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. © 2012 ko et al; licensee biomed central ltd.