K312 and E446 are involved in HCV RNA translation modulation by NS5A domains II and III

•HCV NS5A K312 is involved in down-regulating viral translation by NS5A domain II.•HCV NS5A E446 is involved in down-regulating viral translation by NS5A domain III.•K312A and E446A mutations abrogate viral translation inhibition by full-length NS5A.•HCV NS5A K312 and E446 have differential effects on HCV replication.

HCV NS5A plays a critical role in the HCV life cycle. We previously demonstrated that NS5A down-regulates viral translation through a mechanism requiring the polyU/UC region of the viral 3′UTR and that each of the three domains is capable of carrying out this function individually. In this study, we mapped the regions and amino acid residues within domains II and III involved in the modulation of viral translation. Using a series of deletion and amino acid substitution mutants, we found that K312 and E446 play important roles in the modulation of viral translation by NS5A domains II and III, respectively. In the context of full-length NS5A, mutations of K312 and E446 alone or in combination again abrogate translation down-regulation. In a transient replication assay using HCV subgenomic replicons, the K312A mutation alone does not affect HCV replication throughout a 96-h time course. While the E446A mutation can increase HCV replication at early time points (4–24 h), the K312A and E446A double mutation can enhance viral replication at 24–96 h, suggesting both residues are involved. Our results shed more light on the functions of NS5A.