Herpes Simplex Virus Type 1 Strain Research Articles

In Vitro Synergism of Trifluorothymidine and Ganciclovir against HSV-1

To determine whether trifluorothymidine (TFT) and ganciclovir (GCV) are synergistic against herpes simplex virus type 1 (HSV-1). TFT and GCV activity against 12 strains of HSV-1 (including an acyclovir-resistant strain) was measured by plaque-forming unit (PFU) inhibition. Cellular toxicity was assessed with an MTT dye reduction assay. Synergism was determined by calculating fractional inhibitory concentration (FIC indices) based on PFU reduction. Concentrations of TFT resulting in 50% inhibition of PFUs (IC(50)) of acyclovir-susceptible HSV-1 strains ranged from 3.07 ± 0.36 to 12.52 ± 0.61 μM. GCV IC(50) values ranged from 0.40 ± 0.02 to 1.59 ± 0.14 μM. IC(50) values of TFT and GCV against the acyclovir-resistant strain were 15.40 ± 3.17 and 93.00 ± 9.64 μM, respectively. Concentrations of TFT or GCV resulting in 50% cell cytotoxicity (CC(50)) were 0.99 ± 0.01 and 92.91 ± 8.92 μM, respectively. TFT and GCV combined (10:1) were 10 times more potent against all acyclovir-susceptible HSV-1 strains. For 8 of 12 HSV-1 strains, the IC(50) of TFT and GCV combined was lower than the CC(50) of either drug. For acyclovir-susceptible HSV-1 strains, TFT and GCV combined generated a FIC index of <0.5, suggesting strong synergism between the two drugs. The FIC value for TFT and GCV combined against the acyclovir-resistant HSV-1 strain was 0.84, indicating nonantagonism. TFT and GCV are synergistic against acyclovir-susceptible HSV-1 at concentrations significantly less toxic than if each antiviral were used as a sole agent.

Antiviral activities of aerial subsets of Artemisia species against Herpes Simplex virus type 1 (HSV1) in vitro

Abstract Background: Drug resistance to current anti-herpetic drugs has been increasingly reported. Therefore, there is a need for finding new antiviral agents, in particular from natural sources. Objective: In the present study, antiviral activity of subset extracts obtained from aerial parts of Artemisia including A. incana, A. chamaemelifolia, A. campesteris, A. fragrans, A. annua, A. vulgaris, and A. persica were investigated against Herpes Simplex type I (HSV1). Methods: Different concentrations of extracts (400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 μg/mL) were obtained from subset of each plant separately, and used against KOS strain of HSV1 in HeLa cells. After 24 hours incubation, tetrazolium dye (MTT), was added. The dye absorption by viable cells was measured and compared to the positive control (extract-untreated cells) and acyclovir (as anti-viral agent). Results: The extracts obtained from A. annua had the highest antiviral activity while those of A. chamaemelifolia showed the lowest activity. Conclusion: Subset extracts of A. annua may be an appropriate candidate for further development of anti HSV1 infection.

Drug-resistant herpes simplex virus in HIV infected patients

Herpes simplex virus type 2 (HSV2) infection is a major source of morbidity in human immunodeficiency virus (HIV)-infected patients, since reactivations - whether symptomatic or asymptomatic - are associated with increased HIV viral load and viral shedding. Acyclovir, valacyclovir and famcyclovir are indicated for the treatment of HSV2 in HIV patients. This class of drugs has been shown to enhance survival in HIV-infected individuals. However, with the emergence of drug-resistant strains of HSV2, the rates of resistance among HIV patients are almost ten-fold those in immunocompetent individuals, comparing 0.6% to 6%. These HSV2 infections tend to be more severe and to recur. More ominously, disease progression of HIV is promoted by concurrent infection with HSV2. Intravenous foscarnet and cidofovir may be used for acyclovir-resistant HSV; however, resistance to these drugs has been documented. Newer therapies such as the toll-like receptor agonist imiquimod and immunomodulating dipeptides offer promise for the treatment of HSV2 in HIV-infected individuals.

