Inhibition of cell-associated herpes simplex virus type 2 glycoproteins by delta 9-tetrahydrocannabinol

This study was conducted to define the effect of micromolar concentrations of delta 9-tetrahydrocannabinol (delta 9-THC) on the biosynthesis and expression of herpes simplex virus type 2 (HSV2)-specified glycoproteins. Dose-related reductions in all species of virus glycoproteins were recorded by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [14C]glucosamine-labeled infected Vero cells treated with 10(-7) to 10(-5) M delta 9-THC. A drug dose-related depletion of the mature HSV2 major envelope glycoprotein complex (119-kDa average molecular weight), accompanied by accumulation of immature unglycosylated species, was demonstrated by two-dimensional SDS-PAGE in concert with Western immunoblotting or autoradiography. Light and electron microscopy immunoperoxidase staining revealed that delta 9-THC effected depletion of 119-kDa determinants from the infected cell surface. This depletion occurred concomitantly with accumulation of 119-kDa components at the perinucleus. However, the expression of 119-kDa glycoproteins on the virion envelope was not affected. These results indicate that delta 9-THC inhibits the synthesis, maturation, and cellular transport of HSV2-specified glycoproteins. Decreased expression of virus glycoproteins on the infected cell surface may affect host immune responsiveness to HSV2.     

Delta 9-tetrahydrocannabinol decreases alpha/beta interferon response to herpes simplex virus type 2 in the B6C3F1 mouse

This study was undertaken to determine the effect of delta 9-tetrahydrocannabinol (delta 9-THC) on polyinosinic:polycytidylic acid [poly(I):poly(C)]-induced, and on herpes simplex virus type 2 (HSV-2)-induced, alpha/beta interferon in the B6C3F1 mouse. Animals were administered delta 9-THC, or the diluent, intraperitoneally for 4 consecutive days or at various time intervals prior to administration of the interferon inducer. Poly(I):poly(C) or HSV-2 was injected intravenously on Day 4. Animals receiving poly(I):poly(C) and treated with delta 9-THC at doses ranging from 5 to 100 mg/kg exhibited significantly lower titers of interferon than mice given poly(I):poly(C) and the diluent. Diminished interferon titers occurred in HSV-2-infected animals treated with delta 9-THC in doses exceeding 15 mg/kg when compared to virus-infected animals given the diluent. This suppression of early interferon persisted through 24 hr.     

Use of immunostimulatory sequence-containing oligonucleotides as topical therapy for genital herpes simplex virus type 2 infection

Synthetic oligonucleotides containing CpG motifs in specific sequence contexts have been shown to induce potent immune responses. We have evaluated mucosal administration of two immunostimulatory sequence (ISS)-containing phosphorothioate-stabilized oligonucleotides for antiherpetic efficacy in animal models. The ISS oligonucleotides, suspended in phosphate-buffered saline, were tested in mouse and guinea pig vaginal models of herpes simplex virus type 2 (HSV-2) infection. For comparison, groups of untreated, non-ISS oligonucleotide-treated, and acyclovir-treated animals also were monitored. The results indicated that vaginal epithelial application of ISS (up to 6 h after viral inoculation) with mice lethally challenged with HSV-2 delayed disease onset and reduced the number of animals that developed signs of disease (P = 0.003). ISS application significantly increased survival rates over those of controls (P = 0.0014). The ISS also impacted an established infection in the guinea pig model of HSV-2 disease. A single administration of ISS (21 days after viral inoculation) significantly reduced the frequency and severity of HSV-2 lesions compared to results with non-ISS oligonucleotide-treated and untreated guinea pigs (P < 0.01). HSV-2 is shed from the vaginal cavity of the guinea pig in the absence of lesions, similar to the case with humans. As an additional indication of ISS efficacy, the magnitude of viral shedding also was significantly reduced in ISS-treated animals (P < 0.001). These effects appeared to be immunologically mediated, since ISS had no direct effect on HSV-2 replication in vitro using standard plaque assays. These data suggest that ISS may be useful in the treatment and control of genital herpes in humans.     

Delta 9-tetrahydrocannabinol decreases cytotoxic T lymphocyte activity to herpes simplex virus type 1-infected cells.

