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Journal of Virology, October 2009, p. 10058-10074, Vol. 83, No. 19
0022-538X/09/$08.00+0     doi:10.1128/JVI.02418-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication ^,

Jason Borawski,¹ Philip Troke,¹ Xiaoling Puyang,¹ Veronica Gibaja,¹ ShanChaun Zhao,¹ Craig Mickanin,¹ Juliet Leighton-Davies,¹ Christopher J. Wilson,¹ Vic Myer,¹ Ivan CornellaTaracido,¹ Jeremy Baryza,¹ John Tallarico,¹ Gerard Joberty,² Marcus Bantscheff,² Markus Schirle,¹ Tewis Bouwmeester,^2, Joanna E. Mathy,¹ Kai Lin,¹ Teresa Compton,¹ Mark Labow,¹ Brigitte Wiedmann,¹ and L. Alex Gaither^1*

Novartis Institutes for Biomedical Research, 250 Massachusetts Ave., Cambridge, Massachusetts 02139,¹ Cellzome AG, Meyerhofstrasse 1, D-69117 Heidelberg, Germany²

Received 24 November 2008/ Accepted 29 June 2009

Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.

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* Corresponding author. Mailing address: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, 500 Technology Square, Office 710-05, Cambridge, MA 02139. Phone: (617) 871-7209. Fax: (617) 871-5783. E-mail: alex.gaither{at}novartis.com

Published ahead of print on 15 July 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

Present address: Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland.

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Journal of Virology, October 2009, p. 10058-10074, Vol. 83, No. 19
0022-538X/09/$08.00+0     doi:10.1128/JVI.02418-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.