Signal Peptide Peptidase Cleavage of GB Virus B Core Protein Is Required for Productive Infection in Vivo

Paul Targett-Adams,Torsten Schaller,Graham Hope,Robert E Lanford,Stanley M Lemon,Annette Martin,John Mclauchlan

doi:10.1074/jbc.m605373200

Abstract

Chronic infection by hepatitis C virus (HCV) is a leading cause of liver disease for which better therapies are urgently needed. Because a clearer understanding of the viral life cycle may suggest novel anti-viral approaches, we studied the role of host signal peptide peptidase (SPP) in viral infection. This intramembrane protease cleaves within a C-terminal signal sequence in the viral core protein, but the molecular determinants of cleavage and whether it is required for infection in vivo are unknown. To answer these questions, we studied SPP processing in GB virus B (GBV-B) infection. GBV-B is the closest phylogenetic relative of HCV and offers an accurate surrogate model for HCV infection. We demonstrate that SPP also processes GBV-B core protein and that a serine residue in the hydrophobic region of the signal sequence (present also in HCV) is critical for efficient SPP cleavage. The small size of the serine side chain combined with its ability to form intra- and interhelical hydrogen bonds likely contributes to recognition of the signal sequence as a substrate for SPP. By introducing mutations with differing effects on SPP processing into an infectious GBV-B molecular clone, we demonstrate that SPP processing of the core protein is required for productive infection in primates. These results broaden our understanding of the mechanism and requirements for SPP cleavage and reveal a functional role in vivo for intramembrane proteolysis in host-pathogen interactions. Moreover, they identify SPP as a potential therapeutic target for reducing the impact of HCV infection.

Highlights

Tain signal sequences following proteolysis by signal peptidase [4, 5]
GB virus B (GBV-B) Core Protein Is Processed by signal peptide peptidase (SPP)—The signal sequences located at the C terminus of GBV-B and hepatitis C virus (HCV) core are substrates for cleavage by signal peptidase between amino acid residues 156/157 and 191/192, respectively [28, 29]
To determine whether GBV-B core is a substrate for SPP, a polyprotein encompassing GBV-B core to the N-terminal region of NS2 and including an epitope tag inserted within the core sequence (Fig. 1A) was expressed in BHK cells in the absence or presence of (Z-LL)2 ketone, a SPP inhibitor [27]

Summary

Introduction

Tain signal sequences following proteolysis by signal peptidase [4, 5]. Studies on the biological functions associated with SPP cleavage are very limited. Intrahepatic injection of in vitro transcribed RNA from full-length cDNA of GBV-B gives a pattern of infection that is identical to that following inoculation with infectious material. Such a system enables assessment of GBV-B mutant or GBV-B/HCV chimeric genomes to initiate infection [18, 19, 23]. We characterize the molecular determinants of SPP cleavage and show that SPP processing is required for productive viral infection in nonhuman primates These observations reveal that host SPP has been coopted by a surrogate for HCV for functions that are essential to its life cycle, suggesting novel therapeutic approaches for treatment of HCV infection. The novel role played by SPP in this host-pathogen interaction, which has not been reported previously for other intramembrane-cleaving proteases, offers unique possibilities for unraveling the mechanisms and processes engaged in intramembrane proteolysis

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Conclusion