SPCS1-Dependent E2-p7 processing determines HCV Assembly efficiency.

Nabeel Alzahrani,Olga V Kalinina,Ming-Jhan Wu,Christoph Welsch,Carla F Sousa,Minkyung Yi,Aleem Siddiqui

doi:10.1371/journal.ppat.1010310

Abstract

Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1’s roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.

Highlights

Hepatitis C virus (HCV) is the main causative agent of severe liver diseases, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma [1]
We elucidated that signal peptidase complex subunit 1 (SPCS1) facilitates HCV assembly by enhancing the intrinsically suboptimal, signal peptidase complex (SPC)-mediated processing of the HCV E2-p7 precursor
By performing in silico analyses, we found that the N-terminal domain of the p7, succeeding the cleavage junction, maintains a rigid angle relative to the membrane z-axis, potentially hiding the E2-p7 junction site within the membrane and out of reach from the SPC catalytic site, providing an explanation for the suboptimal processing of this junction

Summary

Introduction

Hepatitis C virus (HCV) is the main causative agent of severe liver diseases, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma [1]. According to the World Health Organization (WHO) report, about 58 million people worldwide are estimated to have chronic HCV infection, leading to 290,000 annual deaths. HCV is a positive-stranded RNA virus belonging to the Hepacivirus genus in the Flaviviridae family. It encodes an open reading frame for a single polyprotein flanked by 5’- and 3’non-coding regions. Host signal peptidase complex (SPC) processes the N-terminal region of HCV polyprotein encoding the structural proteins (Core, E1, and E2) and the two accessory proteins (p7 and NS2) [5,6]. The NS3-NS4A-NS4B-NS5A-NS5B polyprotein is processed by NS3 serine protease together with its co-factor NS4A leading to replicase complex formation involved in viral RNA replication [11,12]

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Conclusion