Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication

HIV-1 Gag, which drives virion assembly, interacts with a plasma membrane (PM)-specific phosphoinositide, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ]. While cellular acidic phospholipid-binding proteins/domains, such as the PI(4,5)P 2 -specific pleckstrin homology domain of phospholipase C␦1 (PH PLC␦1 ), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P 2 interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P 2 with unsaturated or saturated acyl chains affect membrane binding of PH PLC␦ 1 and Gag. Both unsaturated dioleoyl-PI(4,5)P 2 [DO-PI(4,5)P 2 ] and saturated dipalmitoyl-PI(4,5)P 2 [DP-PI(4,5)P 2 ] successfully recruited PH PLC␦ 1 to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P 2 but not DP-PI(4,5)P 2 recruited Gag to GUVs, indicating that PI(4,5)P 2 acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P 2 , cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PH PLC␦ 1 regardless of PI(4,5)P 2 acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P 2 had DO-acyl chains. DP-PI(4,5)P 2 -containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P 2 still promoted recruitment of Gag, but not PH PLC␦ 1 , to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P 2 acyl chains in membrane binding of two PI(4,5)P 2 -binding proteins, Gag and PH PLC␦ 1 . Notably, we observed that nonmyristylated Gag retains the preference for PI(4,5)P 2 containing an unsaturated acyl chain over DP-PI(4,5)P 2 , suggesting that Gag sensitivity to PI(4,5)P 2 acyl chain saturation is determined directly by the matrix-PI(4,5)P 2 interaction, rather than indirectly by a myristate-dependent mechanism. IMPORTANCEBinding of HIV-1 Gag to the plasma membrane is promoted by its interaction with a plasma membrane-localized phospholipid, PI(4,5)P 2 . Many cellular proteins are also recruited to the plasma membrane via PI(4,5)P 2 -interacting domains represented by PH PLC␦ 1 . However, differences and/or similarities between these host proteins and viral Gag protein in the nature of their PI(4,5)P 2 interactions, especially in the context of membrane binding, remain to be determined. Using a novel giant unilamellar vesicle-based system, we found that PI(4,5)P 2 with an unsaturated acyl chain recruited PH PLC␦ 1 and Gag similarly, whereas PI(4,5)P 2 with saturated acyl chains either recruited PH PLC␦ 1 but not Gag or sorted these proteins to different phases of vesicles. To our knowledge, this is the first study to show that PI(4,5)P 2 acyl chains differentially modulate membrane binding of PI(4,5)P 2 -binding proteins. Since Gag membrane binding is essential for progeny...

Section: Figmentioning

confidence: 99%

Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain

Olety

Veatch

Ono

2015

“…We next asked if Pim kinases are involved in HCV internal ribosome entry site (IRES)-mediated translation. To address this question, Huh7.5 cells were treated with various concentrations of SGI-1776 and then transfected with pRL-HL and ␤-galactosidase plasmid as we reported previously (19), and then luciferase activity was determined. We demonstrated that treatment of Pim kinase inhibitor displayed no effects on HCV IRES-dependent translation (Fig.…”

Section: Identification Of Pim1 As An Ns5a Interactor In Protein Arraymentioning

confidence: 99%

“…For the dual-luciferase reporter assay, Huh7.5 cells were transfected with pRL-HL plasmid and pCH110 reference plasmid as we described previously (19). The cells were then incubated with various dosages of Pim kinase inhibitor.…”

mentioning

confidence: 99%

“…Immunoblot analysis was performed as we described previously (19) using the following antibodies: rabbit anti-NS5A, rabbit anti-core, and rabbit anti-NS3 from Byung-Yoon Ahn (Korea University), rabbit anti-Pim1 (Cell signaling), anti-Pim2 (Cell Signaling), anti-Pim3 (Cell Signaling), mouse anti-␤ actin (SigmaAldrich), mouse anti-Myc (Santa Cruz), mouse anti-ubiquitin (Santa Cruz), and mouse anti-Flag (Sigma-Aldrich). Either horseradish peroxidase-conjugated goat anti-rabbit antibody or goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody.…”

mentioning

confidence: 99%

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Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry

Park

Min

Park

et al. 2015

Self Cite

The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ϳ9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry. IMPORTANCEPim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCVinfected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell entry factor but also as a new anti-HCV therapeutic target.

“…TRIB3 promoter constructs (from bp Ϫ2000 to ϩ558) were cloned by using Huh7 genomic DNA and inserted into the pGL3 Basic vector (Promega). Myc-tagged HCV core, NS3, NS4B, NS5A, and NS5B plasmids were described previously (11). For DNA transfection, cells were transfected with the expression plasmid by using a polyethyleneimine reagent (Sigma-Aldrich) as we described previously (11).…”

Section: Methodsmentioning

confidence: 99%

Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection

Tran

Pham

Nguyen

et al. 2016

Self Cite

Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCVinfected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis. IMPORTANCETRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of TRIB3 in virus-infected cells has not yet been demonstrated. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels, and thus, overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating the Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that the TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates the TRIB3-Akt signaling pathway to establish persistent viral infection.