Hepatitis C virus (HCV) nonstructural protein 4B (NS4B), a poorly characterized integral membrane protein, is thought to function as a scaffold for replication complex assembly; however, functional interactions with the other HCV nonstructural proteins within this complex have not been defined. We report that a Con1 chimeric subgenomic replicon containing the NS4B gene from the closely related H77 isolate is defective for RNA replication in a transient assay, suggesting that H77 NS4B is unable to productively interact with the Con1 replication machinery. The H77 NS4B sequences that proved detrimental for Con1 RNA replication resided in the predicted N-and C-terminal cytoplasmic domains as well as the central transmembrane region. Selection for Con1 derivatives that could utilize the entire H77 NS4B or hybrid Con1-H77 NS4B proteins yielded mutants containing single amino acid substitutions in NS3 and NS4A. The second-site mutations in NS3 partially restored the replication of Con1 chimeras containing the N-terminal or transmembrane domains of H77 NS4B. In contrast, the deleterious H77-specific sequences in the C terminus of NS4B, which mapped to a cluster of four amino acids, were completely suppressed by second-site substitutions in NS3. Collectively, these results provide the first evidence for a genetic interaction between NS4B and NS3 important for productive HCV RNA replication.Hepatitis C virus (HCV) is an enveloped positive-sense RNA virus belonging to the genus Hepacivirus of the Flaviviridae family. The 9.6-kb HCV genome encodes a polyprotein of ϳ3,000 amino acids which is translated via an internal ribosome entry site within the 5Ј nontranslated region (NTR). Cleavage of the polyprotein by host enzymes and two viral proteases produces three structural proteins (core, E1, and E2), a small hydrophobic polypeptide named p7, and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B).The HCV proteins NS3, NS4A, NS4B, NS5A, and NS5B are necessary and sufficient for RNA replication. These proteins form a membrane-associated replication complex responsible for synthesizing the positive-strand RNA genome via negativestrand RNA intermediates (9,14,33,38). Although many questions pertaining to the replication complex remain, specific functions for NS3, NS4A, and NS5B have been defined (reviewed in references 1 and 4). NS3 is a multifunctional protein with a serine protease domain spanning the 180 Nterminal amino acids and a RNA helicase/NTPase domain in the remaining C-terminal portion of the protein. The NS4A protein forms a stable complex with the N terminus of NS3 and is required to tether the NS3-4A complex to intracellular membranes and completely fold and activate the NS3 protease domain for cleavages at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions of the viral polyprotein. The NS5B RNAdependent RNA polymerase is the catalytic subunit of the viral replication complex.In contrast to NS3, NS4A, and NS5B, NS4B and the differentially phosphorylated NS5A protein play essential, but as yet undefi...

No abstract

The understanding of human cytomegalovirus (HCMV) biology was long hindered by the inability to perform efficient viral genetic analysis. This hurdle was recently overcome when the genomes of multiple HCMV strains were cloned as infectious bacterial artificial chromosomes (BACs). The BAC system takes advantage of the single‐copy F plasmid of E. coli that can stably carry large pieces of foreign DNA. In this system, a recombinant HCMV virus carrying a modified F plasmid is first generated in eukaryotic cells. Recombinant viral genomes are then isolated and recovered in E. coli as BAC clones. BAC‐captured viral genomes can be manipulated using prokaryotic genetics, and recombinant virus can be reconstituted from BAC transfection in eukaryotic cells. The BAC reverse genetic system provides a reliable and efficient method to introduce genetic alterations into the viral genome in E.coli and subsequently analyze their effects on virus biology in eukaryotic cells. Curr. Protoc. Microbiol. 24:14E.1.1‐14E.1.33. © 2012 by John Wiley & Sons, Inc.

We report the draft genome sequences of five novel members of the family Picornaviridae that were isolated from the stool of rhesus macaques ( Macaca mulatta ) with chronic diarrhea. The strains were named NOLA-1 through NOLA-5 because the macaques were residents of the Tulane National Primate Research Center.