Design and Characterization of Inhibitors of Cell-Mediated Degradation of APOBEC3G That Decrease HIV-1 Infectivity

Abstract

The cytoplasmic human Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) cytidine deaminases G and F (A3G and A3F) can block the spread of human immunodeficiency virus (HIV). HIV counteracts this cell-intrinsic defense through a viral protein called viral infectivity factor (Vif). Vif causes proteasomal degradation of A3G and A3F proteins (A3G/F) in HIV-producing cells to ensure infectivity of virions subsequently released from these cells. Here, we optimized a lead compound reported previously to boost cellular levels of A3G. The modified analogs designed, synthesized, and evaluated here inhibit cell-mediated post-translational degradation of A3G/F, whether Vif is present or not. This increases A3G/F incorporation into Vif-positive virions to decrease viral infectivity. The compounds and processes described here can facilitate the development of new anti-HIV therapeutics whose host-targeted effect may not be evaded by resistance-conferring mutations in HIV Vif.

Keywords: APOBEC3F; APOBEC3G; HIV; HIV cure research; HIV infectivity; HIV-1; Vif.

Figure 1

Structural formulas of compounds previously identified to inhibit Vif-mediated A3G degradation. Methyl groups indicated by a line. (A) IMB-26. Arrow indicates R₂ site. (B) RN-18.

Figure 2

Characterization of puromycin-resistant 293T-cell lines stably expressing A3G-Luc and Vif/A3G-Luc. (A) Relative luciferase activities (RLUs) of two different clones (#5 and #6) transduced by pseudotyped viral particles to stably express different levels of A3G-Luc. A similarly transduced control clonal line lacks A3G-Luc expression. (B) Immunoblots of these three clones probed with an anti-A3G antibody are shown. Lane 1 is A3G-Luc #5. Lane 2 is A3G-Luc #6. Lane 3 is the control line. (C) RLU of clone #5 or clone #6, each with or without stable expression of Vif using the FUGW-Vif vector (indicated as Vif+) and the corresponding empty vector (indicated as EV) control. (D) Immunoblots for A3G, Vif, and β-actin proteins from C are shown. Lane 1 is EV/A3G-Luc #5. Lane 2 is Vif/A3G-Luc #5. Lane 3 is EV/A3G-Luc #6. Lane 4 is Vif/A3G-Luc #6. (E) Luciferase activities produced by A3G-Luc cell clone #5 following transfection with the EV control, the Vif-null NL4.3 plasmid, or the Vif+ NL4.3 plasmid are shown. (F) Luciferase activities produced by A3G-Luc cell clone #6 following transfection with the EV control, the Vif-null NL4.3 plasmid, or the Vif+ NL4.3 plasmid are shown. (G) Relative infectivity of viral particles produced from transfection of Vif+ NL4.3 expression plasmid (indicated as Vif+) into A3G-Luc cell clones #5 and 6, as well as the control line, are shown. Supernatants of Vif+ NL4.3-transfected cultures of each cell clone were normalized by p24 value prior to infection of TZM-bl cells to ensure the same viral input. RLUs were read 24 h after infection of TZM-bl cells. (H) Relative infectivity of viral particles produced from transfection of Vif-null NL4.3 expression plasmid (indicated as Vif-null) into A3G-Luc cell clones #5 and 6, as well as the control line, are shown. Infectivity was assessed as in G.

Figure 3

Structural formulas of compounds selected for further study. The R₂ site is circled in NU-52. Note that methyl groups are shown here, rather than being depicted only as lines, as in Figure 1 and Supplementary Table S1.

Figure 4

NU compounds boost A3G protein levels in uninfected CD4+ T cells and A3F protein levels in A3F-transfected 293T cells. (A) Immunoblot shows resting, uninfected CD4+ T cells treated with NU-52, -302, and -611 (10 μM each). (B) Quantification of band densities in A. (C) Immunoblot shows 293T cells transiently transfected with A3F expression plasmid and treated with DMSO, IMB-26, or NU-52. (D) Quantification of band densities in C.

