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Journal of Virology, February 2006, p. 1886-1896, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1886-1896.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Transient and Stable Knockdown of the Integrase Cofactor LEDGF/p75 Reveals Its Role in the Replication Cycle of Human Immunodeficiency Virus

Linos Vandekerckhove, Frauke Christ, Bénédicte Van Maele, Jan De Rijck, Rik Gijsbers, Chris Van den Haute, Myriam Witvrouw, and Zeger Debyser*

Molecular Medicine, KULeuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium

Received 20 April 2005/ Accepted 18 November 2005

After identifying the interaction between the transcriptional coactivator lens epithelium-derived growth factor (LEDGF/p75) and the human immunodeficiency virus type 1 (HIV-1) integrase (IN), we have now investigated the role of LEDGF/p75 during HIV replication. Transient small interfering RNA-mediated knockdown of LEDGF/p75 in HeLaP4 cells resulted in a three- to fivefold inhibition of HIV-1 (strain NL4.3) replication. Quantitative PCR was used to pinpoint the replication block to the integration step. Next, polyclonal and monoclonal HeLaP4-derived cell lines were selected with a stable knockdown of LEDGF/p75 mediated by a lentiviral vector (lentivector) encoding a short hairpin RNA (shRNA) targeting this protein. Cell lines stably transduced with a lentivector encoding an unrelated hairpin or a double-mismatch hairpin served as controls. Again, a two- to fourfold reduction of HIV-1 replication was observed. The extent of LEDGF/p75 knockdown closely correlated with the reduction of HIV-1 replication. After the back-complementation of LEDGF/p75 in the poly- and monoclonal knockdown cell lines using an shRNA-resistant expression plasmid, viral replication was restored to nearly wild-type levels. The Q168A mutation in integrase has been shown to interfere with the interaction with LEDGF/p75 without reducing the enzymatic activity. Transduction by HIV-1-derived lentivectors carrying the Q168A IN mutant was severely hampered, pointing again to a requirement for LEDGF/p75. Altogether, our data validate LEDGF/p75 as an important cellular cofactor for HIV integration and as a potential target for antiviral drug development.

* Corresponding author. Mailing address: Molecular Medicine, K.U.Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium. Phone: 32 16 33 21 83. Fax: 32 16 33 63 36. E-mail: zeger.debyser{at}med.kuleuven.ac.be.

Journal of Virology, February 2006, p. 1886-1896, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1886-1896.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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