Efficient Rescue of Hepatitis C Virus RNA Replication by<i>trans</i>-Complementation with Nonstructural Protein 5A

Appel, Nicole; Herian, Ulrike; Bartenschlager, Ralf

doi:10.1128/jvi.79.2.896-909.2005

Cited by 86 publications

(115 citation statements)

References 53 publications

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“…The second scenario favors direct feedback inhibition of translation by a viral protein (Gamarnik and Andino 1998). To this end, it is remarkable that we observed stable interactions between NF90/NFAR-1 and NS5A, the only viral protein that operates in trans during formation of the replication complex and which may, in fact, operate as a feedback inhibitor (Grassmann et al 2001;Appel et al 2005;Kalliampakou et al 2005). NS5A has gained much attention since the protein, besides being part of the viral replicase, is thought to modulate the interferon response and physiology of infected cells (Macdonald and Harris 2004).…”

Section: Discussionmentioning

confidence: 94%

Nuclear factors are involved in hepatitis C virus RNA replication

Isken¹,

Baroth²,

Grassmann³

et al. 2007

RNA

146

171

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Unraveling the molecular basis of the life cycle of hepatitis C virus (HCV), a prevalent agent of human liver disease, entails the identification of cell-encoded factors that participate in the replication of the viral RNA genome. This study provides evidence that the so-called NF/NFAR proteins, namely, NF90/NFAR-1, NF110/NFAR-2, NF45, and RNA helicase A (RHA), which mostly belong to the dsRBM protein family, are involved in the HCV RNA replication process. NF/NFAR proteins were shown to specifically bind to replication signals in the HCV genomic 59 and 39 termini and to promote the formation of a looplike structure of the viral RNA. In cells containing replicating HCV RNA, the generally nuclear NF/NFAR proteins accumulate in the cytoplasmic viral replication complexes, and the prototype NFAR protein, NF90/NFAR-1, stably interacts with a viral protein.HCV replication was inhibited in cells where RNAi depleted RHA from the cytoplasm. Likewise, HCV replication was hindered in cells that contained another NF/NFAR protein recruiting virus. The recruitment of NF/NFAR proteins by HCV is assumed to serve two major purposes: to support 59-39 interactions of the viral RNA for the coordination of viral protein and RNA synthesis and to weaken host-defense mechanisms.

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Section: Discussionmentioning

confidence: 94%

Nuclear factors are involved in hepatitis C virus RNA replication

Isken¹,

Baroth²,

Grassmann³

et al. 2007

RNA

146

171

View full text Add to dashboard Cite

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“…are the only viral replication complex components that can be complemented in trans (3,26). Collectively, structural and functional evidence suggests that the NS5A proteins from HCV and related viruses serve similar functions in the viral life cycle.…”

Section: Discussionmentioning

confidence: 99%

Conserved Determinants for Membrane Association of Nonstructural Protein 5A fromHepatitis C Virus and Related Viruses

Brass

Pál

Sapay

et al. 2007

J Virol

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Nonstructural protein 5A (NS5A) is a membrane-associated essential component of the hepatitis C virus (HCV) replication complex. An N-terminal amphipathic alpha helix mediates in-plane membrane association of HCV NS5A and at the same time is likely involved in specific protein-protein interactions required for the assembly of a functional replication complex. The aim of this study was to identify the determinants for membrane association of NS5A from the related GB viruses and pestiviruses. Although primary amino acid sequences differed considerably, putative membrane anchor domains with amphipathic features were predicted in the N-terminal domains of NS5A proteins from these viruses. Confocal laser scanning microscopy, as well as membrane flotation analyses, demonstrated that NS5As from GB virus B (GBV-B), GBV-C, and bovine viral diarrhea virus, the prototype pestivirus, display membrane association characteristics very similar to those of HCV NS5A. The N-terminal 27 to 33 amino acid residues of these NS5A proteins were sufficient for membrane association. Circular dichroism analyses confirmed the capacity of these segments to fold into alpha helices upon association with lipid-like molecules. Despite structural conservation, only very limited exchanges with sequences from related viruses were tolerated in the context of functional HCV RNA replication, suggesting virus-specific interactions of these segments. In conclusion, membrane association of NS5A by an N-terminal amphipathic alpha helix is a feature shared by HCV and related members of the family Flaviviridae. This observation points to conserved roles of the N-terminal amphipathic alpha helices of NS5A in replication complex formation.The family Flaviviridae comprises the genera Flavirus, Hepacivirus, and Pestivirus, as well as the as-yet-unassigned GB virus A (GBV-A), GBV-B, and GBV-C. These enveloped positivestrand RNA viruses express their structural and nonstructural proteins via translation of a single long open reading frame (30). Hepatitis C virus (HCV) is the sole member of the genus Hepacivirus and a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, affecting an estimated 3% of the world's population (44). GB viruses and pestiviruses are more closely related to HCV than the classical flaviviruses. The availability of efficient cell culture systems and animal models makes these related viruses attractive in vitro and in vivo models for HCV. GBV-B, the closest relative of HCV, was recovered from a tamarin inoculated with blood from a surgeon suffering from acute hepatitis (16, 54) and causes acute and chronic hepatitis in New World monkeys, which are believed to represent the natural hosts (10). Subsequent attempts to isolate GBV-B sequences from humans have failed. By contrast, GBV-C persistently infects humans. However, the pathogenic relevance of GBV-C, if any, remains unclear. It does not cause hepatitis but has been associated with prolonged survival in human immunodeficiency virus-infected individuals (62-64). ...

