Faculty Bibliography

Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life to cause herpes zoster, commonly associated with chronic pain. The VZV genome is densely packed and produces multitudes of overlapping transcripts deriving from both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we integrated multiple short-read RNA sequencing approaches with long-read direct RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical ORFs, noncanonical ORFs, and ORF fusions, as well as putative noncoding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated Immediate-Early, were expressed with Late kinetics. Our work showcases the complexity of the VZV transcriptome and provides a comprehensive resource that will facilitate future functional studies of coding RNAs, ncRNAs, and the biological mechanisms underlying the regulation of viral transcription and translation during lytic VZV infection.IMPORTANCE Transcription from herpesviral genomes, executed by the host RNA polymerase II and regulated by viral proteins, results in coordinated viral gene expression to efficiently produce infectious progeny. However, the complete coding potential and regulation of viral gene expression remain ill-defined for the human alphaherpesvirus varicella-zoster virus (VZV), causative agent of both varicella and herpes zoster. Here, we present a comprehensive overview of the VZV transcriptome and the kinetic class of all identified viral transcripts, using two virus strains and two biologically relevant cell types. Additionally, our data provide an overview of how VZV diversifies its transcription from one of the smallest herpesviral genomes. Unexpectedly, the transcript encoding the major viral transactivator protein (pORF62) was expressed with Late kinetics, whereas orthologous transcripts in other alphaherpesviruses are typically expressed during the immediate early phase. Therefore, our work both establishes the architecture of the VZV transcriptome and provides insight into regulation of alphaherpesvirus gene expression.

The varicella-zoster virus (VZV) genome, comprises both unique and repeated regions. The genome also includes reiteration regions, designated R1 to R5, which are tandemly repeating sequences termed elements. These regions represent an understudied feature of the VZV genome. The R4 region is duplicated, with one copy in the internal repeat short (IRs) which we designated R4A and a second copy in the terminal repeat short (TRs) termed R4B. We developed primers to amplify and Sanger sequence these regions, including independent amplification of both R4 regions. Reiteration regions from >80 cases of PCR-confirmed shingles were sequenced and analyzed. Complete genome sequences for the remaining portions of these viruses were determined using Illumina MiSeq. We identified 28 elements not previously reported, including at least one element for each R region. Length heterogeneity was substantial in R3, R4A and R4B. Length heterogeneity between the two copies of R4 was common.

The genomes of DNA viruses encode deceptively complex transcriptomes evolved to maximize coding potential within the confines of a relatively small genome. Defining the full range of viral RNAs produced during an infection is key to understanding the viral replication cycle and its interactions with the host cell. Traditional short-read (Illumina) sequencing approaches are problematic in this setting due to the difficulty of assigning short reads to individual RNAs in regions of transcript overlap and to the biases introduced by the required recoding and amplification steps. Additionally, different methodologies may be required to analyze the 5' and 3' ends of RNAs, which increases both cost and effort. The advent of long-read nanopore sequencing simplifies this approach by providing a single assay that captures and sequences full length RNAs, either in cDNA or native RNA form. The latter is particularly appealing as it reduces known recoding biases whilst allowing more advanced analyses such as estimation of poly(A) tail length and the detection of RNA modifications including N⁶ -methyladenosine. Using herpes simplex virus (HSV)-infected primary fibroblasts as a template, we provide a step-by-step guide to the production of direct RNA sequencing libraries suitable for sequencing using Oxford Nanopore Technologies platforms and provide a simple computational approach to deriving a high-quality annotation of the HSV transcriptome from the resulting sequencing data. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Productive infection of primary fibroblasts with herpes simplex virus Support Protocol: Cell passage and plating of primary fibroblasts Basic Protocol 2: Preparation and sequencing of dRNA-seq libraries from virus-infected cells Basic Protocol 3: Processing, alignment, and analysis of dRNA-seq datasets.

Varicella zoster virus (VZV) is a skin-tropic virus that infects epidermal keratinocytes and causes chickenpox. Although common, VZV infection can be life-threatening, particularly in the immunocompromized. Therefore, understanding VZV-keratinocyte interactions is important to find new treatments beyond vaccination and antiviral drugs. In VZV-infected skin, kallikrein 6 and the ubiquitin ligase MDM2 are upregulated concomitant with keratin 10 (KRT10) downregulation. MDM2 binds to KRT10, targeting it for degradation via the ubiquitin-proteasome pathway. Preventing KRT10 degradation reduced VZV propagation in culture and prevented epidermal disruption in skin explants. KRT10 knockdown induced expression of NR4A1 and enhanced viral propagation in culture. NR4A1 knockdown prevented viral propagation in culture, reduced LC3 levels, and increased LAMP2 expression. We therefore describe a drug-able pathway whereby MDM2 ubiquitinates and degrades KRT10, increasing NR4A1 expression and allowing VZV replication and propagation.

