HSV-1 DNA Polymerase Research Articles

Bridging the Chemical Profiles and Biological Effects of Spathodea campanulata Extracts: A New Contribution on the Road from Natural Treasure to Pharmacy Shelves.

Spathodea campanulata is an important medicinal plant with traditional uses in the tropical zone. In the current work, we aimed to determine the chemical profiles and biological effects of extracts (methanolic and infusion (water)) from the leaves and stem bark of S. campanulata. The chemical components of the tested extracts were identified using LC-ESI-QTOF-MS. Biological effects were tested in terms of antioxidant (radical scavenging, reducing power, and metal chelating), enzyme inhibitory (cholinesterase, amylase, glucosidase, and tyrosinase), antineoplastic, and antiviral activities. Fifty-seven components were identified in the tested extracts, including iridoids, flavonoids, and phenolic acids as the main constituents. In general, the leaves-MeOH extract was the most active in the antioxidant assays (DPPH, ABTS, CUPRAC, FRAP, metal chelating, and phosphomolybdenum). Antineoplastic effects were tested in normal (VERO cell line) and cancer cell lines (FaDu, HeLa, and RKO). The leaf infusion, as well as the extracts obtained from stem bark, showed antineoplastic activity (CC50 119.03–222.07 µg/mL). Antiviral effects were tested against HHV-1 and CVB3, and the leaf methanolic extract (500 µg/mL) exerted antiviral activity towards HHV-1, inhibiting the viral-induced cytopathic effect and reducing the viral infectious titre by 5.11 log and viral load by 1.45 log. In addition, molecular docking was performed to understand the interactions between selected chemical components and viral targets (HSV-1 DNA polymerase, HSV-1 protease, and HSV-1 thymidine kinase). The results presented suggest that S. campanulata may be a bright spot in moving from natural sources to industrial applications, including novel drugs, cosmeceuticals, and nutraceuticals.

Herpesvirus DNA polymerase: Structures, functions, and mechanisms.

Herpesviruses comprise a family of DNA viruses that cause a variety of human and veterinary diseases. During productive infection, mammalian, avian, and reptilian herpesviruses replicate their genomes using a set of conserved viral proteins that include a two subunit DNA polymerase. This enzyme is both a model system for family B DNA polymerases and a target for inhibition by antiviral drugs. This chapter reviews the structure, function, and mechanisms of the polymerase of herpes simplex viruses 1 and 2 (HSV), with only occasional mention of polymerases of other herpesviruses such as human cytomegalovirus (HCMV). Antiviral polymerase inhibitors have had the most success against HSV and HCMV. Detailed structural information regarding HSV DNA polymerase is available, as is much functional information regarding the activities of the catalytic subunit (Pol), which include a DNA polymerization activity that can utilize both DNA and RNA primers, a 3'-5' exonuclease activity, and other activities in DNA synthesis and repair and in pathogenesis, including some remaining to be biochemically defined. Similarly, much is known regarding the accessory subunit, which both resembles and differs from sliding clamp processivity factors such as PCNA, and the interactions of this subunit with Pol and DNA. Both subunits contribute to replication fidelity (or lack thereof). The availability of both pharmacologic and genetic tools not only enabled the initial identification of Pol and the pol gene, but has also helped dissect their functions. Nevertheless, important questions remain for this long-studied enzyme, which is still an attractive target for new drug discovery.

Molecular Prediction for the common causative agents of infectious conjunctivitis among AL-Najaf AL-Ashraf governorate patients in spring and summer.

Aim of the study: is to predict the most common causative agents of infectious conjunctivitis among AL-Najaf AL-Ashraf governorate during spring and summer seasons.Methods : Multiplex PCR was used to amplify viral and bacterial genes related to pathogens suspected of causing infectious conjunctivitis, the samples were collected from patients using cotton eye swabs.Results: A total of 36 (10.29%) patients were chosen from 350 patients who attended private ophthalmic clinic in AL-Najaf AL-Ashraf governorate during spring and summer seasons. Two Cotton swabs were taken from each patient and kept frozen until the day of DNA extraction. Multiplex PCR was used to amplify genes specific for Adenovirus, Herpes Simplex Virus-1 and Herpes Simplex Virus-2, Cytomegalovirus, Varicella Zoster Virus and Chlamydia trachomitis plasmid. The multiplex PCR results found that only ten (27.77%) samples were positive for mixed infection of Adenovirus (140bp), HSV-1and HSV DNA polymerase (208, 211 bp) and only one (2.77%) sample was positive for HSV-1mixed with HSV DNA polymerase .

