Inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase

Genital herpes simplex virus type 2 (HSV-2) is a significant clinical problem. Infection in pregnancy may result in disseminated infection of the newborn with encephalitis. We analyzed the antiviral effects induced by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in cervix carcinoma cells (HeLa) and astrocytoma cells (86HG39). We found that replication of HSV-2 in HeLa cells and in 86HG39 cells is inhibited after stimulation of the cells by IFN-gamma and TNF-alpha. The antiviral effect of IFN-gamma is enhanced in the presence of TNF-alpha, while stimulation by TNF-alpha alone did not induce antiviral activity. We found that IFN-gamma induces a strong activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and in addition, that the IFN-gamma-induced IDO activity was enhanced in the presence of TNF-alpha. Furthermore, we found that the induction of IDO activity is responsible for the inhibition of herpes simplex virus replication, since the presence of excess amounts of l-tryptophan abrogates the antiviral effect induced by IFN-gamma and the combination of IFN-gamma and TNF-alpha. We therefore conclude that the antiviral effect against HSV-2 mediated by type II interferon and TNF-alpha are dependent on IDO activation.     

Alpha/Beta interferon and gamma interferon synergize to inhibit the replication of herpes simplex virus type 1

In vivo evidence suggests that T-cell-derived gamma interferon (IFN-gamma) can directly inhibit the replication of herpes simplex virus type 1 (HSV-1). However, IFN-gamma is a weak inhibitor of HSV-1 replication in vitro. We have found that IFN-gamma synergizes with the innate IFNs (IFN-alpha and -beta) to potently inhibit HSV-1 replication in vitro and in vivo. Treatment of Vero cells with either IFN-beta or IFN-gamma inhibits HSV-1 replication by <20-fold, whereas treatment with both IFN-beta and IFN-gamma inhibits HSV-1 replication by approximately 1,000-fold. Treatment with IFN-beta and IFN-gamma does not prevent HSV-1 entry into Vero cells, and the inhibitory effect can be overcome by increasing the multiplicity of HSV-1 infection. The capacity of IFN-beta and IFN-gamma to synergistically inhibit HSV-1 replication is not virus strain specific and has been observed in three different cell types. For two of the three virus strains tested, IFN-beta and IFN-gamma inhibit HSV-1 replication with a potency that approaches that achieved by a high dose of acyclovir. Pretreatment of mouse eyes with IFN-beta and IFN-gamma reduces HSV-1 replication to nearly undetectable levels, prevents the development of disease, and reduces the latent HSV-1 genome load per trigeminal ganglion by approximately 200-fold. Thus, simultaneous activation of IFN-alpha/beta receptors and IFN-gamma receptors appears to render cells highly resistant to the replication of HSV-1. Because IFN-alpha or IFN-beta is produced by most cells as an innate response to virus infection, the results imply that IFN-gamma secreted by T cells may provide a critical second signal that potently inhibits HSV-1 replication in vivo.     

Herpesvirus infection alters the steady-state levels of cellular polyadenylated RNA in polyoma virus-transformed BHK cells.

Polyoma-transformed BHK cells are permissive for the replication of herpes simplex virus type 1. The effect of herpes infection on the steady-state levels of bulk mRNA sequences in these cells was studied by using cDNA to polyadenylated cytoplasmic RNA from uninfected cells. The principal findings were: (i) herpes simplex virus type 1 infection caused a pronounced reduction in the cytoplasmic levels of moderately abundant mRNAs' (ii) after infection, increased amounts of RNA complementary to the cDNA were isolated as part of the nonadenylated cytoplasmic RNA fraction.     

Effect of interferon on replication of herpes simplex virus types 1 and 2 in human macrophages.

Macrophages derived from human peripheral blood and cultured for 1 week were permissive for the replication of herpes simplex virus (HSV) types 1 and 2. Low titers of interferon (IFN) were produced after virus infection. The yield of infectious virions was reduced by pretreatment of cells with natural and recombinant IFN-alpha and natural IFN-beta. Recombinant and natural IFN-gamma exhibited very low antiviral activity. Treatment of cells with IFN-gamma mixed with IFN-alpha or with IFN-beta did not result in a synergistic inhibition of virus yield. We studied the synthesis of HSV type 1- and HSV type 2-coded proteins in macrophages treated with IFN-beta. Induction of the HSV beta-protein DNA polymerase was strongly inhibited in IFN-treated cells in a dose-dependent manner. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, other beta- and gamma-proteins of HSV were inhibited as well. Immunofluorescence studies revealed a strong inhibition of the expression of immediate early alpha-protein ICP4. The results indicate that IFN acts early during the viral replication cycle to inhibit the synthesis of HSV alpha- and beta-proteins.     

Polyamine metabolism in MRC5 cells infected with different herpesviruses.

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

Gamma interferon impedes the establishment of herpes simplex virus type 1 latent infection but has no impact on its maintenance or reactivation in mice

Murine models of gamma interferon (IFN-gamma) deficiency demonstrate the role of this cytokine in attenuating acute herpes simplex virus (HSV) disease; however, the effect of IFN-gamma on the establishment and maintenance of neuronal latency and viral reactivation is not known. Using the IFN-gamma knockout (GKO) model of IFN-gamma deficiency and sensitive quantitative PCR methods, we show that IFN-gamma significantly reduces the ganglion content of latent HSV-1 in BALB/c mice, which in turn delays viral time to reactivation following UV irradiation. Similar effects were not seen in the C57BL/6 strain. These results indicate that IFN-gamma significantly attenuates latent HSV infection in the mouse model of ocular infection but has no impact on the maintenance of latency or virus reactivation.     

Gamma interferon-induced, nitric oxide-mediated inhibition of vaccinia virus replication.

Gamma interferon (IFN-gamma)-induced nitric oxide synthase (iNOS) and nitric oxide (NO) production in the murine macrophage-like RAW 264.7 cells were previously shown to inhibit the replication of the poxviruses vaccinia virus (VV) and ectromelia virus and herpes simplex virus type 1. In the current study, we performed biochemical analyses to determine the stage in the viral life cycle blocked by IFN-gamma-induced NO. Antibodies specific for temporally expressed viral proteins, a VV-specific DNA probe, and transmission electron microscopy were used to show that the cytokine-induced NO inhibited late protein synthesis, DNA replication, and virus particle formation but not expression of the early proteins analyzed. Essentially similar results were obtained with hydroxyurea and cytosine arabinoside, inhibitors of DNA replication. Enzymatically active iNOS was detected in the lysates of IFN-gamma-treated but not in untreated RAW 264.7 cells. The IFN-gamma-treated RAW 264.7 cells which express iNOS not only were resistant to productive infection but also efficiently blocked the replication of VV in infected bystander cells of epithelial origin. This inhibition was arginine dependent, correlated with nitric production in cultures, and was reversible by the NOS inhibitor N omega-monomethyl-L-arginine.     

Replication of herpes simplex virus type 1 on hydroxyurea-resistant baby hamster kidney cells.

Hydroxyurea-resistant (HUr) baby hamster kidney cells were isolated, subcloned, and characterized. One clonal line, which contained elevated levels of ribonucleotide reductase, lost its HU resistance during passage in the absence of the inhibitor, whereas another clonal line was stably resistant. The replication of herpes simplex virus type 1 on these cells was compared with that of the parvovirus minute virus of mice. Herpes simplex virus type 1 was found to be as sensitive to HU on both lines of HUr baby hamster kidney cells as it was on parental (HU-sensitive) cells, whereas parvovirus replication was about eight times more resistant on HUr baby hamster kidney cells compared with the parental cells. The results suggest that herpes simplex virus type 1 cannot use the cellular reductase and may code for its own.     

Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system

After corneal infection, herpes simplex virus type 1 (HSV-1) invades sensory neurons with cell bodies in the trigeminal ganglion (TG), replicates briefly, and then establishes a latent infection in these neurons. HSV-1 replication in the TG can be detected as early as 2 days after corneal infection, reaches peak titers by 3-5 days after infection, and is undetectable by 7-10 days. During the period of HSV-1 replication, macrophages and gammadelta TCR+ T lymphocytes infiltrate the TG, and TNF-alpha, IFN-gamma, the inducible nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-alpha, IFN-gamma, and the iNOS product nitric oxide (NO) all inhibit HSV-1 replication in vitro. Macrophage and gammadelta TCR+ T cell depletion studies demonstrated that macrophages are the main source of TNF-alpha and iNOS, whereas gammadelta TCR+ T cells produce IFN-gamma. Macrophage depletion, aminoguanidine inhibition of iNOS, and neutralization of TNF-alpha or IFN-gamma all individually and synergistically increased HSV-1 titers in the TG after HSV-1 corneal infection. Moreover, individually depleting macrophages or neutralizing TNF-alpha or IFN-gamma markedly reduced the accumulation of both macrophages and gammadelta TCR+ T cells in the TG. Our findings establish that after primary HSV-1 infection, the bulk of virus replication in the sensory ganglia is controlled by macrophages and gammadelta TCR+ T lymphocytes through their production of antiviral molecules TNF-alpha, NO, and IFN-gamma. Our findings also strongly suggest that cross-regulation between these two cell types is necessary for their accumulation and function in the infected TG.     

Differential effect of arabinofuranosylthymine of the replication of human herpesviruses.

The thymidine analog 1-beta-arabinofuranosylthymine (ara-T) has previously been found to selectively inhibit herpes simplex virus replication. At a relatively nontoxic conentration (50 microgram/ml), ara-T reduced herpes simplex virus yields by 4 to 5 log10. Ara-T was also effective in inhibiting the replication of varicellazoster virus (VZV) in vitro in human embryo fibroblasts, completely preventing VZV-specific cytopathic effects. The inhibition of VZV was reversible upon drug removal at 48 h after addition but was not reversible after 5 days of treatment. ara-T also reduced cell-free virus infectivity and the plaque-forming cell yield of VZV. Compared with the untreated controls, which demonstrated a 1-log10 increase over input plaque-forming cells at 24 h after infection, 50 microgram of ara-T per ml resulted in a 1-log10 decrease. In contrast to herpes simplex virus and VZV, cytomegalovirus replication was relatively resistant to ara-T. Neither cytopathic effects nor the incorporation of [3H]thymidine into acid-insoluble material in cytomegalovirus-infected cells was markedly affected. Analysis of the newly synthesized labeled DNA by CsCl buoyant density determinations indicated that the same relative proportions of cell and virus DNA were synthesized with or without added drug. Interpretation of these results with regard to virus-induced deoxypyrimidine kinase is discussed.     

Mathematical analysis demonstrates that interferons-beta and -gamma interact in a multiplicative manner to disrupt herpes simplex virus replication

Several studies suggest that the innate interferons (IFNs), IFN-alpha and IFN-beta, can act in concert with IFN-gamma to synergistically inhibit the replication of cytomegalovirus and herpes simplex virus type 1 (HSV-1). The significance of this observation is not yet agreed upon in large part because the nature and magnitude of the interaction between IFN-alpha/beta and IFN-gamma is not well defined. In the current study, we resolve this issue by demonstrating three points. First, the hyperbolic tangent function, tanh (x), can be used to describe the individual effects of IFN-beta or IFN-gamma on HSV-1 replication over a 320,000-fold range of IFN concentration. Second, pharmacological methods prove that IFN-beta and IFN-gamma interact in a greater-than-additive manner to inhibit HSV-1 replication. Finally, the potency with which combinations of IFN-beta and IFN-gamma inhibit HSV-1 replication is accurately predicted by multiplying the individual inhibitory effects of each cytokine. Thus, IFN-beta and IFN-gamma interact in a multiplicative manner. We infer that a primary antiviral function of IFN-gamma lies in its capacity to multiply the potency with which IFN-alpha/beta restricts HSV-1 replication in vivo. This hypothesis has important ramifications for understanding how T lymphocyte-secreted cytokines such as IFN-gamma can force herpesviruses into a latent state without destroying the neurons or leukocytes that continue to harbor these viral infections for the lifetime of the host.     

Novobiocin and coumermycin A1 inhibit viral replication and the reactivation of herpes simplex virus type 1 from the trigeminal ganglia of latently infected mice.

Herpes simplex virus type 1 was reactivated from the trigeminal ganglia of latently infected mice in a quantitative and time-dependent manner. Novobiocin and coumermycin A1 reversibly inhibited the reactivation of herpes simplex virus type 1. They did not inhibit viral replication in permissive cells (CV-1) but did inhibit replication in cells of neuronal origin (C1300) and acutely infected trigeminal ganglia.     

