SUMMARY Herpes simplex virus type-1 (HSV-1) undergoes acute primary infection in epithelial cells followed by establishment of latency in the neurons of trigeminal ganglia. The latent virus maintains a life-long dormant state and can reactivate spontaneously in a small fraction of host cells, suggesting transcriptional silencing occurs in neurons. Computer data mining analyses identified a thyroid hormone response element (TRE), the binding site for the thyroid hormone receptor (TR), in the promoter of HSV-1 thymidine kinase (TK). TRs are transcription factors whose activity is dependent on their ligand thyroid hormone (TH or T3). We hypothesize that TR and T3 exert regulation on HSV-1 gene expression in neurons. A neuroblastoma cell line expressing the TR isoform β (N2aTRβ) was utilized for initial in vitro investigation. Liganded TR repressed TK promoter activity but unliganded TR relieved the inhibition. Chromatin immunoprecipitation (ChIP) assays showed that TRs were recruited to TK TRE regardless of the status of T3; hyperacetylated histone H3 at lysine 9 was associated with transcriptionally active promoters. In addition, ChIP results indicated that a repressive mono-methylated H3 modified at lysine 9 was enriched in TK promoter in the presence of TR and T3. T3-treated N2aTRβ cells were suppressive to TK expression and release of infectious viruses at low moi. RT-PCR and plaque assays showed that the TK can be de-repressed and the virus release was increased when the T3 was removed. These results suggest that T3 could regulate HSV-1 gene expression through its receptor via histone modification in cultured neuronal cells.

BackgroundHerpes simplex virus type-1 (HSV-1) infections can cause a number of diseases ranging from simple cold sores to dangerous keratitis and lethal encephalitis. The interaction between virus and host cells, critical for viral replication, is being extensively investigated by many laboratories. In this study, we tested the hypothesis that HSV-1 lytic infection triggers the expression of important multi-functional transcription factor Egr1. The mechanisms of induction are mediated, at least in part, by signaling pathways such as NFκB and CREB.MethodsSIRC, VERO, and 293HEK cell lines were infected with HSV-1, and the Egr-1 transcript and protein were detected by RT-PCR and Western blot, respectively. The localization and expression profile of Egr-1 were investigated further by immunofluorescence microscopy analyses. The recruitment of transcription factors to the Egr-1 promoter during infection was studied by chromatin immunoprecipitation (ChIP). Various inhibitors and dominant-negative mutant were used to assess the mechanisms of Egr-1 induction and their effects were addressed by immunofluorescence microscopy.ResultsWestern blot analyses showed that Egr-1 was absent in uninfected cells; however, the protein was detected 24-72 hours post treatment, and the response was directly proportional to the titer of the virus used for infection. Using recombinant HSV-1 expressing EGFP, Egr-1 was detected only in the infected cells. ChIP assays demonstrated that NFкB and cAMP response element binding protein (CREB) were recruited to the Egr-1 promoter upon infection. Additional studies showed that inhibitors of NFкB and dominant-negative CREB repressed the Egr-1 induction by HSV-1 infection.ConclusionCollectively, these results demonstrate that Egr-1 is expressed rapidly upon HSV-1 infection and that this novel induction could be due to the NFкB/CREB-mediated transactivation. Egr-1 induction might play a key role in the viral gene expression, replication, inflammation, and the disease progression.

Various factors/pathways including hormonal regulation have been suggested to control HSV-1 latency and reactivation. Our computer analysis identified a DNA repeat containing thyroid hormone response elements (TRE) in the regulatory region of HSV-1 LAT. Thyroid hormone (T3) exerts its function via its receptor (TR), a transcriptional factor. Present study investigated the roles of TR and T3 on HSV-1 gene expression using cultured neuoroblastoma cell lines. We demonstrated that liganded TR activated LAT transcription but repressed ICP0 transcription in the presence of LAT TRE. The chromatin immunoprecipitation (ChIP) assays showed that TRs were recruited to LAT TREs independently of T3 and hyperacetylated H4 was associated with promoters that were transcriptionally active. In addition, ChIP results showed that a chromatin insulator protein CTCF was enriched at the LAT TREs in the presence of TR and T3. In addition, chromatin remodeling factor BRG1 complex is found to participate in the T3/TR-mediated LAT activation since overexpression of BRG1 enhanced the LAT transcription and the dominant negative mutant K785R abolished the activation. This is the first report revealing that TR exerted epigenetic regulation on HSV-1 ICP0 expression in neuronal cells and could have a role in the complex processes of HSV-1 latency/reactivation.