Aberrant RNA polymerase initiation and processivity on the genome of a herpes simplex virus 1 mutant lacking ICP27.

Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.

A Revision of Herpes Simplex Virus Type 1 Transcription: First, Repress; Then, Express.

The herpes virus genome bears more than 80 strong transcriptional promoters. Upon entry into the host cell nucleus, these genes are transcribed in an orderly manner, producing five immediate-early (IE) gene products, including ICP0, ICP4, and ICP22, while non-IE genes are mostly silent. The IE gene products are necessary for the transcription of temporal classes following sequentially as early, leaky late, and true late. A recent analysis using precision nuclear run-on followed by deep sequencing (PRO-seq) has revealed an important step preceding all HSV-1 transcription. Specifically, the immediate-early proteins ICP4 and ICP0 enter the cell with the incoming genome to help preclude the nascent antisense, intergenic, and sense transcription of all viral genes. VP16, which is also delivered into the nucleus upon entry, almost immediately reverses this repression on IE genes. The resulting de novo expression of ICP4 and ICP22 further repress antisense, intergenic, and early and late viral gene transcription through different mechanisms before the sequential de-repression of these gene classes later in infection. This early repression, termed transient immediate-early protein-mediated repression (TIEMR), precludes unproductive, antisense, intergenic, and late gene transcription early in infection to ensure the efficient and orderly progression of the viral cascade.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Herpes simplex virus 1 immediate early transcription initiation, pause-release, elongation, and termination in the presence and absence of ICP4.

IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.

Reactivation of Epstein-Barr Virus from Latency Involves Increased RNA Polymerase Activity at CTCF Binding Sites on the Viral Genome.

The ability of Epstein-Barr virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch remain unclear. To investigate transcriptional events during the latent-to-lytic switch, we utilized Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to map cellular RNA polymerase (Pol) activity to single-nucleotide resolution on the host and EBV genome in three different models of EBV latency and reactivation. In latently infected Mutu-I Burkitt lymphoma (BL) cells, Pol activity was enriched at the Qp promoter, the EBER region, and the BHLF1/LF3 transcripts. Upon reactivation with phorbol ester and sodium butyrate, early-phase Pol activity occurred bidirectionally at CTCF sites within the LMP-2A, EBER-1, and RPMS1 loci. PRO-Seq analysis of Akata cells reactivated from latency with anti-IgG and a lymphoblastoid cell line (LCL) reactivated with small molecule C60 showed a similar pattern of early bidirectional transcription initiating around CTCF binding sites, although the specific CTCF sites and viral genes were different for each latency model. The functional importance of CTCF binding, transcription, and reactivation was confirmed using an EBV mutant lacking the LMP-2A CTCF binding site. This virus was unable to reactivate and had disrupted Pol activity at multiple CTCF binding sites relative to the wild-type (WT) virus. Overall, these data suggest that CTCF regulates the viral early transcripts during reactivation from latency. These activities likely help maintain the accessibility of the viral genome to initiate productive replication. IMPORTANCE The ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B cells, and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication. Reactivation occurs once the immediate early (IE) EBV lytic genes are expressed. However, the molecular mechanisms behind the switch from the latent transcriptional program to begin transcription of the IE genes remain unknown. In this study, we mapped RNA Pol positioning and activity during latency and reactivation. Unexpectedly, Pol activity accumulated at distinct regions characteristic of transcription initiation on the EBV genome previously shown to be associated with CTCF. We propose that CTCF binding at these regions retains Pol to maintain a stable latent chromosome conformation and a rapid response to various reactivation signals.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Immediate Early Proteins of Herpes Simplex Virus Transiently Repress Viral Transcription before Subsequent Activation.

