Reduction of Hepatitis C Virus NS5A Hyperphosphorylation by Selective Inhibition of Cellular Kinases Activates Viral RNA Replication in Cell Culture

Neddermann, Petra; Quintavalle, Manuela; Pietro, Chiara Di; Clementi, Angelica; Cerretani, Mauro; Altamura, Sergio; Bartholomew, Linda; Francesco, Raffaele De

doi:10.1128/jvi.78.23.13306-13314.2004

Cited by 129 publications

(152 citation statements)

References 28 publications

Supporting

Mentioning

143

Contrasting

Unclassified

Order By: Relevance

“…Many of these mutations cluster among others, in LCS I of NS5A, and they often reduce hyperphosphorylation arguing that this modification is of disadvantage for efficient RNA replication (8,45). In support of this assumption it was found that treatment of cells carrying non-adapted replicons with an inhibitor of the cellular kinase(s) responsible for NS5A hyperphosphorylation leads to an increase of RNA replication (46). However, when cells carrying adapted replicons were treated with the same kinase inhibitor, replication was reduced.…”

Section: Components Of the Hcv Replication Complexsupporting

confidence: 54%

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

Appel

Schaller

Pénin

et al. 2006

Journal of Biological Chemistry

176

120

View full text Add to dashboard Cite

With an estimated number of about 180 million affected people worldwide and a limited therapy option, infection with the hepatitis C virus (HCV) 2 is an important medical problem. Initial limitations in propagating HCV in cell culture have been overcome step-by-step in the past few years and culminated in the recent development of a system allowing efficient propagation of infectious HCV in tissue culture. Nevertheless, structural and biochemical studies of viral proteins as well as molecular analysis of viral RNA replication are still major challenges. The recent resolution of the three-dimensional structures of some HCV proteins or domains along with elegant reverse genetic and cell biological studies provided first insights into how HCV amplifies its RNA, exploits the cellular machinery, and eventually overcomes innate immune responses. Organization of the Viral GenomeHCV is a positive strand RNA virus that has been classified as the genus Hepacivirus in the Flaviviridae family. The HCV genome is an uncapped, linear molecule with a length of ϳ9600 nucleotides (nt; Fig. 1A). It carries a long open reading frame that is flanked at the 5Ј-and 3Ј-ends by short highly structured non-translated regions (NTRs). The 5Ј-NTR has a length of about 340 nt and contains an internal ribosome entry site (IRES) required for translation of the HCV genome (1). This RNA element binds the 40 S ribosomal subunit in the absence of other translation initiation factors in a way that the initiation codon is placed in the immediate vicinity of the P site (2). Part of the IRES (domain II) overlaps with RNA signals essential for viral replication (Fig. 1A) arguing for a possible role of domain II in regulating a translation-RNA replication switch. The 3Ј-NTR has a tripartite structure composed of an ϳ40-nt-long variable region downstream of the HCV coding sequence, a poly(U/UC) tract of heterogeneous length, and a highly conserved 98-nt-long sequence designated X-tail (Fig. 1A). Genetic studies have shown that a poly(U/UC) tract of at least ϳ25 nt as well as the complete X-tail are required for RNA replication in cell culture and for infectivity of the viral genome in vivo. An additional cis-acting RNA element (CRE) has been identified in the 3Ј-terminal coding region of NS5B (Fig. 1A). This CRE (designated 5BSL3.2) (3) forms a long distance RNA-RNA interaction with SL2 in the X-tail (4), which is indispensable for RNA replication.Expression of the viral proteins from the monocistronic genome is primarily achieved by production of a polyprotein that is proteolytically cleaved into the structural proteins (core, envelope proteins E1 and E2), the hydrophobic peptide p7, and the non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Fig. 1A) (1, 5). Processing of the core to p7 region is mediated by host cell signalases, and in the case of the core protein, in addition by signal peptide peptidase. All remaining cleavages are carried out by two viral proteases: the NS2/3 protease mediating cleavage between NS2 and NS3 and the NS3...

show abstract

Section: Components Of the Hcv Replication Complexsupporting

confidence: 54%

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

Appel

Schaller

Pénin

et al. 2006

Journal of Biological Chemistry

176

120

View full text Add to dashboard Cite

show abstract

“…64 In agreement with this assumption, pharmacological inhibition of NS5A hyperphosphorylation that is mediated by CK1, enhances RNA replication. 61,66,151 Point mutations preventing NS5A hyperphosphorylation enhance RNA replication, but strongly impair virus production, arguing that NS5A phosphorylation status regulates the switch between RNA replication and assembly. 60 How could this regulation be achieved?…”

Section: ©2 0 1 1 L a N D E S B I O S C I E N C E D O N O T D I S Tmentioning

confidence: 99%

“…59,60 It is assumed that newly synthesized viral RNA is transported to LDs by NS5A, 24 whose phosphorylation state possibly regulating the balance between viral RNA replication and particle assembly. 59,60,[64][65][66] NS5B, the RNA-dependent RNA polymerase, is composed of an N-terminal catalytic domain and a C-terminal TM segment anchoring it to intracellular membranes ( Fig. 1).…”

Section: Hcv Proteinsmentioning

confidence: 99%

Hepatitis c virus and host cell lipids: An intimate connection

Alvisi¹,

Madan²,

2011

View full text Add to dashboard Cite

“…Interestingly, its phosphorylation state was found to modulate HCV RNA replication (4,22,43). The crystal structure of domain I, comprising the N-terminal one-third of NS5A, has recently been solved, revealing a novel protein fold, a zinc coordination motif, and a dimerization interface (58).…”

mentioning

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

View full text Add to dashboard Cite

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). ...

show abstract

Reduction of Hepatitis C Virus NS5A Hyperphosphorylation by Selective Inhibition of Cellular Kinases Activates Viral RNA Replication in Cell Culture

Cited by 129 publications

References 28 publications

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

Hepatitis c virus and host cell lipids: An intimate connection

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

Contact Info

Product

Resources

About