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, 90 (3), 1158-68

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

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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

Haidai Hu et al. J Virol.

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 vIRF2DBD) 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 vIRF2DBD 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.

Importance: The 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.

Figures

FIG 1
FIG 1
Genome-wide mapping of KSHV vIRF2-binding sites in 293T cells using ChIP-seq. (A) Summary of vIRF2 ChIP-seq experiments. (B) Peak-calling summary with MACS. (C) Genome distribution of KSHV vIRF2-binding sites. (D) Summary of the top 10 potential vIRF2-binding motifs generated by HOMER.
FIG 2
FIG 2
vIRF2 regulates transcription of genes bound by itself in the promoter region. (A) Schematic of the vIRF2-bound sites within the promoter regions of PIK3C3, HMGCR, and HMGCL. The vIRF2 read depth is plotted as reads per million. (B) Binding of vIRF2 to the promoter regions of PIK3C3, HMGCR, and HMGCL was validated by ChIP-qPCR. The data are shown as means plus standard errors of the mean (SEM); n = 3. *, P < 0.05. (C) mRNA levels of PIK3C3 and HMGCR were significantly upregulated, while that of HMGCL was significantly downregulated, by vIRF2 expression measured by qRT-PCR. GFP, green fluorescent protein. The data are shown as means plus SEM; n = 3. *, P < 0.05.
FIG 3
FIG 3
Structural characteristics of vIRF2DBD. (A) Overall view of vIRF2DBD structure shown as a cartoon. There are two molecules (named vIRF2DBDA, in green, and vIRF2DBDB, in cyan) in the asymmetric unit. (B) Structure-based sequence alignment of vIRF2DBD with vIRF1 and human IRF DBDs, performed using clustal X (version 1.81) and ESPript 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The conserved residues are boxed in blue, and identical and low-conserved residues are shaded in red or appear as red letters, respectively. Arg82 and Arg85 are boxed in red. (C) Superimposition of the two molecules in the asymmetric unit, showing they are essentially the same and the conformations of the putative specificity-determining arginines (Arg82 and Arg85) in α3 of vIRF2 for DNA binding are identical. (D) Molecular surface representation of vIRF2DBD (blue, +7.8 KT; red, −7.8 KT, where KT is the Boltzmann energy at room temperature) colored according to their local electrostatic potentials.
FIG 4
FIG 4
Structural comparisons of vIRF2DBD with vIRF1-DNA and IRF2-DNA complexes. (A) Structural superposition of DBDs between vIRF2 (green) and the vIRF1-DNA complex (orange; PDB ID 4HLY). There were six arginine residues (orange sticks) in DNA recognition helix α3 in vIRF1 (Arg163 and Arg164 can interact with DNA through hydrogen bonds), while there were only two arginine residues (green sticks) potentially involved in DNA binding in α3 of vIRF2DBD. Arg147 and Asn149 in L2 of vIRF1DBD, which made main-chain interactions with the phosphate backbone, corresponded to residues Arg61 and Arg63 in α2 of vIRF2DBDA, respectively. (B) Structural superposition of DBDs between vIRF2 (green) and the IRF2-DNA complex (blue; PDB ID 2IRF). The DNA-binding residues Asn80, Arg82, Cys83, and Asn86 (blue sticks) in α3 of IRF2 correspond to residues Cys79, Gly81, Arg82, and Arg85 (green sticks) in vIRF2DBD.
FIG 5
FIG 5
Interaction of vIRF2DBD with consensus vIRF2-binding motifs in vitro. (A) Validation of direct vIRF2-DNA binding by EMSA using vIRF2DBD and the DNA probe representing vIRF2-binding motif 1. WT vIRF2DBD induced significant gel shift of probe 1 in a dose-dependent manner; however, the R82A, R85A, or R82A/R85A mutant induced much weaker gel shifts for probe 1 than wild-type vIRF2DBD. (B) Purified wild-type vIRF2 DBD and its mutants by Coomassie brilliant blue staining. (C and D) vIRF2 upregulated the artificial motif 1 and motif 2 promoter-driven luciferase reporter activity. 3× Motif1-Luc and 3× Motif2-Luc reporters were constructed by inserting annealed DNA fragments containing 3 tandem copies of representative vIRF2-binding motif 1 or 2 sequences into the pGL6-TA vector. WT vIRF2DBD induced up to 2-fold activation of the luciferase reporter compared to the control, while R82A, R85A, or R82A/R85A had a defect in upregulating reporter activity. The data are shown as means plus SEM; n = 3. *, P < 0.05.
FIG 6
FIG 6
Roles of Arg82 and Arg85 involved in transcription regulation of vIRF2. (A to C) vIRF2 can significantly upregulate the reporter activity of PIK3C and HMGCR promoters or downregulate the activity of the HMGCL promoter, while all the mutations R82A, R85A, and R82A/R85A severely abolished the regulation function of vIRF2. The data are shown as means plus SEM; n = 3. *, P < 0.05. (D) Expression of the wild type and mutants of vIRF2 detected by Western blotting. The results showed Arg82 and Arg85 are required for transcription regulation of vIRF2. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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