Herpes Simplex Virus Type 1 Polymerase Research Articles

Prevalence of Herpesvirus DNA in Corneal Transplant Recipients

Purpose: Graft failure after penetrating keratoplasty (PK) is a serious complication, especially in eyes with herpetic keratitis (HK). This study evaluated the prevalence and graft survival of herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) DNA in recipient corneas during PK. Methods: The retrospective study was performed at the Department of Ophthalmology at University Hospital in Mainz, Germany. We analyzed data from every patient who underwent PK between January 2020 and June 2021. According to our clinical routine, we performed HSV-1 and VZV polymerase chain reaction (PCR) on all excised corneal buttons regardless of the primary clinical diagnosis. Results: We included 112 eyes of 112 consecutive patients who underwent PK. At the time of PK, 91 (81.25%) patients had no history of HK and 21 (18.75%) patients did. The recipient corneas of 91 patients without a history of HK tested positive for HSV-1 DNA in 12 (13.2%) eyes, for VZV DNA in 3 (3.3%) eyes, and for HSV-1 and VZV DNA simultaneously in 2 (2.2%) eyes. The recipient corneas of 21 patients with a preoperative history of HK tested positive for HSV-1 DNA in 13 (61.9%) eyes and VZV DNA in 1 (4.8%) eye. All patients with positive herpes DNA and no history of HK prior to PK received antiherpetic treatment and had a 100% graft survival rate after 1 year. Conclusions: We found herpesvirus DNA in 18.7% of recipient corneas without clinical suspicion or history of herpes keratitis. This suggests the need of routine HSV-1 and VZV PCR testing in all explanted corneas regardless of clinical suspicion, to detect, treat and prevent possible recurrence of herpes infection in corneal grafts and support graft survival.

Herpes Simplex Virus Type 1 Encephalitis: A Descriptive, Cross Sectional Study, 2011 - 2016, Iran

Background: Encephalitis may arise from infectious or non-infectious etiologies. Infections, predominantly viruses, are the most common causes of encephalitis. Herpes simplex virus (HSV) is the most prevalent cause of viral encephalitis in the developed countries. HSV encephalitis occurs equally in both genders and most cases are caused by HSV type 1 (HSV-1). Methods: The current descriptive, cross sectional study was conducted on patients admitted with HSV-1 encephalitis from 2011 to 2016 to Imam-Khomeini hospital complex in Tehran, Iran. The patients with clinical diagnosis of encephalitis whose cerebrospinal fluid (CSF) HSV-1 polymerase chain reaction (PCR) was positive were included. All clinical, laboratory, and imaging characteristics of patients along with their outcomes were evaluated and analyzed. Results: In the current study, of 12 cases with encephalitis manifestations and positive CSF HSV-1 PCR results, 5 were male and 7 were female with mean age of 42 years. Fever and headache were the most common complaints. Headache was more common in the cases admitted earlier (P value = 0.001). The patients were studied by brain magnetic resonance imaging (MRI) and the findings confirmed encephalitis in all the cases. Five cases had unilateral temporal lobe lesions and 6 cases had bilateral lesions. There was a clear relationship between bilateral temporal lobe lesions and higher protein level in CSF (P value = 0.001); 25% of patients (3 cases) needed stay in the intensive care unit (ICU). Three patients died and 5 patients developed disabilities. Conclusions: HSV encephalitis is a devastating disease. Clinical findings along CSF analysis and neuroimaging may result in the appropriate diagnosis. Despite appropriate treatment, undesired outcomes are common.

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

Kinetic Approaches to Understanding the Mechanisms of Fidelity of the Herpes Simplex Virus Type 1 DNA Polymerase

We discuss how the results of presteady-state and steady-state kinetic analysis of the polymerizing and excision activities of herpes simplex virus type 1 (HSV-1) DNA polymerase have led to a better understanding of the mechanisms controlling fidelity of this important model replication polymerase. Despite a poorer misincorporation frequency compared to other replicative polymerases with intrinsic 3′ to 5′ exonuclease (exo) activity, HSV-1 DNA replication fidelity is enhanced by a high kinetic barrier to extending a primer/template containing a mismatch or abasic lesion and by the dynamic ability of the polymerase to switch the primer terminus between the exo and polymerizing active sites. The HSV-1 polymerase with a catalytically inactivated exo activity possesses reduced rates of primer switching and fails to support productive replication, suggesting a novel means to target polymerase for replication inhibition.