HSV-1 DNA polymerase 3'-5' exonuclease-deficient mutant D368A exhibits severely reduced viral DNA synthesis and polymerase expression.

Herpesviruses, including herpes simplex virus-1, encode and express a DNA polymerase that is required for replication of their dsDNA genomes. The catalytic subunit of this enzyme contains a 3'-5' exonuclease that is involved in proofreading during replication. Although certain mutations that severely impair exonuclease activity are not lethal to the virus, it was reported that virus containing the substitution of alanine for aspartate 368 (D368A), which ablates exonuclease activity, could not be recovered, raising the possibility that this activity is essential for viral replication. To investigate this issue, we produced virus containing this mutation (D368A Pol) using a complementing cell line. D368A Pol virus was unable to form plaques on non-complementing cells. Viral DNA synthesis and polymerase activity were severely inhibited in D368A-infected cells, as was expression of the enzyme, suggesting that effects on polymerase expression rather than on exonuclease activity per se largely explain the lethal phenotype of this mutation.

New simplex Type 1 lytic infection pyridine system derivative as a novel goal against Herpes

The pyridine system derivatives have been studied as antiviral drug new models against HIV infection using bioisosterism as a powerful tool. In the last decades, the number of this work, thienopyridine derivatives were evaluated as an HSV-1 inhibitor to since the elevated number of acyclovir resistance in immunocompromised patients could promote a continuous viral pool increase and disease recurrence. In these substances, thienopyridine derivative with radical nitrate in para position (109) was able to inhibit 99% of HSV-1 replication in Vero cells with EC 50 value of 100 pM, and SI value three times higher than acyclovir. In kinetic enzymatic assay this substance was also able to inhibit about 50% of HSV-1 DNA polymerase but in a non-competitive mechanism a new synergic system to control herpes infections. Preliminary in vivo model studies revealed that substance 109 is toxic when administered in 300 g/mL concentrations. In this work, we concluded that thienopyridine derivatives can be considered a promising new anti-HSV-1 drug and can be used for clinical testing.

Clinical efficacy of oral and topical acyclovir in herpes simplex virus stromal necrotizing keratitis.

Purpose:To evaluate the efficacy of systemic and topical antiviral therapy in the treatment of active herpes simplex virus (HSV) necrotizing stromal keratitis (NSK).Design:Prospective interventional case series.Methodology:Patients with a diagnosis of HSV NSK based on history and clinical findings were enrolled in the study. A standard protocol was used for microbiologic investigations. Ten weeks regime of systemic acyclovir and 2 weeks of topical acyclovir was given. Complete ophthalmic examination was performed at every visit. Outcome measures were a reduction in the area of infiltration and improvement in visual acuity.Results:Fifteen patients were enrolled in the study. The mean age of presentation was 51.53 years. The duration of symptoms at presentation ranged from 2 to 8 weeks. HSV1 DNA polymerase chain reaction was positive in 70% cases of those tested. Area of infiltration at trial entry and at the end of 2 weeks of antiviral treatment reduced significantly (P = 0.007). All patients showed a complete resolution of keratitis at the end of study.Conclusion:Topical and systemic acyclovir for treatment of NSK facilitates healing of ulceration. Topical steroids after initial antiviral therapy are safe and decreases inflammation and improve visual recovery. Early initiation of therapy has better outcomes as compared to late presentations.

