Key result
A cell-permeable hairpin peptide (C34) and its 10-amino acid derivative competitively inhibited NS5A-augmented IRES-mediated translation and significantly reduced intracellular viral protein synthesis.
Why the study?
Does a cell-permeable hairpin peptide derived from NS5A inhibit HCV IRES-mediated translation and viral protein synthesis in vitro?
Does a cell-permeable hairpin peptide derived from NS5A inhibit HCV IRES-mediated translation and viral protein synthesis in vitro?
A specific hairpin peptide derived from HCV NS5A inhibits NS5A/HSP70 complex formation, reducing viral translation and protein synthesis, offering a potential target for rational drug design.
This is the authors' abstract. We don't add key points for this paper.
UNLABELLED: NS5A is a key regulator of the hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown that NS5A augments HCV internal ribosomal entry site (IRES)-mediated translation. Furthermore, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit the NS5A-driven augmentation of IRES-mediated translation and infectious virus production. We have also coimmunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization, leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES-mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C-terminal 34 amino acids (C34) as the interaction site. Furthermore, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same-size control peptides from other NS5A domains. C34 also competitively inhibited NS5A-augmented IRES-mediated translation, whereas controls did not. Triple-alanine scan mutagenesis determined that an exposed beta-sheet hairpin in C34 was primarily responsible for NS5A-augmented IRES-mediated translation. Moreover, treatment with a 10-amino acid peptide derivative of C34 suppressed NS5A-augmented IRES-mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. CONCLUSION: These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES-mediated translation, identify a sequence-specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin-mediated NS5A/HSP70 interaction. Identification of this element may allow for further interrogation of NS5A-mediated IRES activity, sequence-specific HSP recognition, and rational drug design. (HEPATOLOGY 2012;55:1662-1672).
Supports NS5A-derived peptides as HCV inhibitors; leaves open in vivo efficacy and clinical translation.
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Khachatoorian et al. (2011) studied Hepatitis C virus (HCV) infection. C34 hairpin peptide and its 10-amino acid derivative vs. Same-size control peptides was evaluated on NS5A-augmented IRES-mediated translation and intracellular viral protein synthesis. A cell-permeable hairpin peptide (C34) and its 10-amino acid derivative competitively inhibited NS5A-augmented IRES-mediated translation and significantly reduced intracellular viral protein synthesis.
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