Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1

Abstract

Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 () or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses.

Genome-Wide Mutagenesis of Hepatitis C Virus Reveals Ability of Genome To Overcome Detrimental Mutations

“…Sanger sequencing of clones derived from 100 ng 0.2AGCT, 50 ng 0.2 AGCT, and 25 ng 0.2 AGCT libraries showed that the sequences had unique mutations. We had made a similar observation earlier: Sanger sequencing of EP-PCR products that were derived from an HEV cDNA clone had unique mutations (15). Table 1 shows a transition-transversion matrix for genome-wide substitutions for all six libraries.…”

“…1A, lanes 1 to 4). Previously, we were able to synthesize and diversify the HEV full-length genome of 7.2 kb by reducing the dATP and dGTP concentrations in EP-PCR (15). In this study, we first attempted to further induce genome-wide mutagenesis by reducing both dATP and dGTP concentrations; however, this failed to provide sustained yields.…”

“…The FL-MRS technique reduces the fidelity of Taq DNA polymerase in a controllable fashion to induce random mutations during the synthesis of full-genome templates and virus transcripts (15). The products of EP-PCR are ideal for the synthesis of transcripts of a defined length, since EP-PCR uses a forward primer located upstream of the T7 promoter and a reverse primer that ends precisely at the last nucleotide of the HCV genome in the cDNA clone.…”

“…This required a molecular technique that allows a massive increase in mutations during the replication of the HCV genome. Recently, we reported a molecular technique, full-length mutant RNA synthesis (FL-MRS) for random introduction of a few to several mutations in a controllable fashion in the hepatitis E virus (HEV) genome (15). The introduction of random mutations in the HCV genome may allow evaluation of the intrinsic mutational tolerance in the sequence space of the HCV genome (16).…”

HCV Replicon Systems: Workhorses of Drug Discovery and Resistance

“…The majority of variants are unable to replicate due to deleterious or lethal effects of the new mutations or are otherwise cleared by the host immune system. However, some variants with beneficial mutations are continuously selected upon due to evolutionary advantages or better adaptation to adverse conditions, such as the presence of a DAA (Agarwal et al, 2018 ; Singh et al, 2020 ). Consequently, natural variants with various levels of susceptibility to DAAs may exist within patients and can be selected after DAA exposure (Ahmed and Felmlee, 2015 ).…”