Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon

100

Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.Hepatitis C virus (HCV) is an enveloped RNA virus that constitutes the genus Hepacivirus within the Flaviviridae family (53). A common feature of this virus group is a single-stranded, linear RNA genome of positive polarity. In the case of HCV the genome has a length of ϳ9,600 nucleotides (nt), and it carries at the 5Ј and 3Ј ends short highly structured nontranslated regions (NTRs) that are important for RNA translation and replication (reviewed in reference 2).

Section: Discussionsupporting

confidence: 63%

Section: Discussionmentioning

confidence: 91%

Efficient Rescue of Hepatitis C Virus RNA Replication bytrans-Complementation with Nonstructural Protein 5A

Appel

Herian

Bartenschlager

2005

100

“…However, a select number of replication and virion assembly defects in the NS3-5B-coding region can be trans-complemented. Early studies using genotype 1b replicons demonstrated transcomplementation of certain NS5A mutations, with most but not all concluding that this required expression of NS5A as an NS3-5A polyprotein (54,59,60). Consistent with such reports, hyperphosphorylation of NS5A, a posttranslational modification associated with replication competency, also requires expression of NS5A as an NS3-5A precursor (61,62).…”

mentioning

confidence: 84%

Genetic Complementation of Hepatitis C Virus Nonstructural Protein Functions Associated with Replication Exhibits Requirements That Differ from Those for Virion Assembly

Herod

Schregel

Hinds

et al. 2014

Within the polyprotein encoded by hepatitis C virus (HCV), the minimum components required for viral RNA replication lie in the NS3-5B region, while virion assembly requires expression of all virus components. Here, we have employed complementation systems to examine the role that HCV polyprotein precursors play in RNA replication and virion assembly. In a trans-complementation assay, an HCV NS3-5A polyprotein precursor was required to facilitate efficient complementation of a replicationdefective mutation in NS5A. However, this requirement for precursor expression was partially alleviated when a second functional copy of NS5A was expressed from an additional upstream cistron within the RNA to be rescued. In contrast, rescue of a virion assembly mutation in NS5A was more limited but exhibited little or no requirement for expression of functional NS5A as a precursor, even when produced in the context of a second replicating helper RNA. Furthermore, expression of NS5A alone from an additional cistron within a replicon construct gave greater rescue of virion assembly in cis than in trans. Combined with the findings of confocal microscope analysis examining the extent to which the two copies of NS5A from the various expression systems colocalize, the results point to NS3-5A playing a role in facilitating the integration of nonstructural (NS) proteins into viral membrane-associated foci, with this representing an early stage in the steps leading to replication complex formation. The data further imply that HCV employs a minor virion assembly pathway that is independent of replication. IMPORTANCEIn hepatitis C virus-infected cells, replication is generally considered an absolute prerequisite for virus particle formation. Here we investigated the role that the viral protein NS5A has in both replication and particle assembly using complementation assays and microscopy. We found that efficient rescue of replication required NS5A to be expressed as part of a larger polyprotein, and this correlated with detection of NS5A at sites where replication occurred. In contrast, rescue of particle assembly did not require expression of NS5A within the context of a polyprotein. Interestingly, although only partial restoration of particle assembly was possible by complementation, that proportion that could be rescued benefitted from expressing NS5A from the same RNA being packaged. Collectively, these findings provide new insight into aspects of polyprotein function. They also support the existence of a minor virion assembly pathway that bypasses replication.

“…The con1 replicon clone has been described (15,16). Reporter replicons for transient assays were made by replacing the neo gene with genes encoding Renilla luciferase or ␤-lactamase (15,18). NS5A mutations in replicon and virus clones were introduced with recombinant PCR and standard cloning techniques and were verified by sequence analysis.…”

Section: Methodsmentioning

confidence: 99%

Distinct Functions of NS5A in Hepatitis C Virus RNA Replication Uncovered by Studies with the NS5A Inhibitor BMS-790052

Fridell

Qiu

Valera

et al. 2011

133

118

BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC 50 s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified druginduced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Secondsite replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interaction between NS5A residues 30 and 92. By using a trans-complementation assay with JFH1 replicons encoding inhibitor-sensitive and inhibitor-resistant NS5A proteins, we provide genetic evidence that NS5A performs the following two distinct functions in HCV RNA replication: a cis-acting function that likely occurs as part of the HCV replication complex and a trans-acting function that may occur outside the replication complex. The cis-acting function is likely performed by basally phosphorylated NS5A, while the trans-acting function likely requires hyperphosphorylation. Our data indicate that BMS-790052 blocks the cis-acting function of NS5A. Since BMS-790052 also impairs JFH1 NS5A hyperphosphorylation, it likely also blocks the trans-acting function.Hepatitis C virus (HCV), a positive-strand RNA virus of the Flaviviridae family, is a leading cause of liver disease and hepatocellular carcinoma (35, 59). The HCV genome encodes a single polyprotein that is cleaved by cellular and viral proteases into at least 10 individual proteins (40, 48). Nonstructural protein 3 (NS3), NS4A, NS4B, NS5A, and NS5B are sufficient for HCV RNA replication in cell culture (6, 30). NS3-4A is the primary viral protease, and NS5B is an RNAdependent RNA polymerase (4,5,14). NS4B, a hydrophobic protein with multiple trans-membrane domains, induces an endoplasmic reticulum-derived membranous web that harbors the HCV replication complex (10, 32). NS5A is also essential for HCV RNA replication, but its precise roles in this process are poorly understood (21, 33).NS5A is an RNA binding protein that interacts with other HCV nonstructural proteins and is capable of altering NS5B polymerase activity in vitro (9,20,41,46,47). NS5A also interacts with a variety of cellular proteins and is potentially involved in modulating multiple aspects of the cellular environment (33, 43). In addition to its role(s) in RNA replication, NS5A is also required for viral assembly (3, 50). NS5A is a modular protein with an N-terminal amphipathic ␣-helix membrane anchor (amino acids [aa] 5 t...