Correlation between NS5A Dimerization and Hepatitis C Virus Replication

Abstract

Background: NS5A is critical for HCV replication, but its role is poorly understood. Results: Cysteines Cys-39, Cys-57, Cys-59, and Cys-80 are vital for NS5A dimerization, RNA binding, and viral replication. Conclusion: NS5A dimerization, RNA binding, and HCV replication are correlated. Significance: This study addresses an important issue in HCV research with NS5A being a major drug target with inhibitors in advanced stages of clinical development.

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

“…In fact, accumulating evidence suggests a functional association between LCS I and NS5A domain I. For example, Hwang et al (13) defined a minimal NS5A RNA binding domain (amino acids 33 to 249) that requires Zn 2ϩ and includes LCS I, and Lim et al (41) recently showed that the LCS I linker region, in addition to domain I, is important for efficient NS5A dimerization. These RNA binding and dimerization studies were carried out with nonphosphorylated NS5A (13,41), implying that NS5A hyperphosphorylation is not required for these activities.…”

“…It will be interesting to determine whether the G337A amino acid substitution affects CypA binding and whether complementation group C is functionally defined by the interaction between NS5A and CypA. Lim et al (41) recently showed that NS5A proteins with C39A and C80A amino acid substitutions retained the ability to bind CypA, suggesting that these mutations do not affect the overall structural integrity of NS5A domain II. These results are consistent with our finding that these alleles can complement the G337A-associated replication defect.…”

“…The inability to successfully trans-complement replicons with these mutations could be explained if the misfolded proteins interact nonproductively with other replication complex components and occlude the integration of helper replicon-encoded NS5A into the defective replication complex. Alternatively, since the C39A and C80A amino acid substitutions are predicted to block NS5A dimerization (41), it is also possible that direct trans-complementation requires the formation of heterodimers between NS5A molecules expressed from the helper and defective replicons.…”

“…For example, Hwang et al (13) defined a minimal NS5A RNA binding domain (amino acids 33 to 249) that requires Zn 2ϩ and includes LCS I, and Lim et al (41) recently showed that the LCS I linker region, in addition to domain I, is important for efficient NS5A dimerization. These RNA binding and dimerization studies were carried out with nonphosphorylated NS5A (13,41), implying that NS5A hyperphosphorylation is not required for these activities. These findings are consistent with a model in which a hypophosphorylated NS5A dimer or multimer binds RNA and is directly involved in RNA replication, possibly as an integral component of the replication complex.…”

“…Complementation group A, defined by the inability to complement a replicon with a P32A allele, includes mutations affecting residues in the N-terminal region of NS5A domain I (P29A, L31A, P32A, C39A, C80A, and V121A). Residues 29, 31, and 32 are highly conserved among sequences in the European HCV database (42) and are located in a proposed hinge region linking the membraneanchoring N-terminal amphipathic ␣-helix to the zinc-binding motif of domain I. Residues C39 and C80 are important for zinc binding, RNA binding, and NS5A dimerization (7,41), and V121, located in ␤-sheet 5 downstream of the zinc-binding motif, is required for an interaction with the cellular protein FKBP8 (38). Among the domain I alleles, P29A, L31A, and P32A were capable of complementing the S232I allele, while C39A, C80A, and V121A were not.…”