Faculty Bibliography

Understanding the molecular mechanisms of herpes simplex virus 1 (HSV-1) latent infection and reactivation in neurons requires the use of in vitro model systems. Establishing a quiescent infection in cultured neurons is problematic, as any infectious virus released can superinfect the cultures. Previous studies have used the viral DNA replication inhibitor acyclovir to prevent superinfection and promote latency establishment. Data from these previous models have shown that reactivation is biphasic, with an initial phase I expression of all classes of lytic genes, which occurs independently of histone demethylase activity and viral DNA replication but is dependent on the cell stress protein DLK. Here, we describe a new model system using HSV-1 Stayput-GFP, a reporter virus that is defective for cell-to-cell spread and establishes latent infections without the need for acyclovir. The establishment of a latent state requires a longer time frame than previous models using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate using established inducers, and as such, a combination of reactivation triggers is required. Using this system, we demonstrate that biphasic reactivation occurs even when latency is established in the absence of acyclovir. Importantly, phase I lytic gene expression still occurs in a histone demethylase and viral DNA replication-independent manner and requires DLK activity. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and not unique to systems that require acyclovir to promote latency establishment. IMPORTANCE Herpes simplex virus-1 (HSV-1) enters a latent infection in neurons and periodically reactivates. Reactivation manifests as a variety of clinical symptoms. Studying latency and reactivation in vitro is invaluable, allowing the molecular mechanisms behind both processes to be targeted by therapeutics that reduce the clinical consequences. Here, we describe a novel in vitro model system using a cell-to-cell spread-defective HSV-1, known as Stayput-GFP, which allows for the study of latency and reactivation at the single neuron level. We anticipate this new model system will be an incredibly valuable tool for studying the establishment and reactivation of HSV-1 latent infection in vitro. Using this model, we find that initial reactivation events are dependent on cellular stress kinase DLK but independent of histone demethylase activity and viral DNA replication. Our data therefore further validate the essential role of DLK in mediating a wave of lytic gene expression unique to reactivation.

MOTIVATION/BACKGROUND:The chemical modification of ribonucleotides regulates the structure, stability, and interactions of RNAs. Profiling of these modifications using short-read (Illumina) sequencing techniques provides high sensitivity but low-to-medium resolution i.e., modifications cannot be assigned to specific transcript isoforms in regions of sequence overlap. An alternative strategy uses current fluctuations in nanopore-based long read direct RNA sequencing (DRS) to infer the location and identity of nucleotides that differ between two experimental conditions. While highly sensitive, these signal-level analyses require high quality transcriptome annotations and thus are best suited to the study of model organisms. By contrast, the detection of RNA modifications in microbial organisms which typically have no or low-quality annotations requires an alternative strategy. Here, we demonstrate that signal fluctuations directly influence error rates during base calling and thus provides an alternative approach for identifying modified nucleotides. RESULTS:DRUMMER (Detection of Ribonucleic acid Modifications Manifested in Error Rates (i) utilizes a range of statistical tests and background noise correction to identify modified nucleotides with high confidence, (ii) operates with similar sensitivity to signal-level analysis approaches, and (iii) correlates very well with orthogonal approaches. Using well-characterized DRS datasets supported by independent meRIP-Seq and miCLIP-Seq datasets we demonstrate that DRUMMER operates with high sensitivity and specificity. AVAILABILITY AND IMPLEMENTATION/METHODS:DRUMMER is written in Python 3 and is available as open source in the GitHub repository: https://github.com/DepledgeLab/DRUMMER. SUPPLEMENTARY INFORMATION/BACKGROUND:Supplementary data are available at Bioinformatics online.

The chemical modification of ribonucleotides plays an integral role in the biology of diverse viruses and their eukaryotic host cells. Mapping the precise identity, location, and abundance of modified ribonucleotides remains a key goal of many studies aimed at characterizing the function and importance of a given modification. While mapping of specific RNA modifications through short-read sequencing approaches has powered a wealth of new discoveries in the past decade, this approach is limited by inherent biases and an absence of linkage information. Moreover, in viral contexts, the challenge is increased due to the compact nature of viral genomes giving rise to many overlapping transcript isoforms that cannot be adequately resolved using short-read sequencing approaches. The recent emergence of nanopore sequencing, specifically the ability to directly sequence native RNAs from virus-infected host cells, provides not just a new methodology for mapping modified ribonucleotides but also a new conceptual framework for what can be derived from the resulting sequencing data. In this minireview, we provide a detailed overview of how nanopore direct RNA sequencing works, the computational approaches applied to identify modified ribonucleotides, and the core concepts underlying both. We further highlight recent studies that have applied this approach to interrogating viral biology and finish by discussing key experimental considerations and how we predict that these methodologies will continue to evolve.

Regulated loading of eIF3-bound 40S ribosomes on capped mRNA is generally dependent upon the translation initiation factor eIF4E; however, mRNA translation often proceeds during physiological stress, such as virus infection, when eIF4E availability and activity are limiting. It remains poorly understood how translation of virus and host mRNAs are regulated during infection stress. While initially sensitive to mTOR inhibition, which limits eIF4E-dependent translation, we show that protein synthesis in human cytomegalovirus (HCMV)-infected cells unexpectedly becomes progressively reliant upon eIF3d. Targeting eIF3d selectively inhibits HCMV replication, reduces polyribosome abundance, and interferes with expression of essential virus genes and a host gene expression signature indicative of chronic ER stress that fosters HCMV reproduction. This reveals a strategy whereby cellular eIF3d-dependent protein production is hijacked to exploit virus-induced ER stress. Moreover, it establishes how switching between eIF4E and eIF3d-responsive cap-dependent translation can differentially tune virus and host gene expression in infected cells.

Infection of immunosuppressed transplant patients with the human γ-herpesvirus Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD), an often fatal complication. Immunosuppressed miniature pigs infected with γ-herpesvirus porcine lymphotropic herpesvirus 1 (PLHV1) develop a similar disease, identifying pigs as a potential preclinical model for PTLD in humans. BILF1 is a G protein-coupled receptor (GPCR) encoded by EBV with constitutive activity linked to tumorigenesis and immunoevasive function downregulating MHC-I. In the present study, we compared BILF1-orthologues encoded by the three known PLHVs (PLHV1-3) with EBV-BILF1 to determine pharmacological suitability of BILF1 orthologues as model system to study EBV-BILF1 druggability. Cell surface localization, constitutive internalization, and MHC-I downregulation as well as membrane proximal constitutive Gα_i signaling patterns were conserved across all BILFs. Only subtle differences between the individual BILFs were observed in downstream transcription factor activation. Using Illumina sequencing, PLHV1 was observed in lymphatic tissue from PTLD-diseased, but not non-diseased pigs. Importantly, these tissues showed enhanced expression of PLHV1-BILF1 supporting its involvement in PTLD infection.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.

Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed direct long-read RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines the value of the SVV non-human primate model to develop new strategies for prevention of herpes zoster.

Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR/Cas9 and BAC technologies. We first established that CRISPR/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells.

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.