Regulation of herpes simplex virus type 1 thymidine kinase gene expression by thyroid hormone receptor in cultured neuronal cells

Abstract

Herpes simplex virus type 1 (HSV-1) undergoes acute infection in epithelial cells followed by establishment of latency in the neurons of trigeminal ganglia. The latent virus maintains a dormant state and can recurs spontaneously, suggesting transcriptional silencing and reactivation occur in neurons. Computer data mining identified a nuclear hormone response element (NRE), the binding site for the thyroid hormone receptor (TR) or other nuclear hormone receptor, in the promoter of HSV-1 thymidine kinase (TK). TRs are transcription factors whose activity is dependent on their ligand thyroid hormone (T(3); triiodothyronine). We hypothesize that TR and T(3) exert regulation on HSV-1 gene expression in neurons. A neuroblastoma cell line expressing the TR isoform beta (N2aTRbeta) was utilized for in vitro investigation. Results showed that liganded TR repressed TK promoter activity but unliganded TR relieved the inhibition. The mutagenesis study demonstrated that one nucleotide mutation at the NRE abolished the T(3)/TR-mediated regulation. N2aTRbeta cells treated with T(3) were suppressive to TK expression and virus release but the removal of T(3) de-repressed TK expression and increased virus release, confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and plaque assays, respectively. Chromatin immunoprecipitation (ChIP) assays showed that TRs were enriched at TK NRE in the presence of T(3). Additional results demonstrated that hyper acetylated histone H4 and monomethylated H3 modified at lysine 9 (H3K9me1) were enriched at transcriptionally active TK promoters but were dissociated from the NRE by T(3)/TR. These results suggest that T(3) could regulate HSV-1 gene expression through its receptor via histone modification in cultured neuronal cells.

Fig. 1. HSV-1 TREs

1A: Negative TREs were identified in the HSV-1 TK promoter. The TREs were organized as inverted repeats with six nucleotides spacing (IR6) and located immediately after the TATA box. Transcription initiation site, Sp1 elements and CAAT box were shown.

Fig. 2. HSV-1 TK transcription was negatively regulated by TR and T₃

N2a and N2aTRβ cells were cotransfected with various plasmids by Nucleofector II with duplication to analyze the regulatory effects of TR and T₃ on TK promoter. Plasmid pEGFP was used as a reference to monitor the transfection efficiency. Plasmid pICP4 was used as normalization control. Fluorescent microscopy was performed using Nikon Eclipse TS100 microscope. Cells were subjected to luciferase assay using Promega Luciferase Assay Kit at 48-hrs post-transfection. The signal was measured by luminometer. The results were normalized by SEAP assay driven by ICP4 promoter. Error bars represent the mean plus or minus the standard deviation.

Fig. 3. HSV-1 TK was repressed by liganded TR during infections

3A: N2a and N2aTRβ cells were infected with wild-type virus 17Syn⁺ at moi=10 “in the absence or presence of T₃ with 50 µg/ml cycloheximide to block the protein synthesis of HSV-1 α genes. The total RNA was purified and subjected to RT-PCR by primers against TK. Actin primers were used as controls. 3B: N2aTRβ cells were infected with wild-type virus 17Syn⁺ at moi=1. The total RNA was purified at 8 hpi and 48 hpi then subjected to RT-PCR using primers against TK and actin. The gel showed that TK transcription was delayed until 48 hpi and was repressed by the addition of T₃ (compare lanes 3 and 4).

Fig. 3. HSV-1 TK was repressed by liganded TR during infections

3A: N2a and N2aTRβ cells were infected with wild-type virus 17Syn⁺ at moi=10 “in the absence or presence of T₃ with 50 µg/ml cycloheximide to block the protein synthesis of HSV-1 α genes. The total RNA was purified and subjected to RT-PCR by primers against TK. Actin primers were used as controls. 3B: N2aTRβ cells were infected with wild-type virus 17Syn⁺ at moi=1. The total RNA was purified at 8 hpi and 48 hpi then subjected to RT-PCR using primers against TK and actin. The gel showed that TK transcription was delayed until 48 hpi and was repressed by the addition of T₃ (compare lanes 3 and 4).

Fig. 4. TRβ is overexpressed in N2aTRβ cells

Protein extracts were purified from N2a and N2aTRβ cells and subjected to gel electrophoresis followed by immunoblot analysis using Abs against TRβ1 and α-tubulin.

Fig. 5. TRβ bound to the TK promoter and induced T₃-mediated histone modification

5A: Cells were transfected with pRL-TK and treated as noted. The transfected cells were subjected to ChIP after 48 h of treatment and immunoprecipitated by Abs against TRβ or acetylated histone H4. 5B: ChIP was performed using the conditions of 5A and immunoprecipitated by anti-mono methylated H3K9 Ab.

Fig. 5. TRβ bound to the TK promoter and induced T₃-mediated histone modification

5A: Cells were transfected with pRL-TK and treated as noted. The transfected cells were subjected to ChIP after 48 h of treatment and immunoprecipitated by Abs against TRβ or acetylated histone H4. 5B: ChIP was performed using the conditions of 5A and immunoprecipitated by anti-mono methylated H3K9 Ab.

Fig. 6. Long-term T3 treatment prevented the TK expression and the washout of T₃ induced the TK expression and virus release

6A: Cells were pre-treated with T₃ for five days and subsequently infected with 17Syn⁺-EGFP viruses at moi of 1. Total RNA was isolated at 48 hpi and subjected to RT-PCR assays using TK primers to study TR-mediated regulation. RT-PCR assays using EGFP primers were performed as internal viral infection control. 6B: The media of infected cells were collected 48 hpi and subjected to plaque assays using CV1 cells to investigate the release of infectious viruses.

Fig. 6. Long-term T3 treatment prevented the TK expression and the washout of T₃ induced the TK expression and virus release

6A: Cells were pre-treated with T₃ for five days and subsequently infected with 17Syn⁺-EGFP viruses at moi of 1. Total RNA was isolated at 48 hpi and subjected to RT-PCR assays using TK primers to study TR-mediated regulation. RT-PCR assays using EGFP primers were performed as internal viral infection control. 6B: The media of infected cells were collected 48 hpi and subjected to plaque assays using CV1 cells to investigate the release of infectious viruses.