Abstract
The virus infectivity factor (Vif) protein facilitates the replication of human immunodeficiency virus type 1 (HIV-1) in primary lymphocytes and macrophages. Its action is strongly dependent on the cellular environment, and it has been proposed that the Vif protein counteracts cellular activities that would otherwise limit HIV-1 replication. Using a glutathione S-transferase pull-down assay, we identified that Vif binds specifically to the Src homology 3 domain of Hck, a tyrosine kinase from the Src family. The interaction between Vif and the full-length Hck was further assessed by co-precipitation assays in vitro and in human cells. The Vif protein repressed the kinase activity of Hck and was not itself a substrate for Hck phosphorylation. Within one single replication cycle of HIV-1, Hck was able to inhibit the production and the infectivity of vif-deleted virus but not that of wild-type virus. Accordingly, HIV-1 vif- replication was delayed in Jurkat T cell clones stably expressing Hck. Our data demonstrate that Hck controls negatively HIV-1 replication and that this inhibition is suppressed by the expression of Vif. Hck, which is present in monocyte-macrophage cells, represents the first identified cellular inhibitor of HIV-1 replication overcome by Vif.
Highlights
The virus infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1)1 is required for efficient virus replication in peripheral blood T lymphocytes in vitro [1,2,3], since vif-deleted mutants are several orders of magnitude less infectious than wild-type (WT) HIV-1 virions [1, 3,4,5]
We have investigated the putative interaction between various Src homology 3 (SH3) domains and HIV-1 Vif protein
We identified a preferential recognition of Vif by the SH3 domain of Hck and by the full-length Hck tyrosine kinase
Summary
Human kidney 293 cells were maintained in Dulbecco’s modified Eagle’s medium (supplemented with 10% fetal calf serum, antibiotics (penicillin/streptomycin, 100 g/ml), and 2 mM glutamine. U937 promonocytes and SupT1, Jurkat JH6.2, C8166, and H9 CD4ϩ T lymphocytes were grown in 1640 RPMI medium supplemented with 10% fetal calf serum, antibiotics, and 2 mM glutamine. For Jurkat cells stably expressing Hck, G418 was added at 2 mg/ml. Human CD4ϩ T lymphocytes and monocytes/macrophages were isolated from peripheral blood mononuclear cells as described [19]
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