The purpose of this study was to examine the effect of delta 9-tetrahydrocannabinol (delta 9-THC), the major psychoactive component of marijuana, on T lymphocyte functional competence against herpes simplex virus Type 1 (HSV1) infection. Spleen cells from C3H/HeJ (H-2k) mice primed with HSV1 and exposed to delta 9-THC were examined for anti-HSV1 cytolytic T lymphocyte (CTL) activity. Flow cytometry was used to determine whether delta 9-THC altered T cytotoxic (Lyt-2+) and T helper (L3T4+) lymphocyte numbers or cell ratios. Nomarski optics microscopy was used to determine whether effector lymphocytes from drug-treated mice were able to bind to virally infected L929 (H-2k) target cells. Cytotoxicity assays demonstrated that CTL from mice exposed to delta 9-THC were deficient in anti-HSV1 cytolytic activity. delta 9-THC in vivo treatment had little effect on the number of T lymphocytes expressing the Lyt-2 or L3T4 antigens. Nomarski optics microscopy revealed that the CTL from the drug-treated mice were able to bind specifically to the HSV1-infected targets. However, delta 9-THC in vivo exposure affected CTL cytoplasmic polarization toward the virus-infected target cell. CTL granule reorientation toward the effector cell-target cell interface following cell conjugation occurred at a lower frequency in co-cultures containing CTL from drug-treated mice. These results suggest that delta 9-THC elicits dysfunction in CTL by altering effector cell-target cell postconjugation events.     

The cotton rat provides a novel model to study genital herpes infection and to evaluate preventive strategies

Prevention of genital herpes and other sexually transmitted infections (STI) is a critical health priority because of the overwhelming impact on women and infants and the epidemiological association with human immunodeficiency virus (HIV)/AIDS. Small animal models are essential for evaluating strategies for prevention or treatment of STI. Neither the murine nor the guinea pig model of genital herpes fully recapitulates human disease. We demonstrate that herpes simplex virus type 2 (HSV-2) readily infects inbred cotton rats (Sigmodon hispidus). Consistent infection does not require pretreatment with medroxyprogesterone, and primary disease resembles that observed in humans. The animals develop genital lesions and fully recover. During primary infection, viral DNA is also detected in liver, lungs, brain, and kidneys. Clinical self-limited recurrences occur spontaneously but may also be induced by dexamethasone. Pretreatment of cotton rats with PRO 2000 gel, a candidate vaginal microbicide being evaluated in clinical trials to prevent HSV and HIV, protects cotton rats from HSV. Together, these studies suggest that the cotton rat may provide an excellent model to study genital herpes and to evaluate preventive strategies.     

Complement-requiring neutralizing antibody in guinea pigs with primary and recurrent genital herpes

Guinea pigs inoculated intravaginally with herpes simplex virus type 2 (HSV-2) strain 1868 produced a serum complement-requiring neutralizing (CRN) antibody during primary acute infection, i.e., 10 days postinoculation. The CRN antibody titers in the guinea pig sera decreased to less than 1:10 after heating at 56 degrees C for 30 min. It was found that 32 units of complement were necessary to obtain a satisfactory HSV-2 neutralizing antibody titer. Nonheated sera significantly reduced virus infectivity titers when mixed with 3.5 log10 PFU of HSV-2 and incubated at 37 degrees C for 20 to 60 min (P less than 0.001), whereas the same sera after heating at 56 degrees C for 30 min showed no inhibitory effect. Only 27.3% of infected guinea pigs had low serum non-CRN antibody titers ranging from 1:20 to 1:40. In addition, no evidence of increase in CRN antibody titers was noted during spontaneous recurrent genital herpes infection.     

Selective ablation of virion host shutoff protein RNase activity attenuates herpes simplex virus 2 in mice

