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A dynamic view of hepatitis C virus replication complexes.

ABSTRACT: Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex (RC). Specific membrane alterations, designated membranous webs, represent predominant sites of HCV RNA replication. The principles governing HCV RC and membranous web formation are poorly understood. Here, we used replicons harboring a green fluorescent protein (GFP) insertion in nonstructural protein 5A (NS5A) to study HCV RCs in live cells. Two distinct patterns of NS5A-GFP were observed. (i) Large structures, representing membranous webs, showed restricted motility, were stable over many hours, were partitioned among daughter cells during cell division, and displayed a static internal architecture without detectable exchange of NS5A-GFP. (ii) In contrast, small structures, presumably representing small RCs, showed fast, saltatory movements over long distances. Both populations were associated with endoplasmic reticulum (ER) tubules, but only small RCs showed ER-independent, microtubule (MT)-dependent transport. We suggest that this MT-dependent transport sustains two distinct RC populations, which are both required during the HCV life cycle.

SUBMITTER: Wolk B

PROVIDER: S-EPMC2573176 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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A dynamic view of hepatitis C virus replication complexes.

Wölk Benno B Büchele Benjamin B Moradpour Darius D Rice Charles M CM

Journal of virology 20080820 21

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex (RC). Specific membrane alterations, designated membranous webs, represent predominant sites of HCV RNA replication. The principles governing HCV RC and membranous web formation are poorly understood. Here, we used replicons harboring a green fluorescent protein (GFP) insertion in nonstructural protein 5A (NS5A) to study HCV RCs in live cells. Two distinct patterns of NS5A-GFP were observed. (i) Large stru ...[more]

PMID: 18715913

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Project description:Hepatitis C virus (HCV) requires proteins from the NS3-NS5B polyprotein to create a replicase unit for replication of its genome. The replicase proteins form membranous compartments in cells to facilitate replication, but little is known about their functional organization within these structures. We recently reported on intragenomic replicons, bicistronic viral transcripts expressing an authentic replicase from open reading frame 2 (ORF2) and a second duplicate nonstructural (NS) polyprotein from ORF1. Using these constructs and other methods, we have assessed the polyprotein requirements for rescue of different lethal point mutations across NS3-5B. Mutations readily tractable to rescue broadly fell into two groupings: those requiring expression of a minimum NS3-5A and those requiring expression of a minimum NS3-5B polyprotein. A cis-acting mutation that blocked NS3 helicase activity, T1299A, was tolerated when introduced into either ORF within the intragenomic replicon, but unlike many other mutations required the other ORF to express a functional NS3-5B. Three mutations were identified as more refractile to rescue: one that blocked cleavage of the NS4B5A boundary (S1977P), another in the NS3 helicase (K1240N), and a third in NS4A (V1665G). Introduced into ORF1, these exhibited a dominant negative phenotype, but with K1240N inhibiting replication as a minimum NS3-5A polyprotein whereas V1665G and S1977P only impaired replication as a NS3-5B polyprotein. Furthermore, an S1977P-mutated NS3-5A polyprotein complemented other defects shown to be dependent on NS3-5A for rescue. Overall, our findings suggest the existence of two interdependent sets of protein complexes supporting RNA replication, distinguishable by the minimum polyprotein requirement needed for their formation.Positive-strand RNA viruses reshape the intracellular membranes of cells to form a compartment within which to replicate their genome, but little is known about the functional organization of viral proteins within this structure. We have complemented protein-encoded defects in HCV by constructing subgenomic HCV transcripts capable of simultaneously expressing both a mutated and functional polyprotein precursor needed for RNA genome replication (intragenomic replicons). Our results reveal that HCV relies on two interdependent sets of protein complexes to support viral replication. They also show that the intragenomic replicon offers a unique way to study replication complex assembly, as it enables improved composite polyprotein complex formation compared to traditional trans-complementation systems. Finally, the differential behavior of distinct NS3 helicase knockout mutations hints that certain conformations of this enzyme might be particularly deleterious for replication.

Project description:UnlabelledThe hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. The S225A mutant exhibited a 10-fold reduction in genome replication, whereas the S225D mutant replicated like the wild type. By confocal microscopy, we show that, in the case of the S225A mutant, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by viruses with other mutations in the low-complexity sequence I (LCS I), namely, S229D, S232A, and S235D, but not by viruses with mutations that caused a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA, and cellular lipids, including phosphatidylinositol 4-phosphate), the mutation in NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluence or another environmental factor and could be partially transcomplemented by wild-type NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex.ImportanceThe mechanisms by which hepatitis C virus replicates its RNA genome remain poorly characterized. We show here that phosphorylation of the viral nonstructural protein NS5A at serine residues is important for the efficient assembly of a complex that is able to replicate the viral genome. This research implicates cellular protein kinases in the control of virus replication and highlights the need to further understand the interplay between the virus and the host cell in order to develop potential avenues for future antiviral therapy.

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A dynamic view of hepatitis C virus replication complexes.

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A dynamic view of hepatitis C virus replication complexes.

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