Regulation of the Varicella Zoster Virus latent-lytic switch

• Applicant: Professor Dr. Daniel Depledge
• Subject Area: Virology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 538763660

VZV is a neurotropic human alphaherpesvirus that establishes lifelong latency in sensory neurons from which it may later reactivate, potentially with severe complications. However, the exact mechanisms that govern reactivation remain elusive. VZV latency is characterized by the sole expression of the VZV latency-associated transcript (VLT) that consists of five exons. Notably however, during reactivation and in productive infections, 3’ end processing of VLT is disrupted and leads instead to the expression of a fusion transcript that consists of VLT and the adjacent ORF63 (termed VLT63). Importantly, we have previously shown that VLT63 is induced in the presence of reactivation stimuli, and that transduction of VZV latently infected sensory neurons with VLT63 is sufficient to induce broad viral gene expression. Identifying the mechanism that regulates VLT 3’ end processing during latency and reactivation is thus the major aim of this proposal. We have previously observed that a U1 snRNP binding site and a polyadenylation signal site colocalize at the 3’ end of VLT exon 5. Prior studies have shown that U1 snRNP binding in proximity to cleavage and polyadenylation (CPA) sites is sufficient to disrupt 3’ end processing. We will thus test the hypothesis that U1 snRNP is a key regulator of VZV latency and reactivation. Our major objectives are to (i) characterize the transcriptional and translational complexity of the VLT63 locus in distinct cellular environments, (ii) mechanistically dissect the role of U1snRNP in regulating VLT/VLT63 isoform abundance, and (iii) characterize the VZV latent/lytic switch in sensory neurons. This will be achieved using a combination of classical molecular biology, advanced sequencing methods (i.e. nanopore sequencing), and computational biology. Given the challenges of working with VZV, a highly cell-associated virus for which there is no useful animal model, we will conduct our studies with the help of several long-established collaborators with complementary expertise in VZV mutagenesis and neuronal latency models. Taken together, this study will definitively address (i) the importance of VLT/VLT63 transcript diversity in both productive and latent/reactivating infections and reveal the underlying factors that enable this diversity, (ii) the mechanistic role of U1snRNP in regulating PCPA across the VZV genome and, more specifically, the VLT63 locus, and (iii) the requirement for VLT and VLT63 in establishing, maintaining, and reactivating from viral latency.

• DFG Programme: Research Grants