Suppressive regulation of KSHV RTA with O-GlcNAcylation

• Published in: Journal of biomedical science Vol. 19; no. 1; p. 12
• Main Authors: Ko, Ying-Chieh, Tsai, Wan-Hua, Wang, Pei-Wen, Wu, I-Lin, Lin, Shu-Yu, Chen, Yu-Lian, Chen, Jen-Yang, Lin, Su-Fang
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 02.02.2012; BioMed Central; BMC
• Subjects: Acylation; Alanine - chemistry; Amino Acid Substitution; Amino acids; Analysis; Blotting, Western; Chromatin; Colleges & universities; Deoxyribonucleic acid; DNA; DNA repair; Epigenetics; Experiments; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation, Viral; Genomes; HEK293 Cells; Herpesvirus 8, Human - genetics; Herpesvirus 8, Human - metabolism; Human herpesvirus 8; Humans; Immediate-Early Proteins - genetics; Immediate-Early Proteins - metabolism; Immunoprecipitation; K-RTA; Kaposi's sarcoma-associated herpesvirus; KSHV; Luciferase; Medical research; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; O-GlcNAcylation; Oligopeptides; PARP1; Peptides - metabolism; Phosphorylation; Plasmids; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases - metabolism; Polyclonal antibodies; Polycomb group (PcG) complex; Proteins; RNA polymerase; RNA, Small Interfering - metabolism; Stem cells; Tandem Mass Spectrometry; Threonine - chemistry; Trans-Activators - genetics; Trans-Activators - metabolism
• ISSN: 1423-0127; 1021-7770; 1423-0127
• DOI: 10.1186/1423-0127-19-12

The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a molecular switch that initiates a productive replication of latent KSHV genomes. KSHV RTA (K-RTA) is composed of 691 amino acids with high Ser and Thr content (17.7%), but to what extent these Ser and Thr are modified in vivo has not been explored. By using tandem mass spectrometric analysis of affinity-purified FLAG tagged K-RTA, we sought to identify Ser and Thr residues that are post-translationally modified in K-RTA. We found that K-RTA is an O-GlcNAcylated protein and Thr-366/Thr-367 is the primary motif with O-GlcNAcylation in vivo. The biological significance of O-GlcNAc modified Thr-366 and Thr-367 was assessed by site-specific amino acid substitution. Replacement of Thr with Ala at amino acid 366 or 367 caused a modest enhancement of K-RTA transactivation activity in a luciferase reporter assay and a cell model for KSHV reactivation. By using co-immunoprecipitation coupled with western blot analysis, we showed that the capacity of K-RTA in associating with endogenous PARP1 was significantly reduced in the Thr-366/Thr-367 O-GlcNAc mutants. PARP1 is a documented negative regulator of K-RTA that can be ascribed by the attachment of large negatively charged polymer onto K-RTA via PARP1's poly (ADP-ribose) polymerase activity. In agreement, shRNA-mediated depletion of O-GlcNAc transferase (OGT) in KSHV infected cells augmented viral reactivation and virus production that was accompanied by diminished K-RTA and PARP1 complexes. KSHV latent-lytic switch K-RTA is modified by cellular O-GlcNAcylation, which imposes a negative effect on K-RTA transactivation activity. This inhibitory effect involves OGT and PARP1, two nutritional sensors recently emerging as chromatin modifiers. Thus, we speculate that the activity of K-RTA on its target genes is continuously checked and modulated by OGT and PARP1 in response to cellular metabolic state.