Inspecting the Ribozyme Region of Hepatitis Delta Virus Genotype 1: Conservation and Variability.

Beatriz Pacin-Ruiz,Anna Galán,Unai Aldama,Josep Gregori,Josep Quer,Mar Riveiro-Barciela,Adriana Palom,Marta Vila,Francisco Rodríguez-Frías,Selene García-García,Rosario Casillas,María Buti,Adrián Najarro,Gloria González-Aseguinolaza,Sara Sopena,Ariadna Rando-Segura,María Francesca Cortese,David Tabernero

doi:10.3390/v14020215

Abstract

The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688–771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715–745. No difference in QS was observed over time. The most variable region was between nt 739–769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence.

Highlights

More than 250 million people worldwide are living with the hepatitis B virus (HBV), and between 15 and 20 million of them are chronically co-infected with hepatitis delta virus (HDV)
HDV genomic RNA replicates through a rolling circle process mediated by cellular RNA polymerase [5], by producing a concatemer of antigenomic monomers
The individual antigenomic molecules are obtained through a self-cleavage process led by the viral ribozyme [6,7]

Summary

Introduction

More than 250 million people worldwide are living with the hepatitis B virus (HBV), and between 15 and 20 million of them are chronically co-infected with hepatitis delta virus (HDV). HDV is composed of an RNA molecule with high intermolecular self-complementarity, giving rise to a rod-like structure. This 1.2 kb genome presents only one reading frame and codes for a protein existing in two isoforms of different length: the short (S-HDAg) isoform consisting of 195 amino acids (aa) and the long (L-HDAg) delta antigen with 214 aa (27 kDa) [4]. The individual antigenomic molecules are obtained through a self-cleavage process led by the viral ribozyme [6,7] Some of these antigenomic monomers are later circularized by a still unclear mechanism [8,9] to be used as templates for the genomic RNA synthesis through another process of rolling circle amplification. The remaining antigenomic molecules enter the transduction process by producing HDAg

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Methods

Conclusion