No abstract

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3 cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3 single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.Hepatitis C virus (HCV), a plus-strand RNA virus, is estimated to infect up to 3% of the world's population (44), causing liver cirrhosis and hepatocellular carcinoma (14). Following entry into the infected cell, the viral RNA directs the translation of a polyprotein that is proteolytically processed to produce 10 individual structural and nonstructural proteins (15, 32). Nonstructural protein 5B (NS5B) is at the C terminus of the polyprotein. NS5B is an RNA-dependent RNA polymerase (RdRp). Based on the paradigms of other RNA virus replication strategies (8), NS5B, along with viral and cellular proteins, forms a replicase that replicates the HCV genome. At present, functional HCV replicase has not been demonstrated in vitro. Therefore, studies of HCV RNA synthesis have focused on recombinant NS5B.Recombinant HCV NS5B can catalyze a number of reactions. In the presence of a primer-template duplex, NS5B catalyzes template-dependent but relatively nonspecific RNA synthesis (5, 23-25, 45, 46). In addition, NS5B has recently been reported to direct de novo (oligonucleotide primer-independent) synthesis (26, 30, 47), a mechanism used for the replication of many plus-strand RNA viruses (8). De novo initiation of RNA synthesis may be especially relevant for HCV since, to our knowledge, it does not contain a VPg-like protein that could mediate protein-primed RNA synthesis, and there is no evidence for a cap-snatching mechanism (32). De novo RNA synthesis directed by HCV NS5B prefers a cytidylate template and the substrate nucleotide GTP (26, 42), although ATP can also be used as an initiation nucleotide (29,42,47). In general, RNA polymerases have a higher K m for the initiation nucleotide than for the same nucleotide during elongating RNA synthesis (for examples, see references ...

No abstract

Solvent conditions where human insulin could be studied by high-resolution NMR were determined. Both low pH and addition of acetonitrile were required to overcome the protein's self-association and to obtain useful spectra. Two hundred eighty-six 1H resonances were located and assigned to specific sites on the protein by using two-dimensional NMR methods. The presence and position of numerous dNN sequential NOE's indicate that the insulin conformation seen in crystallographic studies is largely retained under these solution conditions. Slowly exchanging protons were observed for seven backbone amide protons and were assigned to positions A15 and A16 and to positions B15-B19. These amides all occur within helical regions of the protein [Chawdhury, S.A., Dodson, E.J., Dodson, G.G., Reynolds, C.D., Tolley, S.P., Blundell, T.L., Cleasby, A., Pitts, J.E., Tickle, I.J., & Wood, S.P. (1983) Diabetologia 25, 460-464].

A novel method for the determination of the relative stereochemistry of six-membered chairlike ring molecules by residual dipolar couplings is presented. C-H residual dipolar couplings were used to investigate the relative stereochemistry of 4,6-O-ethylidene-d-glucopyranose. For this and similar systems it is not necessary to acquire redundant dipolar couplings and to calculate the orientation order tensor. The presented methodology is a paradigmatic leap for the determination of the relative stereochemistry or remote stereochemistry in this kind of fused ring system. Residual dipolar coupling data were collected by 1D and 2D direct-measurement heteronuclear multiple quantum coherence (HMQC) spectroscopy. It was demonstrated that direct measurement of HMQC was quick and accurate for small molecules at natural abundance.