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) has endoribonuclease activity and rapidly reduces protein synthesis in infected cells through mRNA degradation. Herpes simplex virus 1 (HSV-1) and HSV-2 vhs mutants are highly attenuated in vivo, but replication and virulence are largely restored to HSV-2 vhs mutants in the absence of a type I interferon (IFN) response. The role of vhs in pathogenesis and the hindrance of the type I IFN response have classically been examined with viruses that completely lack vhs or express a truncated vhs protein. To determine whether RNase activity is the principal mechanism of vhs-mediated type I IFN resistance and virulence, we constructed a HSV-2 point mutant that synthesizes full-length vhs protein lacking RNase activity (RNase(-) virus). Wild-type and mutant HSV-2 vhs proteins coimmunoprecipitated with VP16 and VP22. vhs protein bearing the point mutation was packaged into the virion as efficiently as the wild-type vhs protein. Like a mutant encoding truncated vhs, the RNase(-) virus showed IFN-dependent replication that was restricted compared with that of the wild-type virus. The RNase(-) virus was highly attenuated in wild-type mice infected intravaginally, with reduced mucosal replication, disease severity, and spread to the nervous system comparable to those of the vhs truncation mutant. Surprisingly, in alpha/beta interferon (IFN-alpha/beta) receptor knockout mice, the vhs RNase mutant was more attenuated than the vhs truncation mutant in terms of disease severity and virus titer in vaginal swabs and central nervous system samples, suggesting that non-enzymatically active vhs protein interferes with efficient virus replication. Our results indicate that vhs enzymatic activity plays a complex role in vhs-mediated type I IFN resistance during HSV-2 infection.     

Production of monoclonal antibodies against nucleocapsid proteins of herpes simplex virus types 1 and 2.

We prepared mouse hybrid cell lines which produced antibodies against herpes simplex virus type 1 and 2 nucleocapsids. Cell lines 1D4 and 3E1, respectively, secreted immunoglobulin G1 herpes simplex virus type 1 and immunoglobulin G1 herpes simplex virus type 2 antibodies which immunoprecipitated proteins designated p40 and p45 from homologous nucleocapsid preparations but precipitated no proteins from heterologous preparations. In contrast, guinea pig antisera prepared against either herpes simplex virus type 1 or 2 p40 precipitated p40 and p45 from both homologous and heterologous preparations. These findings suggest that p40 and p45 possess similar antigenic determinants and that the monoclonal antibodies that were tested reacted preferentially with the homologous determinants.     

Thymidine kinase-deficient herpes simplex virus type 2 genital infection in guinea pigs.

In guinea pigs, thymidine kinase-producing strains of herpes simplex virus type 2 replicated to high titer in the vagina and spinal cord, and animals developed severe clinical disease. Infection with thymidine kinase-deficient virus resulted in similar vaginal virus titers; however, animals exhibited little or no clinical illness and only low titers of virus were detected in spinal cord homogenate cultures. Neural and extraneural latent infection as well as recurrent infection were noted in animals inoculated with either thymidine kinase-producing or -deficient viruses. These data suggest that neural pathways are important in the pathogenesis of genital herpes and that virus-coded thymidine kinase may influence virulence but is not required for latency.     

Antiviral activity and dose optimum of recombinant macrophage colony-stimulating factor on herpes simplex genitalis in guinea pigs.

The antiviral activity of recombinant human macrophage CSF (M-CSF) against genital herpes simplex virus type-2 (HSV-2) infection in guinea pigs was investigated. M-CSF stimulates proliferation of human and guinea pig peripheral blood monocytes, specifically the plastic adherent esterase-positive mononuclear cells. When anti-HSV-2 activity of M-CSF was evaluated in guinea pigs by 6 daily injection (s.c.) of M-CSF at various doses (5 x 10(5) to 7 x 10(7) U/kg), we found 2 x 10(6) U/kg to be the optimum dose for protective efficacy against primary HSV-2 infection. Either at a lethal, 5 x 10(5) pfu, or sublethal 5 x 10(4) pfu of virus challenge, animals treated with the optimum regimen of M-CSF exhibited lower herpetic lesion scores (p less than 0.005), and lower mortality (p less than 0.025) than animals in placebo group. M-CSF treatment increased the HSV-infected cell killing activities of plastic-adherent mononuclear cells, indicating that in vivo administration of M-CSF may activate the antiviral effects of guinea pig macrophages that may play a role in protection against severity and mortality of herpetic disease.     

