Transient and stable knockdown of the integrase cofactor LEDGF/p75 reveals its role in the replication cycle of human immunodeficiency virus

被引:169
作者
Vandekerckhove, L
Christ, F
Van Maele, B
De Rijck, J
Gijsbers, R
Van den Haute, C
Witvrouw, M
Debyser, Z
机构
[1] Katholieke Univ Leuven, Louvain, Belgium
[2] IRC, KULAK, Louvain, Belgium
关键词
D O I
10.1128/JVI.80.4.1886-1896.2006
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
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页码:1886 / 1896
页数:11
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