Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations

Geert Leroux-Roels,Charles M Rice,Stephanie Kallis,Philip Meuleman,Gang Long,George Koutsoudakis,Thomas Pietschmann,Ralf Bartenschlager,Volker Lohmann,Nicole Appel,Margarita Zayas

doi:10.1371/journal.ppat.1000475

Abstract

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

Highlights

hepatitis C virus (HCV) is a positive strand RNA virus which belongs to the family Flaviviridae [1]
We show that at least in case of the Con1 isolate, mutations especially in the NS3 helicase, and in nonstructural protein 5A (NS5A) and NS5B lead to strong impairment of virus production whereas Huh-7 cells transfected with the wild type genome or a genome containing one replication enhancing mutations (REMs) in NS4B release substantial amounts of HCV that is infectious in cell culture and in vivo
Interference of REMs with virus production Given the infectivity of the wild type Con1 HCV isolate in vivo on one hand and its poor replicative capacity in transfected Huh-7 cells in vitro on the other hand, we first investigated whether this genome is capable of producing virus particles in cell culture

Summary

Introduction

HCV is a positive strand RNA virus which belongs to the family Flaviviridae [1]. Its genome of about 9.6 kb is composed of the 59non-translated region (NTR), an open reading frame encoding a large polyprotein, and the 39NTR [2] (Fig. 1A). In the N-terminal region, the polyprotein is processed by cellular proteases to yield the structural proteins Core (C), envelope proteins 1 and 2 (E1, E2), and p7. NS4B induces cellular membrane alterations thought to provide a scaffold for the viral replication machinery [3,4]. NS5B is the viral RNA-dependent RNA polymerase whereas NS5A is an RNA binding phosphoprotein involved in RNA replication and virus assembly [5,6,7,8]. Phosphorylation of NS5A appears to be mediated by casein kinase I and II [7,10]. Interference with NS5A hyperphosphorylation by inhibitors of casein kinase I such as H479 enhances viral RNA replication by more than 10-fold arguing that this modification is of disadvantage for high level replication [11]

Methods

Results

Conclusion