Herpes Simplex Virus Type 1 DNA Polymerase Requires the Mammalian Chaperone Hsp90 for Proper Localization to the Nucleus

Many viruses and bacteriophage utilize chaperone systems for DNA replication and viral morphogenesis. We have previously shown that in the herpes simplex virus type 1 (HSV-1)-infected cell nucleus, foci enriched in the Hsp70/Hsp40 chaperone machinery are formed adjacent to viral replication compartments (A. D. Burch and S. K. Weller, J. Virol. 78:7175-7185, 2004). These foci have now been named virus-induced chaperone-enriched (VICE) foci. Since the Hsp90 chaperone machinery is known to engage the Hsp70/Hsp40 system in eukaryotes, the subcellular localization of Hsp90 in HSV-1-infected cells was analyzed. Hsp90 is found within viral replication compartments as well as in the Hsp70/Hsp40-enriched foci. Geldanamycin, an inhibitor of Hsp90, results in decreased HSV-1 yields and blocks viral DNA synthesis. Furthermore, we have found that the viral DNA polymerase is mislocalized to the cytoplasm in both infected and transfected cells in the presence of geldanamycin. Additionally, in the presence of an Hsp90 inhibitor, proteasome-dependent degradation of the viral polymerase was detected by Western blot analysis. These data identify the HSV-1 polymerase as a putative client protein of the Hsp90 chaperone system. Perturbations in this association appear to result in degradation, aberrant folding, and/or intracellular localization of the viral polymerase.

Inhibition of the Herpes Simplex Virus Type 1 DNA Polymerase Induces Hyperphosphorylation of Replication Protein A and Its Accumulation at S-Phase-Specific Sites of DNA Damage during Infection

The treatment of mammalian cells with genotoxic substances can trigger DNA damage responses that include the hyperphosphorylation of replication protein A (RPA), a protein that plays key roles in the recognition, signaling, and repair of damaged DNA. We have previously reported that in the presence of a viral polymerase inhibitor, herpes simplex virus type 1 (HSV-1) infection induces the hyperphosphorylation of RPA (D. E. Wilkinson and S. K. Weller, J. Virol. 78:4783-4796, 2004). We initiated the present study to further characterize this genotoxic response to HSV-1 infection. Here we report that infection in the presence of polymerase inhibitors triggers an S-phase-specific response to DNA damage, as demonstrated by induction of the hyperphosphorylation of RPA and its accumulation within viral foci specific to the S phase of the cell cycle. This DNA damage response occurred in the presence of viral polymerase inhibitors and required the HSV-1 polymerase holoenzyme as well as the viral single-stranded-DNA binding protein. Treatment with an inhibitor of the viral helicase-primase did not induce the hyperphosphorylation of RPA or its accumulation in infected cells. Taken together, these results suggest that the S-phase-specific DNA damage response to infection is dependent on the specific inhibition of the polymerase. Finally, RPA hyperphosphorylation was not induced during productive infection, indicating that active viral replication does not trigger this potentially detrimental stress response.

Interactions of herpes simplex virus type 1 with ND10 and recruitment of PML to replication compartments.

Many of the events required for productive herpes simplex virus type 1 (HSV-1) infection occur within globular nuclear domains called replication compartments, whose formation appears to depend on interactions with cellular nuclear domains 10 (ND10). We have previously demonstrated that the formation of HSV-1 replication compartments involves progression through several stages, including the disruption of intact ND10 (stage I to stage II) and the formation of PML-associated prereplicative sites (stage III) and replication compartments (stage IV) (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). In this paper, we show that some, but not all, PML isoforms are recruited to stage III foci and replication compartments. Genetic experiments showed that the recruitment of PML isoforms to stage III prereplicative sites and replication compartments requires the localization of the HSV-1 polymerase protein (UL30) to these foci but does not require polymerase catalytic activity. We also examined the stages of viral infection under conditions affecting ND10 integrity. Treatment with factors that increase the stability of ND10, arsenic trioxide and the proteasome inhibitor MG132, inhibited viral disruption of ND10, formation of replication compartments, and production of progeny virus. These results strengthen the previously described correlation between ND10 disruption and productive viral infection.

