Poly(C)-binding protein 2 Interacts with sequences Required for Viral Replication in HCV 5’UTR and Directs HCV RNA Replication through Circularizing the Viral Genome

C型肝炎病毒 (HCV) RNA的5’端 (5’UTR) 序列對於調控RNA複製與蛋白轉譯都很重要。HCV的5’端包含了341個核苷酸，其中包含 internal ribosome entry site (IRES) 區域可調控蛋白轉譯功能，而調控RNA複製的功能區域也在5’端之內。調控蛋白轉譯與RNA複製的功能區域是否坐落於5’端的不同區域，而參與其中的蛋白是否也不相同，則需要進一步研究。研究顯示，HCV RNA 5’端的前157個核苷酸區域即足以調控RNA複製，而此區域涵蓋了部份的IRES區域。此複製調控區域內包含了stem-loops 1及stem-loops 2的結構，且對調控RNA複製皆很重要，然而stem-loops 1並不參與HCV蛋白轉譯。此外，蛋白poly(C)-binding protein 2 (PCBP2) 會與HCV 5’端的複製調控區域結合，並與HCV病毒蛋白一同出現在抗界面活性劑的細胞內膜，而此內膜區域為HCV RNA複製複合體出現的位置。利用shRNA抑制PCBP2的表現後，HCV RNA及病毒蛋白的表現量也隨之降低；而在胞外複製試驗中，以PCBP2抗體阻絕PCBP2功能，將使HCV RNA複製功能被抑制。以上實驗皆證明PCBP2直接影響了HCV RNA的複製。另一方面，當PCBP2表現被抑制時，IRES所調控的蛋白轉譯活性也同時減低，表示PCBP2也參與在蛋白轉譯中，以上試驗證明PCBP2同時參與了HCV蛋白轉譯與RNA複製。PCBP2不只與HCV RNA 5’端結合，同時也與3’端結合，在電子顯微鏡下可觀察到PCBP2將RNA兩端連結複合使RNA形成環狀化結構。本研究證實了PCBP2參與在HCV蛋白轉譯與RNA複製的調控性，透過PCBP2的結合，HCV RNA兩末端可結合成為環狀結構。Sequences in the 5’-untranslated region (5’UTR) of hepatitis C virus (HCV) RNA is important for modulating both translation and RNA replication. Translation of the HCV genome depends on an internal ribosome entry site (IRES) located within the 341-nucleotide 5’-UTR, while RNA replication requires a smaller region. A question arises whether the replication and translation functions require different regions of 5’-UTR and different sets of RNA-binding proteins. Here we showed that the 5’-most 157 nucleotides of HCV RNA is the minimum 5’-UTR for RNA replication, which partially overlaps with IRES. Both stem-loops 1 and 2 of 5’-UTR are essential for RNA replication, whereas stem-loop 1 is not required for translation. We also found that poly(C)-binding protein 2 (PCBP2) bound to the replication region of 5’UTR and associated with detergent-resistant membrane fractions, which is the site of HCV replication complex. Knock-down of PCBP2 by shRNA decreased the amounts of HCV RNA and nonstructural proteins. Antibody-mediated blocking of PCBP2 reduced HCV RNA replication in vitro, indicating that PCBP2 is directly involved in HCV RNA replication. Furthermore, PCBP2 knockdown reduced IRES-dependent translation preferentially from a dual reporter plasmid, suggesting that PCBP2 also regulated IRES activity. These findings indicate that PCBP2 participates in both HCV RNA replication and translation. Moreover, PCBP2 interacts with HCV 5’UTR and 3’UTR RNA fragments to form an RNA-protein complex and induces circularization of HCV RNA as revealed by electron microscopy. This study thus demonstrates the mechanism of participation of PCBP2 in HCV translation and replication and provides the physical evidence for HCV RNA circularization through 5’ and 3’-UTR interaction