The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K d , of Ϸ22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.Hepatitis C virus (HCV) belongs to the Flaviviridae family and is responsible for a significant proportion of acute and chronic hepatitis in humans worldwide (7,8). Similar to other flaviviruses, HCV is a small positive-stranded RNA virus with a genome size of Ϸ9.6 kb encoding a single polyprotein (30). This viral polyprotein is processed by both host and virally encoded proteases to generate mature structural and nonstructural proteins essential for virus replication (see references 9, 34, and 39 for review). One of the nonstructural proteins, designated NS5B, is the virally encoded RNA-dependent RNA polymerase (RdRp), which contains the GDD signature motif of RNA polymerases (3).It has been shown that NS5B is a membrane-associated protein, which contains a C-terminal domain comprising Ϸ21 hydrophobic amino acids that is responsible for membrane anchorage (41). NS5B may form a complex with cellular proteins (37) or other HCV nonstructural proteins, including NS3, the viral protease and helicase; NS4A, a cofactor of NS3 protease activity; and NS5A, a phosphoprotein containing a putative interferon sensitivity region (15). Although the HCV replication mechanism is not clearly understood, the essential role of NS5B polymerase in the HCV replication and infection process has been demonstrated in chimpanzees (22). Accordingly, it has been viewed as an attractive target for antiviral intervention.Recombinant HCV NS5B polymerase has been produced and purified from both bacterial and insect cells by several groups (3,11,16,19,25,27,31,41). The availability of highly purified protein has facilitated the biochemical characterization of HCV NS5B polymerase. Similar to other viral RdRps, purified HCV NS5B is able to synthesize RNA using various RNAs as templates in vitro (3,11,16,19,25,27,31,41). In this regard, two RNA synthesis reaction modes have been described for this enzyme: RNA elongation using a preannealed primer and RNA in...

Two-dimensional 1H-15N NMR techniques combined with pulsed hydrogen-deuterium exchange have been used to characterize the folding pathway of T4 lysozyme. In the unfolded state, there is little differential protection of the various amides from hydrogen exchange. In the native folded structure, 84 amides of the 164 residues are sufficiently spectrally resolved and protected from solvent exchange to serve as probes of the folding pathway. These probes are located in both the N-terminal and C-terminal domains of the native folded structure of the protein. The studies described here show that at least one intermediate is formed early during refolding at low denaturant concentrations. This intermediate (or intermediates) forms very rapidly (within the 10-ms temporal resolution of our mixing device) under the conditions used and is completed at least 10 times faster than the overall folding event. The intermediate(s) protect(s) from exchange a subset of amides in the N-terminal and C-terminal regions of the protein. In the final folded states these protected regions correspond to two alpha-helices and a beta-sheet region. These amides are protected from exchange by factors between 20 and 200 as compared to the fully unfolded protein. Protection of this magnitude is consistent with the formation of somewhat exposed secondary structure in these regions and could represent a "molten globule"-like or a "framework"-like structure for the intermediate(s) in which specific parts of the sequence form isolated secondary structures that are not stabilized by extensive tertiary interactions.

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The human U1A protein has two putative RNA binding domains, one at the N-terminal region of the protein (RBD1) and the other at the C-terminal end (RBD2). RBD1 binds tightly and specifically to one of the stem loops of the U1 snRNA, as well as to its own 3'-UTR. In contrast, RBD2 does not appear to associate with any RNA. The two domains share 25% amino acid identity, and both have the same betaalphabeta-betaalphabeta secondary structure fold. In this work, 13C/15N/1H multidimensional NMR methods were used to obtain side-chain assignments for RBD2, and then the tertiary structure was calculated using a distance geometry/simulated annealing algorithm that employs pairwise Gaussian metrization. RBD2 is shown to fold into an alpha/beta sandwich with a four-stranded antiparallel beta-sheet, which is the typical global topology of these domains. Specific structural features of RBD2 include a beta-bulge in beta2, N-capping boxes for both alpha-helices, and an extremely shallow twist of its beta-sheet. The 15N backbone dynamics of these two structurally homologous RBDs are significantly different, compared using order parameters and T2 exchange terms in the Lipari and Szabo model-free formalism. Conformational exchange observed in RBD1, which is absent in RBD2, may correlate to the mechanism of RNA binding.

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