Structure and function analysis of the essential 3′X domain of hepatitis C virus

Jesús Castillo-Martínez,Alfredo Berzal-Herranz,Elisa Oltra,José Gallego,Beatriz Berzal-Herranz,Tamara Ovejero,Cristina Romero-López,Isaías Sanmartín

doi:10.1261/rna.073189.119

Jesús Castillo-Martínez, Alfredo Berzal-Herranz + Show 6 more

Open Access

https://doi.org/10.1261/rna.073189.119

Copy DOI

Abstract

The 3′X domain of hepatitis C virus has been reported to control viral replication and translation by modulating the exposure of a nucleotide segment involved in a distal base-pairing interaction with an upstream 5BSL3.2 domain. To study the mechanism of this molecular switch, we have analyzed the structure of 3′X mutants that favor one of the two previously proposed conformations comprising either two or three stem–loops. Only the two-stem conformation was found to be stable and to allow the establishment of the distal contact with 5BSL3.2, and also the formation of 3′X domain homodimers by means of a universally conserved palindromic sequence. Nucleotide changes disturbing the two-stem conformation resulted in poorer replication and translation levels, explaining the high degree of conservation detected for this sequence. The switch function attributed to the 3′X domain does not occur as a result of a transition between two- and three-stem conformations, but likely through the sequestration of the 5BSL3.2-binding sequence by formation of 3′X homodimers.

Highlights

The Hepatitis C virus (HCV) is an important human pathogen that currently affects around 71 million people worldwide, according to estimates from the World Health Organization
Are connected through domain 5BSL3.2, which establishes a network of distal RNA–RNA contacts involving both the internal ribosome entry site (IRES) and domain 3′X. 5BSL3.2 is part of an essential cis-acting replication element (CRE) located in the NS5B coding sequence (Fig. 1A; RomeroLópez and Berzal-Herranz 2017)
The results indicate that the conformation and the homo- and heteroassociation capacities of the 3′X domain are directly related and have a marked influence on the translation and replication processes of the virus

Summary

INTRODUCTION

The Hepatitis C virus (HCV) is an important human pathogen that currently affects around 71 million people worldwide, according to estimates from the World Health Organization. NMR spectroscopy and gel electrophoresis data indicated that this mutant adopted the two-stem conformation of the wild-type sequence and exhibited the same capacity to bind 5BSL3.2, but was unable to homodimerize (Cantero-Camacho and Gallego 2018) The effect of this mutation on the translation and replication capacities of the virus was analyzed and compared with the effects brought about by the six structural mutants. Mutants U3G, G50C/C52G, and U3G/G50C/C52G were designed to destabilize the two-stem conformation by disrupting the U3:G53, G4:C52, and C6:G50 pairs of subdomain SL2′ These latter mutants were predicted by RNA folding algorithms to form the three-stem structure formerly proposed on the basis of chemical modification techniques (Fig. 1B; Blight and Rice 1997; Ito and Lai 1997; Dutkiewicz and Ciesiolka 2005; Ivanyi-Nagy et al 2006; Romero-López et al 2014).

DISCUSSION

MATERIALS AND METHODS