Hepatitis C virus core protein forms the viral nucleocapsid and plays a critical role in the formation of infectious particles. In this study, we demonstrate that the highly conserved residue G33, located within domain 1 of the core protein, is important for the production of cell culture-infectious virus (HCVcc). Alanine substitution at this position in the JFH1 genome did not alter viral RNA replication but reduced infectivity by ∼2 logs. Virus production by this core mutant could be rescued by compensatory mutations located immediately upstream and downstream of the original G33A mutation. The examination of the helix-loop-helix motif observed in the core protein structure (residues 15 to 41; Protein Data Bank entry 1CWX) indicated that the residues G33 and F24 are in close contact with each other, and that the G33A mutation induces a steric clash with F24. Molecular simulations revealed that the compensatory mutations increase the helix-loop-helix flexibility, allowing rescue of the core active conformation required for efficient virus production. Taken together, these data highlight the plasticity of core domain 1 conformation and illustrate the relationship between its structural tolerance to mutations and virus infectivity.