Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection within peripheral neurons. During latency the viral genomes are maintained as circular episomes and the lytic genes are silenced. Periodically the genomes within some of the neurons reactivate resulting in recurrent clinical disease. A major focus of the proposed research is to determine the viral and cellular factors responsible for regulating HSV-1 latency. During the past project period three major findings were: 1) the histone H3K27triMe demethylases UTX and JMJD3 play a major role in removing repressive heterochromatic histone H3K27triMe marks to facilitate reactivation. In addition we found that while most non-terminally differentiated cells express UTX and JMJD3 constitutively, sensory neurons do not, but these proteins are induced by at least some reactivation stimuli; 2) we identified 2 previously un-reported long non-coding RNAs (TAL and ATAL) that are antisense to each other and the 5' end of the LAT. Significantly, existing LAT promoter mutants reduce the levels of all three transcripts (LAT, TAL and ATAL). In addition, we found that the TAL and ATAL transcripts are differentially expressed in neurons with only partial overlap with neurons expressing the LAT; 3) we have developed a means to knockdown viral and cellular genes in sensory neurons in vivo using AAV vectors. By knocking down the LAT after the establishment of latency we demonstrate that the LAT RNA specifically contributes to reactivation. In order to extend the last two of these findings, we propose the following aims: SA1: Dissect the functional roles that the newly identified LAT region ncRNAs TAL and ATAL play in regulating HSV-1 latency and reactivation; SA2: Characterize the functional roles that the LAT region miRNAs play in regulating HSV-1 phenotypes attributed to the LAT. Finally, it is becoming clear from the work of our lab and others that HSV latency is more dynamic that previously appreciated, and that latent gene expression patterns are heterogeneous. This is highlighted by the finding that the LAT, TAL and ATAL transcripts are expressed in only partially overlapping populations of cells. Therefore in our final aim SA3, we propose to use single cell RNA-seq analyses to identify cell type, viral and host gene signatures of HSV-1 latency and reactivation. For all of these aims we will use well-established mouse and rabbit models of HSV-1 latency and reactivation, but will extend these studies to include a novel human neuronal model of HSV-1 latency and validate these findings, where possible, to analyses of latently infected human trigeminal ganglia. The proposed studies will provide novel insights into the cellular and viral mechanisms regulating HSV-1 latency, and allow the assignment of specific functions to the lncRNAs and miRNAs transcribed from the LAT region. These studies will also provide new critical details concerning the basis of heterogeneity of gene HSV-1 gene expression in different neurons during latency.

Public Health Relevance

/RELEVANCE Herpes simplex virus (HSV) causes cold sores, genital lesions and other serious disease in humans including encephalitis and blindness. While there are antiviral drugs to treat herpes, they are not completely effective and there is no vaccine or cure. The proposed research aims to study how viral non-coding RNAs and cellular factors control HSV latent infection and could identify new targets for better therapies to treat recurrent herpes.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Beisel, Christopher E
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University of Florida
Schools of Medicine
United States
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Watson, Zachary L; Washington, Shannan D; Phelan, Dane M et al. (2018) In Vivo Knockdown of the Herpes Simplex Virus 1 Latency-Associated Transcript Reduces Reactivation from Latency. J Virol 92:
Washington, Shannan D; Edenfield, Samantha I; Lieux, Caroline et al. (2018) Depletion of the insulator protein CTCF results in HSV-1 reactivation in vivo. J Virol :
Morse, Alison M; Calabro, Kaitlyn R; Fear, Justin M et al. (2017) Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing. Viruses 9:
Watson, Zachary L; Ertel, Monica K; Lewin, Alfred S et al. (2016) Adeno-associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Coinfected with Herpes Simplex Virus 1 following Peripheral Inoculation. J Virol 90:7894-901
Messer, Harald G P; Jacobs, Derek; Dhummakupt, Adit et al. (2015) Inhibition of H3K27me3-specific histone demethylases JMJD3 and UTX blocks reactivation of herpes simplex virus 1 in trigeminal ganglion neurons. J Virol 89:3417-20
Yang, Yajie; Fear, Justin; Hu, Jianhong et al. (2014) Leveraging biological replicates to improve analysis in ChIP-seq experiments. Comput Struct Biotechnol J 9:e201401002
Watson, Zachary; Dhummakupt, Adit; Messer, Harald et al. (2013) Role of polycomb proteins in regulating HSV-1 latency. Viruses 5:1740-57
Bertke, Andrea S; Apakupakul, Kathleen; Ma, AyeAye et al. (2012) LAT region factors mediating differential neuronal tropism of HSV-1 and HSV-2 do not act in trans. PLoS One 7:e53281
Bloom, David C; Giordani, Nicole V; Kwiatkowski, Dacia L (2010) Epigenetic regulation of latent HSV-1 gene expression. Biochim Biophys Acta 1799:246-56
Kwiatkowski, Dacia L; Thompson, Hilary W; Bloom, David C (2009) The polycomb group protein Bmi1 binds to the herpes simplex virus 1 latent genome and maintains repressive histone marks during latency. J Virol 83:8173-81

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