-1-induced disease.     

Lymphoid-related CD11c+ CD8alpha+ dendritic cells are involved in enhancing herpes simplex virus type 1 latency

The mechanism(s) by which herpes simplex virus type 1 (HSV-1) latency is established in neurons is not known. In this study, we examined the effect of dendritic cells (DCs) on the level of HSV-1 latency in trigeminal ganglia (TGs) of ocularly infected BALB/c and C57BL/6 mice. We found that immunization of wild-type mice with FMS-like tyrosine kinase 3 ligand (Flt3L) DNA, which increases the number of DCs, increased the amount of latency in infected mice. Conversely, depletion of DCs was associated with reduced latency. Latency was also significantly reduced in Flt3L^−/− and CD8^−/− mice. Interestingly, immunization of Flt3L^−/− but not CD8^−/− mice with Flt3L DNA increased latency. Transfer experiments using DCs expanded ex vivo with Flt3L or granulocyte-macrophage colony-stimulating factor suggested that increased latency was associated with the presence of lymphoid-related (CD11c⁺ CD8α⁺) DCs, while reduced latency was associated with myeloid-related (CD11c⁺ CD8α⁻) DCs. Modulation of DC numbers by Flt3L DNA immunization or depletion did not alter acute virus replication in the eye or TG or eye disease in ocularly infected mice. Our results suggest that CD11c⁺ CD8α⁺ DCs directly or indirectly increase the amount of HSV-1 latency in mouse TGs.