A cell-permeable hairpin peptide inhibits hepatitis C viral nonstructural protein 5A-mediated translation and virus production

Abstract

NS5A is a key regulator of hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown NS5A to augment HCV IRES-mediated translation. Further, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit NS5A-driven augmentation of IRES-mediated translation and infectious virus production. We have also co-immunoprecipitated 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) to be the interaction site. Further, 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, while controls did not. Triple-alanine scan mutagenesis identified an exposed beta-sheet hairpin in C34 to be 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.

Modulation of PI3K-LXRα-dependent lipogenesis mediated by oxidative/nitrosative stress contributes to inhibition of HCV replication by quercetin

“…37 Nevertheless, the molecular mechanisms involved in quercetin-mediated impairment of HCV replication seem to be more complex. Thereby, quercetin may also inhibit the NS5A-driven augmentation of internal ribosomal entry site (IRES)-mediated translation 21,38 and reduce viral production by inhibiting both NS3 and heat-shock proteins essential for HCV replication. 22 In addition to these molecular mechanisms, we investigated the contribution of oxidative/nitrosative stress and lipid metabolism modulation to the inhibition of HCV replication by quercetin.…”

Systematic Analysis of Enhancer and Critical cis -Acting RNA Elements in the Protein-Encoding Region of the Hepatitis C Virus Genome

“…At 96 h p.t., mutant attenuated in infectious-particle production (27). The FNX-Rluc virus is also very sensitive in detecting changes in viral genome replication (27,66,67). The NS3-to-NS5B region was divided into 17 segments (S1 to S17) 285 to 360 nucleotides in size (Table 2 and Fig.…”

Y-Box Binding Protein 1 Stabilizes Hepatitis C Virus NS5A via Phosphorylation-Mediated Interaction with NS5A To Regulate Viral Propagation

“…Both positive and negative roles for NS5A in HCV IRES-mediated translation have been reported with different assay systems and cell lines (17,18,81), implying that specific NS5A interactions with other proteins may determine the effects of NS5A on HCV translation. Given the diverse implication of YB-1, DDX3, NS3, and core in HCV translation ( Fig.…”

“…cDNAs were synthesized using RevertAid First Strand cDNA synthesis kit (Fermentas). Primers used for reverse transcription were oligo(dT) 18 and a previously described primer specific for HCV (50) (5=-CTCCCGGGGCACTCGCAAGC-3=). Realtime PCR was performed using LightCycler FastStart DNA Master SYBR green I (Roche) and LightCycler 2.0 System (Roche).…”

“…Domain III partici-pates in virion assembly (12,13,15). NS5A has also been reported to either positively or negatively regulate HCV IRES-mediated translation (16)(17)(18). By regulating activity of cellular lipid kinase phosphatidylinositol 4-kinase type III alpha (PI4KIII-␣), NS5A has been demonstrated to modulate the formation of a membranous web to support HCV RNA replication (19,20).…”