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. 2014 Mar;88(6):3320–3328. doi: 10.1128/JVI.03833-13

FIG 1.

FIG 1

Separate roles for CBF-β in promoting HIV-1 Vif-CRL5 assembly and Vif stability. (A) The proposed mechanisms by which CBF-β functions in Vif regulation. CBF-β could be involved either in Vif-CRL5 E3 ubiquitin (Ub) ligase complex formation (pathway 2) or in increasing Vif stability (pathway 1). B, ElonginB; C, ElonginC. (B) The stability of Vif was enhanced in CBF-β-silenced cells by the proteasome inhibitor MG132. HEK293T or CBF-β-silenced HEK293T cells were transfected with the Vif-HA (HIV-1 NL4-3) expression vector. After 36 h, cells were treated with 2 μM MG132 for 14 h. Cell lysates were then analyzed by SDS-PAGE and immunoblotting using specific antibodies as indicated. β-Actin was used as an internal control. The data shown represent one of three independent experiments. (C) The Vif-Cul5 interaction was not restored by MG132 treatment in CBF-β-silenced HEK293T cells. HEK293T cells or CBF-β-silenced HEK293T cells were transfected with an expression vector for Vif-HA and treated with MG132. Cell lysates were prepared for immunoblotting or immunoprecipitated (IP) with the anti-HA antibody, and the precipitated samples were analyzed by immunoblotting with anti-HA antibody to detect Vif-HA. Samples were also analyzed using anti-CBF-β, anti-Cul5, or anti-ElonginB antibody. The data shown represent one of three independent experiments. (D) Expression of exogenous CBF-β in CBF-β-silenced HEK293T cells restores the Vif-Cul5 interaction without affecting Vif expression. The data shown represent one of three independent experiments. (E) The CBF-β binding-defective HIV-1 Vif mutant (Trp21 to Ala and Trp38 to Ala substitutions; W21/38A) cannot interact with Cul5 even if its stability is maintained. HEK293T cells were transfected with the indicated expression vectors. Cell lysates were prepared and immunoprecipitated, followed by immunoblot analysis with specific antibodies against Cul5, CBF-β, or the HA tag (to detect Vif-HA). β-Actin was used as an internal control. The data shown represent one of three independent experiments.