Suppressive regulation of KSHV RTA with O-GlcNAcylation.
J Biomed Sci
; 19: 12, 2012 Feb 02.
Article
em En
| MEDLINE
| ID: mdl-22300411
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
Regulação Viral da Expressão Gênica
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Transativadores
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N-Acetilglucosaminiltransferases
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Proteínas Imediatamente Precoces
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Herpesvirus Humano 8
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article