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Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening

Maud Trotard^*,, Charlotte Lepère-Douard^*,, Morgane Régeard, Claire Piquet-Pellorce^*, Dimitri Lavillette^,,||, François-Loic Cosset^,,||, Philippe Gripon^*, and Jacques Le Seyec^*,,1

^* Equipe Associée SERAIC no. 4427, Université de Rennes 1, Rennes, IFR 140, France;

INSERM U522, Rennes, France;

Université de Lyon, UCB-Lyon1, IFR 128, Lyon, France;

INSERM U758, Lyon, France; and

^|| Ecole Normale Supérieure de Lyon, Lyon, France

¹ Correspondence: EA SERAIC no. 4427, Université de Rennes 1, Faculté de Pharmacie, Ave. du Professeur Léon Bernard, 35043 Rennes Cedex, France. E-mail: jacques.leseyec{at}univ-rennes1.fr

The entry pathway of the hepatitis C virus (HCV), a major human pathogen, into the cell is incompletely defined. To better characterize this viral life cycle stage, we screened a small interfering RNA library dedicated to the membrane trafficking and remodeling with the infection model of Huh-7.5.1 cells by HCV pseudoparticles (HCVpp). Results showed that the down-regulation of different factors implied in clathrin-mediated endocytosis (CME) inhibits HCVpp cell infection. In addition, knockdown of the phosphatidylinositol 4-kinase type III- (PI4KIII) prevented infection by HCVpp or by cell-culture grown JFH-1-based HCV. Moreover, the replication activity of an HCV replicon was also affected by the PI4KIII knockdown. Additional investigations on the different members of the PI4K family revealed that the presence of PI4KIIIβ in the host cells influenced their susceptibility to HCVpp infection and their capacity to sustain the HCV replication. The PI4KIII involvement during the HCV life cycle seemed to occur by other ways than the control of the CME or of the membranous expression of HCV receptors. Finally, our library screening completed data on the CME-dependant entry route of HCV and identified 2 kinases, PI4KIII and β, as relevant potential therapeutic targets.—Trotard, M., Lepère-Douard, C., Régeard, M., Piquet-Pellorce, C., Lavillette, D., Cosset, F.-L., Gripon, P., Le Seyec, J. Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening.

Key Words: hepacivirus • virus internalization • endocytosis • replicon • 1-phosphatidylinositol 4-kinase