Figure 5

NU compounds increase incorporation of A3G or A3F into Vif+ virions and decrease Vif+ virion infectivity. (A) Vif+ NL4.3 was co-transfected with an A3G expression plasmid into 293T cells. DMSO or NU compounds (10 μM each) were added 24 h after transfection. One day later, immunoblots of A3G and p24 of pelleted supernatant viral particles were performed from cultures transfected with Vif+ NL4.3 alone (Lane 1) or co-transfected with both Vif+ NL4.3 and A3G followed by treatment with either DMSO (Lane 2), NU-52 (Lane 3), or NU-302 (Lane 4). (B) Relative infectivity of supernatant viruses from the different conditions described in A was determined. Vif+ HIV infectivity was measured by luciferase activity after infection of TZM-bl cells. (C) Immunoblots of A3F and p24 in pelleted supernatant viral particles from 293T cells transfected with Vif+ NL4.3 alone (Lane 1) or co-transfected with both Vif+ NL4-3 and A3F followed by treatment with either DMSO (Lane 2), NU-52 (Lane 3), or NU-302 (Lane 4) are shown. (D) Relative infectivity of supernatant viruses from 293T cells from the different conditions described in C was determined. Vif+ HIV infectivity was measured as in B. (E) Immunoblots of A3G and p24 in pelleted supernatant viral particles from 293T cells stably expressing A3G that were transiently transfected with Vif+ NL4.3 and treated with either DMSO (Lane 1), NU-52 (Lane 2), or NU-302 (Lane 3) are shown. (F) Relative infectivity of supernatant viruses from 293T cells stably expressing A3G from the different conditions described in E was determined. (G) Immunoblots of A3F and p24 in pelleted supernatant viral particles from 293T cells stably expressing A3F that were transiently transfected with Vif+ NL4.3 and treated with either DMSO (Lane 1), NU-52 (Lane 2), or NU-302 (Lane 3) are shown. (H) Relative infectivity of supernatant viruses from 293T cells stably expressing A3F from the conditions described in G was determined. All data shown as means ± SD from ≥3 independent experiments; ANOVA was used to analyze the differences (* p < 0.05, ** p < 0.01, **** p < 0.0001, ns p > 0.05).

Figure 6

NU-611 enhances A3G-mediated reduction in HIV virion infectivity more than NU-52. 293T cells stably expressing A3G (293T IP/G cells) were transfected with Vif+ NL4.3 and then treated with either DMSO control or 15 μM each of either NU-52 or -611. DMSO was plotted as 100% relative infectivity. Data from 8 independent experiments are expressed as means ± SD; ANOVA was used to analyze the differences, **** p < 0.0001.

Figure 7

NU compounds decrease infectivity of Vif+ HIV produced by H9 cells expressing endogenous A3G and A3F after infection. After Vif+ NL4.3 infection, H9 cells were treated with a compound (NU-52, -302, or -611, each at 10 μM) or control (DMSO). TZM-bl cells were infected with p24-normalized H9 culture supernatants and relative infectivity quantified by luciferase activity of TZM-bl cell lysates. Infectivity of virus from DMSO control is plotted as 100%. All data expressed as means ± SD from ≥3 independent experiments; ANOVA was used to analyze the differences (*** p < 0.001, **** p < 0.0001, ns p > 0.05).

Figure 8

Stability of A3G and A3F in the presence of protein synthesis-blocking cycloheximide (CHX) increases with treatment with NU-52 or -302 at 10 μM concentration. (A) Relative luciferase activities are shown from the cell line stably expressing intermediate levels of A3G-Luc (A3G-Luc #6) after treatment with either CHX+DMSO, CHX+NU-52, or CHX+NU-302. (B) Immunoblots from 293T cells transfected with A3G expression plasmid are shown after treatment with either CHX and DMSO (lane 1), CHX plus NU-52 (lane 2), or CHX plus NU-302 (lane 3).

Figure 9

NU-52 and -611 diminish pVHL-mediated degradation of A3G or -F. (A) Immunoblots from cell lysates of 293T cells transfected with pVHL and A3G are shown. (B) Band densities of immunoblots depicted in A are quantified. (C) Immunoblots from cell lysates of 293T cells transfected with pVHL along with A3F are shown. (D) Band densities of immunoblots depicted in C are quantified. Mean ± SD for at least 2 independent immunoblots are shown. ANOVA was used to analyze the differences (* p < 0.05, ** p < 0.01, *** p < 0.001).