No abstract

The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and ⌬pNS3 (helicase domain only) and examined their abilities to interact with the 3-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3 untranslated region (UTR). The results presented here demonstrate that NS3 (and ⌬pNS3) interacts efficiently and specifically with the 3-terminal sequences of both positive-and negative-strand RNA but not with the corresponding complementary 5-terminal RNA sequences. The interaction of NS3 with the 3-terminal negative strand [called 3(؊) UTR 127 ] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3 terminus (nucleotides 5 to 20 from the 3 end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and ⌬pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3(؊) UTR binding, specific interaction of NS3 (or ⌬pNS3) with the 3-terminal sequences of the positive-strand RNA [3(؉) UTR] appears to require the entire 3(؉) UTR of HCV. Deletion of either the 98-nucleotide 3-terminal conserved region or the 5 half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3-terminal sequences of viral positive-and negative-strand RNA in viral replication is discussed.Hepatitis C virus (HCV) is the primary causative agent of parenterally transmitted non-A, non-B hepatitis and affects a significant part of the world's population. HCV infection frequently leads to chronic hepatitis, cirrhosis of the liver, and possibly hepatocellular carcinoma (11,45). HCV, a member of the family Flaviviridae (44), has been a difficult virus to study due to the lack of an appropriate tissue culture system and an adequate, simple, and low-cost animal model. The RNA genome of HCV has recently been cloned, and the singlestranded, plus-polarity RNA genome of the virus is approximately 9,500 nucleotides long and is flanked by untranslated regions (UTR) at both the 5Ј and 3Ј ends (22,28,48). The 5Ј UTR of HCV RNA (341 nucleotides) is highly structured and contains an internal ribosome entry site which extends to nucleotide 370, partially overlapping the structural protein (core) coding sequences (8,20,21,51,53). The secondary structure of the 5Ј UTR appears to be highly conserved among various HCV strai...

The poliovirus-encoded, membrane-associated polypeptide 2C is believed to be required for initiation and elongation of RNA synthesis. We have expressed and purified recombinant, histidine-tagged 2C and examined its ability to bind to the first 100 nucleotides of the poliovirus 5 untranslated region of the positive strand and its complementary 3-terminal negative-strand RNA sequences. Results presented here demonstrate that the 2C polypeptide specifically binds to the 3-terminal sequences of poliovirus negative-strand RNA. Since this region is believed to form a stable cloverleaf structure, a number of mutations were constructed to examine which nucleotides and/or structures within the cloverleaf are essential for 2C binding. Binding of 2C to the 3-terminal cloverleaf of the negative-strand RNA is greatly affected when the conserved sequence, UGUUUU, in stem a of the cloverleaf is altered. Mutational studies suggest that interaction of 2C with the 3-terminal cloverleaf of negative-strand RNA is facilitated when the sequence UGUUUU is present in the context of a double-stranded structure. The implication of 2C binding to negative-strand RNA in viral replication is discussed.

No abstract

The poliovirus-encoded, membrane-associated polypeptide 2C and its precursor, 2BC, is believed to be required for initiation and elongation of viral RNA synthesis. Previous studies have shown that the 2C polypeptide specifically interacts with the 3'-terminal sequence of poliovirus negative-strand RNA. This interaction is facilitated by the presence of the conserved sequence UGUUUU in the "stem a" within the 3'-terminal cloverleaf structure. We show that the 2C precursor, 2BC, also interacts with the 3'-terminal cloverleaf of the negative-strand RNA. We also demonstrate here that interaction of 2C/2BC with the negative strand 3'-terminal sequence not only depends on an intact "stem a" containing the UGUUUU sequence but is equally influenced by the presence of an intact "stem b" within the negative-strand cloverleaf. The results presented here suggest that the spatial configuration of stem a UGUUUU sequence with respect to an intact stem-b is crucial for 2C/2BC interaction with the 3'-terminal negative-strand cloverleaf structure.