Antiviral activity of recombinant cyanovirin-N against HSV-1

In this study, a standard strain of HSV-1 (strain SM(44)) was used to investigate the antiviral activity of the recombinant Cyanovirin-N (CV-N) against Herpes simplex virus type 1 (HSV-1) in vitro and in vivo. Cytopathic effect (CPE) and MTT assays were used to evaluate the effect of CV-N on HSV-1 in Vero cells. The number of copies of HSV-DNA was detected by real-time fluorescence quantitative PCR (FQ-PCR). The results showed that CV-N had a low cytotoxicity on Vero cells with a CC(50) of 359.03 ± 0.56 μg/mL, and that it could not directly inactivate HSV-1 infectivity. CV-N not only reduced the CPE of HSV-1 when added before or after viral infection, with a 50% inhibitory concentration (IC(50)) with 2.26 and 30.16 μg/mL respectively, but it also decreased the copies of HSV-1 DNA in infected host cells. The encephalitis model for HSV-1 infection was conducted in Kunming mice, and treated with three dosages of CV-N (0.5, 5 & 10 mg/kg) which was administered intraperitoneally at 2h, 3d, 5d, 7d post infection. The duration for the appearance of symptoms of encephalitis and the survival days were recorded and brain tissue samples were obtained for pathological examination (HE staining). Compared with the untreated control group, in the 5mg/kg CV-N and 10mg/kg CV-N treated groups, the mice suffered light symptoms and the number of survival days were more than 9 d and 14 d respectively. HE staining also showed that in 5mg/kg CV-N and 10mg/kg CV-N treated groups, the brain cells did not show visible changes, except for a slight inflammation. Our results demonstrated that CV-N has pronounced antiviral activity against HSV-1 both in vitro and in vivo, and it would be a promising new candidate for anti-HSV therapeutics.

Reduction of herpes simplex virus type-2 replication in cell cultures and in rodent models with peptide-conjugated morpholino oligomers

Genital herpes, caused by herpes simplex virus type-2 (HSV-2), is a recurrent, lifelong disease affecting tens of millions of people in the USA alone. HSV-2 can be treated therapeutically with acyclovir (ACV) and its derivatives; however, no treatment can prevent HSV reactivation. Novel topical anti-HSV microbicides are much needed to reduce HSV-2 transmission and to treat primary or reactivated infections, especially for ACV-resistant strains. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are single-stranded DNA analogues that enter cells readily and can reduce target gene expression through steric blockage of complementary messenger RNA (mRNA). We investigated the antiviral activities of PPMOs targeted to the translation start-site regions of the mRNA for two HSV-2 immediate early genes, immediate early protein (ICP)0 and ICP27, and two early genes, unique long gene (UL)30 and UL39. In cell cultures, PPMOs targeting ICP0 or ICP27 mRNA were found to be highly effective against two strains of HSV-2, one of which was ACV-resistant. In vivo, daily topical applications of up to 1 mM ICP27 PPMO caused no gross or microscopic damage to the genital tract of uninfected BALB/c mice or cotton rats. Cotton rats receiving topical application of ICP27 PPMO 24 h after HSV-2 inoculation showed a reduction in genital lesions and a 37.5% reduction in mortality at 14 days post-infection. Mice receiving topical application of 100 μM of an ICP27 and ICP0 PPMO combination before HSV-2 inoculation had no detectable viral replication in the genital tract at 3-5 days post-infection. These results demonstrate that topically applied PPMOs hold promise as candidate antiviral microbicides against HSV-2 genital infection.

Natural zoonotic infections of two marmosets and one domestic rabbit with herpes simplex virus type 1 did not reveal a correlation with a certain gG-, gI- or gE genotype

Infections with herpes simplex virus type 1 (HSV-1) are not restricted to humans but infrequently may be transmitted to certain animal species, in some cases resulting in severe disease, including encephalitis and death. Recent studies demonstrate that human-derived HSV-1 field isolates can be typed according to their gG-gI- and gE gene sequences. We investigated whether HSV-1 infections of animals were predominantly caused by a certain genotype. Isolates derived from two marmosets and one domestic rabbit, however, revealed different genotypes. Despite the very limited number of investigated animal-derived HSV-1 strains, this result does not point towards the existence of certain HSV-1 genotypes with a higher potential of being transmitted to animals.