English

The seroprevalence of herpes simplex virus II (HSV2) was investigated from April, 2011 to May, 2012 among 170 pregnant women attending consultation in the urban health training Yopougon-Attie, Abidjan (Cote d'Ivoire). The anti-HSV2 immunoglobin G (IgG) antibodies were detected by the principle of indirect enzyme-linked immunosorbent assay (ELISA) using the Diagnostic Automation ELISA® kit. Human immunodeficiency virus (HIV) serology was performed using rapid testing according to the national algorithm. Anti HSV2 IgG serology was positive in 96.5% (164/170) of women. HSV2/HIV coinfection was found in 6.7% (11/164) of parturients. No socio-epidemiological factor was related to the seroprevalence of HSV2 infection. Data from this study suggest a serological surveillance of pregnant women, in particular looking for a recent infection by assaying IgM or by real-time quantitative HSV DNA polymerase chain reaction (PCR). Indeed, the high prevalence of HSV2 in this population requires the implementation of a protocol in collaboration with the actors for the health of mother and child. Key words: Pregnant, herpes simplex virus II (HSV2), seroprevalence, herpes simplex virus II-human immunodeficiency virus (HSV2-HIV) coinfection.

Docking of anti-HIV-1 oxoquinoline-acylhydrazone derivatives as potential HSV-1 DNA polymerase inhibitors

Although there are many antiviral drugs available for the treatment of herpes simplex virus (HSV) infections, still the synthesis of new anti-HSV candidates is an important strategy to be pursued, due to the emergency of resistant HSV strains mainly in human immunodeficiency virus (HIV) co-infected patients. Some 1,4-dihydro-4-oxoquinolines, such as PNU-183792 (1), show a broad spectrum antiviral activity against human herpes viruses, inhibiting the viral DNA polymerase (POL) without affecting the human POLs. Thus, on an ongoing antiviral research project, our group has synthesized ribonucleosides containing the 1,4-dihydro-4-oxoquinoline (quinolone) heterocyclic moiety, such as the 6-Cl derivative (2), which is a dual antiviral agent (HSV-1 and HIV-1). Molecular dynamics simulations of the complexes of 1 and 2 with the HSV-1 POL suggest that structural modifications of 2 should increase its experimental anti-HSV-1 activity, since its ribosyl and carboxyl groups are highly hydrophilic to interact with a hydrophobic pocket of this enzyme. Therefore, in this work, comparative molecular docking simulations of 1 and three new synthesized oxoquinoline-acylhydrazone HIV-1 inhibitors (3–5), which do not contain those hydrophilic groups, were carried out, in order to access these modifications in the proposition of new potential anti-HSV-1 agents, but maintaining the anti-HIV-1 activity. Among the docked compounds, the oxoquinoline-acylhydrazone 3 is the best candidate for an anti-HSV-1 agent, and, in addition, it showed anti-HIV-1 activity (EC50=3.4±0.3μM). Compounds 2 and 3 were used as templates in the design of four new oxoquinoline-acylhydrazones (6–9) as potential anti-HSV-1 agents to increase the antiviral activity of 2. Among the docked compounds, oxoquinoline-acylhydrazone 7 was selected as the best candidate for further development of dual anti-HIV/HSV activity.

Recalcitrant Pseudotumoral Anogenital Herpes Simplex Virus Type 2 in HIV-Infected Patients: Evidence for Predominant B-Lymphoplasmocytic Infiltration and Immunomodulators as Effective Therapeutic Strategy

