Identification and characterization of amphiphysin II as a novel cellular interaction partner of the hepatitis C virus NS5A protein

Zech, Birgit; Kurtenbach, Alexander; Krieger, Nicole; Strand, Dennis; Blencke, Stephanie; Morbitzer, Monika; Salassidis, Kostas; Cotten, Matthew; Wissing, Josef; Obert, Sabine; Bartenschlager, Ralf; Herget, Thomas; Daub, Henrik

doi:10.1099/vir.0.18801-0

Cited by 49 publications

(42 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It was only able to bind to the SH3 domain of the Src-family tyrosine kinase, Lyn (16), whereas the PP2.2 motif bound to Lyn, Fyn, Lck, Hck, the adaptor protein Grb2 (25), and amphiphysin II (33). In addition to this, the first proline in this motif (residue 343) is only conserved in genotype 1 isolates of HCV; the lack of this proline would disrupt the SH3-binding consensus, suggesting that if the prolines within PP2.1 did play a role in the viral replication cycle, it was unrelated to the ability to bind SH3 domains.…”

Section: Resultsmentioning

confidence: 99%

“…Of particular interest is the observation that within LCS2 (between domains II and III) (Fig. 1a), NS5A contains two closely spaced polyproline motifs that are able to bind to the SH3 domains of Src-family tyrosine kinases (16), as well as other SH3 domain containing proteins (e.g., Grb2 and amphiphysin II/Bin1) (23,25,33). These motifs, which we have termed PP2.1 and PP2.2 (Fig.…”

mentioning

confidence: 99%

“…1a), conform to the consensus SH3 binding motif Pro-X-X-Pro-X-Arg/Lys, where X is any amino acid (21). Interestingly, although the C-terminal motif (PP2.2) is absolutely conserved in all HCV genotypes, we and others have shown that it is dispensable for HCV RNA replication because alanine substitution of three prolines in this motif (shown to abolish SH3 domain interactions [16]) within a culture-adapted subgenomic replicon had either no effect (19) or resulted in only a modest reduction in replicative capacity (23,33). The N-terminal motif (PP2.1), however, is only conserved in genotype 1 isolates, although within this motif a proline at residue 346 in genotype 1b (342 in genotype 2a) is absolutely conserved throughout all genotypes, which suggests it has an important role in virus replication.…”

mentioning

confidence: 99%

See 2 more Smart Citations

A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

Hughes

Gretton

Shelton

et al. 2009

J Virol

View full text Add to dashboard Cite

We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.Hepatitis C virus (HCV) is an enveloped RNA virus which is estimated to infect some 123 million individuals (24). In the majority of cases the virus establishes a chronic infection that can ultimately result in liver fibrosis, cirrhosis, or hepatocellular carcinoma. Thus, there is great interest in elucidating the mechanisms of viral replication, with a view to developing new chemotherapeutic agents. Since 1999, use of the subgenomic replicon system has led to significant progress in the understanding of the mechanism of viral RNA replication. It has been demonstrated that the five nonstructural proteins-NS3, NS4A, NS4B, NS5A, and NS5B-are necessary and sufficient to replicate an RNA molecule containing the 5Ј and 3Ј untranslated regions (UTRs) of the viral genome. However, apart from the RNA-dependent RNA polymerase (NS5B), the precise details of the roles of each of the nonstructural proteins in the process of RNA replication remain undefined. One problem associated with the subgenomic replicon system is the observation that the replicon RNA undergoes culture adaptation in which, as a result of the error-prone nature of the polymerase, mutations that confer enhanced replicative capacity are selected for in culture. Importantly, it has been shown using the chimpanzee model that, once engineered back into an infectious clone of the virus, such mutations may be attenuating in vivo (5). Recently, the HCV field has been revolutionized by the developmen...

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

Hughes

Gretton

Shelton

et al. 2009

J Virol

View full text Add to dashboard Cite

show abstract

“…both proteins are phosphorylated by the same or similar serine/threonine protein kinase(s) (Reed et al, 1998), both are the only nonstructural (NS) proteins that are complemented in trans (Appel et al, 2005;Grassmann et al, 2001), both share an N-terminal membrane-anchor a-helix (Brass et al, 2007) and, finally, both contain zinc-binding cysteine residues at their N terminus (Tellinghuisen et al, 2005(Tellinghuisen et al, , 2006. Although HCV NS5A has been shown to be associated with a range of cellular proteins involved in cellular signalling pathways (Macdonald & Harris, 2004), such as interferon-induced kinase PKR (Gale et al, 1998), p53 (Lan et al, 2002) and TRAF2 (Park et al, 2002), and with proteins involved in protein trafficking and membrane morphology, such as apolipoprotein A1 (Shi et al, 2002), amphiphysin II (Zech et al, 2003), soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) protein (Tu et al, 1999), human vesicle-associated membrane protein-associated protein subtype B (hVAP-B) (Hamamoto et al, 2005) and Rab-GTPase (Sklan et al, 2007), little is known about the role of NS5A in BVDV RNA replication.…”

Section: Introductionmentioning

confidence: 99%

Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKβ-binding protein

Zahoor

Yamane

Mohamed

et al. 2010

Journal of General Virology

View full text Add to dashboard Cite

Bovine viral diarrhea virus (BVDV) is a positive-sense, single-stranded RNA virus that causes an economically important livestock disease worldwide. Previous studies have suggested that nonstructural protein 5A (NS5A) from hepatitis C virus (HCV) and BVDV plays a similar role during virus infection. Extensive reports are available on HCV NS5A and its interactions with the host cellular proteins; however, the role of NS5A during BVDV infection remains largely unclear. To identify the cellular proteins that interact with the N terminus of NS5A and could be involved in its function, we conducted a yeast two-hybrid screening. As a result, we identified a cellular protein termed bovine NIK-and IKKb-binding protein (NIBP), which is involved in protein trafficking and nuclear factor kappa B (NF-kB) signalling in cells. The interaction of NS5A with NIBP was confirmed both in vitro and in vivo. Complementing our glutathione S-transferase pull-down and immunoprecipitation data are the confocal immunofluorescence results, which indicate that NS5A colocalized with NIBP on the endoplasmic reticulum in the cytoplasm of BVDV-infected cells. Moreover, the minimal residues of NIBP that interact with NS5A were mapped as aa 597-623. In addition, overexpression of NS5A inhibited NF-kB activation in HEK293 and LB9.K cells as determined by luciferase reporter-gene assay. We further showed that inhibition of endogenous NIBP by small interfering RNA molecules enhanced virus replication, indicating the importance of NIBP implications in BVDV pathogenesis. Being the first reported interaction between NIBP and a viral protein, this finding suggests a novel mechanism whereby viruses may subvert host-cell machinery for mediating trafficking as well as NF-kB signalling. INTRODUCTIONPestiviruses are important livestock pathogens that belong to the family Flaviviridae, which also includes the closely related genera Hepacivirus (Hepatitis C virus; HCV) and Flavivirus (Yellow fever virus and West Nile virus). The genus Pestivirus includes the species Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, Border disease virus and Classical swine fever virus. As a well-characterized prototype member of the genus Pestivirus within the family Flaviviridae, BVDV sometimes serves as a surrogate model for HCV life-cycle studies. BVDV is an enveloped virus containing a single, positive-sense RNA of approximately 12.3 kb. The genomic RNA consists of one long open reading frame (ORF), which is flanked by untranslated regions at both ends. The ORF encodes a single polyprotein of about 4000 aa, which is processed co-and posttranslationally by both viral and cellular proteases into at least 11 mature viral proteins (N pro , C, E rns , E1, E2, p7, NS2-3, NS4A, NS4B, NS5A and NS5B) (Lindenbach et al., 2007).BVDV non-structural protein 5A (NS5A) is a phosphoprotein of 56-58 kDa that plays an essential role in BVDV replication (Tellinghuisen et al., 2006) and shares many features with its counterpart HCV NS5A, e.g. both proteins are phosphorylated by the same or simila...

show abstract

“…NS5A seems to have the potential to regulate not only interferon responses but also many other cellular functions, such as mitogenic signalling, apoptosis, the cell cycle and reactive oxygen species signalling, by interacting with a variety of host proteins . These NS5A-interacting proteins include SRCAP (Ghosh et al, 2000), Grb2 (He et al, 2002;Tan et al,1999), p53 (Majumder et al, 2001;Qadri et al, 2002), phosphatidylinositol 3-kinase p85 subunit (He et al, 2002;Street et al, 2004), karyopherin b3 (Chung et al, 2000), apolipoprotein A1 (Shi et al, 2002), amphiphysin II (Zech et al, 2003) and Src family protein tyrosine kinases .…”

Section: Introductionmentioning

confidence: 99%

Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk

Inubushi

Nagano-Fujii

Kitayama

et al. 2008

Journal of General Virology

View full text Add to dashboard Cite

Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-c1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities. INTRODUCTIONHepatitis C virus (HCV) is the major aetiological agent of viral hepatitis worldwide after hepatitis A and B viruses (Choo et al., 1989), with about 170 million people being infected. The majority of HCV-infected individuals develop chronic infection, which may progress to liver cirrhosis and hepatocellular carcinoma (HCC). HCV is a member of the family Flaviviridae and its genome consists of a single-stranded, positive-sense RNA of approximately 9600 nt, which encodes a polyprotein precursor of about 3010 aa. Currently, clinical HCV isolates are classified into six genotypes and more than 60 subtypes (Doi et al., 1996;Mellor et al., 1995;Robertson et al., 1998). The polyprotein is cleaved by signal peptidase, signal peptide peptidase and two virally encoded proteases to generate at least ten mature proteins: core, envelope glycoprotein 1 (E1), E2, p7, non-structural protein 2 (NS2), NS3, NS4A, NS4B, NS5A and NS5B Reed & Rice, 2000).HCV NS5A is part of the replication complex that catalyses replication of the viral genome. NS5A takes two forms, p56 and p58, with different degrees of phosphorylation, which may play distinct roles in the virus replication cycle (Evans 3These authors contributed equally to this work. YXX(L/I)X 6-8 YXX(L/I)] in the cytoplasmic tail of the Fc receptor c-chain or B-cell receptor subunit Iga to be activated after the engagement of immune receptors (Kurosaki et al...

show abstract

Identification and characterization of amphiphysin II as a novel cellular interaction partner of the hepatitis C virus NS5A protein

Cited by 49 publications

References 32 publications

A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKβ-binding protein

Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk

Contact Info

Product

Resources

About