Herpes simplex virus DNA synthesis in a partially purified soluble extract from infected cells.

HEp-2 cells were infected with herpes simplex virus type 1 and extracted with 0.25% Triton X-100 and 0.1 M NaCl. The extract was sedimented on sucrose gradients, and the fractions containing the endogenous DNA polymerizing activity (replication complex) were collected. The properties of the replication complex were partially characterized. Under optimal conditions 375 pmol of dTMP per micrograms of DNA was incorporated, which corresponds to about 50% replication of preexisting viral DNA. The replication complex was shown to contain only DNA of viral origin by its density in CsCl. By using specific assays for DNA polymerases alpha, beta, gamma, and herpes simplex virus, we found that only the viral DNA polymerase copurified with the replication complex.     

Structure of replicating herpes simplex virus DNA.

We have investigated the molecular anatomy of the herpes simplex virus replicative intermediates by cleavage with the restriction endonuclease BglII. We find that in populations of multiply infected cells, pulse-labeled replicating herpes simplex virus DNA contains at least two and probably all four sequence isomers. Also, it contains no detectable termini. In pulse-chase experiments, we show that endless replicative intermediates are the precursors to virion DNA and that maturation is a relatively slow process. The results are discussed in terms of their significance to possible models of herpes simplex virus DNA replication.     

Role of protein-protein interactions during herpes simplex virus type 1 recombination-dependent replication

Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.     

Anti-herpes simplex virus effect of a seed extract from the tropical plant Licania tomentosa (Benth.) Fritsch (Chrysobalanaceae)

Incubation of acyclovir-resistant herpes simplex virus type 1 (ACVr-HSV1), during infection of the HEp-2 cell culture, with an extract prepared from the seeds of Licania tomentosa (Benth.) Fritsch (Chrysobalanaceae) species impaired the productive replication of this virus in a concentration-dependent manner. The extract was able to inhibit extracellular virus (virucidal effect) and also interfered with a very early event of cell infection, at a non-cytotoxic concentration.     

Ectopic expression of gamma interferon in the eye protects transgenic mice from intraocular herpes simplex virus type 1 infections.

Transgenic (rho gamma) mice provide a model for studying the influence of gamma interferon (IFN-gamma) produced in the eye on ocular and cerebral viral infection. To establish this model, we injected BALB/c- and C57BL/6-derived transgenic and nontransgenic mice of different ages intravitreally with herpes simplex virus type 1 (HSV-1) strain F. Eye and brain tissues of these mice were assessed for pathological and immunocytochemical changes. HSV-1 infection induced severe retinitis of the injected eyes and infection of the brain in all mice. In transgenic mice inoculated with HSV-1, the left, nontreated eyes were protected from retinitis, whereas nontransgenic mice developed bilateral retinitis. Additional intravitreal injection of IFN-gamma with the virus protected the noninoculated eyes of nontransgenic mice. Three-week-old nontransgenic mice died from HSV-1 infection, whereas transgenic mice of the same age and nontransgenic mice intravitreally treated with IFN-gamma survived. Ocular IFN-gamma production increased the extent of inflammation in transgenic mice but did not have a significant influence on the growth of HSV-1 until day 3 after inoculation and did not influence the neuroinvasion of this virus. Thus, the effects of IFN-gamma were not caused by an early block of viral replication. Possible mechanisms of IFN-gamma action include activation of the immune response, alteration of the properties of the virus, and direct protection of neurons.     

Rolling circle DNA replication by extracts of herpes simplex virus type 1-infected human cells.

Whole-cell extracts of herpes simplex virus type 1-infected human cells (293 cells) can promote the rolling circle replication of circular duplex DNA molecules. The products of the reaction are longer than monomer unit length and are the result of semiconservative DNA replication by the following criteria: (i) resistance to DpnI and susceptibility to MboI restriction enzymes, (ii) shift in density on a CsCl gradient of the products synthesized in the presence of bromo-dUTP to a position on the gradient consistent with those of molecules composed mainly of one parental DNA strand and one newly synthesized DNA strand, and (iii) the appearance in the electron microscope of molecules consisting of duplex circles with multiunit linear appendages, a characteristic of a rolling circle mode of DNA replication. The reaction requires ATP and is dependent on herpes simplex virus type 1-encoded DNA polymerase.