Genome-Wide Mapping of the Binding Sites and Structural Analysis of Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 2 Reveal that It Is a DNA-Binding Transcription Factor

Abstract

The oncogenic herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is known to encode four viral interferon regulatory factors (vIRF1 to -4) to subvert the host antiviral immune response, but their detailed DNA-binding profiles as transcription factors in the host remain uncharacterized. Here, we first performed genome-wide vIRF2-binding site mapping in the human genome using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq). vIRF2 was capable of binding to the promoter regions of 100 putative target genes. Importantly, we confirmed that vIRF2 can specifically interact with the promoters of the genes encoding PIK3C3, HMGCR, and HMGCL, which are associated with autophagosome formation or tumor progression and metastasis, and regulate their transcription in vivo. The crystal structure of the vIRF2 DNA-binding domain (DBD) (referred to here as vIRF2 DBD ) showed variable loop conformations and positive-charge distributions different from those of vIRF1 and cellular IRFs that are associated with DNA-binding specificities. Structure-based mutagenesis revealed that Arg82 and Arg85 are required for the in vitro DNA-binding activity of vIRF2 DBD and can abolish the transcription regulation function of vIRF2 on the promoter reporter activity of PIK3C3, HMGCR, and HMGCL. Collectively, our study provided unique insights into the DNA-binding potency of vIRF2 and suggested that vIRF2 could act as a transcription factor of its target genes in the host antiviral immune response. IMPORTANCEThe oncogenic herpesvirus KSHV is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV has developed a unique mechanism to subvert the host antiviral immune responses by encoding four homologues of cellular interferon regulatory factors (vIRF1 to -4). However, none of their DNA-binding profiles in the human genome have been characterized until now, and the structural basis for their diverse DNA-binding properties remain poorly understood. In this study, we performed the first genome-wide vIRF2-binding site mapping in the human genome and found vIRF2 can bind to the promoter regions of 100 target cellular genes. X-ray structure analysis and functional studies provided unique insights into its DNA-binding potency and regulation of target gene expression. Our study suggested that vIRF2 could act as a transcription factor of its target genes and contribute to KSHV infection and pathogenesis through versatile functions. K aposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is the etiological agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD) (1-3). Moreover, KS is the first tumor to be associated with HIV infection and the most common AIDS-associated malignancy in sub-Saharan Africa and the second most common cancer in HIV-infected patients (2-5). KSHV has developed a unique mechanism to subvert host antiviral immune responses by encoding four homologu...

Kaposi’s sarcoma-associated herpesvirus vIRF2 protein utilizes an IFN-dependent pathway to regulate viral early gene expression

“…A comparison of the cellular transcriptome in KSHV.WT- and KSHV.ΔvIRF2-infected cells showed that, at protein levels found in KSHV-infected cells, vIRF2 is a transcriptional regulator of different cellular genes (Table 1). This is in line with the fact that it binds to different cellular promoter regions [36]. Among the cellular genes involved in host defense that are differentially expressed in KSHV.WT- vs. KSHV.ΔvIRF2-infected endothelial cells (Table 1), we could identify the IFN-induced proteins with tetratricopeptide repeats 1, 2 and 3 (IFIT1, 2 and 3), whose expression we could show to be directly or indirectly induced by vIRF2 (Fig 7) and which restrict KSHV lytic protein expression (Fig 8).…”

“…Expression of the spliced vIRF2 mRNA has previously been reported to occur only in the lytic replication cycle in different KSHV positive cell lines [26, 65–68]. Recently, it was shown that vIRF2 is able to bind the promoter regions of different cellular genes by means of a DNA-binding domain (DBD; aa 7–114); a nuclear localization signal (aa146-159) has been identified at the N-terminal end of the vIRF2 protein [36, 69].…”

Beyond Viral Interferon Regulatory Factors: Immune Evasion Strategies

“…In addition to vIRF1, genome-wide chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) analysis revealed that vIRF2 is able to bind the promoter regions of numerous cellular genes [45]. Notably, vIRF2 DBD (amino acid 7 to 144) was shown to bind to the promoter regions of PIK3C3, HMGCR, HMGCL and subsequently deregulated their expression levels [45]. Importantly, the consequence of such binding contributed to enhancement of KSHV infection and KSHV-associated pathogenesis.…”

Inactivation of HMGCL promotes proliferation and metastasis of nasopharyngeal carcinoma by suppressing oxidative stress

“…In breast cancer, HMGCL and other enzymes associated with ketone-body production were preferentially expressed in the tumor stroma 35 . The oncogenic Kaposi’s sarcoma-associated herpesvirus was shown to interfere with transcription of HMGCL directly by targeting its promoter region 36 .…”