Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection within sensory 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 our research is to determine how HSV-1 lytic genes are silenced in neurons during latency, and how this process is reversed during reactivation. During the past project period our studies have implicated the polycomb repressor complex (PRC) as an important epigenetic regulator of HSV latency and reactivation. The overall hypothesis to be tested in this proposal is that viral regulation of PRC binding to the HSV genome plays a key role in controlling the degree of suppression of lytic genes during latency in a manner that facilitates reactivation. The experiments proposed here will define the viral elements that are involved in this process.
In Aim 1 we will identify the regions of the HSV-1 genome that recruit the polycomb establishment complex (PRC2). Cellular genes silenced by H3K27triMe recruit PRC2 via cis DNA elements or indirectly through interactions with YY1 or non-coding RNAs (ncRNAs). PRC2 then methylates histone H3 at lysine 27 which leads to heterochromatin formation. Therefore we will determine if PRC2 binds directly to elements on the HSV-1 genome, or whether this binding is mediated through other factors. After heterochromatin is established a second PRC, the PRC maintenance complex (PRC1), recognizes the H3K27triMe mark and maintains the repressed state. RNA immunoprecipitation (RIP) data indicate that the LAT 2.0kb stable intron binds to the PRC1 suggesting that the intron sequesters PRC1 and reduces the degree of H3K27triMe repression on the latent genomes. We hypothesize that this competition with PRC1 binding is a critical factor in maintaining the lytic genes in a more flexible """"""""suppressed but reversible"""""""" heterochromatic state facilitating reactivation.
In Aim 2 we will characterize the binding of PRC1 to the LAT intron and determine if reducing the amount of PRC1 in the cell is suficient to enhance HSV reactivation. Finally, in order for reactivation to occur, the PRC1 repression must be released and the chromatin associated with the HSV-1 lytic promoters must remodel to a transcriptionaly permissive state. We have previously shown that during explant-induced reactivation of latent murine ganglia that there is a transient increase in LAT abundance. The goal of Aim 3 will be to determine how the H3K27triMe mark is removed in order to allow lytic transcription to proceed, and whether the LAT is directly involved in this process. The proposed studies will provide key insight into the mechanism of lytic gene silencing during HSV-1 latency and details concerning the novel role that the LAT intron plays in modulating H3K27triMe. In addition this work may provide new insight into novel mechanisms of PRC regulation of cellular genes.

Public Health Relevance

Herpes simplex virus (HSV) causes cold sores and other serious disease in humans including encephalitis and blindness. While there are antiviral drugs to treat herpes, they don't completely block the infection and there is no cure. This research is aimed at studying a cellular protein complex, PRC, involved in controlling HSV genes during latent infection and could result in identifying new targets to develop better therapies for treating herpes infections.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - B Study Section (VIRB)
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Challberg, Mark D
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University of Florida
Schools of Medicine
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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
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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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