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Results: 3

1.
Fig. 1.

Fig. 1. From: A single amino acid substitution in human APOBEC3G antiretroviral enzyme confers resistance to HIV-1 virion infectivity factor-induced depletion.

Effects of mutations in human APOBEC3G on inhibition of HIV-1 replication in the absence and presence of HIV-1 Vif. (a) Ten mutants containing clusters of amino acid substitutions, starting at the N-terminal end of APOBEC3G, are labeled Apo4, Apo14, Apo15, Apo1, Apo12, Apo12.1, Apo11, Apo2, Apo9, and Apo3 (the amino acid substitutions are listed in Materials and Methods). The clusters of substitution mutations are shown above the sequence in shaded boxes. The HIV-1 Vif-resistant Apo12 mutant is shown in bold and underlined letters. An asterisk next to Apo9 and Apo15 indicates discontinuous sets of mutations. (b) Flow cytometry analysis of the effects of mutations in APOBEC3G on inhibition of HIV-1 replication in the absence (Vif–) and presence (Vif+) of HIV-1 Vif. The proportion of GFP+ cells after infection with HDV-EGFP in the presence of wild-type APOBEC3G and HIV-1 Vif was set to 100%. Error bars represent the SEM of eight experiments for the Apo12 mutant and two experiments for the other mutants.

Hongzhan Xu, et al. Proc Natl Acad Sci U S A. 2004 April 13;101(15):5652-5657.
2.
Fig. 3.

Fig. 3. From: A single amino acid substitution in human APOBEC3G antiretroviral enzyme confers resistance to HIV-1 virion infectivity factor-induced depletion.

A single amino acid substitution renders human APOBEC3G resistant to HIV-1 Vif. (a) Flow cytometry analysis of the effects of amino acid substitutions in APOBEC3G on sensitivity to HIV-1 Vif. The relative proportion of GFP+ cells generated by infection with HDV-EGFP virion in the presence of wild-type or mutant APOBEC3G are shown. The proportion of GFP+ cells generated in the presence of wild-type APOBEC3G in the presence of HIV-1 Vif was set to 100%. The effects of the Apo12 mutant and the single amino acid substitution mutants (D128K, E133Q, and S137I) on viral replication were compared in the absence (Vif–) or presence (Vif+) of HIV-1 Vif. Error bars represent the SEM of two to seven experiments. (b) Sensitivity of wild-type and D128K mutant APOBEC3G to HIV-1, SIVmac239 (SIVmac), and HIV-2 Vif. The proportion of GFP+ cells generated in the presence of wild-type APOBEC3G in the presence of HIV-1 Vif was set to 100%. Error bars represent the SEM of two experiments. (c) Sensitivity of wild-type, Apo12, and D128K mutants of APOBEC3G to SIVagm Vif. The percent of GFP+ cells are shown in the absence of Vif (black bars) and in the presence of SIVagm Vif (white bars). (d) Western blot analysis of the steady-state levels of wild-type APOBEC3G and D128K mutant of APOBEC3G in the absence of any Vif or presence of HIV-1, SIVmac239 (SIVmac), and HIV-2 Vif proteins. The APOBEC3G proteins were detected by using an anti-myc tag antibody, and the tubulin proteins were detected with an anti-tubulin antibody to normalize the amounts of cell lysates analyzed.

Hongzhan Xu, et al. Proc Natl Acad Sci U S A. 2004 April 13;101(15):5652-5657.
3.
Fig. 2.

Fig. 2. From: A single amino acid substitution in human APOBEC3G antiretroviral enzyme confers resistance to HIV-1 virion infectivity factor-induced depletion.

HIV-1 Vif reduces intracellular steady-state levels of wild-type APOBEC3G but not the Vif-resistant Apo12 mutant. (a) Western blotting quantitative analysis of wild-type APOBEC3G degradation in the presence of Vif. Serial dilution of cell lysates transfected with wild-type APOBEC3G in the absence of HIV-1 Vif (APO-WT) are compared to undiluted lysate from cells transfected with wild-type APOBEC3G and pC-Help (APO-WT + Vif). The APOBEC3G proteins were identified by using an anti-myc tag antibody, and the same lysates were analyzed by using an anti-tubulin antibody to ensure that equivalent aliquots were loaded onto gels. The cell lysates after transfection with wild type APOBEC3G and the Apo12 mutant were also analyzed for the presence of Vif by using an anti-Vif antibody. (b) Wild-type and Apo12 mutant APOBEC3G interact with HIV-1 Vif as determined by coimmunoprecipitation. An anti-myc antibody attached to magnetic beads was used to immunoprecipitate the wild-type APOBEC3G and the Apo12 mutant proteins from cell lysates. The cellular proteins that were coimmunoprecipitated were analyzed for the presence of Vif by using an anti-Vif antibody. (c) Flow cytometry analysis of the effect of wild-type APOBEC3G on inhibition of HIV-1 replication by the Apo12 and D128K mutants. Four micrograms of each APOBEC3G plasmid was cotransfected with pHDV-EGFP, C-Help, and HCMV-G for bar graphs labeled APO-WT, Apo12, and D128K, respectively. Two micrograms of each APOBEC3G plasmid was cotransfected with pHDV-EGFP, C-Help, and HCMV-G for bar graphs labeled APO-WT, APO-WT + Apo12, and APO-WT + D128K, respectively. The proportion of GFP+ cells after infection with HDV-EGFP in the presence of wild-type APOBEC3G and HIV-1 Vif was set to 100%.

Hongzhan Xu, et al. Proc Natl Acad Sci U S A. 2004 April 13;101(15):5652-5657.

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