Quasispecies Changes with Distinctive Point Mutations in the Hepatitis C Virus Internal Ribosome Entry Site (IRES) Derived from PBMCs and Plasma.

Luca Mercuri,Emma C Thomson,Joseph Hughes,Peter Karayiannis

doi:10.1155/2018/4835252

Abstract

The 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome contains the internal ribosome entry site (IRES), a highly conserved RNA structure essential for cap-independent translation of the viral polyprotein. HCV, apart from the liver, is thought to be associated with lymphocyte subpopulations of peripheral blood mononuclear cells (PBMCs), in lymph nodes and brain tissue. In this study, RT-PCR, cloning, and sequence analysis were employed to investigate the quasispecies nature of the 5'UTR following extraction of viral RNA from PBMCs and plasma of HCV infected individuals. The nucleotide variation between IRES-derived sequences from PBMCs and plasma indicated the existence of polymorphic sites within the IRES. HCV isolates had divergent variants with unique mutations particularly at positions 107, 204, and 243 of the IRES. Most of the PBMC-derived sequences contained an A-A-A variant at these positions. The mutations associated with the IRESes suggested the presence of unique quasispecies populations in PBMCs compared with plasma.

Highlights

Hepatitis C virus (HCV), a member of the Hepacivirus genus in the Flaviviridae family, is an enveloped virus with an icosahedral capsid that encloses a single-stranded positive sense genomic RNA of approximately 9.5 kb in length [1, 2]
The nucleotide differences among all the 225 sequences derived from peripheral blood mononuclear cells (PBMCs) and all of the 225 sequences derived from plasma samples are highlighted on the secondary RNA structure of the 5’untranslated regions (UTR) HCV (nt. 1-383 genotype 1b (GenBank, AJ238799.1) where numbers indicate internal ribosome entry site (IRES) nucleotide positions relative to strain HCV 1a (GenBank, NC 004102) (Figure 1)
As the two-dimensional structure of HCV genotype 1b 5’UTR was used as an outline to position the nucleotide substitutions detected in HCV IRESes, mainly genotype 1a (Figure 1), the HCV IRES genotype 1b was aligned with consensus sequence genotype 1a to pinpoint any differences between the two IRESes

Summary

Introduction

Hepatitis C virus (HCV), a member of the Hepacivirus genus in the Flaviviridae family, is an enveloped virus with an icosahedral capsid that encloses a single-stranded positive sense genomic RNA of approximately 9.5 kb in length [1, 2]. The presence of HCV RNA in extrahepatic sites has been detected in HCV infected individuals where HCV negative strands (replicative intermediate) have been associated with PBMCs, CD4+, CD8+, CD19+, T-cells, monocytes, and macrophages [12]. The virus has been shown to persist in the PBMCs of patients who had received successful antiviral treatment with Pegylated-IFNα (Peg-IFNα) in combination with ribavirin. In such patients, HCV RNA was associated with PBMCs but not present in the serum [13,14,15]

Objectives

Methods

Results

Discussion

Conclusion