Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Is a Coregulator of K-Rta: Physical Association and Promoter-Dependent Transcriptional Repression

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus that has been implicated in the pathogenesis of Kaposi's sarcoma and B-cell neoplasms. The genomic organization of KSHV is similar to that of Epstein-Barr virus (EBV). EBV encodes two transcriptional factors, Rta and Zta, which functionally interact to transactivate EBV genes during replication and reactivation from latency. KSHV encodes a basic leucine zipper protein (K-bZIP), a homologue of EBV Zta, and K-Rta, the homologue of EBV Rta. EBV Rta and Zta are strong transcriptional transactivators. Although there is ample evidence that K-Rta is a potent transactivator, the role of K-bZIP as a transcriptional factor is much less clear. In this study, we report that K-bZIP modulates K-Rta function. We show that K-bZIP directly interacts with K-Rta in vivo and in vitro. This association is specific, requiring the basic domain (amino acids 122 to 189) of K-bZIP and a specific region (amino acids 499 to 550) of K-Rta, and can be detected with K-bZIP and K-Rta endogenously expressed in BCBL-1 cells treated with tetradecanoyl phorbol acetate. The functional relevance of this association was revealed by the observation that K-bZIP represses the transactivation of the ORF57 promoter by K-Rta in a dose-dependent manner. K-bZIP lacking the interaction domain fails to repress K-Rta-mediated transactivation; this finding attests to the specificity of the repression. Interestingly, this repression is not observed for the promoter of polyadenylated nuclear (PAN) RNA, another target of K-Rta; thus, repression is promoter dependent. Finally, we provide evidence that the modulation of K-Rta by K-bZIP also occurs in vivo during reactivation of the viral genome in BCBL-1 cells. When K-bZIP is overexpressed in BCBL-1 cells, the level of expression of ORF57 but not PAN RNA is repressed. These data support the model that one function of K-bZIP is to modulate the activity of the transcriptional transactivator K-Rta.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Protein Kinase and Its Interaction with K-bZIP

“…Previously, we showed that K-bZIP is a strong transcriptional repressor, which modulates the transcription of K-bZIP and ORF57 promoters mediated by K-Rta (20,29). Based on ChIP assays, K-bZIP is recruited to these two promoters and Ori-Lyt DNA during the early phase of KSHV lytic replication (19).…”

“…Plasmids encoding the full-length K-bZIP and K-Rta genes were described previously (20). This cloning introduced a CpoI site and a Flag tag or T7 tag at the N terminus of each protein as described previously (20).…”

“…Glutathione transferase (GST) fusion proteins were expressed in Escherichia coli strain BL21 transformed with pGEX-ORF36 N-151, pGEX-K-bZIP N-75, pGEX-K-bZIP 76-150 pGEX-K-bZIP 151-C, pGEX-K-bZIP 72-102, pGEX-K-bZIP 87-123, pGEX-KbZIP 87-123 (T111A), pGEX-K-bZIP 113-154, pGEX-K-bZIP 128-154, or pGEX4T-2 (N and C correspond to termini; number represents amino acid position). The procedure of purification of GST fusion proteins was described previously (20).…”

“…To detect low-abundance ORF34 and ORF35 transcripts, RNA probes were generated by T7 RNA polymerase with Strip-EZ RNA kit (Ambion) and incorporated with [␣ 32 -P]UTP. Hybridization was performed at 42°C for DNA probes and at 64°C for RNA probes as described previously (20).…”

“…Beads were washed four times with RIPA buffer and then boiled for 5 min in 20 l of 2ϫ SDS sample buffer. Protein samples from total cell lysates (50 g/lane) or immunoprecipitates were subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membrane as described above and previously (20). For detecting small ubiquitin-like modifier protein (SUMO)-modified K-bZIP, cells were washed twice with PBS and lysed with SUMO lysis buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 20 M N-ethylmalemide [Sigma]) supplemented with a protease inhibitor cocktail (Roche).…”