Loss of the type I interferon pathway increases vulnerability of mice to genital herpes simplex virus 2 infection

The mouse model of genital herpes relies on medoxyprogesterone treatment of female mice to render the vaginal lumen susceptible to inoculation with herpes simplex virus 2 (HSV-2). In the present study, we report that mice deficient in the A1 chain of the type I interferon receptor (CD118(-/-)) are susceptible to HSV-2 in the absence of medroxyprogesterone preconditioning. In the absence of hormone pretreatment, 2,000 PFU of a clinical isolate of HSV-2 was sufficient to establish a productive infection in the vagina of 75% ± 17% and in the spinal cord of 71% ± 14% of CD118(-/-) mice, whereas the same dose of HSV-2 replicated to detectable levels in only 13% ± 13% of vaginal samples and 0% of spinal cord samples from wild-type mice, as determined at day 5 postinfection. The susceptibility to HSV-2 infection in the CD118(-/-) mice was associated with a significant reduction in the infiltration of HSV-specific cytotoxic T lymphocytes into the vaginal tissue, the local production of gamma interferon (IFN-γ), and the expression of T cell-recruiting chemokines CCL5, CXCL9, and CXCL10. Collectively, the results underscore the significant contribution of type I IFNs in resistance to genital HSV-2 infection.     

Modification of the genital herpes infection in a guinea pig model, by prior immunization with bovine herpes mammillitis virus

Bovine herpes mammillitis virus has been shown to partially protect guinea pigs against primary genital herpes simplex virus type 2 infections, further confirming the immunologic cross-reactivity of these two viruses.     

Antigen-presenting liposomes are effective in treatment of recurrent herpes simplex virus genitalis in guinea pigs.

The therapeutic and immunologic effects of a liposome preparation containing both a macrophage activator, muramyl-tripeptide-phosphatidylethanolamine, and a recombinant antigen, glycoprotein D of herpes simplex virus type 1, have been investigated. This preparation was tested in vitro for the ability to stimulate peripheral blood lymphocytes and in vivo for the control of recurrent herpes genitalis in guinea pigs. Our results show that the liposome-antigen-adjuvant preparation is capable of enhancing antigen-specific lymphocyte stimulation, which may be related to the observed 75% suppression of the frequency and severity of reactivation of recurrent herpes simplex virus type 2 genitalis compared with that of placebo controls.     

Alterations in Glycosphingolipid Patterns in a Line of African Green Monkey Kidney Cells Infected with Herpesvirus

The major glycosphingolipids (GSLs) of a line of African green monkey kidney cells (BGM) were characterized as glucosylceramide, lactosylceramide, galactosyl-galactosyl-glucosylceramide, and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide. Neutral GSLs accounted for approximately 80% of the total GSLs isolated. The predominant gangliosides were N-acetylneuraminyl-galactosyl-glucosylceramide, N-acetylgalactosaminyl-N-acetylneuraminyl-galactosyl- glucosylceramide, and galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl -galactosyl-glucosylceramide. The incorporation of labeled galactose into GSLs was compared in mock-infected and herpes simplex virus type 1-infected BGM cells. Herpes simplex virus type 1 infection resulted in a three- to four-fold increase in galactose incorporation into glucosylceramide and a decrease in galactose incorporation into galactosyl-galactosyl-glucosylceramide and N-acetyl-galactosaminyl-galactosyl-galactosyl-glucosylceramide. The virus-induced alteration in the GSL labeling pattern occurred early in infection, before the release of infectious virus, and was not prevented by the presence of cytosine arabinoside. Treatment of uninfected BGM cells with cycloheximide resulted in alterations in the GSL pattern which were similar to those observed in herpes simplex virus type 1-infected cells. These observations suggest that an early virus function such as inhibition of host cell protein synthesis is responsible for the observed alterations of GSL metabolism. Experiments with a syncytium-producing strain of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus indicated that other herpes viruses altered GSL metabolism in a manner similar to herpes simplex virus type 1.     

Immunization with a dominant-negative recombinant Herpes Simplex Virus (HSV) type 1 protects against HSV-2 genital disease in guinea pigs

Background  

CJ9-gD is a novel dominant-negative recombinant herpes simplex virus type 1 (HSV-1) that is completely replication-defective, cannot establish detectable latent infection in vivo, and expresses high levels of the major HSV-1 antigen glycoprotein D immediately following infection. In the present study, CJ9-gD was evaluated as a vaccine against HSV-2 genital infection in guinea pigs.     

