Supplemental Material

This article contains the following supporting material:

• Supplementary figure 1 - A - B. AP-1 depletion decreases the release of HIV-1 particles in the absence of envelope. HeLa cells treated with siRNAs against AP-1µ (si µ1) or luciferase (si Luc) were transfected with HIV-1ΔEnv proviral DNA. A. Depletion of AP-1 and the expression of HIV-1 Gag in cell extracts were analyzed by western blotting with anti-CAp24 and anti-AP-1γ antibodies. B. Secreted HIV-1 CAp24 was quantified by ELISA (mean of four experiments, ± SD). NT: non-transfected cells. C. Infectivity of HIV particles produced by AP-1 depleted cells. Supernatants of HeLa cells depleted for AP-1µ (si µ1) and transfected with HIV-1 proviral DNA, were normalized to CAp24 levels, and used to infect indicator cells. HIV-1 infectivity is expressed as the percentage of control, si Luc (mean of four experiments, ± SD). NT: non-transfected cells. D. The MLV entry is not affected by the absence of AP1. Numbers of infected WT and AP1-/- MEFs were counted after infection with MLV particles containing single-round GFP-encoding genomic vector. The number of infected AP1-/- cells is expressed as percentage of WT. E. Infectivity of MLV particles release by WT and AP-1-/- MEFs. Supernatants of WT and AP-1-/- MEFs chronically-infected with MLV (strain F57) were normalized to Gag levels, and used to infect indicator Dunni cells. MLV infectivity is expressed as the percentage of WT (mean of four experiments, ± SD).
• Supplementary figure 2 - Flag-μ1 is incorporated in AP-1 complex. Co-immunoprecipitation of Flag-μ1 with endogeneous γ-adaptin. 293T cells were transfected wih Flag-μ1. Immunoprecipitation was performed with anti-AP-1γ or with anti-tubulin antibodies as a control. Bound material was analysed by Western blot with anti-Flag (the middle panel), anti-AP-1γ ( the top panel) and anti-tubulin. IgGγ corresponds to heavy chains of antibodies.
• Supplementary figure 3 - Lysotracker co-localizes with MVB marker CD81 in WT and AP-1-/- chronically infected MEFs. WT and AP-1-/- MEFs chronically infected with MLV were labelled with anti-CD81 antibodies (green) and lysotracker (red).
• Supplementary figure 4 - A. Interaction of µ1 and γ1 with proteins involved in MVB pathway. Indicated proteins were fused to DNA-binding (pAS) or DNA-activating domain (pAct) and tested against each other. Number of + indicates the strength of interactions, - no interactions, blank - the interactions were not tested. B. Interaction of µ1, µ2 and µ3 with RSV and HTLV-1 Gag in yeast two-hybrid assays. RSV and HTLV-I Gag were fused to the N-terminus of the Gal4 DNA-binding domain, and tested against µ1, µ2 and µ3. Legend as in Figure 1.