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The herpes simplex virus 1 DNA polymerase contains a highly conserved structural motif found in most family B polymerases and certain RNA-binding proteins. To investigate its importance within cells, we constructed a mutant virus with substitutions in two residues of the motif and a rescued derivative. The substitutions resulted in severe impairment of plaque formation, yields of infectious virus, and viral DNA synthesis while not meaningfully affecting expression of the mutant enzyme, its co-localization with the viral single-stranded DNA binding protein at intranuclear punctate sites in non-complementing cells or in replication compartments in complementing cells, or viral DNA polymerase activity. Taken together, our results indicate that the RNA binding motif plays a crucial role in herpes simplex virus 1 DNA synthesis through a mechanism separate from effects on polymerase activity, thus identifying a distinct essential function of this motif with implications for hypotheses regarding its biochemical functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

IFN responses control acute HSV infection, but their role in regulating HSV latency is poorly understood. To address this we used mice lacking IFN signaling specifically in neural tissues. These mice supported a higher acute viral load in nervous tissue and delayed establishment of latency. While latent HSV-1 genome copies were equivalent, ganglia from neuronal IFN signaling-deficient mice unexpectedly supported reduced reactivation. IFNβ promoted survival of primary sensory neurons after infection with HSV-1, indicating a role for IFN signaling in sustaining neurons. We observed higher levels of latency associated transcripts (LATs) per HSV genome in mice lacking neuronal IFN signaling, consistent with a role for IFN in regulating LAT expression. These data show that neuronal IFN signaling modulates the expression of LAT and may conserve the pool of neurons available to harbor latent HSV-1 genome. The data also show that neuronal IFN signaling is dispensable for the establishment of latency., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Herpesviruses, like most DNA viruses, replicate their genomes in the host cell nucleus. Their DNA is then packaged and assembled into viral nucleocapsids, which, in most cases, are too large to pass through the nuclear pore complex. Instead, herpesviruses use a complex multistep pathway, termed nuclear egress, to exit the nucleus. Key players in this process include two conserved viral proteins that form the nuclear egress complex (NEC). In human cytomegalovirus, these NEC proteins are UL50, embedded in the inner nuclear membrane, and its nucleoplasmic partner UL53. Both are essential for viral nuclear egress. However, other viral components as well as host nuclear envelope proteins may also participate in nuclear egress. Identifying these viral and cellular factors may provide important insight into the herpesvirus lifecycle and its relationship to the underlying, yet still-mysterious, host nuclear egress pathway. We developed an immunoprecipitation-based protocol, described herein, to identify protein-protein interactions involving the NEC from the nuclear fraction of infected cells that express an epitope-tagged version of NEC subunit UL53., (© 2016 Elsevier Inc. All rights reserved.)

A collection of genomic DNA sequences of herpes simplex virus (HSV) strains has been defined and analyzed, and some information is available about genomic stability upon limited passage of viruses in culture. The nature of genomic change upon extensive laboratory passage remains to be determined. In this report we review the history of the HSV-1 KOS laboratory strain and the related KOS1.1 laboratory sub-strain, also called KOS (M), and determine the complete genomic sequence of an early passage stock of the KOS laboratory sub-strain and a laboratory stock of the KOS1.1 sub-strain. The genomes of the two sub-strains are highly similar with only five coding changes, 20 non-coding changes, and about twenty non-ORF sequence changes. The coding changes could potentially explain the KOS1.1 phenotypic properties of increased replication at high temperature and reduced neuroinvasiveness. The study also provides sequence markers to define the provenance of specific laboratory KOS virus stocks., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Several herpes simplex virus 1 microRNAs are encoded within or near the latency associated transcript (LAT) locus, and are expressed abundantly during latency. Some of these microRNAs can repress the expression of important viral proteins and are hypothesized to play important roles in establishing and/or maintaining latent infections. We found that in lytically infected cells and in acutely infected mouse ganglia, expression of LAT-encoded microRNAs was weak and unaffected by a deletion that includes the LAT promoter. In mouse ganglia latently infected with wild type virus, the microRNAs accumulated to high levels, but deletions of the LAT promoter markedly reduced expression of LAT-encoded microRNAs and also miR-H6, which is encoded upstream of LAT and can repress expression of ICP4. Because these LAT deletion mutants establish and maintain latent infections, these microRNAs are not essential for latency, at least in mouse trigeminal ganglia, but may help promote it., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The human cytomegalovirus DNA polymerase subunit UL44 is a phosphoprotein, but its sites and roles of phosphorylation have not been investigated. We compared sites of phosphorylation of UL44 in vitro by the viral protein kinase UL97 and cyclin-dependent kinase 1 with those in infected cells. Transient treatment of infected cells with a UL97 inhibitor greatly reduced labeling of two minor UL44 phosphopeptides. Viruses containing alanine substitutions of most UL44 residues that are phosphorylated in infected cells exhibited at most modest effects on viral DNA synthesis and yield. However, substitution of highly phosphorylated sites adjacent to the nuclear localization signal abolished viral replication. The results taken together are consistent with UL44 being phosphorylated directly by UL97 during infection, and a crucial role for phosphorylation-mediated nuclear localization of UL44 for viral replication, but lend little support to the widely held hypothesis that UL97-mediated phosphorylation of UL44 is crucial for viral DNA synthesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The small molecule salubrinal has antiviral activity against herpes simplex virus-1 (HSV-1) and inhibits dephosphorylation of eIF2 alpha mediated by the HSV-1 protein ICP34.5. We investigated whether salubrinal's activities in infected cells depend on ICP34.5. An ICP34.5 deletion mutant was as sensitive as wild type HSV-1 to salubrinal inhibition of plaque formation in Vero cells. However, salubrinal induced formation of syncytia in infected Vero cells, which was enhanced by ICP34.5 mutations. Expression of HSV-1 US11 with immediate early kinetics, which is known to suppress the effects of ICP34.5 mutations, resulted in slight resistance to salubrinal in murine embryonic fibroblasts, and substantial resistance in those cells when ICP34.5 was additionally mutated. ICP34.5 mutations, but not immediate early expression of US11, prevented salubrinal's ability to increase phosphorylation of eIF2 alpha during HSV-1 infection of Vero cells. Taken together, our data indicate that salubrinal has both ICP34.5-dependent and -independent activities in HSV-1 infected cells.

Small molecules can provide valuable tools to investigate virus biology. We developed a chemical screening approach to identify small molecule inhibitors of poorly understood, pre-early gene expression steps in herpes simplex virus infection, using green fluorescent protein fused to an early protein. Our assay identified ouabain, a cardiac glycoside. Ouabain reversibly decreased viral yield by 100-fold without affecting cellular metabolic activity in an overnight assay. The antiviral potencies of other cardiac glycosides correlated with their potencies against the known target of these compounds, the cellular sodium potassium ATPase. Ouabain had a reduced effect if added 8 h post-infection. It did not inhibit viral attachment or entry, but did reduce the expression of viral immediate-early and early genes by at least 5-fold. Collectively, these results implicate a cellular target that was hitherto not considered important for a stage of HSV replication prior to viral gene expression.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) ordinarily exists in electrophoretically distinct hypophosphorylated and hyperphosphorylated forms. Human cytomegalovirus infection induced forms of this subunit whose electrophoretic mobilities were intermediate without decreases in abundance of the original forms. Phosphatase treatment nearly eliminated the intermediate migrating forms. In vitro, the viral protein kinase, UL97, phosphorylated this subunit, a recombinant protein containing the CTD, and peptides containing the CTD consensus sequence, YSPTSPS. Phosphorylation occurred predominantly on serine 5 and was substantially reduced when either serine 2 or 5 was already phosphorylated. The abundance of the intermediate and hypophosphorylated forms was reduced at most twofold during infections in which UL97 was genetically or pharmacologically inhibited. These results identify a new pattern of RNA polymerase II modification induced by virus infection and a viral enzyme that phosphorylates the CTD in vitro, but only possibly in vivo.