RNA-dependent RNA polymerase, NS5B protein, catalyzes replication of viral genomic RNA, which presumably initiates from the 3-end. We have previously shown that NS5B can utilize the 3-end 98-nucleotide (nt) X region of the hepatitis C virus (HCV) genome as a minimal authentic template. In this study, we used this RNA to characterize the mechanism of RNA synthesis by the recombinant NS5B. We first showed that NS5B formed a complex with the 3-end of HCV RNA by binding to both the poly(U-U/C)-rich and X regions of the 3-untranslated region as well as part of the NS5B-coding sequences. Within the X region, NS5B bound stem II and the single-stranded region connecting stem-loops I and II. Truncation of 40 nt or more from the 3-end of the X region abolished its template activity, whereas X RNA lacking 35 nt or less from the 3-end retained template activity, consistent with the NS5B-binding site mapped. Furthermore, NS5B initiated RNA synthesis from a specific site within the single-stranded loop I. All of the RNA templates that have a double-stranded stem at the 3-end had the same RNA initiation site. However, the addition of single-stranded nucleotides to the 3-end of X RNA or removal of double-stranded structure in stem I generated RNA products of template size. These results indicate that HCV NS5B initiates RNA synthesis from a single-stranded region closest to the 3-end of the X region. These results have implications for the mechanism of HCV RNA replication and the nature of HCV RNA templates in the infected cells. Hepatitis C virus (HCV)1 is the etiological agent of non-A, non-B hepatitis, often causing liver diseases including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1-4). HCV has a positive-sense, single-stranded RNA genome of approximately 9700 nucleotides (nt) in length, which is terminated with a stretch (98 nt) of highly conserved sequence, termed the X region (5-11). The X region folds into a stable secondary structure consisting of three stem-loop domains (12, 13). Upstream of the X region is a stretch of poly(U-U/C)-rich sequences of variable length and highly variable sequences of about 30 -40 nt (5-11). Infectivity assays showed that the X region and U-U/C-rich sequences are required for viral infectivity, but the variable sequences are not (14). As implicated by sequence conservation among all HCV genotypes, the structure and/or sequence of the X region of HCV is important for minusstrand RNA synthesis and translational regulation (15, 16). The replication of HCV RNA is mediated by NS5B, which is an RNA-dependent RNA polymerase (RdRp) (16 -20).The initial step of viral RNA replication is recognition of the 3Ј-end of RNA template by RdRp, which may occur directly or indirectly with the help of cellular proteins (21, 22). For example, Q␤ bacteriophage replicase recognizes the replicable RNA templates with the help of cellular factors, including ribosomal protein S1 and translation elongation factor Tu (23-25), which are also important for template recognition on certain in vitro...

High thymidylate synthase (TS) level in cancer tissue is considered to result in resistance to pemetrexed therapy for advanced stages of nonsquamous non-small cell lung cancers. To further investigate the mechanism of pemetrexed resistance and potential prognostic outcomes in lung cancer, we established pemetrexed-resistant lung adenocarcinoma cell sublines from CL1 harboring a mutated TP53 gene (R248W) and A549 harboring wild-type TP53. We found the TS expression is upregulated in both pemetrexed-resistant sublines and the reduced TS level achieved through shRNA inhibition resulted in higher pemetrexed sensitivity. We also demonstrated that the acquisitions of pemetrexed resistance enhances epithelial–mesenchymal transition (EMT) in vivo with a mice animal model and in vitro with CL1 and A549 sublines, which was associated with upregulation of ZEB1 which, in turn, downregulates E-cadherin and upregulates fibronectin. When ERK1/2 phosphorylation was reduced by an inhibitor (U0126) or siRNA inhibition, both pemetrexed-resistant sublines reduced their migration and invasion abilities. Therefore, the ERK-mediated pathways induce apoptosis with pemetrexed treatment, and may in turn mediate EMT when cancer cells are resistant to pemetrexed. We further demonstrated that the growth of pemetrexed-resistant tumors could be inhibited by vinblastine in vivo and vincristine in vitro. Our data indicate that pemetrexed resistance could be relieved by non-cross-resistant chemotherapeutic drugs such as vinca alkaloids and might be independent to TP53 status. Furthermore, the phosphorylation of ERK was reduced by vincristine. This finding provides a new insight for overcoming pemetrexed resistance and metastasis by application of vinca alkaloids.

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