Herpes Simplex Virus DNA Polymerase Research Articles

Dynamics of the Herpes simplex virus DNA polymerase holoenzyme during DNA synthesis and proof-reading revealed by Cryo-EM.

Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, replicates using seven essential proteins encoded by its genome. Among these, the UL30 DNA polymerase, complexed with the UL42 processivity factor, orchestrates leading and lagging strand replication of the 152 kb viral genome. UL30 polymerase is a prime target for antiviral therapy, and resistance to current drugs can arise in immunocompromised individuals. Using electron cryo-microscopy (cryo-EM), we unveil the dynamic changes of the UL30/UL42 complex with DNA in three distinct states. First, a pre-translocation state with an open fingers domain ready for nucleotide incorporation. Second, a halted elongation state where the fingers close, trapping dATP in the dNTP pocket. Third, a DNA-editing state involving significant conformational changes to allow DNA realignment for exonuclease activity. Additionally, the flexible UL30 C-terminal domain interacts with UL42, forming an extended positively charged surface binding to DNA, thereby enhancing processive synthesis. These findings highlight substantial structural shifts in the polymerase and its DNA interactions during replication, offering insights for future antiviral drug development.

Potential of hydroxybenzoic acids from Graptopetalum paraguayense for inhibiting of herpes simplex virus DNA polymerase – metabolome profiling, molecular docking and quantum-chemical analysis

According to our previous investigation the total methanol extract from Graptopetalum paraguayense E. Walther demonstrates a significant inhibitory effect on herpes simplex virus type 1 (HSV-1). To clarify what causes this inhibitory activity on HSV-1, a metabolic profile of the plant was performed. Three main fractions: non-polar substances, polar metabolites and phenolic compounds were obtained and gas chromatography–mass spectrometry (GC-MS) analysis was carried out. Since it is well known that phenolic compounds show a significant anti-herpes effect and that viral DNA polymerase (DNApol) appears to play a key role in HSV virus replication, we present a docking and quantum-chemical analysis of the binding of these compounds to viral DNApol amino acids. Fourteen different phenolic acids found by GC-MS analyses, were used in molecular docking simulations. According to the interaction energies of all fourteen ligands in the DNApol pockets based on docking results, density functional theory (DFT) calculations were performed on the five optimally interacting with the receptor acids. It was found that hydroxybenzoic acids from phenolic fraction of Graptopetalum paraguayense E. Walther show a good binding affinity to the amino acids from the active site of the HSV DNApol, but significantly lower than that of acyclovir. The mode of action on virus replication of acyclovir (by DNApol) is different from that of the plant phenolic acids one, probably.

Acyclovir-Resistant Herpes Simplex Virus 1 Keratitis: A Concerning and Emerging Clinical Challenge

To describe the clinical and virological profiles of patients with herpes simplex keratitis (HSK) caused by acyclovir-resistant (ACVR) strains of herpes simplex virus 1 (HSV-1). Multicenter retrospective case series. HSV-1 resistance to ACV was confirmed using sequencing of genes encoding HSV-1 thymidine kinase (TK) and DNA polymerase (DNA pol). Data were collected on the number of HSK episodes before and after the diagnosis of resistance, ocular findings including the type of HSK, immune status of patients, antiviral treatments, and HSV-1 genotypic resistance profiles. This study evaluated 18 HSK patients (13 male and 5 female, aged 66.8 ± 4.7 years) with ACVR HSV-1-positive ocular samples. Genotypic resistance testing was performed because of frequent recurrences despite adequate antiviral prophylaxis (AVP) (n=13, 72%), or poor response to suppressive antiviral therapy (n=5, 28%). Resistance mutations were found in the TK (n=15, 83%) or in the DNA pol gene (n=3, 17%). Prior to the diagnosis of resistance, the duration of disease was 29.8 ± 20.4 years, with more than 10 HSK recurrences in 15 patients (83%). The number of recurrences between the first episode and the diagnosis of resistance was significantly lower in immunocompromised patients (n=6, 33%) than in immunocompetent patients (n=12; 67%) (11.5 ± 4.9 vs 16.4 ± 1.9, P=.05). HSV-1 resistance to ACV must be suspected in HSK patients with recurrences despite AVP and/or in cases that respond poorly to a suppressive antiviral regimen. Immunocompromised patients and/or those with longstanding disease may be particularly at risk for developing resistance.