In patients with human immunodeficiency virus (HIV) infection, genital herpetic lesions may be extensive and tend to persist for longer periods; in addition, atypical hypertrophic, ulcerative, or pseudotumor forms have been reported, frequently showing resistance to acyclovir (ACV) treatment. Between 2003 and 2011, 10 HIV-1-infected patients presenting with chronic pseudotumoral anogenital herpes simplex type 2 (HSV-2) infections were studied. All patients developed chronic, hypertrophic HSV-2 anogenital lesions with multilesional presentation in 7 cases and involvement of 2 anatomical sites in 6 of them. At the time of diagnosis, the median CD3(+)CD4(+) absolute blood count was 480.5 cells/µL (range, 165-632 cells/µL), whereas the plasma HIV load was undetectable in all cases. Histopathologic analysis of lesion biopsies showed a moderately dense dermal polytypic plasma cell infiltrate. Detection of HSV-2 by culture and/or polymerase chain reaction was positive for all patients, with evidence for ACV-resistant strains in 6 of 8 cases. In addition, viral resistance to ACV was found only in HSV-2 isolated from ulcerative lesions, whereas purely pseudotumoral ones harbored sensitive strains. Durable control was observed with HSV DNA polymerase inhibitors in only 2 cases, and the immunomodulators imiquimod and thalidomide allowed 5 patients to reach sustained complete response. HSV-2-related pseudolymphoma in HIV-infected patients is characterized by a predominant polyclonal lymphoplasmacytic infiltration, and is frequently refractory to antiherpetic drugs. Immunomodulatory therapeutic strategies using thalidomide showed consistent efficacy, and should be considered early during the course of disease.

A Multi-targeted Drug Candidate with Dual Anti-HIV and Anti-HSV Activity

Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens, in particular herpes simplex virus type 2 (HSV-2). The resulting coinfection is involved in a vicious circle of mutual facilitations. Therefore, an important task is to develop a compound that is highly potent against both viruses to suppress their transmission and replication. Here, we report on the discovery of such a compound, designated PMEO-DAPym. We compared its properties with those of the structurally related and clinically used acyclic nucleoside phosphonates (ANPs) tenofovir and adefovir. We demonstrated the potent anti-HIV and -HSV activity of this drug in a diverse set of clinically relevant in vitro, ex vivo, and in vivo systems including (i) CD4+ T-lymphocyte (CEM) cell cultures, (ii) embryonic lung (HEL) cell cultures, (iii) organotypic epithelial raft cultures of primary human keratinocytes (PHKs), (iv) primary human monocyte/macrophage (M/M) cell cultures, (v) human ex vivo lymphoid tissue, and (vi) athymic nude mice. Upon conversion to its diphosphate metabolite, PMEO-DAPym markedly inhibits both HIV-1 reverse transcriptase (RT) and HSV DNA polymerase. However, in striking contrast to tenofovir and adefovir, it also acts as an efficient immunomodulator, inducing β-chemokines in PBMC cultures, in particular the CCR5 agonists MIP-1β, MIP-1α and RANTES but not the CXCR4 agonist SDF-1, without the need to be intracellularly metabolized. Such specific β-chemokine upregulation required new mRNA synthesis. The upregulation of β-chemokines was shown to be associated with a pronounced downmodulation of the HIV-1 coreceptor CCR5 which may result in prevention of HIV entry. PMEO-DAPym belongs conceptually to a new class of efficient multitargeted antivirals for concomitant dual-viral (HSV/HIV) infection therapy through inhibition of virus-specific pathways (i.e. the viral polymerases) and HIV transmission prevention through interference with host pathways (i.e. CCR5 receptor down regulation).

Structure based molecular design, synthesis and biological evaluation of α-pyrone analogs as anti-HSV agent

Several options for treating Herpes Simplex Virus type 1 and type 2 are available. However, non-specific inhibition and drug resistance warrants the discovery of new anti-herpetic compounds with better therapeutic profile or different mode of action. The non-nucleoside inhibitors of HSV DNA polymerase target the site that is less important for the binding of a natural nucleoside or nucleoside inhibitors. In the present study, we have explored the possibility to find a new lead molecule based on α-pyrone analogs as non-nucleoside inhibitors using structure based modeling approach. The designed molecules were synthesized and evaluated for anti-HSV activity using MTT assay. The compound 5h with EC50 7.4μg/ml and CC50 52.5μg/ml was moderately active against HSV when compared to acyclovir. A plaque reduction assay was also carried out and results reveal that 5h is more effective against HSV-1 with better selective index of 12.8 than against HSV-2 (SI=3.6). The synthesized compounds were also evaluated for anti-HIV activity, but none were active.