Development of an oncolytic herpes simplex virus using a tumor-specific HIF-responsive promoter

We exploited the differential activation of hypoxia-inducible factor (HIF)-dependent gene expression in tumors versus normal tissue for the design of a targeted oncolytic Herpes simplex virus type-1 (HSV-1). A gene that is essential for viral replication, ICP4, was placed under the regulation of a HIF-responsive promoter and then introduced into the thymidine kinase locus (UL23) of HSV d120 which contains partial deletions in the two endogenous ICP4 genes. Recombinant HIF-HSV were isolated and their derivation from d120 was verified by expression of a truncated, nonfunctional form of ICP4 protein. Disruption of the UL23 locus was confirmed by loss of thymidine kinase expression and resistance to acyclovir. Unexpectedly, HIF-HSV expressed ICP4 and induced tumor cell lysis at similar levels under normoxia and hypoxia. The lack of HIF-dependent ICP4 transgene expression by HIF-HSV was due to two factors that have not previously been reported- reversion of the ICP4 gene region to its wild-type configuration and increased HIF-transcriptional activity under normoxia when cells were infected with any strain of HSV-1. The findings that an oncolytic HSV-1 is genetically unstable and can activate a tumor-related promoter in a non-specific manner have important implications for any proposed use of this virus in cancer therapy.

Herpes simplex virus type 1 strain KOS carries a defective US9 and a mutated US8A gene

The membrane protein encoded by the US9 gene of alphaherpesviruses plays an important role during virion assembly and transport in neurons. Here, we demonstrate that in herpes simplex virus type 1 (HSV-1) strain KOS, due to base substitutions, the predicted TATA-box of US9 is mutated, and a premature stop is present at codon 58 of US9, which contains 91 codons in other HSV-1 strains. The TATA-box mutation also removes the native stop codon of the adjacent US8A gene, leading to extension of the coding region from 160 to 191 codons. Northern blot analyses revealed reduced transcription of US9 in cells infected with HSV-1 KOS. Moreover, a US9-specific antiserum did not detect any gene products in Western blot and immunofluorescence analyses of KOS-infected cells, indicating that the truncated protein is not stable. In contrast, Western blot reactions of a pUS8A-specific antiserum confirmed enlargement of this protein in HSV-1 KOS.

Immunization with Different Viral Antigens Alters the Pattern of T Cell Exhaustion and Latency in Herpes Simplex Virus Type 1-Infected Mice

We have shown previously that immunization with herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) exacerbated corneal scarring (CS) in ocularly infected mice. In this study, we investigated whether higher levels of CS were correlated with higher levels of latency and T cell exhaustion in gK-immunized mice. BALB/c mice were vaccinated with baculovirus-expressed gK or gD or mock immunized. Twenty-one days after the third immunization, mice were ocularly infected with 2 × 10(4) PFU/eye of virulent HSV-1 strain McKrae. On day 5 postinfection, virus replication in the eye was measured, and on day 30 postinfection, infiltration of the trigeminal ganglia (TG) by CD4, CD8, programmed death 1 (PD-1), and T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was monitored by immunohistochemistry and quantitative real-time PCR (qRT-PCR). This study demonstrated that higher levels of CS were correlated with higher levels of latency, and this was associated with the presence of significantly higher numbers of CD4(+)PD-1(+) and CD8(+)PD-1(+) cells in the TG of the gK-immunized group than in both the gD- and mock-immunized groups. Levels of exhaustion associated with Tim-3 were the same among gK- and mock-vaccinated groups but higher than levels in the gD-vaccinated group. In this study, we have shown for the first time that both PD-1 and Tim-3 contribute to T cell exhaustion and an increase of latency in the TG of latently infected mice.