Susceptibility Evaluation of Clinically Isolated HSV-1 Strains to Acyclovir: A Phenotypic and Genotypic Study

Background: Mutations in herpes simplex virus Thymidine kinase (TK, UL23) and DNA polymerase (pol, UL30) genes may confer resistance to acyclovir (ACV). Phenotypic resistance must be determined along with genotypic resistance to achieve complete acyclovir susceptibility. Objectives: The present study aimed to characterize HSV-1 clinical isolates from outpatients and organ transplant recipients in terms of phenotypic ACV resistance. Moreover, genotypic resistance to ACV was assessed through sequencing the viral TK and pol genes amplified from virus-infected cell DNA. Methods: Twenty-six HSV-1 clinical isolates collected between 2016 and 2019 were examined for drug susceptibility. The samples were collected from eyes, oropharyngeal, facial, and other skin parts of immunocompetent and immunocompromised individuals. Phenotypic susceptibility was determined by using three different concentrations of ACV. The results were expressed based on the ability of ACV in reducing viral plaques by 50%. Genotyping was carried out by polymerase chain reaction and sequencing of TK and pol genes. Results: All the strains were characterized as sensitive at 0.01 and 0.05 µg.ml-1 concentrations to ACV. Seventy percent inhibition was observed at ≥ 0.1 µg.mL-1 of ACV for three isolates (two from patients who received transplants and one from an outpatient). Nine natural polymorphisms were detected in the TK gene and 31 in the Pol gene. Furthermore, four susceptible-associated mutations in the DNA pol gene were analyzed. A substitution was encoded in the conserved region of the pol Exo III motif (M553L), and nine amino acid substitutions in TK were detected. The phylogenetic analysis of partial genome sequences revealed high diversity in the TK and pol genes of HSV-1. Conclusions: A higher number of mutations were observed in patients who received transplants and underwent long-term treatment compared with outpatients. The high genetic variability of HSV-1 TK and DNA pol was not associated with phenotypic resistance.

Herpes Simplex Virus-1 infection in human primary corneal epithelial cells is blocked by a stapled peptide that targets processive DNA synthesis

PurposeAcyclovir is most commonly used for treating ocular Herpes Keratitis, a leading cause of infectious blindness. However, emerging resistance to Acyclovir resulting from mutations in the thymidine kinase gene of Herpes Simplex Virus −1 (HSV-1), has prompted the need for new therapeutics directed against a different viral protein. One novel target is the HSV-1 Processivity Factor which is essential for tethering HSV-1 Polymerase to the viral genome to enable long-chain DNA synthesis. MethodsA series of peptides, based on the crystal structure of the C-terminus of HSV-1 Polymerase, were constructed with hydrocarbon staples to retain their alpha-helical conformation. The stapled peptides were tested for blocking both HSV-1 DNA synthesis and infection. The most effective peptide was further optimized by replacing its negative N-terminus with two hydrophobic valine residues. This di-valine stapled peptide was tested for inhibiting HSV-1 infection of human primary corneal epithelial cells. ResultsThe stapled peptides blocked HSV-1 DNA synthesis and HSV-1 infection. The unstapled control peptide had no inhibitory effects. Specificity of the stapled peptides was confirmed by their inabilities to block infection by an unrelated virus. Significantly, the optimized di-valine stapled peptide effectively blocked HSV-1 infection in human primary corneal epithelial cells with selectivity index of 11.6. ConclusionsHydrocarbon stapled peptides that simulate the α-helix from the C-terminus of HSV-1 DNA polymerase can specifically block DNA synthesis and infection of HSV-1 in human primary corneal epithelial cells. These stapled peptides provide a foundation for developing a topical therapeutic for treating human ocular Herpes Keratitis.

Multiple In vitro biological effects of phenolic compounds from Morus alba root bark.

