Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets

Abstract

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein.

Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein

“…Domain 2 was hypothesized to interact with lipid droplets and confer stability to the HCV core protein (12). Although a mutation in the signal sequence of core protein that renders it resistant to SPP proteolysis restored retention on lipid droplets and overall stability (30), deletion of domain 2 from HCV core protein leads to diffusion in the cytoplasm and degradation after processing by signal peptidase (30). Indeed, these data suggest that mature HCV core protein is retained on lipid droplets via domain 2, but they do not necessarily indicate that the region is required for the ER retention of HCV core protein.…”

“…Martoglio and colleagues reported that HCV core protein is processed by SPP after cleavage by host signal peptidase and that Ala 180 , Ser 183 , and Cys 184 residues in the signal sequence of HCV core protein of type 1a Glasgow strain are essential for SPP proteolysis, as they maintain the structure of the breaking ␣-helix (23,30). To determine the amino acids essential for SPP cleavage of the signal sequence of type 1b HCV core protein, Flag-Core-E1-HA and its substitution mutants were expressed in 293T cells (Fig.…”

“…HCV polyprotein is cleaved between residues 191 and 192 by host signal peptidase to generate C-terminal and N-terminal polypeptides encompassing the core and E1 proteins, respectively. For the full maturation of HCV core protein, the Cterminal signal-anchor sequence was thought to be further processed by an unidentified microsomal protease (25,30,31,43,53), and the 21-kDa isoform of core protein is predominantly detected both in cultured cells by transfection with expression plasmid and in viral particles obtained from sera of patients with hepatitis C (53). These results suggest that p21 is the mature form of HCV core protein (53).…”

“…SPP is located in the ER membrane and promotes intramembrane proteolysis of signal peptides. The chemical compound (Z-LL) 2 -keton inhibits processing of signal peptides by SPP, and it was shown to suppress intramembrane proteolysis of major histocompatibility complex class I molecules, preprolactin, HCV core protein, and others (21,30,51). Replacement of Asp 265 with Ala in SPP resulted in a loss of catalytic function, although this mutant could bind to TBL 4 K, a derivative of (Z-LL) 2 -keton (50).…”

Virus de l’hépatite C et gouttelettes lipidiques

“…Des études complémentaires ont montré que le clivage de la protéine de core par une protéase cellulaire, la SPP (signal peptide peptidase) ( Figure 1A et 1B) est nécessaire pour permettre la migration de cette protéine vers la surface des gouttelettes [9]. Après ce clivage, la protéine de core du VHC reste ancrée dans la membrane du RE ou associée aux gouttelettes grâce à la présence d'un domaine carboxy-terminal hydrophobe, sans équivalent dans la protéine de core des autres Flavivirus (à l'exception du virus GBV-B, très proche phylogénétiquement du VHC) [10].…”