Transient and stable knockdown of the integrase cofactor LEDGF/p75 reveals its role in the replication cycle of human immunodeficiency virus.
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.
Vandekerckhove
Linos
L
Molecular Medicine, K.U.Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium.
Christ
Frauke
F
Van Maele
Bénédicte
B
De Rijck
Jan
J
Gijsbers
Rik
R
Van den Haute
Chris
C
Witvrouw
Myriam
M
Debyser
Zeger
Z
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000502:physiology*; D019943:Amino Acid Substitution; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D020868:Gene Silencing; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D016655:HIV Core Protein p24 / Q000032:analysis; D019427:HIV Integrase / Q000235:genetics / Q000502:physiology*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D016086:Lentivirus; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000032:analysis; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014161:Transduction, Genetic; D016662:Virus Integration; D014779:Virus Replication
D048868:Adaptor Proteins, Signal Transducing; D004279:DNA, Viral; D016655:HIV Core Protein p24; C504649:PSIP1 protein, human; D034741:RNA, Small Interfering; D012367:RNA, Viral; D014157:Transcription Factors; D019427:HIV Integrase