Morus alba L. is used in traditional Chinese medicine for the treatment of various diseases, including bacterial infections and inflammation. As a rich source of phenolic compounds, the plant is an object of many phytochemical and pharmacological studies. The aim of the study was to isolate and evaluate possible parallel antiviral, antibacterial, and anti-inflammatory activities of phenolic mulberry compounds. Extensive chromatographic separation of mulberry root bark extract and in vitro biological screening of 26 constituents identified promising candidates for further pharmacological research. Selected compounds were screened for anti-infective and anti-inflammatory activities. Antiviral activity was determined by the plaque number reduction assay and by the titer reduction assay, antibacterial using broth microdilution method, and anti-inflammatory activity using COX Colorimetric inhibitor screening assay kit. One compound was evaluated in vivo in carrageenan-induced paw-edema in mice. Five prenylated compounds 1, 2, 8, 9, and 11, together with a simple phenolic ester 13, exhibited inhibitory activity against the replication of herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2), with IC50 values ranging from 0.64 to 1.93 μg/mL, and EC50 values 0.93 and 1.61 μg/mL. Molecular docking studies demonstrated the effects of the active compounds by targeting HSV-1 DNA polymerase and HSV-2 protease. In antibacterial assay, compounds 1, 4, 11, and 17 diminished the growth of all of the Gram-positive strains tested, with MIC values of 1-16 μg/mL. The anti-inflammatory ability of several compounds to inhibit cyclooxygenase 2 (COX-2) was tested in vitro, and compound 16 displayed greater activity than the indomethacin, positive control. Mulberrofuran B (11) showed anti-inflammatory activity in vivo against carrageenan-induced paw-edema in mice. Experimental investigation showed promising antiviral, antibacterial, and/or anti-inflammatory activities of the phenolic mulberry constituents, often with multiple inhibitory effects that might be used as a potential source of new medicine.

Herpes Simplex Virus 1 DNA Polymerase RNase H Activity Acts in a 3'-to-5' Direction and Is Dependent on the 3'-to-5' Exonuclease Active Site.

The catalytic subunit (Pol) of herpes simplex virus 1 (HSV-1) DNA polymerase has been extensively studied both as a model for other family B DNA polymerases and for its differences from these enzymes as an antiviral target. Among the activities of HSV-1 Pol is an intrinsic RNase H activity that cleaves RNA from RNA-DNA hybrids. There has long been a controversy regarding whether this activity is due to the 3'-to-5' exonuclease of Pol or whether it is a separate activity, possibly acting on 5' RNA termini. To investigate this issue, we compared wild-type HSV-1 Pol and a 3'-to-5' exonuclease-deficient mutant, D368A Pol, for DNA polymerase activity, 3'-to-5' exonuclease activity, and RNase H activity in vitro Additionally, we assessed the RNase H activity using differentially end-labeled templates with 5' or 3' RNA termini. The mutant enzyme was at most modestly impaired for DNA polymerase activity but was drastically impaired for 3'-to-5' exonuclease activity, with no activity detected even at high enzyme-to-DNA substrate ratios. Importantly, the mutant showed no detectable ability to excise RNA with either a 3' or 5' terminus, while the wild-type HSV-1 Pol was able to cleave RNA from the annealed RNA-DNA hairpin template, but only detectably with a 3' RNA terminus in a 3'-to-5' direction and at a rate lower than that of the exonuclease activity. These results suggest that HSV-1 Pol does not have an RNase H separable from its 3'-to-5' exonuclease activity and that this activity prefers DNA degradation over degradation of RNA from RNA-DNA hybrids.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a member of the Herpesviridae family of DNA viruses, several of which cause morbidity and mortality in humans. Although the HSV-1 DNA polymerase has been studied for decades and is a crucial target for antivirals against HSV-1 infection, several of its functions remain to be elucidated. A hypothesis suggesting the existence of a 5'-to-3' RNase H activity intrinsic to this enzyme that could remove RNA primers from Okazaki fragments has been particularly controversial. In this study, we were unable to identify RNase H activity of HSV-1 DNA polymerase on RNA-DNA hybrids with 5' RNA termini. We detected RNase H activity on hybrids with 3' termini, but this was due to the 3'-to-5' exonuclease. Thus, HSV-1 is unlikely to use this method to remove RNA primers during DNA replication but may use pathways similar to those used in eukaryotic Okazaki fragment maturation.

The Anti-Human Immunodeficiency Virus Drug Tenofovir, a Reverse Transcriptase Inhibitor, Also Targets the Herpes Simplex Virus DNA Polymerase.

