Sirtuin 6 Attenuates Kaposi's Sarcoma–associated herpesvirus (KSHV) Reactivation via Suppressing the Ori-Lyt Activity and Expression of RTA

Abstract

Kaposi's sarcoma–associated herpesvirus (KSHV, also called human herpesvirus 8, HHV-8), upon being reactivated, causes serious diseases in immunocompromised individuals. Its reactivation has not been fully understood, especially how the cellular regulating mechanisms play roles in KSHV gene expression and viral DNA replication. In searching for the cellular factors that regulate KSHV gene expression, we found that several histone deacytelases (HDACs) and sirtuins (SIRTs), including HDAC 2, 7, 8 and 11, and SIRT 4 and 6, repress KSHV ori-Lyt promoter activity. Interestingly, the nuclear protein, SIRT6 presents the greatest inhibitory effect on ori-Lyt promoter activity. A more detailed investigation reveals that SIRT6 exerted repressive effects on multiple promoter of KSHV. As a consequence of inhibiting the KSHV promoters, SIRT6 not only represses viral protein production, but also inhibits viral DNA replication as investigated in a KSHV-contained cell line, SLK-iBAC-gfpK52. Depletion of the SIRT6 protein using the siRNA cannot directly reactivate KSHV from the SLK-iBAC-gfpK52, but makes it more effective in reactivating KSHV by a low amount of the reactivator (Doxycyclin) and enhances the viral DNA replication in KSHV infection system. We performed DNA chromatin immunoprecipitation (ChIP) assays for SIRT6 in SLK-iBAC-gfpK52 cell line to determine whether SIRT6 interacts with KSHV genome in order to play the regulatory effects. Our results suggest that SIRT6 interacts with KSHV ori-Lyt and ORF50 promoters. Furthermore, the SIRT6-KSHV DNA interaction is significantly negated by reactivation. Therefore, we identified a cellular regulator, SIRT6 that represses KSHV replication through interacting with KSHV DNA and inhibiting viral gene expression.

IMPORTANCE Kaposi's sarcoma–associated herpesvirus (KSHV) is a pathogen causing cancer in the immune-deficiency population. The reactivation of KSHV from latency is important for it to be carcinogenic. Our finding that SIRT6 plays inhibitory effects on KSHV reactivation through interaction with viral genome and suppressing viral gene expression is important because it might lead to a strategy of interfering with KSHV reactivation. Overexpression of SIRT6 represses several KSHV promoters' activities leading to a reduced gene expression and DNA replication of KSHV in a KSHV BAC-containing cell line. Depletion of SIRT6 favors reactivation of KSHV from SLK-iBACV-gfpK52. More importantly, we reveal that SIRT6 interacts with KSHV DNA. Whether the interaction of SIRT6 with KSHV DNA occurs at a global level will be further studied in the future.