Suppressive regulation of KSHV RTA with O-GlcNAcylation.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
National Institute of Cancer Research, National Health Research Institutes, Miaoli County, Taiwan.
Ying-Chieh
YC
Ko
Wan-Hua
WH
Tsai
Pei-Wen
PW
Wang
I-Lin
IL
Wu
Shu-Yu
SY
Lin
Yu-Lian
YL
Chen
Jen-Yang
JY
Chen
Su-Fang
SF
Lin
D000215:Acylation; D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D015153:Blotting, Western; D008401:Gas Chromatography-Mass Spectrometry; D015967:Gene Expression Regulation, Viral; D057809:HEK293 Cells; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D047468:Immunoprecipitation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000737:chemistry; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
D017874:Immediate-Early Proteins; D009842:Oligopeptides; D010455:Peptides; D034741:RNA, Small Interfering; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D013912:Threonine; C085706:FLAG peptide; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D000409:Alanine