Small-molecule inhibition of HIV-1 Vif

Abstract

The HIV-1 protein Vif, essential for in vivo viral replication, targets the human DNA-editing enzyme, APOBEC3G (A3G), which inhibits replication of retroviruses and hepatitis B virus. As Vif has no known cellular homologs, it is an attractive, yet unrealized, target for antiviral intervention. Although zinc chelation inhibits Vif and enhances viral sensitivity to A3G, this effect is unrelated to the interaction of Vif with A3G. We identify a small molecule, RN-18, that antagonizes Vif function and inhibits HIV-1 replication only in the presence of A3G. RN-18 increases cellular A3G levels in a Vif-dependent manner and increases A3G incorporation into virions without inhibiting general proteasome-mediated protein degradation. RN-18 enhances Vif degradation only in the presence of A3G, reduces viral infectivity by increasing A3G incorporation into virions and enhances cytidine deamination of the viral genome. These results demonstrate that the HIV-1 Vif-A3G axis is a valid target for developing small molecule-based new therapies for HIV infection or for enhancing innate immunity against viruses.

Figure 1

Small molecules that inhibit HIV-1 Vif. (a) Small-molecule structures. Structures of small molecules isolated from a 30,000-compound library and exhibiting dose-dependent anti-Vif activities. (b) IC₅₀ values. ++++, 0–10 μM; +++, 10 μM–30 μM; ++, 30–100 μM; +, >100 μM; ND, not determined.

Figure 2

The small-molecule Vif antagonist, RN-18, inhibits HIV-1 replication in nonpermissive cells but not in permissive cells. (a–d) CD⁺ T-cell lines H9 (a), CEM (b), MT4 (c) or CEM-SS (d) were treated overnight with varying concentrations of RN-18 before infection with HIV-1 (X4-tropic HIV-1 variant [HIV-1_LAI]) at 2 × 10⁵ c.p.m. reverse transcriptase (RT) per well. All cells were maintained for 14–21 d in the presence or absence of RN-18, and viral replication was monitored every other day by RT activity in culture supernatants. (e,f) For comparison, replication profiles of wild-type and Vif-deleted HIV-1 in nonpermissive (H9, e) and permissive (MT4, f) cells are shown.

Figure 3

The Vif antagonist, RN-18, increases APOBEC3G abundance in HIV-1 producer cells and virions, but does not affect APOBEC3B levels. 293T cells co-expressing HIV-1 (pNL4-3LucR⁻E⁻) and various HA-tagged APOBEC3 expression vectors were cultured for 24 h in the presence (+) or absence (−) of 50 μM RN-18. (a) Total producer-cell lysates were analyzed by immunoblotting with antibodies against the HA tag and HIV-1 antigen, p24. (b) HIV-1 virions from producer-cell supernatants were concentrated, lysed and analyzed by immunoblotting with antibodies against HA tag, HIV-1 Vif and p24. (c) Levels of APOBEC3G and APOBEC3F were determined by immunoblotting with antibodies against HA tag, Vif and cyclin T1 (internal control). (d) Levels of APOBEC3C and APOBEC3B were determined as previously specified.

Figure 4

The Vif antagonist, RN-18, enhances APOBEC3G expression in nonpermissive cells and in a Vif-dependent manner. (a,b) H9 (nonpermissive) and MT4 (permissive) CD⁺ T-cells were infected with pNL4-3 LucR⁻E⁻ (a) or ΔVifpNL4-3 LucR-E (b) HIV-1. Immediately after infection, cells were washed and incubated for 48 h in complete medium containing 0 or 50 μM RN-18. Total cell lysates were analyzed by immunoblotting for cyclin T1, endogenous APOBEC3G, wild-type HIV-1 and p24. (c) RN-18 does not increase endogenous APOBEC3G levels in H9 cells. The nonpermissive H9 cells were incubated 48 h in complete medium containing 0 or 50 μM RN-18 and total cell lysates analyzed by immunoblotting for APOBEC3G and cyclin T1. (d) The Vif antagonist, RN-18, is not a general proteasome inhibitor. 293T cells were treated for 24 h with increasing doses of RN-18 (0 to 50 μM in 0.5% DMSO). Total cell lysates were analyzed by immunoblotting for cyclin T1 and the cell cycle inhibitor, p21. As a positive control (d), 293T cells were treated for 8 h with 0 or 10 μM of a proteasome inhibitor cocktail (MG132, ALLN and lacatacystin in 0.5% DMSO), and cell lysates were analyzed as in c.