Molecular modeling studies of 1,4-dihydro-4-oxoquinoline ribonucleosides with anti-HSV-1 activity

Abstract Eight human herpes viruses ( e.g. , herpes simplex, varicella-zoster, Epstein-Barr, cytomegalovirus, Kaposi’s sarcoma) are responsible for several diseases from sub-clinic manifestations to fatal infections, mostly in immunocompromised patients. The major limitations of the currently available antiviral drug therapy are drug resistance, host toxicity, and narrow spectrum of activity. However, some non-nucleoside 1,4-dihydro-4-oxoquinoline derivatives ( e.g. , PNU-183792) [4] shows broad spectrum antiviral activity. We have developed molecular modeling studies, including molecular docking and molecular dynamics simulations, based on a model proposed by Liu and co-workers [14] in order to understand the mechanism of action of a 6-chloro substituted 1,4-dihydro-4-oxoquinoline ribonucleoside, synthesized by the synthetic group, which showed anti-HSV-1 activity [9] . The molecular docking simulations confirmed the Liu’s model showing that the ligand needs to dislocate template residues from the active site in order to interact with the viral DNA polymerase enzyme, reinforcing that the interaction with the Val823 residue is pivotal for the inhibitory activity of non-nucleoside 1,4-dihydro-4-oxoquinoline derivatives, such as PNU-183792, with the HSV-1. The molecular dynamics simulations showed that the 6-chloro-benzyl group of PNU-183792 maintains its interaction with residues of the HSV-1 DNA polymerase hydrophobic pocket, considered important according to the Liu’s model, and also showed that the methyl group bounded to the nitrogen atom from PNU-183792 is probably contributing to a push–pull effect with the carbonyl group.

Topical tenofovir, a microbicide effective against HIV, inhibits herpes simplex virus-2 replication.

The HIV reverse-transcriptase inhibitor, tenofovir, was recently formulated into a vaginal gel for use as a microbicide. In human trials, a 1% tenofovir gel inhibited HIV sexual transmission by 39% and, surprisingly, herpes simplex virus-2 (HSV-2) transmission by 51%. We demonstrate that the concentration achieved intravaginally with a 1% tenofovir topical gel has direct antiherpetic activity. Tenofovir inhibits the replication of HSV clinical isolates in human embryonic fibroblasts, keratinocytes, and organotypic epithelial 3D rafts, decreases HSV replication in human lymphoid and cervicovaginal tissues ex vivo, and delays HSV-induced lesions and death in topically treated HSV-infected mice. The active tenofovir metabolite inhibits HSV DNA-polymerase and HIV reverse-transcriptase. To exert dual antiviral effects, tenofovir requires topical administration to achieve a drug concentration higher than systemic levels achieved by oral treatment. These findings indicate that a single topical treatment, like tenofovir, can inhibit the transmission of HIV and its copathogens.

No selection of nucleoside reverse transcriptase inhibitor resistance associated mutations by acyclovir suppressive therapy in herpes simplex virus-2/HIV-1 dually infected persons