Herpes simplex virus 1 (HSV-1) utilizes cellular RNA polymerase II (Pol) to transcribe its genes in one of two phases. In the latent phase, viral transcription is highly restricted, but during the productive lytic phase, more than 80 genes are expressed in a temporally coordinated cascade. In this study, we used Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to characterize early viral transcriptional events using HSV-1 immediate early (IE) gene mutants, corresponding genetically repaired viruses, and wild-type virus. Unexpectedly, in the absence of the IE genes ICP4, ICP22, and ICP0 at 1.5 hours postinfection (hpi), we observed high levels of aberrant transcriptional activity across the mutant viral genomes but substantially less on either wild-type or the congenic repaired virus genomes. This feature was particularly prominent in the absence of ICP4 expression. Cycloheximide treatment during infection with both the ICP4 and ICP22 mutants and their respective genetic repairs did not alter the relative distribution of Pol activity, but it increased overall activity across both viral genomes, indicating that both virion components and at least some de novo protein synthesis were required for full repression. Overall, these data reveal that prior to their role in transcriptional activation, IE gene products and virion components first repress transcription and that the HSV-1 lytic transcriptional cascade is mediated through subsequent derepression steps. IMPORTANCE HSV-1 transcription during productive replication is believed to comprise a series of activation steps leading to a specific sequence of gene expression. Here, we show that virion components and IE gene products ICP0, ICP4, and ICP22 first repress viral gene transcription to various degrees before subsequently activating specific gene subsets. It follows that the entire HSV transcriptional program involves a series of steps to sequentially reverse this repression. This previously uncharacterized repressive activity of IE genes very early in infection may represent an important checkpoint allowing HSV-1 to orchestrate either the robust lytic transcriptional cascade or the more restricted transcriptional program during latency.

ICP22 of Herpes Simplex Virus 1 Decreases RNA Polymerase Processivity.

To determine the role of ICP22 in transcription, we performed precise nuclear run-on followed by deep sequencing (PRO-seq) and global nuclear run-on with sequencing (GRO-seq) in cells infected with a viral mutant lacking the entire ICP22-encoding α22 (US1/US1.5) gene and a virus derived from this mutant bearing a restored α22 gene. At 3 h postinfection (hpi), the lack of ICP22 reduced RNA polymerase (Pol) promoter proximal pausing (PPP) on the immediate early α4, α0, and α27 genes. Diminished PPP at these sites accompanied increased Pol processivity across the entire herpes simplex virus 1 (HSV-1) genome in GRO-seq assays, resulting in substantial increases in antisense and intergenic transcription. The diminished PPP on α gene promoters at 3 hpi was distinguishable from effects caused by treatment with a viral DNA polymerase inhibitor at this time. The ICP22 mutant had multiple defects at 6 hpi, including lower viral DNA replication, reduced Pol activity on viral genes, and increased Pol activity on cellular genes. The lack of ICP22 also increased PPP release from most cellular genes, while a minority of cellular genes exhibited decreased PPP release. Taken together, these data indicate that ICP22 acts to negatively regulate transcriptional elongation on viral genes in part to limit antisense and intergenic transcription on the highly compact viral genome. This regulatory function directly or indirectly helps to retain Pol activity on the viral genome later in infection. IMPORTANCE The longstanding observation that ICP22 reduces RNA polymerase II (Pol II) serine 2 phosphorylation, which initiates transcriptional elongation, is puzzling because this phosphorylation is essential for viral replication. The current study helps explain this apparent paradox because it demonstrates significant advantages in negatively regulating transcriptional elongation, including the reduction of antisense and intergenic transcription. Delays in elongation would be expected to facilitate the ordered assembly and functions of transcriptional initiation, elongation, and termination complexes. Such limiting functions are likely to be important in herpesvirus genomes that are otherwise highly transcriptionally active and compact, comprising mostly short, intronless genes near neighboring genes of opposite sense and containing numerous 3'-nested sets of genes that share transcriptional termination signals but differ at transcriptional start sites on the same template strand.

RNA Polymerase II Promoter-Proximal Pausing and Release to Elongation Are Key Steps Regulating Herpes Simplex Virus 1 Transcription.

