Cooperation between Viral Interferon Regulatory Factor 4 and RTA To Activate a Subset of Kaposi's Sarcoma-Associated Herpesvirus Lytic Promoters

Abstract

The four Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded interferon (IFN) regulatory factor homologues (vIRF1 to vIRF4) are used to counter innate immune defenses and suppress p53. The vIRF genes are arranged in tandem but differ in function and expression. In KSHV-infected effusion lymphoma lines, K10.5/vIRF3 and K11/vIRF2 mRNAs are readily detected during latency, whereas K9/vIRF1 and K10/vIRF4 mRNAs are upregulated during reactivation. Here we show that the K10/vIRF4 promoter responds to the lytic switch protein RTA in KSHV-infected cells but is essentially unresponsive in uninfected cells. Coexpression of RTA with vIRF4 is sufficient to restore regulation, a property not shared by other vIRFs. The K9/vIRF1 promoter behaves similarly, and production of infectious virus is enhanced by the presence of vIRF4. Synergy requires the DNAbinding domain (DBD) and C-terminal IRF homology regions of vIRF4. Mutations of arginine residues within the putative DNA recognition helix of vIRF4 or the invariant cysteines of the adjacent CxxC motif abolish cooperation with RTA, in the latter case by preventing self-association. The oligomerization and transactivation functions of RTA are also essential for synergy. The K10/ vIRF4 promoter contains two transcription start sites (TSSs), and a 105-bp fragment containing the proximal promoter is responsive to vIRF4/RTA. Binding of a cellular factor(s) to this fragment is altered when both viral proteins are present, suggesting a possible mechanism for transcriptional synergy. Reliance on coregulators encoded by either the host or viral genome provides an elegant strategy for expanding the regulatory potential of a master regulator, such as RTA.

Quantitative Analysis of the KSHV Transcriptome Following Primary Infection of Blood and Lymphatic Endothelial Cells

“…vIRF-4 has been shown to cooperate with ORF50 in late gene expression [67], while Kaposin A is a membrane-associated protein through its two hydrophobic domains [68], suggesting functional similarities with the other Cluster 5 genes. In addition, the transcription/regulatory genes (dark green dots) in Cluster 5 included ORFs 18, 30 and 31, which are all involved in the regulation of late gene expression, and ORFs 18 and 30 have been specifically implicated in the regulation of the majority of genes in Cluster 5 [69,70].…”

Comprehensive Mapping and Analysis of Kaposi's Sarcoma-Associated Herpesvirus 3′ UTRs Identify Differential Posttranscriptional Control of Gene Expression in Lytic versus Latent Infection

“…To ensure that the effects that we observed were due to viral lytic replication (versus nonspecific effects of the drug regimen utilized to induce TREx-RTA BCBL-1 cells into lytic infection), we conducted the same experiment on the ORF 24 and ORF 74 3= UTR reporters in the presence of an shRNA directed against the ORF 50 transcript (3,77). The ORF 50 transcript gives rise to the master lytic switch protein that is necessary for KSHV lytic replication (13,(78)(79)(80)(81). When RTA levels were knocked down, we observed a stark decrease (Ͼ2.5ϫ) in the effects observed ( Fig.…”

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Targets Expression of Cellular IRF4 and the Myc Gene To Facilitate Lytic Replication

“…vIRF4 also interacts with the poly(A)-binding protein, potentially interfering with host translation (22). Finally, vIRF4 was recently found to be a positive regulator of RTA function itself (23). These observations indicate that vIRF4 potentially functions as a regulator by cooperation with either host or viral proteins to promote an efficient KSHV life cycle.…”