Abstract
Human immunodeficiency virus type 1 (HIV-1)-encoded Vif protein is important for viral replication and infectivity. Vif is a cytoplasmic protein that acts during virus assembly by an unknown mechanism, enhancing viral infectivity. The action of Vif in producer cells is essential for the completion of proviral DNA synthesis following virus entry. Therefore, Vif is considered to be an important alternative therapeutic target for inhibition of viral infectivity at the level of viral assembly and reverse transcription. To gain insight into this process, we developed a Vif-specific single-chain antibody and expressed it intracellularly in the cytoplasm. This intrabody efficiently bound Vif protein and neutralized its infectivity-enhancing function. Intrabody-expressing cells were shown to be highly refractory to challenge with different strains of HIV-1 and HIV-1-infected cells. Inhibition of Vif by intrabody expression in the donor cell produced viral particles that do not complete reverse transcription in the recipient cell. The anti-Vif scFv was shown to be specific for Vif protein because its function was observed only in nonpermissive cells (H9, CEM, and U38). Moreover, transduction of peripheral blood mononuclear cells with an HIV-derived retroviral vector expressing Vif intrabody was shown to confer resistance to laboratory-adapted and primary HIV strains. This study provides biochemical evidence for the role of Vif in the HIV-1 lifecycle and validates Vif as a target for the control of HIV-1 infection.
Highlights
Human immunodeficiency virus type 1 (HIV-1)1 is a complex c Supported by a Bolsa de Investigacao Científica from Fundacao para a Ciencia e Tecnologia
When anti-thyroglobulin scFv was expressed together with anti-Vif scFv, no obvious co-localization was detected. These results demonstrate that Vif protein is recognized in the cytoplasm by the intracellular expression of anti-Vif scFv and that its interaction co-localizes Vif and scFv 4BL throughout the cytoplasm
ScFv-based strategies directed against Vif may be an effective approach to inhibit these two crucial steps of the viral replication cycle (36 –38)
Summary
Cells—H9, U38, Jurkat, SupT1, and CEM cells were grown in RPMI 1640 medium containing 10% fetal bovine serum and antibiotics. HeLa and 293T cells were grown in Dulbecco’s modified minimal essential medium supplemented with 10% fetal calf serum. Viruses—Plasmids coding for HIV-1NL4–3, pHIV-1NL4–3⌬vif, and HIV-2ROD were obtained from the AIDS Research and Reference Reagent Program. Plasmids for the trans-complementation assay were described previously [15]. The coding sequence for anti-thyroglobulin scFv is cloned in plasmid pHEN obtained from Griffiths et al [41]. PHXBnPLAP-IRES-Nϩ was obtained from the AIDS Research and Reference Reagent Program. HRP-conjugated anti-M13 phage antibody was obtained from Amersham Biosciences. Proteins—Vif protein was derived from HIV-1HXB2 strain and purified as described [45, 46]. HIV-1 protease was obtained from the AIDS Research and Reference Reagent Program
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