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (Pol II). Expression of viral immediate early (α) genes is followed sequentially by early (ß), late (γ1), and true late (γ2) genes. We used precision nuclear run-on with deep sequencing to map and to quantify Pol II on the HSV-1(F) genome with single-nucleotide resolution. Approximately 30% of total Pol II relocated to viral genomes within 3 h postinfection (hpi), when it occupied genes of all temporal classes. At that time, Pol II on α genes accumulated most heavily at promoter-proximal pause (PPP) sites located ∼60 nucleotides downstream of the transcriptional start site, while ß genes bore Pol II more evenly across gene bodies. At 6 hpi, Pol II increased on γ1 and γ2 genes while Pol II pausing remained prominent on α genes. At that time, average cytoplasmic mRNA expression from α and ß genes decreased, relative to levels at 3 hpi, while γ1 relative expression increased slightly and γ2 expression increased more substantially. Cycloheximide treatment during the first 3 h reduced the amount of Pol II associated with the viral genome and confined most of the remaining Pol II to α gene PPP sites. Inhibition of both cyclin-dependent kinase 9 activity and viral DNA replication reduced Pol II on the viral genome and restricted much of the remaining Pol II to PPP sites.IMPORTANCE These data suggest that viral transcription is regulated not only by Pol II recruitment to viral genes but also by control of elongation into viral gene bodies. We provide a detailed map of Pol II occupancy on the HSV-1 genome that clarifies features of the viral transcriptome, including the first identification of Pol II PPP sites. The data indicate that Pol II is recruited to late genes early in infection. Comparing α and ß gene occupancy at PPP sites and gene bodies suggests that Pol II is released more efficiently into the bodies of ß genes than α genes at 3 hpi and that repression of α gene expression late in infection is mediated by prolonged promoter-proximal pausing. In addition, DNA replication is required to maintain full Pol II occupancy on viral DNA and to promote elongation on late genes later in infection.

The Coxsackievirus and Adenovirus Receptor, a Required Host Factor for Recovirus Infection, Is a Putative Enteric Calicivirus Receptor.

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in both developed and developing countries. Studies of HuNoV host cell interactions are limited by the lack of a simple, robust cell culture system. Due to their diverse HuNoV-like biological features, including histo-blood group antigen (HBGA) binding, rhesus enteric caliciviruses (ReCVs) are viable surrogate models for HuNoVs. In addition, several ReCV strains can be propagated to high titers in standard nonhuman primate cell lines while causing lytic infection and cell death. To identify the ReCV entry receptor, we performed CRISPR/Cas9 library screening in Vero cells, which identified the coxsackievirus and adenovirus receptor (CAR) as a candidate ReCV entry receptor. We showed that short interfering RNA, anti-human CAR (hCAR) monoclonal antibody RmcB treatment, and recombinant hCAR ectodomain blocked ReCV replication in LLC-MK2 cells. CRISPR/Cas9-targeted knockout of CAR in LLC-MK2 and Vero cells made these cell lines resistant to ReCV infection, and susceptibility to infection could be restored by transient expression of CAR. CHO cells do not express CAR or HBGAs and are resistant to ReCV infection. Recombinant CHO cells stably expressing hCAR or the type B HBGA alone did not support ReCV infection. However, CHO cells expressing both hCAR and the type B HBGA were susceptible to ReCV infection. In summary, we have demonstrated that CAR is required for ReCV infection and most likely is a functional ReCV receptor, but HBGAs are also necessary for infection.IMPORTANCE Because of the lack of a simple and robust human norovirus (HuNoV) cell culture system surrogate, caliciviruses still represent valuable research tools for norovirus research. Due to their remarkable biological similarities to HuNoVs, including the utilization of HBGAs as putative attachment receptors, we used rhesus enteric caliciviruses (ReCVs) to study enteric calicivirus host cell interactions. Using CRISPR/Cas9 library screening and functional assays, we identified and validated the coxsackievirus and adenovirus receptor (CAR) as a functional proteinaceous receptor for ReCVs. Our work demonstrated that CAR and HBGAs both are necessary to convert a nonsusceptible cell line to being susceptible to ReCV infection. Follow-up studies to evaluate the involvement of CAR in HuNoV infections are ongoing.

