Multifaceted counter-APOBEC3G mechanisms employed by HIV-1 Vif

Abstract

In the absence of human immunodeficiency virus type 1 (HIV-1) Vif protein, the host antiviral deaminase apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) restricts the production of infectious HIV-1 by deamination of dC residues in the negative single-stranded DNA produced by reverse transcription. The Vif protein averts the lethal threat of deamination by precluding the packaging of A3G into assembling virions by mediating proteasomal degradation of A3G. In spite of this robust Vif activity, residual A3G molecules that escape degradation and incorporate into newly assembled virions are potentially deleterious to the virus. We hypothesized that virion-associated Vif inhibits A3G enzymatic activity and therefore prevents lethal mutagenesis of the newly synthesized viral DNA. Here, we show that (i) Vif-proficient HIV-1 particles released from H9 cells contain A3G with lower specific activity compared with Δvif-virus-associated A3G, (ii) encapsidated HIV-1 Vif inhibits the deamination activity of recombinant A3G, and (iii) purified HIV-1 Vif protein and the Vif-derived peptide Vif25-39 inhibit A3G activity in vitro at nanomolar concentrations in an uncompetitive manner. Our results manifest the potentiality of Vif to control the deamination threat in virions or in the pre-integration complexes following entry to target cells. Hence, virion-associated Vif could serve as a last line of defense, protecting the virus against A3G antiviral activity.

Fig. 1. HIV-1 produced by H9 cells incorporates enzymatically active A3G protein

A. Western blots of wild-type or HIV-1Δvif viruses produced by H9 cells. Equal amounts of viral proteins, (20 ng of p24, as measured by p24 antigen capture test) were loaded onto each slot of SDS-PAGE. Endogenous A3G and viral p24 CA proteins (upper and lower panels, respectively) were detected by using specific antibodies. Virus harvested from 293T cells transiently co-transfected with HIV-1 Δvif DNA and pcDNA3-A3G-MycHis was used as positive control (PC). B. 30 ng of p24 of HIV-1 wt and Δvif virions produced by H9 and SupT1 cells were loaded into the slots of SDS-gels. The presence of A3G and Vif proteins in those virus preparations was determined by using polyclonal anti-A3G and anti-Vif antibodies. C. Deamination of synthetic ss-deoxynucleotide substrate by virus-associated A3G. Equal amounts of HIV-1 wt and Δvif viruses (1.25 ng of p24) were added to the reactions containing increasing amounts of the substrate (ranging from 0.01 to 0.2 fmol), as indicated. S, an 80 nt-long substrate used for the deamination assay; P, a 40 nt-long product of the restriction reaction.

Fig. 2. Inhibition of A3G activity by Vif

A. Recombinant A3G protein (rec. A3G) (0.75 fmol) was mixed with wt HIV-1 or Δvif virus from SupT1 cells (2.5 ng of p24 per reaction). Reactions were carried out on 1 fmol of A3G oligonucleotide substrate. As negative controls, deamination reactions were loaded with viruses from SupT1 (no A3G). All reactions contained 2.5 ng of p24 (as measured by p24 CA antigen capture test). B. Purified A3G (0.35 fmol) was incubated with the ss-deoxy-oligonucleotide substrate in the presence of purified Vif for 15 min. Lane 1, positive control (dU containing oligonucleotide); Lane 2, negative control (no A3G); Lane 3, sample containing 10 μM BSA; Lane 4, sample containing the elution fraction of Ni-NTA purification from non Vif-expressing bacteria (amount equal to lane 10); Lanes 5-10, dose-dependent inhibition of A3G deamination by increasing Vif concentrations, as indicated. A graphic representation of the Vif-mediated inhibition is shown on the bottom. Values represent the average of triplicates; SD values were less than +/−0.8. C. Purified wt and D128K A3G enzymes (0.2 fmol) were incubated with purified Vif protein at indicated concentrations for 10 min at RT, followed by addition of 10 fmol ss-deoxy-oligonucleotide substrate for 30 min. The plot presents the relative deaminase activity, while 100 percent of activity was adjusted to deamination without Vif.

Fig. 3. Inhibition of A3G deaminase activity by Vif-derived peptides

Fifteen-mer Vif-derived peptides covering the complete Vif sequence were assessed for the inhibition of A3G. The standard deamination reaction was carried out in the presence of 10 μM (A) or 1 μM (B) of each peptide, or an RSV-derived peptide (P.C., positive control). PAGE analyses of the cleaved deamination products are shown above the charts. C. the effect of the Vif-derived peptide concentration on A3G mediated deamination. Values represent the average of triplicates; SD values were less than +/− 0.5 where not indicated.

Fig. 4. Determination of the Vif and Vif-derived peptides mode of inhibition

Deamination of an ss-deoxyoligonucleotide as function of the substrate concentration in the presence of Vif (A) and the Vif-derived peptides Vif105-119 (B) and Vif25-39 (C) was determined and is shown by double-reciprocal (double-inverse) plot. The Vif and Vif-derived peptides concentrations used are indicated. Values represent the average of triplicates; SD values were less than +/−0.4.