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“…As a consequence of the membrane compartmentalization and sequestration of viral replication complexes, diffusion of virus-encoded replication proteins between replication complexes encoded by different viral genomes may be limited, leading to a preference for cis-dominant replication functions. For HCV, cis-acting RNA replication functions have been reported for NS3, NS4A, and NS5B, while transacting functions have been reported for NS5A and NS4B (25)(26)(27)(28).…”

Section: H Epatitis C Virus (Hcv) Is An Enveloped Positive-sense Singmentioning

confidence: 99%

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Fridell

Valera

Qiu

et al. 2013

J Virol

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Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion assembly, and exists in hypoand hyperphosphorylated forms. Accumulated data suggest that phosphorylation is involved in modulating NS5A functions. We performed a mutational analysis of highly conserved serine residues in the linker region between domains I and II of genotype 2a JFH1 NS5A. As with genotype 1b Con1 NS5A, we found that specific serine residues were important for efficient hyperphosphorylation of JFH1 NS5A. However, in contrast with Con1 replicons, we observed a positive correlation between hyperphosphorylation and JFH1 replicon replication. We previously demonstrated trans-complementation of a hyperphosphorylation-deficient, replication-defective JFH1 replicon. Our results suggested that the defective NS5A encoded by this replicon, while lacking one NS5A function, was capable of performing a separate replication function. In this report, we examined an additional set of replication-defective NS5A mutations in trans-complementation assays. While some behaved similarly to the S232I replicon, others displayed a unique trans-complementation phenotype, suggesting that NS5A trans-complementation can occur by two distinct modes. Moreover, we were able, for the first time, to demonstrate intragenic complementation of replication-defective NS5A alleles. Our results identified three complementation groups: group A, comprising mutations within NS5A domain I; group B, comprising mutations affecting serine residues important for hyperphosphorylation and a subset of the domain I mutations; and group C, comprising a single mutation within the C-terminal region of domain II. We postulate that these complementation groups define three distinct and genetically separable functions of NS5A in RNA replication. H epatitis C virus (HCV) is an enveloped positive-sense singlestranded RNA virus of the genus Hepacivirus in the familyFlaviviridae. The ϳ9.6-kb HCV genome encodes an ϳ3,000-amino-acid (aa) polyprotein that is cleaved by viral and cellular proteases into 10 mature proteins: core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (reviewed in references 1 and 2). Core is the viral capsid protein, while E1 and E2 are highly glycosylated envelope proteins important for viral entry. P7, a small transmembrane ion channel protein, and NS2, a membrane-associated protease, are required for assembly and release of infectious virions. The remaining nonstructural (NS) proteins are essential components of the HCV RNA replication complex. NS5B is an RNAdependent RNA polymerase that catalyzes synthesis of positiveand negative-strand RNA. NS3 and its cofactor NS4A function as a serine protease responsible for releasing mature NS proteins from the polyprotein precursor. NS3 also possesses RNA helicase and NTPase activities essential for RNA replication. NS4B is an integral membrane protein likely involved in formation of intracellular membranous compartments where viral replication occurs. The final viral component of the replication c...

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

Cited by 86 publications

References 53 publications

Nuclear factors are involved in hepatitis C virus RNA replication

Nuclear factors are involved in hepatitis C virus RNA replication

Conserved Determinants for Membrane Association of Nonstructural Protein 5A fromHepatitis C Virus and Related Viruses

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

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