Herpes simplex virus type 1 and human DNA polymerase interactions with 2′-deoxyguanosine 5′-triphosphate analogues: Kinetics of incorporation into DNA and induction of inhibition

The ability of herpes simplex virus type 1 (HSV-1) DNA polymerase, HeLa polymerase α, and HeLa polymerase β to utilize several dGTP analogues has been investigated using a defined synthetic template primer. The relative efficiencies of the triphosphates of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir triphosphate, ACVTP), 9-[(1,3-dihydroxy-2-propoxy)methyl] guanine (ganciclovir triphosphate, DHPGTP), and 2′,3′-dideoxyguanosine (ddGTP) as substrates for the three polymerases were: HSV-1 polymerase, dGTP ≫ ACVTP ≅ DHPGTP ≫ ddGTP; polymerase α, dGTP ≫ ACVTP ≅ DHPGTP ≫ ddGTP; polymerase β, ddGTP ≫ dGTP ≫ ACVTP ≅ DHPGTP. The potent inhibition of HSV-1 polymerase by ACVTP has been shown previously to be due to the formation of a dead-end complex upon binding of the next 2′-deoxynucleoside 5′-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3′ end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405–7411). This mechanism was shown here to be a general mechanism for inhibition of polymerases by the obligate chain terminators, ACVTP and ddGTP. The ACVTP-induced inhibition was 30-fold more potent with HSV-1 polymerase than with polymerase α. This difference may contribute to the antiviral selectivity of this nucleotide analogue. The effect of ganciclovir monophosphate incorporation (a nonobligate chain terminator) on subsequent primer extension was also evaluated. With HSV-1 polymerase and polymerase α, although there was a considerable reduction in the efficiency of utilization of the 3′-DHPGMP-terminal primer, contrasting kinetic behavior was observed. With HSV-1 polymerase, insertion of DHPGTP resulted in a significant reduction in Vmax for subsequent nucleotide incorporations. In contrast, with polymerase α, a relatively small decrease in Vmax was accompanied by increased Km values for subsequent nucleotide incorporations.

Distinctive properties of DNA polymerases induced by herpes simplex virus type-1 and Epstein-Barr virus

The properties of DNA polymerases induced by two human herpesviruses, herpes simplex virus type-1 (HSV-1) and Epstein-Barr virus (EBV), have been compared. The HSV-1 and EBV polymerases can be distinguished from one another by differences in the elution profiles in phosphocellulose and single-stranded DNA cellulose columns. Although both enzymes require monovalent cations for optimum activity, the HSV-1 enzyme requires ammonium sulfate whereas the EBV enzyme activity is inhibited by it; on the other hand, the EBV polymerase requires KCl. Other reaction requirements are also different for the two viral enzymes. Thus, when the EBV DNA polymerase was assayed under conditions optimum for the HSV-1 DNA polymerase, only 15% of its activity was expressed. Differences were also noted in sensitivities of the two viral enzymes to the 5′-triphosphates of nucleoside analogs with antiherpesvirus activity such as BVdU, IVdU, ACV, FIAC and IdUrd. The HSV-1 polymerase was more sensitive than the EBV DNA polymerase to inhibition by phosphonoacetate, phosphonoformate, aphidicolin and N-ethylmaleimide. However, the EBV DNA polymerase was more sensitive than HSV-1 DNA polymerase to heat treatment at 42°C. Thus, the marked differences between the two viral enzymes can be useful in identifying enzyme activities in cells producing the virus and also in studying the biochemical mechanism of action of some of the antiviral agents.