  ResearchMDM2 is a novel E3 ligase for HIV-1 VifTaisuke Izumi1 1 Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan 2 Japanese Foundation for AIDS Prevention, 1-3-12 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan 3 Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan 4 Department of Molecular Cell Biology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan 5 CREST, Japan Science Technology Corporation, Kawaguchi, Saitama 332-0012, Japan 6 Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan 7 Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan 8 Laboratory of Disease Control, Tukuba Primate Research Center, National Institute of Biomedical Innovation, Hachimandai-1, Tsukuba, Ibaraki 305-0843, Japan 9 Laboratory of Viral Pathgenesis, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan
Retrovirology 2009, 6:1doi:10.1186/1742-4690-6-1
Additional filesAdditional file 1: Supplementary figure 1 – the stability of Vif protein in p53-/- MEF and p53-/-MDM2-/- MEF cells. MEF cells were transfected with pDON/Vif or pcDNA3/HA-A3G. Twenty-two hours after transfection, the cells were treated with cycloheximide (CHX) for the indicated times, and cell lysates were subjected to immunoblotting with the indicated Abs. Format: PDF Size: 73KB Download file This file can be viewed with: Adobe Acrobat Reader Additional file 2: Supplementary figure 2 – immunopurified MDM2 induced the polyubiquitination of Vif in vitro. (A) MDM2 as well as Cul5 induced the polyubiquitination of Vif. HEK293T cells were transfected with expression vectors for His-MDM2 and His-Cul5. His-tagged proteins were purified using Ni-NTA agarose and subjected to in vitro ubiquitination assays as described in a legend to Fig. 4A. Reactions were subjected to immunoblotting with anti-Vif Ab. Arrows indicate GST-Ub-conjugated Vif. Asterisks indicate non-specific bands associated with GST-Vif protein recognized by anti-Vif Ab, as they are seen in lanes 1 and 3. (B) MDM2 induced the polyubiquitination of Vif Wt but not that of Δ22 that was defective for binding MDM2. Filled asterisks indicate non-specific bands associated with GST-Vif protein, while white asterisks indicate those associated with GST-Vif Δ22. Format: PDF Size: 62KB Download file This file can be viewed with: Adobe Acrobat Reader Additional file 3: Supplementary figure 3 – the overexpression of MDM2 inhibited HIV-1 replication in the presence of A3F. Single round infection assays were performed in the presence or absence of A3F as described in a legend to Fig. 5A. Values are presented as averages of more than 3 independent experiments. Format: PDF Size: 50KB Download file This file can be viewed with: Adobe Acrobat Reader |
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