As highlighted in PA-19-237, there is a gap in knowledge about non-coding RNAs and their functions for many pathogens, and the human herpesvirus Varicella Zoster Virus (VZV) is one such pathogen. VZV is one of only two of nine human herpesviruses in which microRNAs have not been reported. Our searches identified a family of novel VZV-encoded small non-coding RNAs (VZVsncRNA), suggesting an exciting possibility that VZV regulates its infectious process using sncRNA. Our overarching premise is that better knowledge of sncRNA could form the basis for novel therapies for VZV disease, which is still a major public health concern. While childhood ?chickenpox? is no longer common in the US due to varicella vaccination, most adults still harbor wild-type VZV in their ganglia in a latent state, with potential to reactivate. When it does, Herpes Zoster develops, a painful morbid and debilitating disease encountered by a third in their lifetime that is frequently complicated, most often by chronic pain. There is still some half of a million HZ cases annually. We detected these VZVsncRNA in both infected epithelial cells and lytic infected neurons. Importantly, cells transfected with locked nucleic acid (LNA) inhibitors to them alter VZV spread and growth. Here we focus on four VZVsncRNA which are antisense to the VZV Latency Transcript (VLT) found in latently infected human ganglia and productive infections, as LNA inhibitors to them reduce VZV virus production (indicating the sncRNA promote infection). A combination of three LNA inhibitors effectively reduced VZV growth by more than 90%, not only supporting a functional role in infection but also establishing a promising novel therapeutic strategy. We hypothesize that these sncRNA regulate epithelial productive infections and in neurons, the lytic/latency decisions that must be made.
Aim 1 addresses how these four VZVsncRNA influence VZV growth in epithelial cells using LNA modulators, in single and in combination, to determine how they change viral expression patterns. To determine if they target virus or host, we will develop VZV altered in sequence encoding the VZVsncRNA and using them in conjunction with epithelial cell lines expressing the VZVsncRNA. Our hypothesis predicts that specific virus mutation will prevent expression of sncRNA from influencing VZV infection; if not, it will indicate sncRNA may target host pathways important for VZV. Our primary hypothesis is that sncRNA target VLT RNA to influence expression of the critical pro-lytic ORF61 protein.
Our second aim will test the hypotheses that the four VZVsncRNA promote lytic infection or enhance reactivation from latency in neurons. We will exploit our human neuron culture system that hosts a model VZV latent state and experimental reactivation from it. We will develop AAV to express VZVsncRNA in latently infected neurons and determine if reactivation efficiency is favored. Globally, our studies will set the stage for understanding a novel gene regulatory mechanism for VZV, their possible functions in interacting with VLT and the potential for targeting sncRNA as a future therapeutic approach for treating Herpes zoster.

Public Health Relevance

Most adults have VZV in their nervous system and are at risk for herpes zoster, a debilitating disease with potential life-changing consequences. This project examines how small non-coding RNAs influence VZV growth and re-emergence from latency, and may establish a basis of a novel antiviral strategy to VZV disease.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Virology - B Study Section (VIRB)
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Beisel, Christopher E
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University of Pittsburgh
Internal Medicine/Medicine
Schools of Medicine
United States
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