Interferon regulatory factor 3-dependent pathways are critical for control of herpes simplex virus type 1 central nervous system infection.

The initiation of the immune response at the cellular level relies on specific recognition molecules to rapidly signal viral infection via interferon (IFN) regulatory factor 3 (IRF-3)-dependent pathways. The absence of IRF-3 would be expected to render such pathways inoperative and thereby significantly affect viral infection. Unexpectedly, a previous study found no significant change in herpes simplex virus (HSV) pathogenesis in IRF-3(-/-) mice following intravenous HSV type 1 (HSV-1) challenge (K. Honda, H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, Y. Ohba, A. Takaoka, N. Yoshida, and T. Taniguchi, Nature 434:772-777, 2005). In contrast, the present study demonstrated that IRF-3(-/-) mice are significantly more susceptible to HSV infection via the corneal and intracranial routes. Following corneal infection with 2 x 10(6) PFU of HSV-1 strain McKrae, 50% of wild-type mice survived, compared to 10% of IRF-3-deficient mice. Significantly increased viral replication and inflammatory cytokine production were observed in brain tissues of IRF-3(-/-) mice compared to control mice, with a concomitant deficit in production of both IFN-beta and IFN-alpha. These data demonstrate a critical role for IRF-3 in control of central nervous system infection following HSV-1 challenge. Furthermore, this work underscores the necessity to evaluate multiple routes of infection and animal models in order to fully determine the role of host resistance factors in pathogenesis.

Interferon-λ in immunocompetent individuals with a history of recurrent herpes labialis

Herpes labialis (HL) is the most common manifestation of recurrent oral herpes simplex virus type-1 (HSV-1) infection. Between 20% and 40% of the population is affected by recurrent HL. The biological basis for the difference between HSV-1-infected individuals who do and who do not suffer recurrences, has long been investigated. Interferon (IFN)-alpha and IFN-lambda are essential for antiviral immunity, but the precise role of IFN-lambda in vivo is not yet well understood. Healthy immunocompetent patients with or without a history of recurrent HL were recruited from the Policlinico of the University of Rome Tor Vergata (Rome, Italy), and HSV-1-seronegative individuals were recruited from the Department of Experimental Medicine of the University of Rome Tor Vergata, between July 2007 and December 2008. Participants were interviewed by medically trained investigators and underwent a blood test. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood of patients and stimulated in vitro with intact HSV-1 strain F1 (1 plaque-forming unit/cell). PBMC supernatants were assayed for IFN-alpha, IFN-gamma and IFN-lambda production by ELISA at 24 and 48 h after viral challenge. PBMC from patients with a history of recurrent HL produced markedly lower levels of IFN-lambda and marginally lower levels of IFN-alpha and IFN-gamma than those from the history-negative HSV-1-seropositive controls. Among individuals with HL recurrences, those with more frequent and severe manifestations showed a significant trend towards lower levels of IFN-lambda production. A reduced IFN-lambda response might correlate with the development of recurrent HSV-1 infection in immunocompetent individuals. Testing for IFN-lambda response might be useful to predict individual patterns of antiviral response, contributing to more successful therapeutic or prophylactic interventions.

Microsatellite analysis of HSV-1 isolates: From oropharynx reactivation toward lung infection in patients undergoing mechanical ventilation

