Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B
Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B
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DOI：
10.1128/jvi.01540-15
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发表时间：
2016-03-01
影响因子：
5.4
通讯作者：
Aizaki, Hideki
中科院分区：
文献类型：
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作者：
Kong, Lingbao;Fujimoto, Akira;Aizaki, Hideki
有人提出，丙型肝炎病毒（HCV） NS4B蛋白可触发膜性HCV复制区，但其潜在的分子机制尚不完全清楚。本研究通过串联亲和纯化和蛋白质组学分析筛选ns4b相关膜蛋白，鉴定出202个宿主蛋白。随后用小干扰RNA筛选复制子细胞，发现催乳素调节元件结合（PREB）是一种新的HCV宿主辅助因子。通过免疫沉淀、免疫荧光和近端结扎实验证实了PREB和NS4B之间的相互作用。PREB在HCV复制子细胞中与双链RNA和新合成的三磷酸溴吡啶标记的HCV RNA共定位。此外，在存在NS4B表达的Huh7细胞中，PREB转移到HCV复制复合物所在的耐洗涤剂膜（DRMs）上。然而，缺乏NS4B结合区（PREBd3）的PREB突变体不能与双链RNA共定位，在NS4B存在下也不能向DRM转移。这些结果表明PREB通过与NS4B相互作用定位在HCV复制复合体上。PREB沉默抑制了膜状HCV复制室的形成，增加了DRMs中HCV复制酶蛋白和RNA的蛋白酶和核酸酶敏感性。总的来说，这些数据表明PREB通过参与膜性复制室的形成并通过与NS4B相互作用维持其适当的结构来促进HCV RNA复制。此外，体外和体内HCV感染诱导了PREB。我们的发现为HCV宿主辅助因子提供了新的见解。
It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors.