NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling

Abstract To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (Ay/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.

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Src Homology 3 Domain of Hepatitis C Virus NS5A Protein Interacts With Bin1 and Is Important for Apoptosis and Infectivity

Gastroenterology ◽

10.1053/j.gastro.2005.12.030 ◽

2006 ◽

Vol 130 (3) ◽

pp. 794-809 ◽

Cited By ~ 43

Author(s):

Santosh K. Nanda ◽

David Herion ◽

T. Jake Liang

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Src Homology 3 ◽

Ns5a Protein ◽

Src Homology 3 Domain ◽

Src Homology

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The Small 11-kDa Protein from B19 Parvovirus Binds Growth Factor Receptor-Binding Protein 2 in Vitro in a Src Homology 3 Domain/Ligand-Dependent Manner

Virology ◽

10.1006/viro.2001.1217 ◽

2001 ◽

Vol 291 (2) ◽

pp. 285-291 ◽

Cited By ~ 22

Author(s):

Mannie M.Y. Fan ◽

Lillian Tamburic ◽

Cynthia Shippam-Brett ◽

Darren B. Zagrodney ◽

Caroline R. Astell

Keyword(s):

Growth Factor ◽

Receptor Binding ◽

Growth Factor Receptor ◽

Dependent Manner ◽

Src Homology 3 ◽

B19 Parvovirus ◽

Src Homology 3 Domain ◽

Src Homology ◽

Receptor Binding Protein

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SGIP1α, but Not SGIP1, is an Ortholog of FCHo Proteins and Functions as an Endocytic Regulator

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.801420 ◽

2021 ◽

Vol 9 ◽

Author(s):

Sang-Eun Lee ◽

Eunji Cho ◽

Soomin Jeong ◽

Yejij Song ◽

Seokjo Kang ◽

...

Keyword(s):

Energy Homeostasis ◽

Growth Factor Receptor ◽

Domain Growth ◽

Interacting Protein ◽

Src Homology 3 ◽

Functional Consequences ◽

Src Homology 3 Domain ◽

Src Homology ◽

Growth Factor Receptor Bound

Src homology 3-domain growth factor receptor-bound 2-like interacting protein 1 (SGIP1), originally known as a regulator of energy homeostasis, was later found to be an ortholog of Fer/Cip4 homology domain-only (FCHo) proteins and to function during endocytosis. SGIP1α is a longer splicing variant in mouse brains that contains additional regions in the membrane phospholipid-binding domain (MP) and C-terminal region, but functional consequences with or without additional regions between SGIP1 and SGIP1α remain elusive. Moreover, many previous studies have either inadvertently used SGIP1 instead of SGIP1α or used the different isoforms with or without additional regions indiscriminately, resulting in further confusion. Here, we report that the additional region in the MP is essential for SGIP1α to deform membrane into tubules and for homo-oligomerization, and SGIP1, which lacks this region, fails to perform these functions. Moreover, only SGIP1α rescued endocytic defects caused by FCHo knock-down. Thus, our results indicate that SGIP1α, but not SGIP1, is the functional ortholog of FCHos, and SGIP1 and SGIP1α are not functionally redundant. These findings suggest that caution should be taken in interpreting the role of SGIP1 in endocytosis.

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Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation

Journal of Virology ◽

10.1128/jvi.00567-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7385-7400 ◽

Cited By ~ 4

Author(s):

Wei Xiong ◽

Jie Yang ◽

Mingzhen Wang ◽

Hailong Wang ◽

Zhipeng Rao ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Casein Kinase ◽

Genome Replication ◽

Sh3 Domains ◽

Content Type ◽

Src Homology 3 ◽

Src Homology

ABSTRACTHepatitis C virus (HCV) nonstructural protein 5A (NS5A) is essential for HCV genome replication and virion production and is involved in the regulation of multiple host signaling pathways. As a proline-rich protein, NS5A is capable of interacting with various host proteins containing Src homology 3 (SH3) domains. Previous studies have suggested that vinexin, a member of the sorbin homology (SoHo) adaptor family, might be a potential binding partner of NS5A by yeast two-hybrid screening. However, firm evidence for this interaction is lacking, and the significance of vinexin in the HCV life cycle remains unclear. In this study, we demonstrated that endogenously and exogenously expressed vinexin β coimmunoprecipitated with NS5A derived from different HCV genotypes. Two residues, tryptophan (W307) and tyrosine (Y325), in the third SH3 domain of vinexin β and conserved Pro-X-X-Pro-X-Arg motifs at the C terminus of NS5A were indispensable for the vinexin-NS5A interaction. Furthermore, downregulation of endogenous vinexin β significantly suppressed NS5A hyperphosphorylation and decreased HCV replication, which could be rescued by expressing a vinexin β short hairpin RNA-resistant mutant. We also found that vinexin β modulated the hyperphosphorylation of NS5A in a casein kinase 1α-dependent on manner. Taken together, our findings suggest that vinexin β modulates NS5A phosphorylation via its interaction with NS5A, thereby regulating HCV replication, implicating vinexin β in the viral life cycle.IMPORTANCEHepatitis C virus (HCV) nonstructural protein NS5A is a phosphoprotein, and its phosphorylation states are usually modulated by host kinases and other viral nonstructural elements. Additionally, cellular factors containing Src homology 3 (SH3) domains have been reported to interact with proline-rich regions of NS5A. However, it is unclear whether there are any relationships between NS5A phosphorylation and the NS5A-SH3 interaction, and little is known about the significance of this interaction in the HCV life cycle. In this work, we demonstrate that vinexin β modulates NS5A hyperphosphorylation through the NS5A-vinexin β interaction. Hyperphosphorylated NS5A induced by vinexin β is casein kinase 1α dependent and is also crucial for HCV propagation. Overall, our findings not only elucidate the relationships between NS5A phosphorylation and the NS5A-SH3 interaction but also shed new mechanistic insight onFlaviviridaeNS5A (NS5) phosphorylation. We believe that our results may afford the potential to offer an antiviral therapeutic strategy.

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Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

Journal of Virology ◽

10.1128/jvi.73.2.1649-1654.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1649-1654 ◽

Cited By ~ 172

Author(s):

Eric Ferrari ◽

Jacquelyn Wright-Minogue ◽

Jane W. S. Fang ◽

Bahige M. Baroudy ◽

Johnson Y. N. Lau ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Nonstructural Protein ◽

Full Length ◽

Dependent Manner ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Hydrophobic Domain ◽

Hcv Ns5b

ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

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Interaction of Grb2 SH3 domain with UVRAG in an Alzheimer’s disease–like scenario

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0001 ◽

2014 ◽

Vol 92 (3) ◽

pp. 219-225 ◽

Cited By ~ 1

Author(s):

Kasturi Roy ◽

Oishee Chakrabarti ◽

Debashis Mukhopadhyay

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Growth Factor Receptor ◽

Adaptor Protein ◽

Sh3 Domain ◽

Binding Motifs ◽

Src Homology 3 ◽

Src Homology ◽

Cellular Compartmentalization

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein which participates in trafficking pathways alongside its role in signaling. Proteins important for actin remodeling and cellular compartmentalization contain SRC Homology 3 (SH3) binding motifs that interact with Grb2. While studying the Grb2–amyloid precursor protein (APP) intracellular domain (AICD) interaction in Alzheimer’s disease cell line models, it was seen that Grb2 colocalized to compartments that mature into autophagosomes. The entrapping of AICD in the Grb2 vesicles and its clearance via autophagosomes was a survival contrivance on the part of the cell. Here, we report that Grb2, when in excess, interacts with ultraviolet radiation resistance-associated gene protein (UVRAG) under excess conditions of AICD–Grb2 or Grb2. The N-terminal SH3 domain of Grb2 specifically interacts with UVRAG, unlike the C-terminal SH3 domain. This interaction helps to understand the role of Grb2 in the autophagic maturation of vesicles.

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Cochaperone Activity of Human Butyrate-Induced Transcript 1 Facilitates Hepatitis C Virus Replication through an Hsp90-Dependent Pathway

Journal of Virology ◽

10.1128/jvi.01035-09 ◽

2009 ◽

Vol 83 (20) ◽

pp. 10427-10436 ◽

Cited By ~ 30

Author(s):

Shuhei Taguwa ◽

Hiroto Kambara ◽

Hiroko Omori ◽

Hideki Tani ◽

Takayuki Abe ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Heat Shock Protein 90 ◽

Dependent Manner ◽

Replication Complex ◽

Fk506 Binding Protein ◽

Unfolded Protein ◽

Hsp90 Chaperone ◽

Protein Response

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of the replication complex consisting of several host and viral proteins. We have previously reported that human butyrate-induced transcript 1 (hB-ind1) recruits heat shock protein 90 (Hsp90) and FK506-binding protein 8 (FKBP8) to the replication complex through interaction with NS5A. To gain more insights into the biological functions of hB-ind1 in HCV replication, we assessed the potential cochaperone-like activity of hB-ind1, because it has significant homology with cochaperone p23, which regulates Hsp90 chaperone activity. The chimeric p23 in which the cochaperone domain was replaced with the p23-like domain of hB-ind1 exhibited cochaperone activity comparable to that of the authentic p23, inhibiting the glucocorticoid receptor signaling in an Hsp90-dependent manner. Conversely, the chimeric hB-ind1 in which the p23-like domain was replaced with the cochaperone domain of p23 resulted in the same level of recovery of HCV propagation as seen in the authentic hB-ind1 in cells with knockdown of the endogenous hB-ind1. Immunofluorescence analyses revealed that hB-ind1 was colocalized with NS5A, FKBP8, and double-stranded RNA in the HCV replicon cells. HCV replicon cells exhibited a more potent unfolded-protein response (UPR) than the parental and the cured cells upon treatment with an inhibitor for Hsp90. These results suggest that an Hsp90-dependent chaperone pathway incorporating hB-ind1 is involved in protein folding in the membranous web for the circumvention of the UPR and that it facilitates HCV replication.

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