Hepatitis C virus infection induces the expression of amphiregulin, a factor related to the activation of cellular survival pathways and required for efficient viral assembly

Guo

et al. 2012

J Virol

Self Cite

c Similar to other positive-sense, single-stranded RNA viruses, hepatitis C virus (HCV) replicates its genome in a remodeled intracellular membranous structure known as the membranous web (MW). To date, the process of MW formation remains unclear. It is generally acknowledged that HCV nonstructural protein 4B (NS4B) can induce MW formation through interaction with the cytosolic endoplasmic reticulum (ER) membrane. Many host proteins, such as phosphatidylinositol 4-kinase III␣ (PI4KIII␣), have been identified as critical factors required for this process. We now report a new factor, the cytosolic phospholipase A2 gamma (PLA2G4C), which contributes to MW formation, HCV replication, and assembly. The PLA2G4C gene was identified as a host gene with upregulated expression upon HCV infection. Knockdown of PLA2G4C in HCV-infected cells or HCV repliconcontaining cells by small interfering RNA (siRNA) significantly suppressed HCV replication and assembly. In addition, the chemical inhibitor methyl arachidonyl fluorophosphonate (MAFP), which specifically inhibits PLA2, reduced HCV replication and assembly. Electron microscopy demonstrated that MW structure formation was defective after PLA2G4C knockdown in HCV replicon-containing cells. Further analysis by immunostaining and immunoprecipitation assays indicated that PLA2G4C colocalized with the HCV proteins NS4B and NS5A in cells infected with JFH-1 and interacted with NS4B. In addition, PLA2G4C was able to transport the HCV nonstructural proteins from replication sites to lipid droplets, the site for HCV assembly. These data suggest that PLA2G4C plays an important role in the HCV life cycle and might represent a potential target for anti-HCV therapy.

Section: Resultsmentioning

confidence: 99%

Cytosolic Phospholipase A2 Gamma Is Involved in Hepatitis C Virus Replication and Assembly

Guo

et al. 2012

J Virol

Self Cite

“…The JFH1-HA virus with a hemagglutinin (HA) epitope at the N terminus of NS2 was constructed as previously described (15). The viral titer in the culture supernatants and cell lysates was determined by a modified endpoint dilution assay as previously described (30,34).…”

Section: Methodsmentioning

confidence: 99%

“…Huh7.5.1 cells (kindly provided by Frank Chisari) and Huh7 cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Invitrogen) supplemented with 2 mM L-glutamine, nonessential amino acids, and 10% fetal bovine serum (FBS) (Invitrogen). The subgenomic HCV replicon cell lines (Huh7.5.1-SGR [30] and Con1 [34] containing subgenomic genotype 2a and 1b HCV, respectively) were grown in the same medium supplemented with 500 g ml Ϫ1 G418. Virus production.…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylserine-Specific Phospholipase A1 Involved in Hepatitis C Virus Assembly through NS2 Complex Formation

Guo

Yang

et al. 2015

J Virol

Self Cite

Several members of the phospholipase family have been reported to be involved in hepatitis C virus (HCV) replication. Here, we identified another phospholipase, phosphatidylserine-specific phospholipase A1 (PLA1A), as a host factor involved in HCV assembly. PLA1A was upregulated by HCV infection, and PLA1A knockdown significantly reduced J399EM (genotype 2a) HCV propagation at the assembly step but not the entry, RNA replication, and protein translation steps of the life cycle. Protein localization and interaction analysis further revealed a role of PLA1A in the interaction of NS2-E2 and NS2-NS5A, as the formation of the NS2-E2 and NS2-NS5A complexes was weakened in the absence of PLA1A. In addition, PLA1A stabilized the NS2/NS5A dotted structure during infection. These data suggest that PLA1A plays an important role in bridging the membrane-associated NS2-E2 complex and the NS5A-associated replication complex via its interaction with E2, NS2, and NS5A, which leads to a coordinating interaction between the structural and nonstructural proteins and facilitates viral assembly. IMPORTANCEHepatitis C virus (HCV) genomic replication is driven by the replication complex and occurs at the membranous web, while the lipid droplet is the organelle in which virion assembly is initiated. In this study, we identified phosphatidylserine-specific phospholipase A1 (PLA1A), a member of phospholipase A 1 family, as a novel host factor involved in the assembly process of HCV. PLA1A, which is induced by HCV infection at a late infection stage, interacts with HCV E2, NS2, and NS5A proteins and enhances and stabilizes the NS2-E2 and NS2-NS5A complex formation, which is essential for viral assembly. Thus, PLA1A is an important host factor which is involved in the initiation of the viral assembly in close proximity to Core-decorated lipid droplets through bringing together the HCV replication complex and envelope complex. Hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting approximately 185 million people worldwide (1). HCV is a positive single-stranded RNA virus belonging to the Flaviviridae family. The HCV 9.6-kb genome contains a large open reading frame encoding a single polyprotein that is processed into its structural proteins (Core, E1, and E2) and nonstructural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) by host and viral proteinases (2). The structural proteins are components of the virion, while nonstructural proteins NS3 to NS5B compose the minimal viral replicase governing RNA replication (3, 4).The overall HCV life cycle has been well defined since the development of an infectious HCV cell culture system (5-7). HCV genomic replication is driven by the replication complex (RC) and occurs at the membranous web, a rearranged membrane structure induced by virus infection (8-10). Recent progress regarding the study of the assembly process demonstrates that the lipid droplet (LD) is the organelle in which virion assembly is initiated (11) and that, in addition to the structural prote...

“…The mixtures were centrifuged, washed with IP buffer four times, denatured by boiling in SDS-PAGE sample buffer for 5-10 min, and subjected to SDS-PAGE. Immunoblot (IB) analysis was performed as previously described (35). An Ultra ECL kit, Western Lightning Ultra, was purchased from PerkinElmer.…”

Section: Immunoprecipitation Coimmunoprecipitation and Immunoblot Amentioning

confidence: 99%

An Alternative Splicing Isoform of MITA Antagonizes MITA-Mediated Induction of Type I IFNs

Chen

The Journal of Immunology

Zhu

et al. 2014

Self Cite

Mediator of IFN regulatory transcription factor 3 activation (MITA) is an important adaptor protein to mediate the induction of type I IFNs. In this study, we identified an alternatively spliced isoform of MITA lacking exon 7, termed MITA-related protein (MRP). MRP shares the N-terminal portion aa 1–253 with MITA but possesses a unique 30-aa sequence at the carboxyl terminal part, therefore lacking the conserved domains including TANK-binding kinase 1 (TBK1) and cyclic diguanylate binding domain. MRP is expressed in multiple tissues and distinct cell lines. Overexpression of MRP inhibited MITA-mediated activation of IFN-β promoter by sendai virus infection and cyclic diguanylate treatment but enhanced that in HSV-1 infection. Interestingly, MRP expression was reduced after Sendai virus infection but was upregulated after HSV-1 infection. Overexpression of MRP inhibited MITA-mediated induction of IFN-β via TBK1-IFN regulatory transcription factor 3 by disrupting the MITA-TBK1 interaction. However, NF-κB pathway was still activated by MRP, as MRP retained the ability to interact with inducible inhibitor of NF-κB (iκB) kinase. Thus, MRP acts as a dominant negative regulator of MITA-mediated induction of IFN production.