Background According to recent reports, herpes simplex virus type 1 (HSV-1) induces bronchopneumonitis (BPn) in immunocompetent patients undergoing prolonged mechanical ventilation (MV), whose respiratory functions deteriorate with a poor outcome. HSV-1 BPn is associated with HSV symptomatic or symptomless reactivation in the oropharynx. Objectives We sought to systematically and genetically characterize HSV-1 strains isolated from immunocompetent patients receiving prolonged MV and to characterize the genetic relationship of strains sequentially isolated from oropharyngeal samples (OPS) and broncho-alveolar liquids (BAL) to determine the natural course of HSV BPn. Study design In this molecular epidemiological study, microsatellite technology was used to determine genetic relationships between 211 HSV-1 strains isolated from OPS and/or BAL from 106 patients receiving MV. Results Microsatellite haplotypes of HSV-1 strains sequentially isolated from the same individual were identical, and HSV-1 isolates from the lung were genetically indistinguishable from strains isolated from the oral cavity. Each patient was characterized by their own HSV-1 microsatellite haplotype, and no nosocomial transmission of strains between patients was observed. Conclusion Our results demonstrate that, in patients who receive MV, the HSV-1 pulmonary infection results from the reactivation of genetically related HSV-1 in the oropharynx, which progressively infects the lower respiratory tract.

Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses

Abstract The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6–99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.

Phenoxazine Derivatives Suppress the Infections Caused by Herpes Simplex Virus Type-1 and Herpes Simplex Virus Type-2 Intravaginally Inoculated Into Mice

We examined the in vivo antiviral activities of 2-amino-4,4α-dihydro-4α-7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4α-dihydro-4α-8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-aminophenoxazine-3-one (Phx-3) against herpes viruses. The virus yield three days after administration, changes in the 6-degree’s lesion scores, and the morbidity were assessed after herpes simplex virus type-1 (HSV-1) [acyclovir (ACV)-sensitive KOS strain or ACV-resistant A4-3 strain] or HSV-2 (ACV-sensitive UW 268 strain) was inoculated intravaginally to mice with administration of Phx-1, Phx-2, Phx-3, or ACV (0.2 mg per administration, 3 times daily) for 8 days starting from 1 day before virus inoculation to 7 days after infection. Phx-1, Phx-2, and Phx-3 extensively suppressed the virus yield of HSV-1. Only Phx-2 exerted moderate inhibitory effects against HSV-2 in mice. The lesion scores, as clinical signs manifested by infection of the KOS strain of HSV-1, were extensively suppressed by intravaginal application of Phx-1, Phx-2, or Phx-3. The lesion scores in HSV-2–infected mice indicated moderate suppression, when Phx-1, Phx-2, or Phx-3 was applied. Without treatment by one of the compounds, none of the HSV-1– infected mice died, but all the HSV-2–infected ones did. However, by the administration of Phx-1, Phx-2, or Phx-3 fairly improved the survival rates of the HSV-2–infected mice. Phx-2 showed dose-dependent anti-HSV-2 efficacy when administered at doses of 0.2 and 1 mg per administration. The present in vivo data suggest that the Phx-1, Phx-2, and Phx-3 are attractive candidates for agents to prevent both replication of HSV and aggravation of lesions caused by these viruses.

Identification of virus resistant tumor cell subpopulations in three-dimensional uveal melanoma cultures

To better understand melanoma resistance to herpes simplex virus type 1 (HSV-1)-mediated oncolysis, traditional two-dimensional (2D) cultures and extracellular matrix (ECM) containing three-dimensional (3D) cultures of OCM1 and C918 uveal melanoma cells were infected with an HSV-1 strain that expresses the green fluorescent protein (GFP) marker during replication. Although 2D cultures were completely destroyed within a few days of HSV-1 inoculation, viable GFP-negative tumor cells remained detectable in 3D cultures for several weeks. Tumor cells with increased resistance to HSV-1 included cells that formed vasculogenic mimicry patterns and multicellular spheroids and cells that invaded Matrigel individually. Mechanisms of tumor resistance against HSV-1 in the 3D environment included impaired virus spread in the ECM and ECM-mediated inhibition of viral replication after viral entry into tumor cells. Observations also suggested that HSV-1 established quiescent infection in some tumor cells present in multicellular spheroids and that this could revert to productive viral infection when the tumor growth pattern changed. These findings indicate that 3D tumor cell cultures can be used to identify distinct tumor cell populations with increased resistance to HSV-1 and to explore mechanisms of ECM-mediated tumor resistance to oncolytic virotherapy.

