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Journal of Biomedical Science

, 19:12

First Online: 02 February 2012Received: 10 January 2012Accepted: 02 February 2012DOI: 10.1186-1423-0127-19-12

Cite this article as: Ko, YC., Tsai, WH., Wang, PW. et al. J Biomed Sci 2012 19: 12. doi:10.1186-1423-0127-19-12 ABSTRACT

BackgroundThe 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.

MethodsBy 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.

ResultsWe 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.

ConclusionsKSHV 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.

KeywordsKSHV K-RTA O-GlcNAcylation PARP1 Polycomb group PcG complex Electronic supplementary materialThe online version of this article doi:10.1186-1423-0127-19-12 contains supplementary material, which is available to authorized users.

Ying-Chieh Ko, Wan-Hua Tsai contributed equally to this work.

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Autor: Ying-Chieh Ko - Wan-Hua Tsai - Pei-Wen Wang - I-Lin Wu - Shu-Yu Lin - Yu-Lian Chen - Jen-Yang Chen - Su-Fang Lin

Fuente: https://link.springer.com/







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