Genital herpes simplex virus type 2 (HSV-2) shedding has been associated with increased concentrations of HIV-1 in the genital tract and plasma [1,2]. Recent data from randomized controlled trials suggest that daily HSV-2 suppressive therapy with acyclovir (ACV) or valacyclovir over periods of up to 3 months may reduce plasma and genital HIV RNA load by 0.3–0.5 log10 copies/ml among HIV/HSV-2 coinfected individuals not receiving antiretroviral therapy (ART) [3–7]. However, a recent trial of suppressive therapy [ACV 400 mg twice daily (b.i.d.)] delivered to the HIV/HSV-2 seropositive partner in HIV discordant couples found no decrease in the rate of HIV transmission to the HIV negative partner [8]. ACV is an acyclic guanosine analog that inhibits HSV DNA polymerase after conversion to its triphosphate form. As the first phosphorylation is synthesized selectively by HSV thymidine kinase [9], ACV is mainly active in HSV-infected cells and was therefore thought to have no or poor antiretroviral activity. The postulated mechanism of impact on HIV is that herpes suppressive therapy is able to prevent HIV replication by disrupting direct HSV-2/HIV-1 interaction and, indirectly, by reducing the systemic and local immune activation induced by HSV-2 [10]. However, recent in-vitro experiments have suggested that ACV may directly inhibit HIV replication and induce the selection of mutations in the HIV reverse transcriptase known to confer resistance to nucleoside reverse transcriptase inhibitors (NRTIs) [11,12]. ACV has been shown to select the mutation V75I, associated with the reverse transcriptase multidrug resistance complex, and also, to a lesser extent, T69N and M184I, which typically precedes the emergence of the M184V mutation that confers a high level of resistance to lamivudine/emtricitabine. These results suggest that the use of ACV may select HIV variants harboring mutations associated with resistance to NRTI. If confirmed in vivo, this may impact the choice and efficacy of antiretroviral regimens among HIV patients who have previously been treated with ACV, and would suggest that the use of ACV in ART-naïve HIV-positive patients should be carefully considered. To determine the impact of ACV suppressive therapy on the selection of mutations in the HIV reverse transcriptase gene in vivo, we analyzed plasma samples from dually HIV/HSV-2-infected women receiving ACV suppressive therapy who were enrolled in a randomized placebo-controlled trial of ACV 400 mg b.i.d. for HSV suppression in Tanzania [13,14]. The detection of mutations was performed by HIV reverse transcriptase gene sequence analysis on plasma samples selected from women with a reported high-ACV adherence (defined as taking >90% tablets by tablet counts) and HIV RNA load more than 3 log10 copies/ml after 12 months of ACV suppressive therapy, and who were not on ART. Among 21 selected samples, none of the mutations V75I, T69N and M184I were detected. No other mutations known to confer resistance to NRTI were detected in the reverse transcriptase gene. In four samples, mutations at the codon 69 (69A n = 2, 69P n = 1, T69S n = 1) were detected. T69A and T69P are not known to be associated with NRTI resistance, but T69S in combination with other mutations may be associated with tenofovir resistance. This mutation may have been selected owing to ACV exposure or owing to natural polymorphism. These results suggest that b.i.d. HSV-2 suppressive therapy with a standard dose of 400 mg of ACV for 12 months did not induce the selection of NRTI resistance mutations in HIV-infected women not receiving ART. Because the threshold of resistant variant detection with sequence analysis is above 20%, we cannot completely rule out the presence of minor populations containing NRTI resistance mutations. However, we selected samples from women with high-ACV adherence to guarantee the highest selective pressure of ACV, and restricted the laboratory analyses to those who had been on ACV therapy for 12 months in order to be able to detect late emergence of ACV-associated NRTI resistance mutations. Thus, the selection of mutations in the reverse transcriptase gene by ACV exposure seems unlikely in our population. Considering the bioavailability of oral ACV, plasma concentrations after 400 mg b.i.d. should not exceed 1 μmol/l, five-fold lower than the half maximal inhibitory concentration for HIV [12] and such concentrations might not be sufficient to achieve antiretroviral activity. Our results do not confirm the in-vitro data obtained studies using high-dose ACV [12], but corroborate results recently presented on patients receiving ACV or valacyclovir for periods of 8–24 months [15]. These reassuring results for the use of 400 mg b.i.d. ACV in HSV-2/HIV coinfected patients deserve further confirmation especially if higher dosage of ACV is used in HIV-infected individuals.

Hematemesis Due to Severe HSV Esophagitis: A Rare Occurrence in Immunocompetent Patients