The dependence of viruses on the host cell to complete their replicative cycle renders cellular functions potential targets for novel anti-virals. We screened a panel of broad acting cellular ion channel inhibitors for activity against human cytomegalovirus (HCMV) and identified the voltage-gated chloride ion channel inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) as a potent inhibitor of HCMV replication. Time of addition studies demonstrated that DIDS inhibited entry via a direct interaction with the virion that impeded binding to the plasma membrane. Synthesis and analysis of pharmacological variants of DIDS suggested that intrinsic cysteine, and not lysine, reactivity was important for activity against HCMV.Although sequencing of a DIDS-resistant HCMV revealed enrichment of a mutation within UL100 (encoding for glycoprotein M) and a specific truncation of glycoprotein RL13, these did not explain the DIDS resistance phenotype. Specifically, only the introduction of the RL13 mutant partially pheno-copied the DIDS resistance phenotype. Serendipitously, the entry of DIDS-resistant HCMV also became independent of heparan sulfate proteoglycans (HSPGs) suggesting that evasion of DIDS lowered dependence on an initial interaction with HSPGs. Intriguingly, the DIDS-resistant virus demonstrated increased sensitivity to antibody neutralisation, which mapped, in part, to the presence of the gM mutation.Taken together the data characterise the anti-viral activity of a novel HCMV inhibitor that drives HCMV infection to occur independent of HSPGs and the generation of increased sensitivity to humoral immunity. The data also demonstrate that compounds with cysteine reactivity have the potential to act as anti-viral compounds against HCMV via direct engagement of virions.IMPORTANCE Human cytomegalovirus (HCMV) is major pathogen of non-immunocompetent individuals which remains in need of new therapeutic options. Here we have identified a potent antiviral compound (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, DIDS), its mechanism of action and the chemical properties required for its activity. In doing so, the data argue that cysteine-reactive compounds could have the capacity to be developed for anti-HCMV activity. Importantly, the data show that entry of DIDS resistant virus became independent of heparan sulfate proteoglycans (HSPGs) but, concomitantly, became more sensitive to neutralising antibody responses. This serendipitous observation suggests that retention of an interaction with HSPGs during the entry process in vivo may be evolutionarily advantageous through better evasion of humoral responses directed against HCMV virions.

Herpes zoster (HZ) is caused by reactivation of varicella zoster virus (VZV) that established latency in sensory and autonomic neurons during primary infection. In the Shingles Prevention Study (SPS), a large efficacy trial of live attenuated Oka/Merck zoster vaccine (ZVL), PCR-confirmed second episodes of HZ occurred in two of 660 placebo and one of 321 ZVL recipients with documented HZ during a mean follow-up of 3.13 years. An additional two ZVL recipients experienced a second episode of HZ in the Long-Term Persistence Substudy. All episodes of HZ were caused by wild-type VZV. The first and second episodes of HZ occurred in different dermatomes in each of these five participants, with contralateral recurrences in two. Time between first and second episodes ranged from 12 to 28 months. One of the five participants, who was immunocompetent on study enrollment, was immunocompromised at the onset of his first and second episodes of HZ. VZV DNA isolated from rash lesions from the first and second episodes of HZ was used to sequence the full-length VZV genomes. For the unique-sequence regions of the VZV genome, we employed target enrichment of VZV DNA, followed by deep sequencing. For the reiteration regions, we used PCR amplification and Sanger sequencing. Our analysis and comparison of the VZV genomes from the first and second episodes of HZ in each of the five participants indicate that both episodes were caused by the same VZV strain. This is consistent with the extraordinary stability of VZV during the replication phase of varicella and the subsequent establishment of latency in sensory ganglia throughout the body. Our observations also indicate that VZV is stable during the persistence of latency and the subsequent reactivation and replication that results in HZ.

Herpes Simplex Virus type 1 (HSV-1) chronically infects over 70 per cent of the global population. Clinical manifestations are largely restricted to recurrent epidermal vesicles. However, HSV-1 also leads to encephalitis, the infection of the brain parenchyma, with high associated rates of mortality and morbidity. In this study, we performed target enrichment followed by direct sequencing of HSV-1 genomes, using target enrichment methods on the cerebrospinal fluid (CSF) of clinical encephalitis patients and from skin swabs of epidermal vesicles on non-encephalopathic patients. Phylogenetic analysis revealed high inter-host diversity and little population structure. In contrast, samples from different lesions in the same patient clustered with similar patterns of allelic variants. Comparison of consensus genome sequences shows HSV-1 has been freely recombining, except for distinct islands of linkage disequilibrium (LD). This suggests functional constraints prevent recombination between certain genes, notably those encoding pairs of interacting proteins. Distinct LD patterns characterised subsets of viruses recovered from CSF and skin lesions, which may reflect different evolutionary constraints in different body compartments. Functions of genes under differential constraint related to immunity or tropism and provide new hypotheses on tissue-specific mechanisms of viral infection and latency.

