上海巴斯德所等发现卡波氏肉瘤病毒编码基因

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11月4日，国际学术期刊Journal of Virology 在线发表了中国科学院上海巴斯德研究所肿瘤病毒研究组的研究论文：Genome-wide mapping of the binding sites and structural analysis of KSHV vIRF2 reveal it is a DNA-binding transcription factor（《卡波氏肉瘤相关疱疹病毒编码的vIRF2作为转录因子在基因组上DNA结合位点的检测和vIRF2蛋白质结合的解析》）。

卡波氏肉瘤病毒（Kaposi’s sarcoma associated herpesvirus, KSHV）又称人类疱疹病毒8型，可以引发卡波氏肉瘤、原发外渗淋巴瘤以及多中心卡斯特曼疾病。KSHV参与编码与四个人类干扰素调节因子同源的蛋白vIRFs。vIRFs在KSHV的生命周期以及肿瘤的形成过程中起到重要的作用。因此，进一步了解KSHV vIRFs的功能性可以为肿瘤病毒的致瘤机理研究提供新的线索。

vIRF2在KSHV的生命周期中主要对干扰素信号通路进行抑制，从而帮助病毒更好地建立持续感染。例如，vIRF2可作为负调控因子，抑制IRF-1的表达，从而对干扰素β（INFβ）的信号通路进行抑制。此外，vIRF2也可以通过与ISGF3复合体中蛋白相互作用，抑制下游ISG基因的表达，从而抑制干扰素信号通路。前期研究表明，KSHV的vIRF2对基因表达的调控主要通过蛋白-蛋白相互作用完成，但尚不清楚vIRF2是否可以作为转录因子对宿主基因进行调控。

上海巴斯德所肿瘤病毒研究组研究生胡海岱、董家珍在研究员蓝柯的指导下，与中科院高能物理研究所董宇辉研究组合作，通过CHIP-seq手段对vIRF2在人类基因组上DNA结合位点进行检测并预测了vIRF2潜在靶基因的启动子结合位点；通过凝胶迁移实验（EMSA）和q-PCR实验，证实了vIRF2作为转录因子可以对宿主PIK3C3等基因进行调控。为了深入研究vIRF2与DNA的结合能力，研究人员通过蛋白质结构解析，确认了vIRF2的DNA结合区域(vIRF2DBD)蛋白构象，并发现vIRF2DBD蛋白α3区域的82位和85位的精氨酸对于vIRF2的DNA结合能力是必要的；对这两个位点的氨基酸进行突变后，发现了突变后的基因会影响vIRF2作为转录因子对宿主基因的调控能力。该研究首次证实了KSHV编码的vIRF2具有DNA结合能力并能作为转录因子对宿主基因进行调控，对KSHV的感染与致瘤研究具有一定的功能性意义。

该研究得到了国家自然科学基金重点项目及国家“973”计划等项目的经费支持。

图：vIRF2的结构特点。（A）vIRF2和vIRF1以及人类IRFs二级结构比对；（B）vIRF2DBD蛋白质构象全景图；(C)vIRF2DBD分子表。

来源：上海巴斯德研究所

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【标题】：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
【作者】：Hu, H.; Dong, J.; Liang, D. (...)
【来源】：J Virol, 2015, 90(3), 1158-1168
【摘要】：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.