RNA-dependent RNA polymerase (RdRp) encoded by positive-strand RNA viruses is critical to the replication of viral RNA genome. Like other positive-strand RNA viruses, replication of hepatitis C virus (HCV) RNA is mediated through a negative-strand intermediate, which is generated through copying the positivestrand genomic RNA. Although it has been demonstrated that HCV NS5B alone can direct RNA replication through a copy-back primer at the 3 end, de novo initiation of RNA synthesis is likely to be the mode of RNA replication in infected cells. In this study, we demonstrate that a recombinant HCV NS5B protein has the ability to initiate de novo RNA synthesis in vitro. The NS5B used HCV 3 X-tail RNA (98 nucleotides) as the template to synthesize an RNA product of monomer size, which can be labeled by [␥-32 P]nucleoside triphosphate. The de novo initiation activity was further confirmed by using small synthetic RNAs ending with dideoxynucleotides at the 3 termini. In addition, HCV NS5B preferred GTP as the initiation nucleotide. The optimal conditions for the de novo initiation activity have been determined. Identification and characterization of the de novo priming or initiation activity by HCV NS5B provides an opportunity to screen for inhibitors that specifically target the initiation step.Hepatitis C virus (HCV) is recognized as the causative agent for most cases of non-A and non-B hepatitis (5, 12), with an estimated prevalence of 170 million worldwide (21). Upon first exposure to HCV, about 10 to 20% of infected individuals develop acute clinical hepatitis, while others appear to resolve the infection spontaneously. In most cases (80 to 90%), however, the virus establishes a chronic infection that persists for decades, which may lead to more severe disease states such as cirrhosis and hepatocellular carcinoma (18,19). Currently, there is no broadly effective treatment for the debilitating progression of chronic HCV.HCV is a positive-strand RNA virus belonging to the Flaviviridae family (15, 16). The genome of HCV encodes a single open reading frame which is translated into a polyprotein of about 3,010 amino acids (for reviews, see references 3 and 17). This polyprotein is subsequently processed by host as well as virally encoded proteases into at least 10 separate proteins with the following order (from the amino to the carboxy terminus): NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. The nonstructural proteins are believed to provide catalytic machinery for viral replication. One key enzyme encoded by HCV is NS5B, which has been shown to possess an RNAdependent RNA polymerase (RdRp) activity (1, 4, 6-8, 13, 14). NS5B is thus believed to be an essential component in the HCV replication complex.By itself, HCV NS5B RdRp appears to lack specificity for HCV RNA and can copy-back heterologous nonviral RNA or elongate on an oligonucleotide primer annealed to a homopolymeric RNA template (4,6,7,13,14). This lack of specificity for HCV RNA may reflect the notion that additional viral or host factors are required fo...

Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cellshas been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5B⌬CT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primerindependent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (⌬CT179 and ⌬CT218) that lacked RdRp activities.

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Human interleukin (IL)-5 gene transcription is regulated by several transcription factor binding sites, including CLE 0, GATA, and a region from position -123 to -92 known as response element (RE)-II. By expression cloning, a partial protein was identified that bound to concatamers of RE-II. Recombinant protein derived from this initial complementary DNA (cDNA) encoding the partial protein specifically bound to RE-II-containing oligonucleotides in electromobility shift assays. The complete sequence (3,649 bp) was determined by 5' rapid amplification of cDNA ends and comparisons to existing ESTs, and found to be identical to the 3' half of Wolf-Hirschhorn syndrome candidate 1, (WHSC1; also known as Multiple Myeloma SET domain [MMSET]). The full-length protein contains an SET domain and two plant homeodomain-type zinc fingers. Transcription initiation of RE-II binding protein (RE-IIBP) messenger RNA (mRNA) uniquely occurred within the middle of WHSC1 near a region that exhibits complex mRNA splicing. RE-IIBP reactive polyclonal antisera identified proteins in human T-cell nuclear protein extracts of 62 and 66 kD that were consistent with the length of the longest open reading frame in RE-IIBP. In contrast, WHSC1 is predicted to encode a protein of 136 kD. In activated human Jurkat and murine D10.G4.1 T cells, expression of full-length and truncated forms of RE-IIBP repressed RE-II promoter activity of a 5X-RE-II luciferase reporter construct by as much as 75%. In addition, RE-IIBP expressed in activated D10.G4.1 T cells inhibited endogenous murine IL-5 production. The repressor activity of RE-IIBP is consistent with the presence of an SET domain that is found in other proteins that act as gene silencers.

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