The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear

Abstract

HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the (2226)DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using co-immunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The (2226)DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope.

Keywords: ABCA1; Co-immunoprecipitation; HIV-1; Interaction; Molecular modeling; Nef; Yeast 2-hybrid.

Figure 1. Analysis of Nef-ABCA1 interaction using yeast 2-hybrid system. Figure 1. Analysis of Nef-ABCA1 interaction using yeast two-hybrid system

A - Western blot analysis using anti-cMyc antibody of yeast lysates from strain Y187 transformed with pGBKT7 expressing the GAL4 DNA-binding domain (BD) alone (lane 1) or fused to Nef (lane 2) or to the D2′ domain of ABCA1 (lane 3). B - Western blot analysis using anti-HA antibody of yeast lysates from strain AH109 transformed with pACT2 expressing the GAL4 activation domain (AD) alone (lane 1) or fused to Nef (lane 2) or to the D2′ domain of ABCA1 (lane 3). Arrows indicate the expected positions of fusion proteins. Positions of ladder marker proteins and their molecular weights in kDa are indicated in the left lanes of A and B. C - Yeast strains Y187 expressing BD or BD-Nef were mated with each of the AH109 strains expressing AD or AD-D2′ and grown on leucine- and tryptophan-deficient medium for 5 days for mating control. D – Yeast strains AH109 expressing AD or AD-Nef were mated with Y187 strains expressing BD or BD-D2′ and grown on leucine- and tryptophan-deficient medium for 5 days for mating control. E - Mated yeast from C were grown on leucine-, tryptophan- and histidine-deficient medium supplemented with 2.5 mM 3-aminotriazol for 7 days. F – Mated yeast from D were grown on leucine-, tryptophan- and histidine-deficient medium for 7 days. AH109 strain expressing AD-T (SV40) and Y187 strain expressing BD-p53 served as a positive control for mating (C, D) and interaction (E, F).

Figure 2. Analysis of Nef-ABCA1 interaction using co-immunoprecipitation

A - HEK 293T cells were transfected with vectors expressing Nef and either FLAG-tagged ABCA1, ABCA1 fragments D1 or D2, or Hcf1. Expression was tested in cell lysates with anti-FLAG or anti-Nef antibody. B – Transfected cells from A were lysed, immunoprecipitated with anti-FLAG beads, and proteins eluted with FLAG peptide were analyzed by Western blotting using anti-Nef antibody.

Figure 3. Model of the hypothetical ABCA1 – Nef complex

The model accounts for demonstrated interaction between ABCA1 and Nef [1], loss of this interaction when ²²²⁶DDDHLK is mutated [9], and weak interaction between cytoplasmic domains and Nef (this report) and can be used to discover other interaction interfaces. Yellow– ABCA1 D1 cytoplasmic domain (⁸⁹⁹Val - ¹¹²⁰Gln), cyan– ABCA1 D2 cytoplasmic domain (¹⁹⁰⁸Gln - ²²⁶¹Val), green – HIV-1 Nef. The D2 fragment ¹⁹⁰⁸Gln - ¹⁹²¹Tyr was deleted in the construct used for the 2-hybrid assay. The D1 fragment ¹⁰⁰⁹Lys-¹⁰²⁷Lys predicted to participate in the interaction with Nef, and the D2 motif ²²²⁶DDDHLK are shown in blue. Other color-coding: red – N-termini, black – C-termini of ABCA1 fragments.