Suppressive Regulation of KSHV RTA with O-GlcNAcylation

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.

Inhibition of O-Linked N -Acetylglucosamine Transferase Reduces Replication of Herpes Simplex Virus and Human Cytomegalovirus

“…Also, increasing O-GlcNAcylation by overexpression of OGT reduced KSHV replication (49). In another study, increased O-GlcNAcylation of the KSHV major replication and transcriptional activator (RTA) reduced its ability to transactivate viral genes (34). Consistent with this study, we observed that OGT inhibition did not prevent reactivation of latent KSHV, as measured by the levels of lytic gene expression (results not shown).…”

“…Several studies implicate O-GlcNAcylation as an important modification that can regulate the replication cycle of viruses. O-GlcNAc modification has been found on many viral proteins, including HCMV UL32 tegument protein (45), adenovirus fiber protein (46), baculovirus tegument protein gp41 (47), rotavirus NS26 protein (48), and several KSHV proteins involved in DNA replication (34,49). A few studies suggest that high O-GlcNAc levels have an inhibitory effect on viral replication.…”

Downregulation of Poly(ADP-Ribose) Polymerase 1 by a Viral Processivity Factor Facilitates Lytic Replication of Gammaherpesvirus

“…Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant nuclear protein that catalyzes the poly(ADP-ribosyl)ation of target proteins acting in DNA repair, cell cycle control, apoptosis, and gene expression (5)(6)(7). In KSHV and MHV-68 infections, PARP-1 interacts with the poly(ADP-ribosyl)ate replication and transcription activator (RTA), the molecular switch of lytic replication, thereby repressing lytic viral replication (8)(9)(10)(11). PARP-1 is also known to be a component of the viral replication complex, increasing viral genome replication (9,12).…”

KSHV Reactivation and Novel Implications of Protein Isomerization on Lytic Switch Control

“…Rta protein has an apparent molecular weight of 73.7–120 kilodalton (kDa), a difference indicative of its extensive posttranslational modifications, predominantly phosphorylation (20 kDa alone) [ 7 , 34 , 37 , 42 , 43 , 44 ]. Rta is also ADP-ribosylated [ 45 ], and may contain other modifications.…”