Genital herpes is an important cofactor for acquisition of human immunodeficiency virus (HIV) infection, and effective prophylaxis is a helpful strategy to halt both HIV and herpes simplex virus (HSV) transmission. The antiretroviral agent tenofovir, formulated as a vaginal microbicide gel, was shown to reduce the risk of HIV and HSV type 2 (HSV-2) acquisition. HSV type 1 (HSV-1) and HSV-2 mutants were selected for resistance to tenofovir and PMEO-DAPy (6-phosphonylmethoxyethoxy-2,4-diaminopyrimidine, an acyclic nucleoside phosphonate with dual anti-HSV and anti-HIV activity) by stepwise dose escalation. Several plaque-purified viruses were characterized phenotypically (drug resistance profiling) and genotypically (sequencing of the viral DNA polymerase gene). Tenofovir resistant and PMEO-DAPy-resistant viruses harbored specific amino acid substitutions associated with resistance not only to tenofovir and PMEO-DAPy but also to acyclovir and foscarnet. These amino acid changes (A719V, S724N, and L802F [HSV-1] and M789T and A724V [HSV-2]) were also found in clinical isolates recovered from patients refractory to acyclovir and/or foscarnet therapy or in laboratory-derived strains. A total of 10 (HSV-1) and 18 (HSV-2) well-characterized DNA polymerase mutants had decreased susceptibility to tenofovir and PMEO-DAPy. Tenofovir and PMEO-DAPy target the HSV DNA polymerase, and clinical isolates with DNA polymerase mutations emerging under acyclovir and/or foscarnet therapy showed cross-resistance to tenofovir and PMEO-DAPy.

Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus-1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA.

We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease, or transfer of the DNA from the polymerase to the exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2'-hydroxyl did not affect exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.

Erythema multiforme syndrome associated with acute acquired cytomegalovirus infection.

A 7-year-old boy was admitted to the pediatric ward (University of Chieti) for the onset of multiple erythematous target lesions, some with vesicles in the middle, located on the ears, face (Figure 1 A), in the oral cavity (Figure 1 B), on both hands, feet and lower limbs. Four days prior to admission the patient presented prodromal symptoms, such as fever (maximum temperature 38°C), sore throat and asthenia. All these features supported a diagnosis of erythema multiforme (EM) secondary to an infection. Blood examination revealed lymphopenia (1.28 × 10,000/µl), neutrophilia (6.10 × 10,000/µl), erythrocyte sedimentation rate of 15 mm/h (normal range: 1–12 mm/h) and alanine aminotransferase of 80 U/l (normal range: 11–66). Serum antibodies against Mycoplasma pneumoniae, Adenovirus, Varicella Zoster, Epstein-Barr virus and Herpes Simplex Virus (HSV) 1 and 2 were negative. In contrast, cytomegalovirus (CMV) IgM antibody titers were positive (23.6 U/ml; normal range: 0 to 18), whereas IgG antibody values were normal (< 5 U/ml; normal range: 0 to 12). Four weeks later, the levels of anti-CMV IgG antibodies rose (190 U/ml) with normalization of anti-CMV IgM levels. During hospitalization, management included intravenous fluids and electrolyte infusion for skin and mucosae dehydration and for the poor oral intake due to an intense oral burning sensation; methylprednisolone (0.8 mg/kg/day); intravenous antibiotic therapy (ceftriaxone) to prevent skin bacterial infection, because the patient (unable to take oral drugs for the several lesions in the oral cavity) presented fever and neutrophilia (6.10 × 10,000/µl) and mild elevation of erythrocyte sedimentation rate (15 mm/h).

Pritelivir for HSV-2 Genital Infections

Nucleoside analogues, which inhibit herpes simplex virus (HSV) DNA polymerase following phosphorylation by the viral thymidine kinase, have been a

Clinical features, diagnosis, and treatment of erythema multiforme: a review for the practicing dermatologist

Erythema multiforme (EM) is an uncommon, immune-mediated disorder that presents with cutaneous or mucosal lesions or both. In herpes simplex virus (HSV)-associated EM, the findings are thought to result from cell-mediated immune reaction against viral antigen-positive cells that contain the HSV DNA polymerase gene (pol). The target lesion, with concentric zones of color change, represents the characteristic cutaneous finding seen in this disorder. Although EM can be induced by various factors, HSV infection continues to be the most common inciting factor. Histopathologic testing and other laboratory investigations may be used to confirm the diagnosis of EM and to differentiate it from other clinical imitators. Imitators of EM include urticaria, Stevens-Johnson syndrome, fixed drug eruption, bullous pemphigoid, paraneoplastic pemphigus, Sweet's syndrome, Rowell's syndrome, polymorphus light eruption, and cutaneous small-vessel vasculitis. Because disease severity and mucosal involvement differ among patients, treatment should be tailored to each patient, with careful consideration of treatment risk vs benefit. Mild cutaneous involvement of EM can be managed primarily with a goal of achieving symptomatic improvement; however, patients with HSV-associated recurrent EM and idiopathic recurrent EM require treatment with antiviral prophylaxis. Inpatient hospitalization may be required for patients with severe mucosal involvement that causes poor oral intake and subsequent fluid and electrolyte imbalance. With this review, we strive to provide guidance to the practicing dermatologist in the evaluation and treatment of a patient with EM.

Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A

BackgroundHerpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug.ResultsWithaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured.ConclusionsWe have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus.

Genotypic characterization of herpes simplex virus DNA polymerase UL42 processivity factor

The herpes simplex virus (HSV) DNA polymerase is composed of the UL30 catalytic subunit and the UL42 processivity factor. The UL42 subunit increases the processivity of the polymerase along the DNA template during replication. The molecular mechanisms of HSV resistance to drugs interfering with viral DNA synthesis reported so far mainly rely on modifications of the viral thymidine kinase and DNA polymerase. We aimed to extensively describe the genetic variations of HSV UL42 processivity factor and to evaluate its potential involvement in resistance to antivirals. The full-length UL42 gene sequence of HSV was investigated among two laboratory strains (KOS and gHSV-2), 94 drug-sensitive clinical isolates and 25 phenotypically ACV-resistant clinical isolates. This work provided extensive data about natural variability of UL42 processivity factor among both HSV-1 and HSV-2 strains and showed that this viral protein is highly conserved among HSV strains, with a weaker variability for HSV-2. The analysis of 25 HSV clinical isolates exhibiting ACV-resistance documented most of the previously reported mutations related to UL42 natural polymorphism in addition to some unpreviously described polymorphisms. Surprisingly, a single-base deletion in UL42 gene sequence leading to a frameshift in the C-terminal region was identified among 3 HSV clinical isolates. From this preliminary study, UL42 processivity factor did not seem to be likely involved in HSV resistance to antivirals.

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIalpha-XRCC1, performs uracil-initiated 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 show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions -1 and -2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.

Rapidly Cleared Episodes of Oral and Anogenital Herpes Simplex Virus Shedding in HIV-Infected Adults

To determine whether rapidly cleared episodes of herpes simplex virus (HSV) reactivation occur in HIV-infected adults. Twenty HSV-2-seropositive, HIV-seropositive adults, including 9 (45%) who were also HSV-1 seropositive, collected oral and anogenital swabs for HSV DNA polymerase chain reaction 4 times a day for 60 days. Samples were positive for HSV if we detected > or =150 copies of HSV DNA/mL of specimen. Median HSV shedding episode duration was 7.5 (range 4-253) hours for oral and 11 (range 4-328) hours for anogenital reactivation. Thirty-five percent of oral and 29% of anogenital reactivations lasted < or =6 hours, and 59% of oral and 53% of anogenital reactivations lasted < or =12 hours. Seven of 9 participants who shed orally and 10 of 15 who shed anogenitally had > or =1 reactivation lasting < or =6 hours. The median maximum level of HSV DNA detected in an episode increased with episode duration for both oral and anogenital episodes. Concurrent oral and anogenital shedding occurred more frequently than expected: oral HSV shedding was detected on 17% of time points with anogenital but 1% of time points without anogenital, shedding (P < 0.001). Rapidly cleared episodes of oral and anogenital HSV shedding occur in HIV-infected persons, supporting the hypothesis that frequent anogenital mucosal immune activation caused by HSV-2 is present in HIV coinfected persons, potentially contributing to HIV infectiousness.

The Herpes simplex virus‐1 DNA polymerase exhibits 3′ phosphatase activity

Herpes simplex virus‐1 (HSV‐1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We recently demonstrated that the HSV‐1 DNA polymerase (UL30) exhibits AP and 5′ dRP lyase activities. Moreover, UL30 in conjunction with the viral uracil DNA glycosylase as well as cellular AP endonuclease and DNA ligase IIIα/XRCC1, perform uracil‐initiated Base Excision Repair (BER). As an alternative route to repairing base adducts and abasic sites, and as a mechanism to process ssDNA breaks we have investigated the ability of UL30 to process various 3′ ends. Interestingly, we find that UL30 can degrade 3′ sugar phosphate as well as 3′ phosphate terminated DNA. Moreover, UL30 exhibits a 3′ phosphatase activity that removes 3′ phosphate prior to 3′–5′ exonuclease action. These novel activities would allow UL30 to process AP sites following β‐elimination though incision by its inherent AP lyase activity or DNA breaks that result in 3′ phosphate terminated DNA following βδ‐elimination or X‐ray irradiation. Further studies will investigate how UL30 can repair this damage in conjunction with other factors. These findings further highlight the role of DNA repair in the maintenance of a viral genome with implications on both lytic replication and reactivation from latency.This work was supported by grant GM 062643 from the NIH.

