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.

Abbreviations: early growth response gene 1 (Egr-1); herpes simplex virus type 1 (HSV-1); immediate-early gene(s) (IE gene(s)); infected cell protein No. 22 (ICP22); infected cell protein No. 4 (ICP4); histone deacetylase complexes (HDACs); NGFI-A/EGR1-binding protein (Nab2); chromatin immunoprecipitation (ChIP); secreted alkaline phosphatase (SEAP) The molecular mechanisms mediating herpes simplex virus type 1 (HSV-1) gene silencing during latent infection are not clear. Five copies of early growth response gene 1 (Egr-1) binding elements were identified in the intron of HSV-1 ICP22 (infected cell protein No. 22) gene, leading to the hypothesis that Egr-1 binds to the viral genome and regulates the viral gene expression. Transient co-transfection assays indicated that Egr-1 negatively regulated the transcription of both full-length and intron-removed ICP22 promoters. The same assays also revealed that Egr-1 repressed ICP4 (infected cell protein No. 4) promoter activity in a dose-dependent manner but showed less inhibition when the intron was removed. Histone deacetylation was not involved in this regulation since histone deacetylase inhibitor trichostatin A did not exhibit any effect on Egr-1-mediated repression. Chromatin immunoprecipitation assays showed that Egr-1 reduced the binding of Sp1 to the promoters and that the co-repressor Nab2 (NGFI-A/EGR1-binding protein) was recruited to the proximity of ICP4 in the presence of Egr-1. These results suggested that the multifunctional transcription factor Egr-1 can repress HSV-1 immediate-early gene expression through the recruitment of co-repressor Nab2 and reduction of Sp1 occupancy, and thus may play a critical role in HSV-1 gene silencing during latency.

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.