The Glasgow s17 syn+ strain of herpes simplex virus 1 (HSV1) is arguably the best characterized strain and has provided the reference sequence for HSV1 genetic studies. Here we show that our original s17 syn+ stock was a mixed population from which we have isolated a minor variant that, unlike other strains in the laboratory, fails to be efficiently released from infected cells and spreads predominantly by direct cell-to-cell transmission. Analysis of other s17-derived viruses that had been isolated elsewhere revealed a number with the same release phenotype. Second-generation sequencing of 8 plaque-purified s17-derived viruses revealed sequences that vary by 50 single-nucleotide polymorphisms (SNPs), including approximately 10 coding SNPs. This compared to interstrain variations of around 800 SNPs in strain Sc16, of which a quarter were coding changes. Amongst the variations found within s17, we identified 13 variants of glycoprotein C within the original stock of virus that were predominantly a consequence of altered homopolymeric runs of C residues. Characterization of seven isolates coding for different forms of gC indicated that all were expressed, despite six of them lacking a transmembrane domain. While the release phenotype did not correlate directly with any of these identified gC variations, further demonstration that nine clinical isolates of HSV1 also fail to spread through extracellular release raises the possibility that propagation in tissue culture had altered the HSV1 s17 transmission phenotype. Hence, the s17 intrastrain variation identified here offers an excellent model for understanding both HSV1 transmission and tissue culture adaptation.

During latent infections with herpes simplex virus 1 (HSV-1), viral transcription is restricted and the genomes are mostly maintained in silenced chromatin, whereas in lytically infected cells all viral genes are transcribed and the genomes are dynamically chromatinized. Histones in the viral chromatin bear markers of silenced chromatin at early times in lytic infection or of active transcription at later times. The virion protein VP16 activates transcription of the immediate-early (IE) genes by recruiting transcription activators and chromatin remodelers to their promoters. Two IE proteins, ICP0 and ICP4 which modulate chromatin epigenetics, then activate transcription of early and late genes. Although chromatin is involved in the mechanism of activation of HSV- transcription, its precise role is not entirely understood. In the cellular genome, chromatin dynamics often modulate transcription competence whereas promoter-specific transcription factors determine transcription activity. Here, biophysical fractionation of serially digested HSV-1 chromatin followed by short-read deep sequencing indicates that nuclear HSV-1 DNA has different biophysical properties than protein-free or encapsidated HSV-1 DNA. The entire HSV-1 genomes in infected cells were equally accessible. The accessibility of transcribed or non-transcribed genes under any given condition did not differ, and each gene was entirely sampled in both the most and least accessible chromatin. However, HSV-1 genomes fractionated differently under conditions of generalized or restricted transcription. Approximately 1/3 of the HSV-1 DNA including fully sampled genes resolved to the most accessible chromatin when HSV-1 transcription was active, but such enrichment was reduced to only 3% under conditions of restricted HSV-1 transcription. Short sequences of restricted accessibility separated genes with different transcription levels. Chromatin dynamics thus provide a first level of regulation on HSV-1 transcription, dictating the transcriptional competency of the genomes during lytic infections, whereas the transcription of individual genes is then most likely activated by specific transcription factors. Moreover, genes transcribed to different levels are separated by short sequences with limited accessibility.

Epstein-Barr virus (EBV) is one of the most common viral infections in humans and persists within its host for life. EBV therefore represents an extremely successful virus that has evolved complex strategies to evade the host's innate and adaptive immune response during both initial and persistent stages of infection. Here, we conducted a comparative genomics analysis on 223 whole genome sequences of world-wide EBV strains. We recover extensive genome-wide linkage disequilibrium (LD) despite pervasive genetic recombination. This pattern is explained by the global EBV population being subdivided into three main sub-populations, one primarily found in East Asia, one in Southeast Asia and Oceania, and the third including most of the other globally distributed genomes we analyzed. Additionally, sites in LD were overrepresented in immunogenic genes. Taken together, our results suggest that host immune selection and local adaptation to different human host populations has shaped the genome-wide patterns of genetic diversity in EBV.