Characterization of Hepatitis C Virus Deletion Mutants Circulating in Chronically Infected Patients

Abstract

Hepatitis C virus (HCV) has a linear positive-stranded RNA genome of ϳ9,600 nucleotides in length and displays a high level of sequence diversity caused by high mutation rates and recombination. However, when we performed long distance reverse transcription-PCRs on HCV RNA isolated from serum of chronic HCV patients, not only full-length HCV genomes but also HCV RNAs which varied in size from 7,600 to 8,346 nucleotides and contained large in-frame deletions between E1 and NS2 were amplified. Carefully designed control experiments indicated that these deletion mutants are a bona fide natural RNA species, most likely packaged in virions. Moreover, deletion mutants were detected in sera of patients infected with different HCV genotypes. We observed that 7/37 (18.9%) of genotype 1, 5/43 (11.6%) of genotype 3, and 4/13 (30.7%) of genotype 6 samples contained HCV deletion mutant genomes. These observations further exemplify HCV's huge genetic diversity and warrant studies to explore their biological relevance.Hepatitis C virus (HCV) is an enveloped virus that belongs to the family Flaviviridae. Its linear positive-stranded RNA genome of approximately 9.6 kb in length encodes both structural (core, E1, and E2) and nonstructural (p7, N2, NS3, NS4a/b, and NS5a/b) proteins in a single open reading frame, flanked by short conserved untranslated regions (UTRs) located at the 5Ј and 3Ј ends of the genome that are required for viral replication and protein translation (5,7,8). One of the most striking characteristics of HCV is its capacity to cause a chronic liver disease in a high percentage of individuals (24). To do so, HCV encodes proteins which promote persistence, and sequence variation, especially in the envelope genes (E1 and E2), results in escape from adaptive immune responses (9, 27).Lack of proofreading ability of the viral RNA-dependent RNA polymerase is the driving force behind HCV's genetic diversity. As a result of the large amount (10 12 ) of virions produced each day in chronic hepatitis C patients and the rate of incorrect nucleotide insertions, which reaches the order of 10 Ϫ3 to 10 Ϫ4 base substitutions per site per year, HCV quasispecies are generated (22,26). Recombination may be another mechanism by which genetic diversity is driven, given the recent identification of naturally occurring intergenotypic recombinant viruses (13, 21). Because of the huge genetic diversity, HCVs are currently categorized into six major genotypes and more than 80 subtypes (25).HCV genetic variation has been studied in relation to epidemiology, response to antiviral therapy, and clinical parameters, using different techniques that have focused on short genomic regions. However, analysis of full-length viral genomes may be necessary to better understand the characteristics of HCV. Previously, we analyzed sera from HCV RNA-positive blood donors from Ho Chi Minh City, Vietnam, in order to analyze the molecular heterogeneity of HCV in Southeast Asia (21). Based on sequence analysis of core and NS5b regions in a set of sera, two ...

Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans -Packaged In Vitro To Generate Infectious Defective Particles

“…Because of this huge genetic diversity, HCV is currently classified into six major genotypes and more than 80 subtypes (44). Recombination may be another mechanism exploited by HCV to increase genetic diversity: naturally occurring intergenotypic recombinant viruses that often have their recombination points in the trans-membrane domains of NS2 were recently identified (20,21,26,27,33,35).Recent publications have reported the presence of natural HCV subgenomic RNAs in serum and liver of infected patients, mostly containing large in-frame deletions from E1 up to NS2, always found together with the full-length wild-type (wt) RNAs (5,16,36,54). These mutant viral genomes persist for a long time (at least 2 years), and sequence analysis suggests that subgenomic (the predominant species during this period) and full-length HCV evolve independently (54).…”

“…Analysis of the genetic structure of 18 independent subgenomic deletion-containing RNAs (5,16,36,54) strongly suggests that the possibility of recombination and/or deletion is restricted to specific regions.…”

“…Recent publications have reported the presence of natural HCV subgenomic RNAs in serum and liver of infected patients, mostly containing large in-frame deletions from E1 up to NS2, always found together with the full-length wild-type (wt) RNAs (5,16,36,54). These mutant viral genomes persist for a long time (at least 2 years), and sequence analysis suggests that subgenomic (the predominant species during this period) and full-length HCV evolve independently (54).…”

Genetic Analysis of Hepatitis C Virus with Defective Genome and Its Infectivity in Vitro

“…Most DI viruses occur spontaneously in the course of cell culture infected with a high titer of wild-type viruses. Hepatitis C virus (HCV) with a defective genome has been found in liver and serum specimens of some HCV patients (4,8,15). HCV has a plus-strand RNA genome that encodes the viral core, E1, E2, and p7 structural proteins and NS2, NS3, NS4A, NS4B, NS5A, and NS5B nonstructural proteins (10).…”

The hepatitis C virus core gene yields restriction fragments of variable length and polymorphism

“…Currently, the global spread of HCV has accelerated with the advent of more methods and frequency of transmission routes of populations 21 which probably accounts for the appearance of pattern III HCV. However, we have found three patterns designated as II, IV, and V which may have arisen from mutation by recombination in the local population 22,23 . Mutations of the core region impact on clinical outcomes [24][25][26] .…”