Purpose: To include HSV esophagitis in the differential diagnosis of non-cirrhotic patients presenting with hematemesis. A 69-year-old man with past medical history of coronary artery disease, Fournier's gangrene treated surgically, diabetes mellitus II and heartburn presented to the emergency room with nausea and vomiting. While in the ER, he started to have hematemesis with hemoglobin dropping from 13.9 gm/dl to 9.4 gm/dl overnight. A nasogastric tube drained a large amount of bright red blood. He had tachycardia but his blood pressure remained stable. He was given intravenous fluids, blood products and proton pump inhibitors. An esophagogastroduodenoscopy (EGD) showed severe erosive esophagitis involving 50-75% of the esophageal mucosa. The ulcerated mucosa was covered with whitish exudates and clotted blood, consistent with a recent bleed. Esophageal biopsies showed cytopathic changes and immunohistochemical staining of esophageal biopsies was positive for HSV. No evidence of a fungal infection was found. The patient was started on acyclovir. He did not have recurrent hematemesis and was discharged home after a few days. Herpes simplex esophagitis (HSV-E) is usually caused by HSV 1. It has been described in case reports and is considered to be rare in immunocompetent patients. In patients less than 40 years old, 77% of the patients with HSV-E are men, but sex distribution is equal in older patients. Presenting complaints usually include odynophagia, dysphagia, fever, epigastric pain, nausea and vomiting. Fistula formation, food impaction and hemorrhage are among the rare complications of HSV-E. Although a barium swallow can show non-specific ulcer(s) in the esophagus, the diagnosis is usually established with EGD and biopsies/brushings that should be obtained from the edges of the ulcer where viral cytopathic effect is most likely to be present. Histological findings of multinucleated giant cells with ground glass nuclei and eosinophilic inclusions, and immunohistochemistry are helpful in establishing the diagnosis. HSV DNA polymerase chain reaction is considered to be the most sensitive test. Although HSV-E is considered to be a self limiting condition in immunocompetent patients, the treatment with acyclovir hastens the recovery and shortens the illness and is recommended in patient with complications. Recurrent disease is considered to be very rare. Conclusion: HSV-E is a rare condition among immunocompetent patients that can cause life- threatening complications. Thus, it should be included in the differential diagnosis while working up a patient presenting with various upper gastrointestinal and esophageal symptoms, including hematemesis.

Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase

It has recently been demonstrated that the anti-herpetic drug acyclovir (ACV) also displays antiviral activity against the human immunodeficiency virus type 1 (HIV-1). The triphosphate form of ACV is accepted by HIV-1 reverse transcriptase (RT), and subsequent incorporation leads to classical chain termination. Like all approved nucleoside analogue RT inhibitors (NRTIs), the selective pressure of ACV is associated with the emergence of resistance. The V75I mutation in HIV-1 RT appears to be dominant in this regard. By itself, this mutation is usually not associated with resistance to currently approved NRTIs. Here we studied the underlying biochemical mechanism. We demonstrate that V75I is also selected under the selective pressure of a monophosphorylated prodrug that was designed to bypass the bottleneck in drug activation to the triphosphate form (ACV-TP). Pre-steady-state kinetics reveal that V75I discriminates against the inhibitor at the level of catalysis, whereas binding of the inhibitor remains largely unaffected. The incorporated ACV-monophosphate (ACV-MP) is vulnerable to excision in the presence of the pyrophosphate donor ATP. V75I compromises binding of the next nucleotide that can otherwise provide a certain degree of protection from excision. Collectively, the results of this study suggest that ACV is sensitive to two different resistance pathways, which warrants further investigation regarding the detailed resistance profile of ACV. Such studies will be crucial in assessing the potential clinical utility of ACV and its derivatives in combination with established NRTIs.

Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1

Herpes simplex virus-1 is a large double-stranded DNA virus that is self-sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV-1 DNA polymerase (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities that are integral to base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we have reconstituted a system with purified HSV-1 and human proteins that perform all the steps of uracil DNA glycosylase-initiated base excision repair. In this system nucleotide incorporation is dependent on the HSV-1 uracil DNA glycosylase (UL2), human AP endonuclease, and the HSV-1 DNA polymerase. Completion of base excision repair can be mediated by T4 DNA ligase as well as human DNA ligase I or ligase IIIalpha-XRCC1 complex. Of these, ligase IIIalpha-XRCC1 is the most efficient. Moreover, ligase IIIalpha-XRCC1 confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV-1 DNA polymerase processivity factor (UL42) and prevents base excision repair from occurring with heterologous DNA polymerases. Completion of base excision repair in this system is also dependent on the incorporation of the correct nucleotide. These findings demonstrate that the HSV-1 proteins in combination with cellular factors that are not encoded by the virus are capable of performing base excision repair. These results have implications on the role of base excision repair in viral genome maintenance during lytic replication and reactivation from latency.

