|dc.description.abstract||Hepatitis C virus (HCV) non-structural protein NS5A is a multifunctional protein and despite lacking enzymatic activity has critical roles in viral replication and assembly. The role of NS5A in HCV RNA translation has not been well studied. In an attempt to better understand the role of HCV NS5A in RNA translation, our previous work showed that HCV-1b NS5A downregulates viral RNA translation by binding to the poly(U/UC) region in the 3’UTR. All three domains are capable of individually downregulating translation, albeit with a lesser effect than the full-length wild-type NS5A.
There are multiple HCV genotypes and NS5A from different genotypes may or may not carry out the same function. Therefore, to determine whether the role of NS5A is conserved in other genotypes, we studied the effect of HCV-2a NS5A on monocistronic HCV-2a RNA reporters and replication defective genomic RNA with or without poly(U/UC) region at the 3’UTR. We found that although HCV-2a NS5A also downregulates viral translation, it does not require the poly(U/UC) region in 3’UTR. The translation downregulation by HCV-2a NS5A was predominantly mediated by domain I. Our results elucidated that HCV-2a NS5A modulates viral translation through a mechanism different from HCV-1b NS5A.
NS5A is a phospho-protein and exists as hypo- and hyper-phosphorylated NS5A. The hyperphosphorylation of NS5A is mediated through the phosphorylation of the conserved serine residues cluster in the low complexity sequence LCS I. The serine residues are S222, S225, S229, S232, S235 and S238. Phosphorylation on these serine residues has been found to be important for HCV replication and viral assembly. To further understand the significance of NS5A hyperphosphorylation on HCV life cycle, we investigated the role of HCV-1b NS5A hyperphosphorylation on translation by analyzing the effects of phospho-ablative and phospho-mimetic mutants of the six serine residues on HCV-1b genomic RNA translation. We showed that phosphorylation of S222, S225, S235 is not involved in translation downregulation by NS5A. In contrast, alanine mutations at S229 or S238 can no longer downregulate translation, whereas S229D or S238D mutations have no effect. Interestingly, S232D, but not S232A, abrogates translation downregulation by NS5A.
NS5A exists as a dimer and its dimerization is important for regulating its function. Therefore, we studied the effect of phospho-mutants of S229, S232, and S238 on dimerization in a protein-protein interaction assay and showed that phospho-mimetic S229D or S238D mutations enhance NS5A dimerization, whereas the phospho-ablative mutations of them have no effect. In contrast, neither phospho-ablative nor phospho-mimetic mutations of S232 affect dimerization. In conclusion, these results indicated that phosphorylation of NS5A at S229, S232, and S238 is involved in viral translation regulation and NS5A dimerization.
In summary, these findings suggest that NS5A downregulates the translation of HCV RNA however, the mechanism may differ within the genotypes. In addition, hyperphosphorylation of NS5A is involved in regulation of HCV translation and NS5A dimerization. These results aid in the understanding the mechanism involved in regulation of viral translation by NS5A and may help in the development of pan-genotypic novel antiviral targets.||