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.
Highlights
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
293TetNLSm and 293TetER [21] were immunoprecipitated with RL2, an antibody recognizing the O-GlcNAc epitope, followed by western blot analysis using the M2-FLAG antibody. We found that both KRTA and its mutant defective in nuclear localization signal (NLSm), but not EBV Rta, were recognized by RL2 (Figure 1D), indicating that KSHV RTA (K-RTA) is O-GlcNAcylated
Given that O-GlcNAcylation is a dynamic process keenly responding to glucose fluctuation, we speculate that the activity of K-RTA is closely controlled by the metabolic state of the host cell
Summary
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. The replication and transcription activator K-RTA ( known as ORF50 or Lyta) is the immediate-early protein of Kaposi’s sarcoma-associated herpesvirus (KSHV) that orchestrates and completes a KSHV lytic cycle of replication in many cell backgrounds. O-GlcNAcylation is a dynamic process that is catalyzed by O-GlcNAc transferase (OGT) and reversed by OGlcNAcase ( known as OGA, NCOAT, MGEA5) [9]. Because both O-GlcNAcylation and O-phosphorylation act on the side chains of Ser and Thr residues, interplays between the two reactions have long been suspected. Large-scale proteomic analysis revealed that the crosstalk between O-GlcNAcylation and O-phosphorylation can be derived from direct competition for a structural occupancy or by alteration of each other’s enzyme activity via reciprocal modifications [12]
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