Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is involved in genome replication and virus assembly. NS5B contains a distinct loop (loop Lambda2) at the beginning of the nucleoside triphosphate tunnel with a highly conserved lysine (K151). In this study, reverse genetic analysis revealed that substitution of Jc1 NS5B K151 for alanine (K151A) and aspartic acid (K151D) affected genome replication and infectious virus production. However, genome replication and virus production by Jc1 containing NS5B K151R remained unaltered. A major deletion in loop Lambda2 abolished RNA replication, suggesting a role for this structural domain in NS5B polymerase activity. In conclusion, this study demonstrated that the conserved K151 modulates infectious virus production; and loop Lambda2 is essential for the polymerase activity of NS5B.