Targeting of Hepatitis C Virus Core Protein to Mitochondria through a Novel C-Terminal Localization Motif

Abstract

The hepatitis C virus (HCV) core protein represents the first 191 amino acids of the viral precursor polyprotein and is cotranslationally inserted into the membrane of the endoplasmic reticulum (ER). Processing at position 179 by a recently identified intramembrane signal peptide peptidase leads to the generation and potential cytosolic release of a 179-amino-acid matured form of the core protein. Using confocal microscopy, we observed that a fraction of the mature core protein colocalized with mitochondrial markers in coreexpressing HeLa cells and in Huh-7 cells containing the full-length HCV replicon. Subcellular fractionation confirmed this observation and showed that the core protein associates with purified mitochondrial fractions devoid of ER contaminants. The core protein also fractionated with mitochondrion-associated membranes, a site of physical contact between the ER and mitochondria. Using immunoelectron microscopy and in vitro mitochondrial import assays, we showed that the core protein is located on the mitochondrial outer membrane. A stretch of 10 amino acids within the hydrophobic C terminus of the processed core protein conferred mitochondrial localization when it was fused to green fluorescent protein. The location of the core protein in the outer mitochondrial membrane suggests that it could modulate apoptosis or lipid transfer, both of which are associated with this subcellular compartment, during HCV infection.Hepatitis C virus (HCV), the major causative agent of non-A, non-B hepatitis, is estimated to infect 3% of the world's population (11). Viral infection persists in ϳ80% of infected individuals, causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 13). Analyses of viral kinetics showed that HCV particles are continuously produced and cleared during chronic infection (46, 54). The lack of an efficient infectious cell culture model has prevented a comprehensive understanding of the viral life cycle. Important progress was made with the development of autonomously replicating HCV RNAs which produce nonstructural viral proteins upon transfection into hepatoma cells (7,27). Although these subgenomic replicons were recently extended to the full-length HCV genome encoding all viral proteins, no viral particle formation was observed (52).The HCV genome, a single plus-stranded RNA molecule of 9,600 nucleotides, encodes a single precursor polyprotein of about 3,000 amino acids (for reviews, see references 6 and 58). The precursor consists of N-terminal structural and C-terminal nonstructural components. During and after translation, the precursor protein undergoes an ordered series of proteolytic cleavages by viral and host proteases that generate the individual viral proteins. The functional properties of these proteins have been deduced by analogy with related RNA viruses and by cDNA expression experiments in mammalian cells (for a review, see reference 65).There are at least three viral proteins that have structural properties, including the core protein and the envelop...

Hepatitis C virus proteins interfere with the activation of chemokine gene promoters and downregulate chemokine gene expression

“…The core protein showed a partial co-localization with Cardif in mitochondria in the UCp7con-9 cell line. Previous studies have shown that the core protein is located in lipid storage vesicles (Barba et al, 1997) and also in the outer membrane of mitochondria (Schwer et al, 2004). In the UHCV-11 cell line, the staining for NS3 was weaker compared with that seen in UNS3-4A-24 cells.…”

Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein

“…Mitochondria were purified using Nycodenz (Nycomed Pharma, Zürich, Switzerland) according to the protocols reported by Okado-Matsumoto et al 17 For transient transfection experiments, HepG2 cells were transfected with a core-expression plasmid using TransIT-LT1 (Mirus Bio, Madison, WI). Huh7 cells harboring HCV genotype 1b full-genomic (RCYM1) 18 or subgenomic replicon (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), and livers of 3-month-old core-gene transgenic mice 2 were also used for the analysis.…”

“…2,5,6 In addition, the double structure of mitochondrial membranes is disrupted in hepatocytes of core-gene transgenic mice. [2][3][4] Evidence suggests that the core protein modulates some mitochondrial functions, including fatty acid ␤-oxidation, the impairment of which may induce lipid abnormalities and hepatic steatosis.…”

Mitochondrial and Secretory Human Cytomegalovirus UL37 Proteins Traffic into Mitochondrion-Associated Membranes of Human Cells

“…Although numerous proteins localize to both the ER and the mitochondria (bcl-2, cytochrome), sequential trafficking of proteins from the ER to mitochondria has been rarely found (12,27,41). More often, the case is that separate but competitive trafficking of proteins occurs into either secretory apparatus or mitochondria (16,22,39).…”

“…Because this protein is anchored in the membrane by three transmem-brane domains, we hypothesized its sequential trafficking from ER to mitochondria occurs through a continuous lipid bilayer connecting the two organelles. Although localization of proteins in the ER and in mitochondria is frequently observed (3,9,23), few cellular (12) and viral (27,41) proteins are known to traffic sequentially from the ER to mitochondria. Consequently, this trafficking pathway connecting the ER to mitochondria is poorly understood.…”