Faculty Bibliography

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125-241 kbp contain 70-170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. VZV transcription during latency is restricted to the latency-associated transcript (VLT) and RNA 63 (encoding ORF63) in naturally VZV-infected human trigeminal ganglia (TG). While significantly more abundant, VLT levels positively correlated with RNA 63 suggesting co-regulated transcription during latency. Here, we identify VLT-ORF63 fusion transcripts and confirm VLT-ORF63, but not RNA 63, expression in human TG neurons. During in vitro latency, VLT is transcribed, whereas VLT-ORF63 expression is induced by reactivation stimuli. One isoform of VLT-ORF63, encoding a fusion protein combining VLT and ORF63 proteins, induces broad viral gene transcription. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m⁶A) to mRNAs. While m⁶A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m⁶A mapping using short-read sequencing, here we profile m⁶A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m⁶A detection. Although both early and late viral transcripts contain m⁶A, depletion of m⁶A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m⁶A discovery within individual transcripts at nucleotide resolution, and highlight the role of m⁶A in regulating splicing of a viral pathogen.

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.