Previous studies have demonstrated the absence of viral replication of Vif ؊ mutants in stimulated primary blood mononuclear cells (PBMC). Human immunodeficiency virus type 1 strain NDK Vif ؊ mutants were propagated on the semipermissive CEM cell line, and the viral stock obtained was compared with the wild-type virus during a single cycle in PBMC. The Vif ؊ virus was able to enter PBMC with the same efficiency as the wild type, as demonstrated by quantification of the strong-stop cDNA, and retrotranscription was observed for both viruses within 4 h postinfection. Using a PCR assay with an Alu-long terminal repeat pair of primers, we detected integration for both the wild-type and Vif ؊ viruses. We then used qualitative and quantitative reverse transcription-mediated PCR techniques to study the steady-state level of intracellular and extracellular viral RNAs. All mRNA species were detected in PBMC infected with the wild-type virus or with the Vif ؊ virus 36 h postinfection. Furthermore, quantification of viral RNA released from infected cells demonstrated similar levels of virus produced after a unique cycle of replication. However, the Vif ؊ virus obtained after one replication cycle in PBMC was unable to initiate retrotranscription in permissive target cells. These data strongly suggest that the failure to infect target cells is due to a defect in the formation of the viral particle in PBMC.
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.
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