Dataset Information

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

ABSTRACT: Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

SUBMITTER: Kaul A

PROVIDER: S-EPMC2718831 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

Kaul Artur A Stauffer Sarah S Berger Carola C Pertel Thomas T Schmitt Jennifer J Kallis Stephanie S Zayas Margarita M Lohmann Volker V Luban Jeremy J Bartenschlager Ralf R

PLoS pathogens 20090814 8

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting ...[more]

PMID: 19680534

Similar Datasets

Project description:UNLABELLED:Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis- and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses. IMPORTANCE:Like all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends on cis cleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses.

Project description:Hepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstrated in vitro processing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.IMPORTANCE Hepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.

Project description:In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.

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.

Dataset Information

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

Publications

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets