To gain an insight into the impact of mutations on viability of hepatitis C virus (HCV) genome, we created a set of full-genome mutant libraries, differing from the parent sequence as well as each other by using a random mutagenesis approach; the proportion of mutations increased across these libraries with declining template amount or dATP concentration. Replication efficiency of full-genome mutant libraries ranged between 71 and 329 foci forming unit (FFU) per 105 Huh7.5 cells. Mutant libraries with low proportions of mutations demonstrated low replication capability, whereas those with high proportions of mutations had their replication capability restored. Hepatoma cells transfected with selected mutant libraries, low (4 mutations per 10,000 bp copied), moderate (33 mutations) and high (66 mutations) proportions of mutations, and their progeny were subjected to serial passage. Predominant virus variants (mutants) from these mutant libraries (Mutantl, Mutantm and Mutanth , respectively) were evaluated for changes in growth kinetics and particle-to-FFU-unit ratio, virus protein expression and modulation of host cell protein synthesis. Compared to the parent, Mutantm and Mutantl produced >3.0 log higher extracellular progeny per ml and, Mutanth produced progeny at rate 1.0-log lower. More than 80% of mutations were in nonstructural part of mutant genomes, majority were nonsynonymous, and a moderate to large proportion were in the conserved regions. Our results suggest that HCV genome has the ability to overcome lethal/deleterious mutations because of the high reproduction rate, but highly selected for random, beneficial mutations. [ABSTRACT FROM AUTHOR]