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Lipid droplet binding of hepatitis C virus core protein genotype 3.

ABSTRACT: Background. Hepatitis C virus (HCV) genotype 3 is known to cause steatosis (fatty liver) that is more frequent and severe than other genotypes. We previously identified sequence elements within genotype 3 HCV Core domain 3 that were sufficient for lipid accumulation. Aims. We examined various genotype 3 Core domains for lipid droplet localization and compared the lipid droplet binding regions of domain 2 with a genotype 1 isolate. Methods. We generated HCV Core domain constructs fused with green fluorescent protein and performed immunofluorescence to visualize lipid droplets. Results. Constructs containing HCV Core domain 2 are appropriately localized to lipid droplets with varying degrees of efficiency. When compared to genotype 1, there are polymorphisms within domain 2 that do not appear to alter lipid droplet localization. Conclusions. In summary, the differences in a steatosis-associated HCV Core genotype 3 isolate do not appear to involve altered lipid droplet localization.

SUBMITTER: Qiang G

PROVIDER: S-EPMC3401521 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Lipid droplet binding of hepatitis C virus core protein genotype 3.

Qiang Guan G Jhaveri Ravi R

ISRN gastroenterology 20120711

Background. Hepatitis C virus (HCV) genotype 3 is known to cause steatosis (fatty liver) that is more frequent and severe than other genotypes. We previously identified sequence elements within genotype 3 HCV Core domain 3 that were sufficient for lipid accumulation. Aims. We examined various genotype 3 Core domains for lipid droplet localization and compared the lipid droplet binding regions of domain 2 with a genotype 1 isolate. Methods. We generated HCV Core domain constructs fused with green ...[more]

PMID: 22844606

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Project description:AIM:To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS:HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. RESULTS:C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION:The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression.

Project description:Retinoid (vitamin A) is an essential diet constituent that governs a broad range of biological processes. Its biologically active metabolite, all-trans retinoic acid (ATRA), exhibits a potent antiviral property by enhancing both innate and adaptive antiviral immunity against a variety of viral pathogens, such as, but not limited to, HIV, respiratory syncytial virus (RSV), herpes simplex virus (HSV), and measles. Even though the hepatocyte is highly enriched with retinoid and its metabolite ATRA, it supports the establishment of efficient hepatitis C virus (HCV) replication. Here, we demonstrate the hepatocyte-specific cell-intrinsic mechanism by which ATRA exerts either a proviral or antiviral effect, depending on how it engages cellular retinoic acid binding proteins (CRABPs). We found that the engagement of CRABP1 by ATRA potently supported viral infection by promoting the accumulation of lipid droplets (LDs), which robustly enhanced the formation of a replication complex on the LD-associated endoplasmic reticulum (ER) membrane. In contrast, ATRA binding to CRABP2 potently inhibited HCV via suppression of LD accumulation. However, this antiviral effect of CRABP2 was abrogated due to the functional and quantitative predominance of CRABP1 in the hepatocytes. In summary, our study demonstrates that CRABPs serve as an on-off switch that modulates the efficiency of the HCV life cycle and elucidates how HCV evades the antiviral properties of ATRA via the exploitation of CRABP1 functionality.IMPORTANCE ATRA, a biologically active metabolite of vitamin A, exerts pleiotropic biological effects, including the activation of both innate and adaptive immunity, thereby serving as a potent antimicrobial compound against numerous viral pathogens. Despite the enrichment of hepatocytes with vitamin A, HCV still establishes an efficient viral life cycle. Here, we discovered that the hepatocellular response to ATRA creates either a proviral or an antiviral environment depending on its engagement with CRABP1 or -2, respectively. CRABP1 supports the robust replication of HCV, while CRABP2 potently inhibits the efficiency of viral replication. Our biochemical, genetic, and microscopic analyses reveal that the pro- and antiviral effects of CRABPs are mediated by modulation of LD abundance, where HCV establishes the platform for viral replication and assembly on the LD-associated ER membrane. This study uncovered a cell-intrinsic mechanism by which HCV exploits the proviral function of CRABP1 to establish an efficient viral life cycle.

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Lipid droplet binding of hepatitis C virus core protein genotype 3.

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Lipid droplet binding of hepatitis C virus core protein genotype 3.

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