Clinical management of herpes simplex virus infections: past, present, and future.

Infection with herpes simplex virus (HSV) types 1 and 2 is ubiquitous in the human population. Most commonly, virus replication is limited to the epithelia and establishes latency in enervating sensory neurons, reactivating periodically to produce localized recurrent lesions. However, these viruses can also cause severe disease such as recurrent keratitis leading potentially to blindness, as well as encephalitis, and systemic disease in neonates and immunocompromised patients. Although antiviral therapy has allowed continual and substantial improvement in the management of both primary and recurrent infections, resistance to currently available drugs and long-term toxicity pose a current and future threat that should be addressed through the development of new antiviral compounds directed against new targets. The development of several promising HSV vaccines has been terminated recently because of modest or controversial therapeutic effects in humans. Nevertheless, several exciting vaccine candidates remain in the pipeline and are effective in animal models; these must also be tested in humans for sufficient therapeutic effects to warrant continued development. Approaches using compounds that modulate the chromatin state of the viral genome to suppress infection and reactivation or induce enhanced antiviral immunity have potential. In addition, technologies such as CRISPR/Cas9 have the potential to edit latent viral DNA in sensory neurons, potentially curing the neuron and patient of latent infection. It is hoped that development on all three fronts-antivirals, vaccines, and gene editing-will lead to substantially less HSV morbidity in the future.

The HIV Integrase Inhibitor Raltegravir Inhibits Felid Alphaherpesvirus 1 Replication by Targeting both DNA Replication and Late Gene Expression.

Alphaherpesvirus-associated ocular infections in humans caused by human alphaherpesvirus 1 (HHV-1) remain challenging to treat due to the frequency of drug application required and the potential for the selection of drug-resistant viruses. Repurposing on-the-market drugs is a viable strategy to accelerate the pace of drug development. It has been reported that the human immunodeficiency virus (HIV) integrase inhibitor raltegravir inhibits HHV-1 replication by targeting the DNA polymerase accessory factor and limits terminase-mediated genome cleavage of human betaherpesvirus 5 (HHV-5). We have previously shown, both in vitro and in vivo, that raltegravir can also inhibit the replication of felid alphaherpesvirus 1 (FeHV-1), a common ocular pathogen of cats with a pathogenesis similar to that of HHV-1 ocular disease. In contrast to what was reported for HHV-1, we were unable to select for a raltegravir-resistant FeHV-1 strain in order to define any basis for drug action. A candidate-based approach to explore the mode of action of raltegravir against FeHV-1 showed that raltegravir did not impact FeHV-1 terminase function, as described for HHV-5. Instead, raltegravir inhibited DNA replication, similarly to HHV-1, but by targeting the initiation of viral DNA replication rather than elongation. In addition, we found that raltegravir specifically repressed late gene expression independently of DNA replication, and both activities are consistent with inhibition of ICP8. Taken together, these results suggest that raltegravir could be a valuable therapeutic agent against herpesviruses.IMPORTANCE The rise of drug-resistant herpesviruses is a longstanding concern, particularly among immunocompromised patients. Therefore, therapies targeting viral proteins other than the DNA polymerase that may be less likely to lead to drug-resistant viruses are urgently needed. Using FeHV-1, an alphaherpesvirus closely related to HHV-1 that similarly causes ocular herpes in its natural host, we found that the HIV integrase inhibitor raltegravir targets different stages of the virus life cycle beyond DNA replication and that it does so without developing drug resistance under the conditions tested. This shows that the drug could provide a viable strategy for the treatment of herpesvirus infections.

Herpes Simplex Virus 1 Dramatically Alters Loading and Positioning of RNA Polymerase II on Host Genes Early in Infection.

