Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif

Abstract

The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs.

Figure 1.

(A) Schematic representation of HIV-1 Vif domains required for interactions with viral and cellular partners. (B) Secondary structure of RNA sequences TAR, and its DNA analog, dTAR. Primary structure of fragment B2 encompassing genomic nucleotides 539–547, and its DNA analog dB2.

Figure 2.

Size exclusion chromatography on wild-type and mutant Vif proteins. Elution profiles of Vif wild-type (A) and Vif AALA (B) are represented together with protein standards. (C) The molecular weights (log) are plotted against elution volumes calibrated using molecular weight standards. Measured molecular weights of Vif wild-type and Vif AALA give 209 kDa and 76 kDa, respectively, corresponding to about 9 and 3 Vif proteins in each complex.

Figure 3.

Diffusion light scattering profiles of wild-type Vif (A) and Vif AALA (B) proteins. (A) Mean radius for wild-type protein was 5.3 ± 0.6 nm, which corresponds to polymers containing 5 to 8 Vif proteins. (B) Vif AALA proteins showed a mean radius of 3.2 ± 0.3 nm, mainly corresponding to dimers of Vif.

Figure 4.

Influence of Vif oligomerization on its secondary structure content and fluorescence properties. (A) Far-UV-CD spectra of wild-type (green) and AALA mutant (blue) Vif proteins. Proteins were prepared at a final concentration of 10 µM. (B) Emission fluorescence spectra of free Trp amino acid (dashed line), free Vif AALA protein in solution (solid line) and in complex with TAR fragment (dotted line). Excitation wavelength was set at 295 nm.

Figure 5.

Ribonucleoprotein complexes were characterized by DLS. Increasing concentration of wild-type Vif were added to various amounts of oligoribonucleotides TAR and B2 (R RNA/protein ratio varying between 0 and 2). Similarly, TAR and B2 were added to Vif AALA. Protein/nucleic acid complex formation resulted in increasing complex radius.