Drug-resistant viral variants are a major issue in the use of direct-acting antiviral agents in chronic hepatitis C. Ketoamides are potent inhibitors of the NS3 protease, with V55A identified as mutation associated with resistance to boceprevir. Underlying molecular mechanisms are only partially understood. We applied a comprehensive sequence analysis to characterize the natural variability at Val55 within dominant worldwide patient strains. A residue-interaction network and molecular dynamics simulation were applied to identify mechanisms for ketoamide resistance and viral fitness in Val55 variants. An infectious H77S.3 cell culture system was used for variant phenotype characterization. We measured antiviral 50% effective concentration (EC₅₀) and fold changes, as well as RNA replication and infectious virus yields from viral RNAs containing variants. Val55 was found highly conserved throughout all hepatitis C virus (HCV) genotypes. The conservative V55A and V55I variants were identified from HCV genotype 1a strains with no variants in genotype 1b. Topology measures from a residue-interaction network of the protease structure suggest a potential Val55 key role for modulation of molecular changes in the protease ligand-binding site. Molecular dynamics showed variants with constricted binding pockets and a loss of H-bonded interactions upon boceprevir binding to the variant proteases. These effects might explain low-level boceprevir resistance in the V55A variant, as well as the Val55 variant, reduced RNA replication capacity. Higher structural flexibility was found in the wild-type protease, whereas variants showed lower flexibility. Reduced structural flexibility could impact the Val55 variant's ability to adapt for NS3 domain-domain interaction and might explain the virus yield drop observed in variant strains.