Herpes simplex virus 1 (HSV-1) transcription is mediated by cellular RNA polymerase II (Pol II). Recent studies investigating how Pol II transcription of host genes is altered after HSV-1 are conflicting. Chromatin immunoprecipitation sequencing (ChIP-seq) studies suggest that Pol II is almost completely removed from host genes at 4 h postinfection (hpi), while 4-thiouridine (4SU) labeling experiments show that host transcription termination is extended at 7 hpi, implying that a significant amount of Pol II remains associated with host genes in infected cells. To address this discrepancy, we used precision nuclear run-on analysis (PRO-seq) to determine the location of Pol II to single-base-pair resolution in combination with quantitative reverse transcription-PCR (qRT-PCR) analysis at 3 hpi. HSV-1 decreased Pol II on approximately two-thirds of cellular genes but increased Pol II on others. For more than 85% of genes for which transcriptional termination could be statistically assessed, Pol II was displaced to positions downstream of the normal termination zone, suggesting extensive termination defects. Pol II amounts at the promoter, promoter-proximal pause site, and gene body were also modulated in a gene-specific manner. qRT-PCR of selected RNAs showed that HSV-1-induced extension of the termination zone strongly correlated with decreased RNA and mRNA accumulation. However, HSV-1-induced increases of Pol II occupancy on genes without termination zone extension correlated with increased cytoplasmic mRNA. Functional grouping of genes with increased Pol II occupancy suggested an upregulation of exosome secretion and downregulation of apoptosis, both of which are potentially beneficial to virus production.IMPORTANCE This study provides a map of RNA polymerase II location on host genes after infection with HSV-1 with greater detail than previous ChIP-seq studies and rectifies discrepancies between ChIP-seq data and 4SU labeling experiments with HSV-1. The data show the effects that a given change in RNA Pol II location on host genes has on the abundance of different RNA types, including nuclear, polyadenylated mRNA and cytoplasmic, polyadenylated mRNA. It gives a clearer understanding of how HSV-1 augments host transcription of some genes to provide an environment favorable to HSV-1 replication.

Broad anti-herpesviral activity of α-hydroxytropolones.

Herpesviruses are ubiquitous in animals and cause economic losses concomitant with many diseases. Most of the domestic animal herpesviruses are within the subfamily Alphaherpesvirinae, which includes human herpes simplex virus 1 (HSV-1). Suppression of HSV-1 replication has been reported with α-hydroxytropolones (αHTs), aromatic ring compounds that have broad bioactivity due to potent chelating activity. It is postulated that αHTs inhibit enzymes within the nucleotidyltransferase superfamily (NTS). These enzymes require divalent cations for nucleic acid cleavage activity. Potential targets include the nuclease component of the herpesvirus terminase (pUL15C), a highly conserved NTS-like enzyme that cleaves viral DNA into genomic lengths prior to packaging into capsids. Inhibition of pUL15C activity in biochemical assays by various αHTs previously revealed a spectrum of potencies. Interestingly, the most potent anti-pUL15C αHT inhibited HSV-1 replication to a limited extent in cell culture. The aim of this study was to evaluate three different αHT molecules with varying biochemical anti-pUL15C activity for a capacity to inhibit replication of veterinary herpesviruses (BoHV-1, EHV-1, and FHV-1) and HSV-1. Given the known discordant potencies between anti-pUL15C and HSV-1 replication inhibition, a second objective was to elucidate the mechanism of action of these compounds. The results show that αHTs broadly inhibit herpesviruses, with similar inhibitory effect against HSV-1, BoHV-1, EHV-1, and FHV-1. Based on immunoblotting, Southern blotting, and real-time qPCR, the compounds were found to specifically inhibit viral DNA replication. Thus, αHTs represent a new class of broadly active anti-herpesviral compounds with potential veterinary applications.

A Domain of Herpes Simplex Virus pUL33 Required To Release Monomeric Viral Genomes from Cleaved Concatemeric DNA.

Monomeric herpesvirus DNA is cleaved from concatemers and inserted into preformed capsids through the actions of the viral terminase. The terminase of herpes simplex virus (HSV) is composed of three subunits encoded by UL15, UL28, and UL33. The UL33-encoded protein (pUL33) interacts with pUL28, but its precise role in the DNA cleavage and packaging reaction is unclear. To investigate the function of pUL33, we generated a panel of recombinant viruses with either deletions or substitutions in the most conserved regions of UL33 using a bacterial artificial chromosome system. Deletion of 11 amino acids (residues 50 to 60 or residues 110 to 120) precluded viral replication, whereas the truncation of the last 10 amino acids from the pUL33 C terminus did not affect viral replication or the interaction of pUL33 with pUL28. Mutations that replaced the lysine at codon 110 and the arginine at codon 111 with alanine codons failed to replicate, and the pUL33 mutant interacted with pUL28 less efficiently. Interestingly, genomic termini of the large (L) and small (S) components were detected readily in cells infected with these mutants, indicating that concatemeric DNA was cleaved efficiently. However, the release of monomeric genomes as assessed by pulsed-field gel electrophoresis was greatly diminished, and DNA-containing capsids were not observed. These results suggest that pUL33 is necessary for one of the two viral DNA cleavage events required to release individual genomes from concatemeric viral DNA.IMPORTANCE This paper shows a role for pUL33 in one of the two DNA cleavage events required to release monomeric genomes from concatemeric viral DNA. This is the first time that such a phenotype has been observed and is the first identification of a function of this protein relevant to DNA packaging other than its interaction with other terminase components.

Herpesvirus Nuclear Egress.

Herpesviruses assemble and package their genomes into capsids in the nucleus, but complete final assembly of the mature virion in the cell cytoplasm. This requires passage of the genome-containing capsid across the double-membrane nuclear envelope. Herpesviruses have evolved a mechanism that relies on a pair of conserved viral gene products to shuttle the capsids from the nucleus to the cytoplasm by way of envelopment and de-envelopment at the inner and outer nuclear membranes, respectively. This complex process requires orchestration of the activities of viral and cellular factors to alter the architecture of the nuclear membrane, select capsids at the appropriate stage for egress, and accomplish efficient membrane budding and fusion events. The last few years have seen major advances in our understanding of the membrane budding mechanism and helped clarify the roles of viral and cellular proteins in the other, more mysterious steps. Here, we summarize and place into context this recent research and, hopefully, clarify both the major advances and major gaps in our understanding.

Vertex-Specific Proteins pUL17 and pUL25 Mechanically Reinforce Herpes Simplex Virus Capsids.

Using atomic force microscopy imaging and nanoindentation measurements, we investigated the effect of the minor capsid proteins pUL17 and pUL25 on the structural stability of icosahedral herpes simplex virus capsids. pUL17 and pUL25, which form the capsid vertex-specific component (CVSC), particularly contributed to capsid resilience along the 5-fold and 2-fold but not along the 3-fold icosahedral axes. Our detailed analyses, including quantitative mass spectrometry of the protein composition of the capsids, revealed that both pUL17 and pUL25 are required to stabilize the capsid shells at the vertices. This indicates that herpesviruses withstand the internal pressure that is generated during DNA genome packaging by locally reinforcing the mechanical sturdiness of the vertices, the most stressed part of the capsids.IMPORTANCE In this study, the structural, material properties of herpes simplex virus 1 were investigated. The capsid of herpes simplex virus is built up of a variety of proteins, and we scrutinized the influence of two of these proteins on the stability of the capsid. For this, we used a scanning force microscope that makes detailed, topographic images of the particles and that is able to perform mechanical deformation measurements. Using this approach, we revealed that both studied proteins play an essential role in viral stability. These new insights support us in forming a complete view on viral structure and furthermore could possibly help not only to develop specific antivirals but also to build protein shells with improved stability for drug delivery purposes.