Release of autoinhibition converts ESCRT-III components into potent inhibitors of HIV-1 budding -- Zamborlini et al. 103 (50): 19140 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Zambortini et al. 10.1073/pnas.0603788103.

Supporting Figures

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Supporting Figure 6
Supporting Figure 7
Supporting Figure 8

Supporting Figure 6

Fig. 6. Truncated, but not full-length, CHMP4B-FLAG blocks HIV-1 particle production. 293T cells were transfected with 1.5 mg of proviral DNA and 500 ng of an expression vector for CHMP4B_1-153FLAG or full-length CHMP4B-FLAG, or of the empty vector. Virion production and Gag expression levels were compared by Western blotting with anti-CA serum, and CHMP4B expression levels were compared by Western blotting with anti-FLAG. WT, wild type.

Supporting Figure 7

Fig. 7. CHMP3_1-150FLAG does not inhibit ligand-induced EGFR degradation. HeLa cells stably expressing the tetracycline repressor (tetR) alone or together with CHMP3_1-150FLAG under the control of a tetracycline-inducible promoter were exposed to 2 mg/ml doxycycline for 24 h, starved in serum-free medium for 6 h, and stimulated with 100 ng/ml EGF (Cell Signaling Technology, Danvers, MA) for 0, 10, or 30 min as indicated. Total cell lysates were then analyzed by Western blotting with rabbit anti-EGFR antibody (Cell Signaling Technology). The retroviral vectors used to express tetR (pCXneo/TR-2) and CHMP3_1-150FLAG [pC4bsrR(TO)] were kindly provided by Tsuyoshi Akagi.

Supporting Figure 8

Fig. 8. Inhibition of HIV-1 particle production by the N-terminal basic domain of CHMP3 and subcellular localization of CHMP3 truncation mutants. (A) CHMP3_1-113FLAG inhibits HIV-1 particle production if expressed at detectable levels. 293T cells were transfected with WT HIV-1 proviral DNA (1.5 mg) and vectors expressing CHMP3_1-113FLAG (2.5 mg), CHMP3_1-150FLAG (0.5 mg), or the empty vector (2.5 mg). HIV-1 Gag proteins and the CHMP3 truncation mutants were detected by Western blotting with antibodies against CA and FLAG, respectively. (B) Fluorescence microscopic images showing the subcellular localizations of full-length and truncated CHMP3 proteins. (C) Western blots showing that CHMP3_1-113RFP fusion proteins potently inhibit HIV-1 release. 293T cells were transfected with WT HIV-1 proviral DNA (1.5 mg) and vectors expressing RFP, CHMP3_1-113RFP, or CHMP3_1-1132RFP (0.5 mg). HIV-1 Gag proteins and RFP fusion proteins were detected by Western blotting with anti-CA and anti-DsRed serum, respectively.