Broad and Dynamic Diversification of Infectious Hepatitis C Virus in a Cell Culture Environment

Abstract

Previous studies documented that long-term hepatitis C virus (HCV) replication in human hepatoma Huh-7.5 cells resulted in viral fitness gain, expansion of the mutant spectrum, and several phenotypic alterations. In the present work, we show that mutational waves (changes in frequency of individual mutations) occurred continuously and became more prominent as the virus gained fitness. They were accompanied by an increasing proportion of heterogeneous genomic sites that affected 1 position in the initial HCV population and 19 and 69 positions at passages 100 and 200, respectively. Analysis of biological clones of HCV showed that these dynamic events affected infectious genomes, since part of the fluctuating mutations became incorporated into viable genomes. While 17 mutations were scored in 3 biological clones isolated from the initial population, the number reached 72 in 3 biological clones from the population at passage 200. Biological clones differed in their responses to antiviral inhibitors, indicating a phenotypic impact of viral dynamics. Thus, HCV adaptation to a specific constant environment (cell culture without external influences) broadens the mutant repertoire and does not focus the population toward a limited number of dominant genomes. A retrospective examination of mutant spectra of foot-and-mouth disease virus passaged in cell cultures suggests a parallel behavior here described for HCV. We propose that virus diversification in a constant environment has its basis in the availability of multiple alternative mutational pathways for fitness gain. This mechanism of broad diversification should also apply to other replicative systems characterized by high mutation rates and large population sizes. IMPORTANCE The study shows that extensive replication of an RNA virus in a constant biological environment does not limit exploration of sequence space and adaptive options. There was no convergence toward a restricted set of adapted genomes. Mutational waves and mutant spectrum broadening affected infectious genomes. Therefore, profound modifications of mutant spectrum composition and consensus sequence diversification are not exclusively dependent on environmental alterations or the intervention of population bottlenecks.

Dissimilar conservation pattern in hepatitis C virus mutant spectra, consensus sequences, and data banks

“…Two-hundred serial passages of a clonal [derived by transcription of plasmid Jc1FLAG2(p7-nsGLuc2A) (abbreviated Jc1Luc)] HCV population [28] in Huh-7.5 cells identified 145 different mutations —located within genomic nucleotides 7649 to 8653— that participated in mutational waves based on deep sequencing data of 39 HCV populations [19] (Fig. 1A) [residue numbers are according to HCV reference isolate JFH-1 (accession number #AB047639); mutations and deduced amino acid substitutions are given in Table S1 (http://babia.cbm.uam.es/~lab121/SupplMatGarcia-Crespo)].…”

“…Experimental design, and HCV genome analysis. (A) Clonal HCV population HCVcc prepared by transcription of plasmid Jc1Luc [28], followed by RNA electroporation into Huh-7 Lunet cells, was amplified to produce HCV p0, and the population subjected to 200 passages in Huh-7.5 cells, as described [17–19]. The initial population HCV p0 and the populations at passages 100 (HCV p100) and 200 (HCV p200) were passaged four additional times in Huh-7.5 cells in triplicate [replicas (a), (b), (c)].…”

“…Mutant frequencies in quasispecies have been obtained in cell culture thanks to the availability of infectious molecular clones of HCV that permit experimental evolution designs with this important pathogen [14–18]. In a recent study we examined mutant spectrum variations upon subjecting a clonal HCV population to 200 serial infections of Huh-7.5 cells, using fresh cells at each passage, thereby preventing co-evolution of the host cells [19]. Under these conditions, a variation of mutant frequency of several individual genomic residues even between successive passages was consistently observed in three biological replicas.…”

“…Under these conditions, a variation of mutant frequency of several individual genomic residues even between successive passages was consistently observed in three biological replicas. Because mutant frequency variations were continuous, we termed them “mutational waves” to emphasize a difference with those residues whose frequency remained constant with passage number [17,19]. Mutational waves were characterized by deep sequencing analysis of 1,005 genomic positions from the NS5A-NS5B-coding region, and they involved 145 different mutations.…”

“…Mutational waves were characterized by deep sequencing analysis of 1,005 genomic positions from the NS5A-NS5B-coding region, and they involved 145 different mutations. The dependability of the deep sequencing analyses was sustained on experimental and bioinformatics controls that yielded reliable cut-off mutant frequency values of 0.5% [19–21] (see also Materials and Methods). Furthermore, Sanger sequencing of the HCV p0, HCV p100, HCV p200 and their derivatives at passage 4 identified 114 heterogeneous sites (two different nucleotides at the same genomic position) within the NS2- to NS5B-coding region, thus confirming mutant frequency variations by two independent procedures [19].…”