Acyclovir Susceptibility and Genetic Characteristics of Sequential Herpes Simplex Virus Type 1 Corneal Isolates from Patients with Recurrent Herpetic Keratitis

The incidence and clinical significance of herpes simplex virus type 1 (HSV-1) acyclovir resistance were determined in patients with recurrent herpetic keratitis (RHK). Sequential corneal isolates (n = 39) from 15 immunocompetent patients with RHK were assayed for acyclovir susceptibility and genotyped by analyzing the hypervariable regions of the HSV-1 genes US1 and US12. The thymidine kinase (TK) gene of each isolate was sequenced, and the proportion of acyclovir-resistant viruses within isolates was determined. Uniform acyclovir-resistant or acyclovir-sensitive sequential isolates were identified in 4 and 2 patients, respectively. Notably, the acyclovir susceptibility of sequential isolates changed from acyclovir sensitive to acyclovir resistant (5 patients) or from acyclovir resistant to acyclovir sensitive (3 patients). The acyclovir-resistant phenotype of the isolates correlated with the patient's unresponsiveness to acyclovir therapy. Combined analyses of the TK gene and genotype of sequential isolates showed that acyclovir-sensitive isolates contained multiple acyclovir-resistant variants of the same virus and that an identical acyclovir-resistant HSV-1 strain reappeared in the patient's cornea during RHK episodes. Corneal HSV-1 isolates are mixtures of acyclovir-sensitive and acyclovir-resistant viruses that share the same genotype but have different TK sequences. Recovery of the same acyclovir-resistant virus during consecutive herpetic keratitis episodes suggests that acyclovir-resistant HSV-1 establishes latency and reactivates intermittently to cause acyclovir-refractory RHK.

Construction of, and T-helper (Th)1/Th2 immune responses to, a herpes simplex virus type 2 glycoprotein D-cytotoxic T-lymphocyte epitope DNA vaccine

Recombinant cytotoxic T lymphocyte (CTL) epitope DNA vaccines offer an attractive approach for the induction of robust cellular and humoral immune responses directed against human pathogen target antigens. To construct a pVAX-gD-CTL vector expressing the glycoprotein (g)D and gB CTL epitopes from herpes simplex virus type 2 (HSV2) and evaluate it in mice for immunogenicity and protective efficacy against intraperitoneal challenge with the HSV2 strain Sav. The gD gene transcript and gB CTL epitope were inserted into the pVAX vector to obtain the recombinant plasmid pVAX-gD-CTL. An in vitro study was then conducted to detect the expression of gD by immunocytochemistry and western blotting. BALB/c mice were immunized with this DNA vaccine, then the CTL activity and expression of anti-HSV2 gD IgG, interferon-gamma and interleukin-4 by lactate dehydrogenase release assay and ELISA, respectively. The protection given to the mice was assayed by a fatal dose of virus. The pVAX-gD-CTL vector was successfully constructed and could express gD in COS-7 cells. Immunogenicity of gD, anti-HSV2 gD neutralizing serum IgG antibody and robust Th1-polarized immune responses were found. Furthermore, mice were prophylactically protected from challenge with a high dose of HSV2. In summary, pVAX-gD-CTL vector was successfully used to elicit potent Th1-like cellular and humoral immune responses that were protective against HSV2 disease in mice.

Inhibition of Herpesvirus Replication by 5-Substituted 4′-Thiopyrimidine Nucleosides