Prophylactic and therapeutic treatment with acyclovir of genital herpes in the guinea pig

The antiviral drug, acyclovir, was tested on experimentally infected guinea pigs with either of two herpes simplex virus type 1 (HSV-1) isolates following intravaginal inoculation. The drug was continuously infused subcutaneously utilizing an osmotic pump. Infusion was begun either prior to virus inoculation (prophylactic) or after virus inoculation at the time of first appearance of lesions (therapeutic). Prophylactic treatment markedly reduced the severity of the genital lesions, the appearance of acute neurologic sequellae, and the virus excretion in the genital tract of guinea pigs infected with either of the two strains tested. Therapeutic acyclovir treatment, however, did not decrease the incidence of acute neurologic sequellae with one of the two HSV-1 strains tested, nor did it reduce the severity of the genital lesions of either strain. These neurologic sequellae may be due to insufficient levels of ACV in the central nervous system as the concentration of ACV in the dorsal root ganglia was found to exceed that of the plasma, but only trace amounts of acyclovir were present in the brain and spinal cord. Continuous perfusion of ACV gave far higher tissue levels than intermittent injections. These findings suggest that prophylactic ACV is far more effective than therapeutic treatment for genital herpes in the guinea pig model.     

Terminally Repeated Sequences on a Herpesvirus Genome Are Deleted following Circularization but Are Reconstituted by Duplication during Cleavage and Packaging of Concatemeric DNA

The mechanisms underlying cleavage of herpesvirus genomes from replicative concatemers are unknown. Evidence from herpes simplex virus type 1 suggests that cleavage occurs by a nonduplicative process; however, additional evidence suggests that terminal repeats may also be duplicated during the cleavage process. This issue has been difficult to resolve due to the variable numbers of reiterated terminal repeats that the herpes simplex virus type 1 genome can contain. Guinea pig cytomegalovirus is a herpesvirus with a simple terminal repeat arrangement that defines two genome types. Type II genomes have a single copy of a 1-kb terminal repeat at both their left and right termini, whereas type I genomes have only one copy at their left termini and lack the repeat at their right termini. In a previous study, we constructed a recombinant guinea pig cytomegalovirus in which certain cis elements were disrupted such that only type II genomes were produced. Here we show that double repeats that are formed by circularization of infecting genomes are rapidly converted to single repeats, such that the junctions between genomes within replicative concatemers formed late in infection almost exclusively contain single copies of the terminal repeat. Therefore, for the recombinant virus, each cleavage event begins with a single repeat within a concatemer yet produces two repeats, one at each of the resulting termini, demonstrating that terminal repeat duplication occurs in conjunction with cleavage. For wild-type guinea pig cytomegalovirus, the formation of type I genomes further suggests that cleavage can also occur by a nonduplicative process and that duplicative and nonduplicative cleavage can occur concurrently. Other herpesviruses having terminal repeats, such as the herpes simplex viruses and human cytomegalovirus, may also utilize repeat duplication and deletion; however, the biological importance of these events remains unknown.     

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.     

Truncation of the carboxy-terminal 28 amino acids of glycoprotein B specified by herpes simplex virus type 1 mutant amb1511-7 causes extensive cell fusion.

Three amber mutations were introduced proximal to the syn3 locus of the herpes simplex virus type 1 glycoprotein B (gB) gene specifying gB derivatives lacking the carboxy-terminal 28, 49, or 64 amino acids. A complementation system that utilized gBs expressed in COS cells to complement gB-null virus K delta T was established. The 49- or 64-amino-acid-truncated gBs failed to complement gB-null virus K delta T, while the 28-amino-acid-truncated gB complemented K delta T efficiently. Mutant herpes simplex virus type 1 KOS (amb1511-7) specifying the 28-amino-acid-truncated gB fused Vero cells extensively.     

Herpes simplex virus type 2 UL24 gene is a virulence determinant in murine and guinea pig disease models

A herpes simplex virus type 2 (HSV-2) UL24 beta-glucuronidase (UL24-betagluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-betagluc yielded cytopathic effect with syncytium formation. UL24-betagluc replicated to wild-type viral titers in three different cell lines. UL24-betagluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD50) of the parental virus. Furthermore, few UL24-betagluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-betagluc at a dose equivalent to the LD50 of parental virus (approximately 5 x 10(3) PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-betagluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.