Volume 8 issue 3 March 2007
Host ABCE1 is at plasma membrane HIV assembly sites and its dissociation from Gag is linked to subsequent events of virus production
Julia E. Dooher¹, Bobbie L. Schneider², Jonathan C. Reed³, and Jaisri R. Lingappa¹, ⁴

Legends to Supplementary Figures

Figure S1. HIV Gag is associated with ABCE1 independently of ribosomes or polysomes. Cos-1 cells expressing HIV (lanes 3-5) or mock plasmid (lanes 6-8) were pulse radiolabeled with ³⁵S-methionine/cysteine and chased with unlabeled media. Lysates were harvested under native conditions using (A) buffer lacking EDTA (lanes 3 and 6), (B) buffer containing 10 mM EDTA (lanes 4 and 7), or (C) buffer containing RNase A but no EDTA (lanes 5 and 8), and were immunoprecipitated with αABCE1. Markers (M) for the position of Gag and ABCE1 are shown. Dot indicates position of coimmunoprecipitated p55Gag found in lanes 3-5.

Figure 1(.jpg)

Figure S2. ABCE1 is not present in substantial quantities in virions released from cells expressing HIVPro-. (A) Cos-1 cells were transfected to express HIV, HIVPro-, HIVΔNCΔp6, or mock plasmid, as shown by Western blotting (WB) aliquots of cell lysate with αGag. (B) Cell lysates were subjected to immunoprecipitation with αABCE1 under native conditions. followed by Western blotting with either αABCE1 (lanes 5 – 8, top panel) or αGag (lanes 5 - 8, bottom panel) to show the relative ratio of ABCE1 and Gag present in intracellular complexes. VLPs were collected by sedimenting medium from these cells through a sucrose cushion. ABCE1 was not detected in VLP preparations (lanes 9 - 12) which contained amounts of Gag equal to or greater than those found in the immunoprecipitated intracellular complexes. Immunoprecipitation of VLP aliquots with αABCE1 also failed to reveal ABCE1 in VLPs (data not shown). All lanes in panel B were analyzed in parallel with the dividing line indicating splicing of lanes from a single film. Molecular weight markers are shown to the left in all panels. HC indicates IgG heavy chain detected in immunoprecipitations.

Figure 2(.jpg)

Figure S3. Chelation of divalent cations mimics effect of ATP depletion in enhancing Gag present in ~80/150S complexes. Human H9 cells stably expressing the HIV-1 provirus were harvested in non-detergent buffer containing Mg⁺², Mg⁺² and apyrase (which depletes ATP), or EDTA and analyzed by velocity sedimentation and Western blotting for Gag. Positions of ~10S and ~80/150S complexes are indicated. Data are representative of more than 4 experiments.

Figure 3(.jpg)