Structural Analysis of Hepatitis C Virus Core-E1 Signal Peptide and Requirements for Cleavage of the Genotype 3a Signal Sequence by Signal Peptide Peptidase

Abstract

bThe maturation of the hepatitis C virus (HCV) core protein requires proteolytic processing by two host proteases: signal peptidase (SP) and the intramembrane-cleaving protease signal peptide peptidase (SPP). Previous work on HCV genotype 1a (GT1a) and GT2a has identified crucial residues required for efficient signal peptide processing by SPP, which in turn has an effect on the production of infectious virus particles. Here we demonstrate that the JFH1 GT2a core-E1 signal peptide can be adapted to the GT3a sequence without affecting the production of infectious HCV. Through mutagenesis studies, we identified crucial residues required for core-E1 signal peptide processing, including a GT3a sequence-specific histidine (His) at position 187. In addition, the stable knockdown of intracellular SPP levels in HuH-7 cells significantly affects HCV virus titers, further demonstrating the requirement for SPP for the maturation of core and the production of infectious HCV particles. Finally, our nuclear magnetic resonance (NMR) structural analysis of a synthetic HCV JFH1 GT2a core-E1 signal peptide provides an essential structural template for a further understanding of core processing as well as the first model for an SPP substrate within its membrane environment. Our findings give deeper insights into the mechanisms of intramembrane-cleaving proteases and the impact on viral infections.

Modulation of Triglyceride and Cholesterol Ester Synthesis Impairs Assembly of Infectious Hepatitis C Virus

“…The pJFH1 plasmid and LD540 were gifts from Takaji Wakita (National Institute of Infectious Diseases, Tokyo, Japan) and Christoph Thiele (University of Bonn), respectively. Antibodies used to detect HCV core (rabbit antiserum 4210), E2 (AP33; a gift from Arvind Patel, Glasgow University), dsRNA (J2; supplied by SCICONS, Hungary), NS5A (sheep antiserum; a gift from Mark Harris, Leeds University), NS3 (mouse antibody; a gift from Thomas Pietschmann, TWINCORE, Hannover, Germany), human apoE (clone EP1374Y; supplied by Abcam), and human Perilipin-2 (PLIN2) have been described previously (25, 41–44). For detection of PLIN2 by indirect immunofluorescence, an antibody raised in guinea pig against the protein was used (Progen).…”

Roles of regulated intramembrane proteolysis in virus infection and antiviral immunity

“…This cleavage liberates the core protein from the ER, allowing it to associate with lipid droplets where it assembles viral particles into a virus-lipoprotein complex (Figure 3) [12,69-72]. Consistent with these observations, inhibition of SPP-catalyzed maturation of the core protein blocked production of infectious HCV particles [68,73]. …”

“…It is localized at cytosolic side of the ER membrane and is linked to the remainder of the viral protein via a signal peptide-like transmembrane sequence at its COOH terminus (Figures 1 and 3). After cleavage by signal peptidase in the ER lumen, SPP cleaves the signal peptide-like transmembrane domain [12,67,68] (Figures 1 and 3). This cleavage liberates the core protein from the ER, allowing it to associate with lipid droplets where it assembles viral particles into a virus-lipoprotein complex (Figure 3) [12,69-72].…”

Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein

“…During the following cleavage, the core protein remains anchored to the ER through a C-terminal hydrophobic region (Grakoui et al, 1993) being further processed at its C-terminus by a signal peptide peptidase resulting in the mature protein (Liu et al, 1997; Yasui et al, 1998). The C-terminus of mature core protein is not precisely elucidated but probably lies between residues 170 and 182 (Kopp et al, 2010; Oehler et al, 2012). Although core protein has been shown to possess a nuclear localization signal (Suzuki et al, 1995; Yan et al, 2005), it is located exclusively in the cytoplasm in cells infected with cell culture-derived HCV, consistent with the cytoplasmic life cycle of HCV (Lindenbach & Rice, 2001).…”