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.     

Ocular avirulence of a herpes simplex virus type 1 strain is associated with heightened sensitivity to alpha/beta interferon.

BALB/c mice infected on the scarified cornea with herpes simplex virus type 1 strain 35 [HSV-1(35)] rarely developed ocular disease even at challenge doses as high as 10(7) PFU per eye. In contrast, HSV-1(RE) consistently induced stromal keratitis at an inoculum of 2 x 10(4) PFU. The goal of this study was to determine the reason for the difference in virulence between the two HSV strains. Both HSV-1 strains replicated to similar titers in excised corneal "buttons." However, after in vivo infection of the cornea, the growth of strain 35 was evident only during the first 24 h postinfection, whereas the replication of strain RE persisted for at least 4 days. In vitro tests revealed that HSV-1(35) was greater than 10 times more sensitive to alpha/beta interferon (IFN-alpha/beta) than HSV-1(RE). Both strains induced comparable serum levels of IFN after intraperitoneal inoculation. The kinetics of HSV-1(35) clearance from the eye was markedly altered by treatment with rabbit anti-IFN-alpha/beta. Virus titers exceeding 10(4) PFU per eye could be demonstrated 4 to 5 days postinfection in mice given a single inoculation of antiserum 1 h after infection. Furthermore, anti-IFN treatment in 3-week-old mice infected with HSV-1(35) led to the development of clinically apparent corneal disease which subsequently progressed to stromal keratitis in the majority of recipients. These results indicate that the striking difference in the capacity of HSV-1(35) and HSV-1(RE) to induce corneal disease was related to the inherently greater sensitivity of strain 35 to IFN-alpha/beta produced by the host in response to infection.     

Interleukin-12- and gamma interferon-dependent innate immunity are essential and sufficient for long-term survival of passively immunized mice infected with herpes simplex virus type 1

Interferon (IFN) type I (alpha/beta IFN [IFN-alpha/beta]) is very important in directly controlling herpes simplex virus type I (HSV-1) replication as well as in guiding and upregulating specific immunity against this virus. By contrast, the roles of IFN type II (IFN-gamma) and antibodies in the defense against HSV-1 are not clear. Mice without a functional IFN system and no mature B and T cells (AGR mice) did not survive HSV-1 infection in the presence or absence of neutralizing antibodies to the virus. Mice without a functional IFN type I system and with no mature B and T cells (AR129 mice) were unable to control infection with as little as 10 PFU of HSV-1 strain F. By contrast, in the presence of passively administered neutralizing murine antibodies to HSV-1, some AR129 mice survived infection with up to 10(4) PFU of HSV-1. This acute immune response was dependent on the presence of interleukin-12 (IL-12) p75. Interestingly, some virus-infected mice stayed healthy for several months, at which time antibody to HSV-1 was no longer detectable. Treatment of these virus-exposed mice with dexamethasone led to death in approximately 40% of the mice. HSV-1 was found in brains of mice that did not survive dexamethasone treatment, whereas HSV-1 was absent in those that survived the treatment. We conclude that in the presence of passively administered HSV-1-specific antibodies, the IL-12-induced IFN-gamma-dependent innate immune response is able to control low doses of virus infection. Surprisingly, in a significant proportion of these mice, HSV-1 appears to persist in the absence of antibodies and specific immunity.     

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.     

Herpes simplex virus 2 causes apoptotic infection in monocytoid cells

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

The role of natural killer cells and interferon in resistance to acute infection of mice with herpes simplex virus type 1

The relative roles of interferon (IFN) and natural killer (NK) cells in herpes simplex virus type 1 (HSV-1) infection of mice were examined. Adoptive transfer of adult mouse leukocytes into 4- to 6-day-old suckling mice protected the recipients from HSV-1 infection, as judged by viral titers in the spleen 2 days postinfection. Protection was mediated by several classes of leukocytes, including those depleted of NK cell activity by antibody to asialo GM1 and those depleted of macrophages by size separation. Mice receiving these leukocytes produced significantly higher levels of IFN 6 hr postinfection (early IFN) than did HSV-1-infected mice not receiving donor leukocytes. Antibody to IFN, under conditions that blocked early but not late IFN synthesis, greatly enhanced HSV-1 synthesis in mice receiving leukocytes and completely removed the protective effect mediated by leukocytes. High doses of anti-asialo GM1 blocked both NK cell activity and early IFN production and resulted in high titers of HSV-1. This effect on virus synthesis was not seen if mice were given antibody 1 day postinfection. Lower doses of anti-asialo GM1, which still depleted NK cell activity but had no effect on early IFN production, did not enhance HSV-1 synthesis. Depletion of NK cell activity with a low dose of antibody had no effect on the reduced HSV-1 synthesis resulting from prophylactic IFN treatment or on the enhanced HSV-1 synthesis resulting from antibody to IFN treatment. Thus, resistance to acute HSV-1 infection in mice correlates with early IFN production but not with NK cell activity, suggesting that NK cells are not major mediators of natural resistance in this model and that the antiviral effect of IFN is not mediated by NK cells.     

Antiapoptotic activity of herpes simplex virus type 2: the role of US3 protein kinase gene

In order to determine the ability of herpes simplex virus type 2 (HSV-2) to suppress apoptosis, we examined the effect of HSV-2 infection on apoptosis induced in HEp-2 cells by treatment with 1 M sorbitol. Although a wild-type strain of HSV-2 induced apoptosis in a significant fraction of the infected cells, HSV-2 could suppress sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that HSV-2, like HSV-1, has an antiapoptosis gene. Characterization of the cells infected with a US3-deletion mutant of HSV-2 revealed the necessity of a US3 gene in the antiapoptotic activity of this virus.     

Role of specific innate immune responses in herpes simplex virus infection of the central nervous system

Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.     

