The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production.
The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Nonstructural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70. We confirmed an NS5A/HSP interaction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A. A transient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion.


CONCLUSIONS
Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA 90095-1732, USA.
Oscar
O
Gonzalez
Vanessa
V
Fontanes
Santanu
S
Raychaudhuri
Rachel
R
Loo
Joseph
J
Loo
Vaithilingaraja
V
Arumugaswami
Ren
R
Sun
Asim
A
Dasgupta
Samuel W
SW
French
D002460:Cell Line; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D011794:Quercetin / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
D006360:Heat-Shock Proteins; D011794:Quercetin; D034741:RNA, Small Interfering; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase