The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3 NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3 NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3 end of the HCV genome that is essential for replication. The hepatitis C virus (HCV) is the only member of theHepacivirinae within the Flaviviridae family (46). These viruses possess an RNA genome of positive polarity that in the case of HCV has a length of about 9,600 nucleotides (nt). Its genome carries a single long open reading frame (ORF) that is flanked at either end by highly conserved nontranslated regions (NTRs) (2, 37). The 5Ј NTR harbors an internal ribosome entry site (IRES) directing the synthesis of the viral polyprotein which is cleaved into 10 different products. RNA replication takes place in the cytoplasm in a distinct compartment (12) and requires viral proteins NS3 to NS5B as well as host cell factors (28,43). In addition, cisacting replication elements (CREs) are important for the synthesis of RNA progeny. Such CREs may reside at various positions in an RNA genome but are most often found in the NTRs. In the case of HCV, the minimal signals that are essential for RNA replication have been mapped within the NTRs. The 5Ј NTR is composed of four distinct domains, with domains 1 and 2 being sufficient for RNA replication (10, 19). The 3Ј NTR has a tripartite structure and is composed of a highly variable region immediately downstream of the stop codon of the polyprotein, a polypyrimidine [poly(U/UC)] tract of variable length, and a highly conserved 98-nt-long RNA element designated the X-tail (22,41,42,52). The latter has the potential to form three stemloop (SL) structures, of which the most 3Ј terminal, designated SL1, has been confirmed experimentally (5,15). In contrast, the structures of the two other SLs that are located upstream are much less clear (5,15,56). Both the X-tai...

The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.