The multimerization of human immunodeficiency virus type I Vif protein: a requirement for Vif function in the viral life cycle

Abstract

The Vif (virion infectivity factor protein of human immunodeficiency virus type I (HIV-1) is essential for viral replication in vivo and productive infection of peripheral blood mononuclear cells, macrophages, and H9 T-cells. However, the molecular mechanism(s) of Vif remains unknown and needs to be further determined. In this report, we show that, like many other proteins encoded by HIV-1, Vif proteins possess a strong tendency toward self-association. In relatively native conditions, Vif proteins formed multimers in vitro, including dimers, trimers, or tetramers. Through in vivo binding assays such as coimmunoprecipitation and the mammalian two-hybrid system, we also demonstrated that Vif proteins could interact with each other within a cell, indicating that the multimerization of Vif proteins is not simply due to fortuitous aggregation. Further studies indicated that the domain affecting Vif self-association is located at the C terminus of this protein, especially the proline-enriched 151-164 region. Moreover, we found that a Vif mutant with deletion at amino acid 151-164 was unable to rescue the infectivity of vif-defective viruses generated from H9 T-cells, suggesting that the multimerization of Vif proteins could be important for Vif function in the viral life cycle. Our studies identified a new feature of Vif and should accelerate our understanding of its role in HIV-1 pathogenesis.

Fig. 1. Vif self-association in a cell-free system

A, in vitro translated, ³⁵S-labeled HIV-1_NL4–3 Vif proteins were allowed to bind with GST-Vif conjugated on beads. After binding, the bead-associated ³⁵S-labeled Vif was analyzed via SDS-PAGE and direct autoradiography. B, Vif proteins form dimers and multimers in native or mild-denatured loading buffer. In vitro-translated ³⁵S-labeled HIV-1_NL4–3 Vif proteins were loaded directly onto a 4–20% Tris-HCl gel (SDS-free) with native loading buffer (62.5 mm Tris-HCl (pH 6.8), 20% glycerol) plus SDS at different concentrations. Electrophoresis was performed with a Tris/glycine running buffer containing 0.05% SDS, followed by autoradiography. βME, β-mercaptoethanol.

Fig. 2. The effect of Vif mutants on Vif-Vif interactions

A, a series of deletions along the Vif 192 amino acids were generated via PCR-based mutagenesis and in vitro translation. The in vitro-translated, ³⁵S-labeled HIV-1_NL4–3 Vif protein and its mutants were allowed to bind to GST-Vif conjugated on agarose beads. The bead-associated, ³⁵S-labeled Vif protein and its mutants were subjected to SDS-PAGE and visualized by direct autoradiography. The values were obtained by quantitation with densitometry of the autoradiographs. The ratio of bound Vif versus the input was then calculated. The ratio of GST-Vif-bound ³⁵S-labeled wild-type Vif and ³⁵S-labeled wild-type Vif input was further set as 100% (with the standard deviations). The relative binding ability of Vif mutants was thus determined. In most cases, the data reflect at least five independent experiments. WT, wild-type. B, in vitro-translated ³⁵S-labeled HIV-1_NL4–3 Vif protein and its mutants (50,000 cpm count for each) were loaded directly onto a 4–20% Tris-HCl gel (SDS-free), with loading buffer (62.5 mm Tris-HCl (pH 6.8), 20% glycerol) plus 0.1% SDS. Electrophoresis was performed with a Tris/glycine running buffer containing 0.05% SDS, followed by autoradiography.

Fig. 3. Coimmunoprecipitation method to study Vif-Vif interactions within cells

COS-1 cells were transfected with vectors harboring FLAG or c-Myc tagged Vif. After 54 h of incubation at 5% CO₂, 37 °C, 20 μg of total cell lysates were resolved by 15% Tris-HCl gel. The Vif proteins were detected by Western blotting (WB) using an M2 anti-FLAG monoclonal antibody and A14 anti-c-Myc polyclonal antibody, respectively. For coimmunoprecipitation, the whole-cell lysates from the same batch were subjected to immunoprecipitation (IP) with A14 anti-c-Myc polyclonal antibody. Immunoprecipitates were resolved at 15% Tris-HCl gel, transferred onto a membrane, and then detected using an M2 anti-FLAG antibody.

Fig. 4. Mammalian two-hybrid system to study Vif-Vif interaction

A, a schematic map showing the plasmids utilized in the experiments. B, COS-1 cells were transfected with plasmids combined with various vectors. After 48 h, cell lysates were harvested and subjected to CAT analyses.

Fig. 5. Viral infectivity affected by Vif or Vif mutants

The pCI-Neo constructs, containing wild-type vif gene or its mutants, pNL4–3ΔenvΔvif plasmid and pMD.G (containing VSV envelope (env)), were cotransfected into H9 cells to generate the pseudotyped viral particles. After concentration via ultracentrifugation, the viral particles were normalized by HIV-1 p24 antigen. In the presence of polybrene (8 μg/ml), the viruses were used to infect HelaCD4-CAT cells. After 48 h, the cell lysates were collected and subjected to CAT analyses. Lane 1, pNL4–3; lane 2, pNL4–3ΔenvΔvif, VSV env plus wild-type vif; lane 3, pNL4–3ΔenvΔvif, VSV env plus vifΔ 151–164; lane 4, pNL4–3ΔenvΔvif, VSV env plus vifΔ 144–150; lane 5, pNL4–3ΔenvΔvif, VSV env plus pCI-Neo vector only. The value of wild-type vif complementation was set as 100%. The relative values of the other samples were calculated accordingly. This figure is representative of three independent experiments. Values are means ± standard deviations.