Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins

Abstract

Virion infectivity factor (Vif) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) in vivo, but its function remains uncertain. Recently, we have shown that Vif proteins are able to form multimers, including dimers, trimers, or tetramers. Because the multimerization of Vif proteins is required for Vif function in the viral life cycle, we propose that it could be a novel target for anti-HIV-1 therapeutics. Through a phage peptide display method, we have identified a set of 12-mer peptides containing a PXP motif that binds to HIV-1 Vif protein. These proline-enriched peptides potently inhibited the Vif-Vif interaction in vitro. We have also screened a set of synthesized Vif peptides (15-mer), which covers all the amino acids of the HIV-1 Vif protein sequence, for their ability to inhibit the Vif-Vif interaction in vitro. We demonstrated that Vif-derived proline-enriched peptides that contain the (161)PPLP(164) domain are able to inhibit the Vif-Vif interaction. Conversely, the deletion of the (161)PPLP(164) domain of Vif protein will significantly impair the capability of Vif proteins to interact with each other, indicating that the (161)PPLP(164) domain plays a key role in Vif multimerization. All these results demonstrate that the proline-enriched peptides block the multimerization of Vif through interfering with the polyproline interfaces of Vif formed by (161)PPLP(164) domain. Moreover, these peptides which inhibit the Vif-Vif interaction in vitro potently inhibit HIV-1 replication in the "nonpermissive" T-cells. We propose that this study starts a novel strategy to develop structural diverse inhibitors of Vif such as peptidomimetics or small organic molecules.

Fig. 1

A, relative affinity comparisons between PXP motif-containing peptides. GST fusion protein (100 μg/ml) of Vif, Vif-(Δ151–192), and GST only were coated onto the 96-well plate. The phages clones isolated through the GST-Vif-containing column were serially diluted and added. After incubation to allow phage-Vif binding, excess phages were washed off. Anti-M13 phage antibody conjugated with horseradish peroxide was added to bind the phages that were captured by GST-Vif. After washing, the substrate was added, and color development was allowed to proceed. The phages captured by GST-Vif were, therefore, semi-quantitated. Optical density at 405 nm equal to or greater than 0.15 was considered to be positive. The phage sample number (VMI) is the same as shown in Table I. B, in vitro binding affinity of peptides to Vif. Various peptides (10⁻⁷, 5 × 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ m) were added to the mixture of ³⁵S-labeled Vif and GST-Vif-conjugated agarose beads. The ³⁵S-labeled Vif binding to GST-Vif was dissociated from beads by adding 2% SDS loading buffer and then analyzed by SDS-PAGE followed by autoradiography and quantitation using PhosphorImager. IC₅₀ is the concentration of the peptides inhibiting 50% ³⁵S-labeled Vif binding to GST-Vif in GST pull-down assays.

Fig. 2. The inhibition of HIV-1 Vif (15-mer) peptides upon Vif-Vif binding

HIV-1 consensus B Vif (15-mer) peptides (100 μm) were added to the mixture of ³⁵S-labeled Vif- and GST-Vif-conjugated agarose beads. The ³⁵S-labeled Vif binding to GST-Vif was dissociated from beads by adding 2% SDS loading buffer and then analyzed by SDS-PAGE followed by autoradiography and quantitation using a PhosphorImager

Fig. 3. Deletion of PPLP eliminates Vif-Vif interaction

GST-Vif- or GST-Vif(ΔPPLP)-conjugated agarose beads were mixed with ³⁵S-labeled Vif or its mutants in binding buffer and incubated at 4 °C for 1 h. The ³⁵S-labeled Vif or its mutants remaining on beads were dissociated from beads by adding 2%SDS loading buffer and then analyzed by SDS-PAGE followed by autoradiography and quantitation using a Phosphor-Imager. A, GST-Vif/³⁵S-Vif; B, GST-Vif/³⁵S-Vif-(Δ151–192); C, GST-Vif/³⁵S-Vif-(Δ151–164); D, GST-Vif/³⁵S-Vif(ΔPPLP); E, GST-Vif(ΔPPLP)/³⁵S-Vif; F, GST-Vif(ΔPPLP)/³⁵S-Vif(ΔPPLP).

Fig. 4. The in vitro inhibition by the peptides on Vif-Vif or Vif-Hck binding

Various peptides (100 μm) were added to the mixtures of ³⁵S-labeled Vif and GST-Vif- or GST-Hck-conjugated agarose beads. The ³⁵S-labeled Vif binding to GST-Vif or GST-Hck was dissociated from beads by adding 2% SDS containing loading buffer and then analyzed by SDS-PAGE followed by autoradiography and quantitation using a PhosphorImager.

Fig. 5

A, internalization of peptides. H9 cells were incubated with biotinylated peptides VMI 9 and Ant-VMI 9 for 30 min. The excess peptides were then washed off. After fixing, the internalized peptides were detected with streptavidin-fluorescein isothiocyanate followed by visualization with fluorescence microscopy. A, Ant-VMI 9, fluorescence; B, Ant-VMI 9, phase-contrast; C, VMI 9, fluorescence; D, VMI 9, phase contrast. B, Ant fusion peptides inhibit HIV-1 replication. H9 cells were infected by virions at 37 °C HIV-1_NL4–3 for 4 h. The infected H9 cells (1 × 10⁶) were then cultured in duplicate in 2 ml of RPMI 1640 medium plus 10% fetal bovine serum without or with peptides (50 μm). Portions of the supernatants (0.5 ml) were collected every 3–4 days. The HIV-1 p24 antigen levels were determined by ELISA. This data represent three independent experiments.