Herpes Simplex Virus Type 1 Renders Infected Cells Resistant to Cytotoxic T-Lymphocyte-Induced Apoptosis

Many viruses interfere with apoptosis of infected cells, presumably preventing cellular apoptosis as a direct response to viral infection. Since cytotoxic T lymphocytes (CTL) induce apoptosis of infected cells as part of the “lethal hit,” inhibition of apoptosis could represent an effective immune evasion strategy. We report here herpes simplex virus type 1 (HSV-1) interference with CTL-induced apoptosis of infected cells and show that HSV-1 inhibits the nuclear manifestations of apoptosis but not the membrane changes. The HL-60 cell line (human promyelocytic leukemia) undergoes apoptosis in response to many stimuli, including incubation with ethanol. After HSV-1 infection (strains E115 and 17+), ethanol-treated cells did not produce oligonucleosomal DNA fragments characteristic of apoptosis, as assayed by gel electrophoresis and enzyme-linked immunosorbent assay. Inhibition was detected 2 h after infection and increased over time. Importantly, HSV-1-infected cells were resistant to apoptosis induced by antigen-specific CD4⁺ CTL, despite the fact that CTL recognition and degranulation in response to infected targets remained intact. Unlike HSV-1, HSV-2 (strains 333 and HG52) did not inhibit DNA fragmentation. In contrast to the inhibition of DNA fragmentation by HSV-1, none of the HSV-1 or -2 strains interfered with the ethanol-induced exposure of surface phosphatidylserine characteristic of apoptosis, as determined by annexin V binding. These results demonstrate that genes of HSV-1 inhibit the nuclear manifestations of apoptosis but not the membrane manifestations, suggesting that these may be mediated via separate pathways. They also suggest that HSV-1 inhibition of CTL-induced apoptosis may be an important mechanism of immune evasion.     

In vivo significance of NK cell on resistance against virus (HSV-1) infections in mice

Susceptibility to infection with herpes simplex virus type 1 (HSV-1) was examined in euthymic as well as athymic nude mice which were continuously depleted of natural killer (NK) cell activity by i.v. injection of anti-asialo GM1. In those NK cell activity-depleted mice, the mortality rate of infection with HSV-1 and the virus titers in the brain, liver, and spleen were notably higher than in the control mice. The enhanced susceptibility was demonstrated only in the mice receiving anti-asialo GM1 and HSV-1 simultaneously, but not in the mice in which NK cell deletion was postponed by injecting the antisera 5 days after the virus inoculation. Interferon (IFN) production of peritoneal exudate cells was also reduced in the anti-asialo GM1-injected mice. The decline of resistance against HSV-1 infection proved to be primarily due to deletion of NK cells, but not due to the inability to produce IFN, because repeated injections of IFN increased the NK cell activity and prolonged the life of HSV-1-infected mice with an intact NK cell activity. In the NK cell activity-depleted mice, however, neither the NK cell activity nor the life span was improved by the administration of IFN.     

Dendritic cells from mice neonatally vaccinated with modified vaccinia virus Ankara transfer resistance against herpes simplex virus type I to naive one-week-old mice

Modified vaccinia Ankara (MVA) is an attenuated virus. MVA induces the production of IFN and Flt3-L (FL), which results in the expansion of dendritic cells (DC) and enhanced resistance against viral infections. We report on the interplay among IFN, FL, and DC in the resistance against heterologous virus after injection of neonatal mice with MVA. The induction of serum FL was tested on day 2, and the expansion of DC was tested 1 wk after treatment with MVA. At this time point the resistance against infection with heterologous virus was also determined. After MVA treatment, serum FL was enhanced, and DC, including plasmacytoid cells in spleen, were increased in number. Mice that lacked functional IFN type I and II systems failed to increase both the concentration of FL and the number of DC. Treatment with MVA enhanced resistance against HSV-1 in wild-type animals 100-fold, but animals without a functional IFN system were not protected. Transfer of CD11c(+) cells from MVA-treated mice into naive animals protected against lethal infection with HSV-1. Thus, although the increased resistance could be largely attributed to the increase in activation of IFN-producing plasmacytoid cells, this, in turn, depends on a complex interplay between the DC and T cell systems involving both FL and IFNs.     

Glycoprotein gE of herpes simplex virus type 1: effects of anti-gE on virion infectivity and on virus-induced fc-binding receptors.

An Fc-binding glycoprotein, designated gE, was detected previously in cells infected with herpes simplex virus type 1 (HSV-1) and in virion preparations isolated from infected cells. For the studies reported here, we purified gE from HSV-1 strain HFEM(syn) by affinity chromatography and preparative electrophoresis and then immunized a rabbit to produce an antiserum to glycoprotein gE. We found that this antiserum selectively precipitated gE and its precursors from detergent-solubilized extracts of HSV-1 strain HFEM(syn)-infected HEp-2 cells, from extracts of other cell lines infected with the same virus, and from extracts of HEp-2 cells infected with several other HSV-1 strains. The antiserum did not precipitate any proteins from uninfected cells. The several forms of gE detected by immunoprecipitation accumulated in variable quantities in different cells infected with the different virus strains and also varied slightly with respect to electrophoretic mobility, suggesting some differences in the gE's from different HSV-1 strains and some effects of the host cell on the nature and extent of post-translational processing. One of the electrophoretic forms of gE previously detected in purified preparations of virions could be precipitated by anti-gE from extracts of purified HSV-1 strain HFEM(syn) virions. Moreover, anti-gE neutralized HSV-1 infectivity, but only in the presence of complement. Finally, F(ab')2 fragments of the anti-gE immunoglobulin partially inhibited the binding of 125I-labeled immunoglobulin G to the Fc receptors on HSV-1-infected cells.