With the exception of nucleoside analogs, few direct acting antivirals in clinical development are active across the six major hepatitis C virus genotypes. We report novel consensus sequence chimeras for genotypes 2b, 3a, 4a, 5a, and 6a NS5B and show variable susceptibilities over a panel of NS5B inhibitors. Tegobuvir (GS-9190) had EC(50)s of <16 nM against genotype 1 and >100 nM for other genotypes tested here. An NS5B F445C mutation engineered into the GT3a, 4a, and 6a chimeric replicons lowered the tegobuvir EC(50) to levels comparable to those for genotype 1a, but did not considerably alter the EC(50) of site 2 or nucleoside analog inhibitors. These data support the use of HCV chimeras in profiling direct acting antivirals across genotypes and specifically determines the impact of the C445F NS5B polymorphism on tegobuvir potency against genotypes 3a, 4a, and 6a.

Replication of the segmented double-stranded (ds)RNA genome of rotavirus requires the viral RNA-dependent RNA polymerase (RdRP) to use 11 different (+)RNAs as templates for (-) strand synthesis. Complementary sequences proximal to the 5\' and 3\' termini are predicted to direct cyclization of the (+)RNAs by forming panhandle structures from which short highly conserved terminal sequences protrude as single-stranded tails. Cell-free replication assays indicate that such structural organization of the 5\'- and 3\'-ends is required for efficient dsRNA synthesis. Multiple specifically recognized elements exist at the 3\'-end that promote dsRNA synthesis including RdRP-recruitment signals and a (-) strand initiation sequence. In contrast to the 3\'-end, the role of the 5\'-end has been less well defined. In this study, we determined that the 5\'-end contains a base-specific recognition signal that plays an important role in the assembly of the RdRP and cofactors into a stable initiation complex for (-) strand synthesis. The 5\' recognition signal is associated with the G2 residue of the 5\'-consensus sequence, a residue that shows absolute conservation among all rotavirus groups (A, B, and C) examined to date. From our results, we suggest that rotavirus (+)RNA cyclization, although likely initiated by 5\'- 3\' nucleotide complementarity, may be stabilized by RdRP-dependent bridging. Given that synthesis of the (-) strand on the (+)RNA template will disrupt 5\'-3\' nucleotide interactions, RdRP-dependent bridging may be the sole mechanism by which the dsRNA product can be held in the necessary cyclized conformation required for efficient multiple rounds of transcription.

We have defined a replication origin, ORI305, within chromosome III of Saccharomyces cerevisiae by means of mutational analysis. cis-acting elements required for origin activity in the chromosome, as assayed by two-dimensional gel electrophoresis of replication intermediates, are the same as those required for the function of an autonomously replicating sequence, ARS305, in a plasmid. Essential elements include (i) an 11 bp sequence that is a near match to the ARS consensus and (ii) a broad sequence directly 3\' to the consensus near match. Origin function is inactivated by point mutations in the essential near match sequence, suggesting that the sequence contributes to specifying the origin in the chromosome. Other consensus near matches with different sequences are present but are not required. The essential 3\'-flanking sequence exhibits DNA helical instability and is sensitive to deletion mutations that stabilize the DNA helix. The wild-type 3\'-flanking sequence can be functionally substituted by dissimilar sequences that also exhibit helical instability. The requirement for DNA helical instability indicates that the essential 3\'-flanking sequence serves as a DNA unwinding element in the chromosome.

Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements. To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence. The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis. Deletion of ARS306 deleted the associated replication origin. The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability. These results demonstrate that chromosomal origin function is a property of the ARS element itself.