Phase Ib Trial of Oncolytic Herpes Virus G207 Shows Safety of Multiple Injections and Documents Viral Replication

The prognosis for patients with glioblastoma remains poor despite numerous phase I–II clinical trials on systemic chemotherapies such as the vascular endothelial growth factor–neutralizing antibody bevacizumab.1 Indeed, only two chemotherapies—implantable carmustine-containing Gliadel wafers and temozolomide2—have been approved for glioblastoma by the Food and Drug Administration, because these two agents showed benefit in phase III clinical trials by prolonging median survival by approximately 8 weeks. Therefore, there is a continued need to explore new treatments or to further refine and optimize older treatments for this type of brain cancer.

Herpes Simplex Virus (HSV) Suppression with Valacyclovir Reduces Rectal and Blood Plasma HIV‐1 Levels in HIV‐1/HSV‐2–Seropositive Men: A Randomized, Double‐Blind, Placebo‐Controlled Crossover Trial

Herpes simplex virus type 2 (HSV-2) infection is common among human immunodeficiency virus (HIV)-infected persons, and HSV reactivation increases plasma and genital HIV-1 levels. We studied HIV-1 levels during HSV suppression in coinfected persons in a placebo-controlled crossover trial. Twenty antiretroviral therapy (ART)-naive HIV-1/HSV-2-seropositive men who have sex with men in Lima, Peru, with CD4 cell counts >200 cells/ microL were randomized to receive either valacyclovir at 500 mg twice daily or placebo for 8 weeks, after which they underwent a 2-week washout period and then received the alternative regimen for 8 weeks. Specimens included daily anogenital swabs (for HSV DNA polymerase chain reaction [PCR]), thrice weekly rectal mucosal secretions (for HIV-1 RNA and HSV DNA PCR) obtained by anoscopy, and weekly plasma (for HIV-1 RNA PCR). Outcomes were rectal and plasma HIV-1 RNA levels by treatment arm. HIV-1 was detected in 73% of 844 rectal and 99% of 288 plasma specimens. HSV was detected in 29% and 4% of mucocutaneous specimens obtained during placebo and valacyclovir administration, respectively (P<.001). Valacyclovir resulted in a 0.16 (95% confidence interval [CI], 0.07-0.25; P=.0008; 33% decrease) log(10) copies/mL lower mean within-subject rectal HIV-1 level and a 0.33 (95% CI, 0.23-0.42; P<.0001; 53% decrease) log(10) copies/mL lower plasma HIV-1 level, compared with values for placebo. Valacyclovir significantly reduces rectal and plasma HIV-1 levels in HIV-1/HSV-2-coinfected men. HSV suppression may provide clinical benefits to persons not receiving highly active ART as well as public health benefits.

Inhibition of HSV-1 replication and HSV DNA polymerase by the chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(β- d-ribofuranosyl) quinoline-3-carboxylic acid and its aglycone

We describe in this paper that the synthetic chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(β- d-ribofuranosyl) quinoline-3-carboxylic acid (compound A) and its free aglycogene base (compound B) inhibit, with low cytotoxicity, the replication of herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). Compound A inhibited HSV-1 replication in Vero cells with an EC 50 of 1.3 and 1.4 μM for an acyclovir (ACV)-sensitive strain and an ACV-resistant strain of this virus, respectively. Additionally, it inhibited HSV-2 replication with an EC 50 of 1.1 μM. Compound B also inhibited the ACV-sensitive and -resistant HSV-1 strains, and HSV-2 at EC 50 values of 1.7, 1.9 and 1.6 μM, respectively. Time-of-addition assays, performed with compound A, suggested that this molecule at an early time point of the HSV replication cycle. Kinetic assays demonstrated that compounds A and B inhibit the HSV DNA polymerase activity in a noncompetitive fashion, with a K i equal to 0.1 and 0.2 μM, respectively. Taken together, our results suggest that compounds A and B represent promising lead molecules for further anti-HSV drug design.