A series of 4'-thionucleosides were synthesized and evaluated for activities against orthopoxviruses and herpesviruses. We reported previously that one analog, 5-iodo-4'-thio-2'-deoxyuridine (4'-thioIDU), exhibits good activity both in vitro and in vivo against two orthopoxviruses. This compound also has good activity in cell culture against many of the herpesviruses. It inhibited the replication of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus with 50% effective concentrations (EC(50)s) of 0.1, 0.5, and 2 microM, respectively. It also inhibited the replication of human cytomegalovirus (HCMV) with an EC(50) of 5.9 microM but did not selectively inhibit Epstein-Barr virus, human herpesvirus 6, or human herpesvirus 8. While acyclovir-resistant strains of HSV-1 and HSV-2 were comparatively resistant to 4'-thioIDU, it retained modest activity (EC(50)s of 4 to 12 microM) against these strains. Some ganciclovir-resistant strains of HCMV also exhibited reduced susceptibilities to the compound, which appeared to be related to the specific mutations in the DNA polymerase, consistent with the observed incorporation of the compound into viral DNA. The activity of 4'-thioIDU was also evaluated using mice infected intranasally with the MS strain of HSV-2. Although there was no decrease in final mortality rates, the mean length of survival after inoculation increased significantly (P < 0.05) for all animals receiving 4'-thioIDU. The findings from the studies presented here suggest that 4'-thioIDU is a good inhibitor of some herpesviruses, as well as orthopoxviruses, and this class of compounds warrants further study as a therapy for infections with these viruses.

Two Small RNAs Encoded within the First 1.5 Kilobases of the Herpes Simplex Virus Type 1 Latency-Associated Transcript Can Inhibit Productive Infection and Cooperate To Inhibit Apoptosis

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected trigeminal ganglionic sensory neurons. Expression of the first 1.5 kb of LAT coding sequences is sufficient for the wild-type reactivation phenotype in small animal models of infection. The ability of the first 1.5 kb of LAT coding sequences to inhibit apoptosis is important for the latency-reactivation cycle. Several studies have also concluded that LAT inhibits productive infection. To date, a functional LAT protein has not been identified, suggesting that LAT is a regulatory RNA. Two small RNAs (sRNAs) were previously identified within the first 1.5 kb of LAT coding sequences. In this study, we demonstrated that both LAT sRNAs were expressed in the trigeminal ganglia of mice latently infected with an HSV-1 strain that expresses LAT but not when mice were infected with a LAT null mutant. LAT sRNA1 and sRNA2 cooperated to inhibit cold shock-induced apoptosis in mouse neuroblastoma cells. LAT sRNA1, but not LAT sRNA2, inhibited apoptosis less efficiently than both sRNAs. When rabbit skin cells were cotransfected with plasmids that express LAT sRNA1 and HSV-1 genomic DNA, the amount of infectious virus released was reduced approximately 3 logs. Although LAT sRNA2 was less effective at inhibiting virus production, it inhibited expression of infected cell protein 4 (ICP4). Neither LAT sRNA had an obvious effect on ICP0 expression. These studies suggested that expression of two LAT sRNAs plays a role in the latency-reactivation cycle by inhibiting apoptosis and productive infection.

One-step column purification of herpes simplex virus 1 helicase–primase subcomplex using C-terminally his-tagged UL5 subunit

A protocol for purification of the two-subunit complex of herpes simplex virus type 1 (HSV-1) helicase-primase by metal affinity chromatography is presented. In order to bind the enzyme complex consisting of UL5 and UL52 gene functions to the affinity column, the C-terminus of the UL5 gene of HSV-1 strain ANG was fused in-frame with a sequence encoding six histidines, resulting in a His6-tagged DNA helicase (UL5his) when expressed via recombinant baculovirus. In addition, hybridoma cell lines producing anti-UL5 IgG were generated for screening of DNA helicase expression. Initial purification trials revealed that the presence of low concentrations of imidazole in the wash buffers interfered with the binding of the helicase-primase subunit complex to the metal affinity resin. Alternative means, such as high salt, altered pH, and substitution of imidazole by histidine tetrapeptide (His4), were tested. From those, the addition of His4 in combination with an acidic pH turned out to be very efficient for the removal of protein contaminants from a Ni2+-NTA (nitrilotriacidic acid) affinity resin. By applying only one column step, the present protocol yields a helicase-primase preparation, which is suitable for inhibitor screening and further functional studies. The final preparation is free of interfering enzyme activities, and exerts each of the enzymatic functions described for a two subunit complex, i.e., DNA-dependent ATPase, DNA primase, and DNA helicase activities.