Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis.

Véronique Pène,Francis Harper,Arielle R Rosenberg,Gérard Pierron,Matthieu Lemasson,Ranjit Ray

doi:10.1371/journal.pone.0175810

Abstract

In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.

Highlights

Hepatitis C virus (HCV), a major causative agent of chronic hepatitis in human, was the first described member of the genus Hepacivirus belonging to the Flaviviridae family
Huh-7.5.1 cells were transfected with the wild-type (WT) or mutated versions of HCV RNAs Con1/C3, and lysed at various time points post-transfection for western blot analysis with monoclonal antibodies (mAb) against HCV core protein (Fig 1B)
In cells transfected with WT or ΔE1E2 constructs, core protein was detected as a unique band with an apparent molecular mass of 21 kDa, corresponding to the mature protein usually referred to as p21

Summary

Introduction

Hepatitis C virus (HCV), a major causative agent of chronic hepatitis in human, was the first described member of the genus Hepacivirus belonging to the Flaviviridae family. HCV is an enveloped virus with a single-strand positive RNA genome This genome encodes a single polyprotein precursor that undergoes a series of proteolytic cleavages to generate functional viral proteins (Fig 1A). Cleavage by host cell signal peptidase (SP) at the luminal side of the ER separates E1 from p23, the so-called immature form of core protein containing 191 residues [2, 3]. This complete form of HCV core protein is anchored in the ER lipid bilayer by the Cterminal signal peptide [4]. It is established that SP-catalyzed cleavage at core-E1 junction is a prerequisite for SPP-catalyzed cleavage [5, 6]

Methods

Results

Conclusion