Finger Domain Mutation Affects Enzyme Activity, DNA Replication Efficiency, and Fidelity of an Exonuclease-Deficient DNA Polymerase of Herpes Simplex Virus Type 1

The catalytic subunit of herpes simplex virus DNA polymerase (Pol), a member of the B family polymerases, possesses both polymerase and exonuclease activities. We previously demonstrated that a recombinant virus (YD12) containing a double mutation within conserved exonuclease motif III of the Pol was highly mutagenic and rapidly evolved to contain an additional leucine-to-phenylalanine mutation at residue 774 (L774F), which is located within the finger subdomain of the polymerase domain. We further demonstrated that the recombinant L774F virus replicated DNA with increased fidelity and that the L774F mutant Pol exhibited altered enzyme kinetics and impaired polymerase activity to extension from mismatched primer termini. In this study, we demonstrated that addition of the L774F mutation to the YD12 Pol did not restore the exonuclease deficiency. However, the polymerase activity of the YD12 Pol to extension from mismatched primer termini and on the nucleotide incorporation pattern was altered upon addition of the L774F mutation. The L774F mutation-containing YD12 Pol also supported the growth of viral progeny and replicated DNA more efficiently and more accurately than did the YD12 Pol. Together, these studies demonstrate that a herpes simplex virus Pol mutant with a highly mutagenic ability can rapidly acquire additional mutations, which may be selected for their survival and outgrowth. Furthermore, the studies demonstrate that the polymerase activity of HSV-1 Pol on primer extension is influenced by sequence context and that herpes simplex virus type 1 Pol may dissociate more frequently at G.C sites during the polymerization reaction. The implications of the findings are discussed.

Healthcare Seeking and Sexual Behavior Among Patients with Symptomatic Newly Acquired Genital Herpes

Symptoms among patients with first episode herpes simplex virus (HSV) likely influence health seeking and sexual behavior. An improved understanding of this relationship provides insight into the experience of having genital herpes and has implications for counseling. To describe the healthcare seeking and sexual behavior in patients with symptomatic laboratory confirmed first episode HSV infection. Two hundred thirty-six patients (94 men and 142 women) with newly acquired genital herpes were asked to complete a demographic and sexual history questionnaire. To confirm initial HSV diagnosis, swabs of lesions were collected for viral culture and HSV DNA polymerase chain reaction and blood was drawn for confirmation of HSV serostatus using the Western blot. Women reported pain and men reported lesions as the most frequent and bothersome symptom or sign causing each to seek healthcare. Forty-three percent of all participants missed some work or school because of their symptoms; women missed more school or work, sought care sooner, and saw more providers than men. Before diagnosis, most respondents (67%) suspected genital herpes was the etiology of the symptoms. Twenty-seven percent reported having sex after noticing their symptoms, though those who missed more school or work were less likely to engage in sexual intercourse. Men and women have different experiences with first episode HSV, but morbidity is substantial, especially among women. Both men and women may continue to engage in sexual activity after onset of genital herpes, emphasizing the need for providers to counsel their patients to avoid exposing partners to the infection.

The Antiherpetic Drug Acyclovir Inhibits HIV Replication and Selects the V75I Reverse Transcriptase Multidrug Resistance Mutation

The antiviral drug acyclovir is a guanosine nucleoside analog that potently inhibits herpes simplex virus (HSV) replication. Acyclovir treatment in patients coinfected with HSV and human immunodeficiency virus (HIV) has been observed to alter disease course and decrease HIV viral load, a finding that has been attributed to indirect effects of HSV suppression on HIV replication. Based on this hypothesis, several clinical studies have recently investigated the use of acyclovir for treatment of patients coinfected with HSV and HIV or for prophylaxis against HIV transmission. In this report, we use a single round HIV infectivity assay to show that acyclovir directly inhibits HIV infection with an IC50 of approximately 5 microm. The target of acyclovir in HIV-infected cells is validated as HIV reverse transcriptase (RT) by the emergence of the RT variant V75I under the selective pressure of acyclovir. The V75I mutation is part of the multidrug resistance pathway that enhances viral resistance to many of the best RT inhibitors approved for the treatment of HIV. Biochemical analyses demonstrate that acyclovir triphosphate is a chain terminator substrate for HIV RT and can compete with dGTP for incorporation into DNA. Although acyclovir may prove a useful lead for development of new HIV treatments, the selection of resistant mutants raises a cautionary note to the use of acyclovir monotherapy in patients coinfected with HSV and HIV.