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. 2004 Dec 1;18(23):2867-72.
doi: 10.1101/gad.1250204.

"V体育平台登录" Selective assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase complex through a novel SOCS box and upstream cysteines

Yunkai Yu  1 , Zuoxiang Xiao, Elana S Ehrlich, Xianghui Yu, Xiao-Fang Yu
Affiliations

VSports最新版本 - Selective assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase complex through a novel SOCS box and upstream cysteines

VSports注册入口 - Yunkai Yu et al. Genes Dev. .

Abstract

APOBEC3G, which induces hypermutations in newly synthesized viral DNA, is suppressed by HIV-1 Vif, acting through Cul5-ElonginB-ElonginC E3 ubiquitin ligase VSports手机版. We have now characterized a novel SOCS box in HIV-1 Vif that mediates its interaction with ElonginC. In this SOCS box, alanine replaces the consensus cysteine in the previously identified SOCS box. This new motif was necessary but insufficient for interaction with Cul5-ElonginB-ElonginC, as two highly conserved Cys residues outside the SOCS box were required to interact with Cul5 but not ElonginC. Therefore, selective assembly with Cul5 versus Cul2 E3 may require protein interfaces besides the SOCS-box-ElonginC interaction. .

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V体育官网 - Figures

Figure 1.
Figure 1.
HIV-1 Vif contains a putative SOCS-box motif. (A) Vif of HIV-1 (M, O, and N groups) and closely related SIVcpz viruses contain a SOCS-box-like motif. (B) The interaction interface between VHL and ElonginC was modeled based on structure data from Stebbins et al. (1999). VHL amino acids are in yellow, and those of ElonginC are in cyan. (C) As in the VHL-ElonginC interaction, HIV-1 Vif is predicted to form a hydrophobic interface with ElonginC. Vif amino acids are in yellow, and those of ElonginC are in cyan.
Figure 2.
Figure 2.
A critical cysteine in the SOCS box is functionally replaced with an alanine in HIV-1 Vif. (A) Cul5-elonginB-elonginC complexes were coimmunoprecipitated with HIV-1 wild-type (WT) Vif (lane 2) and mutant Vif A149C (lane 4) but not mutant Vif A149L (lane 3). (B) Coimmunoprecipitation of h-APO3G with wild-type (lane 2), mutant A149C (lane 3), and mutant A149L Vif (lane 4). (C) Degradation of h-APO3G by wild-type and Vif mutants. 293 cells were cotransfected with phAPO3G-HA plus VR1012, pVif-myc, pVifA149C-myc, or pVifA149L-myc. Equal amounts of cell extract were analyzed by immunoblotting using antibodies against HA for the detection of APOBEC3G, myc for the detection of Vif, and Ribosomal P19 antigen for total protein loading control. (D) Evaluation of Vif function. Viruses were produced from 293/h-APO3G cells cotransfected with HXB2ΔVif plus the control vector VR1012, pHIV-1Vif (wild type [WT]), pVifA149C, pVifA149L, pVifA149T, or pVifA149S. Virus infectivity was examined using MAGI-CCR5 cells. Virus input was normalized by the level of p24. The infectivity of HXB2ΔVif plus wild-type Vif produced from 293/h-APO3G cells was set as 100%. The results are the average of five independent experiments.
Figure 3.
Figure 3.
The hydrophobic interface between HIV-1 Vif and ElonginC is critical for their interaction. (A) Interaction of wild-type (WT) and mutant ElonginC with HIV-1 Vif. 293 cells were cotransfected with pHIV-1Vif plus pEloC-HA, pEloCΔ2-HA, or pEloCΔ4-HA. Equal amounts of cell extract were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting using antibodies against HA (top panel), Vif (middle panel) or ElonginB (bottom panel). (B) Interaction of ElonginC with wild-type or mutant VifΔSLQ. (C) L145 of HIV-1 Vif is critical for its interaction with ElonginC. 293 cells were cotransfected with phAPO3G-HA plus pVif-myc or pVifL145A-myc. Equal amounts of cell extract were immunoprecipitated with anti-myc antibody and analyzed by immunoblotting using antibodies against Vif (top panel), HA (middle panel), or ElonginC (bottom panel). (D) Reduced degradation of hAPO3G by VifL145A. Equal amounts of cell extract from C were analyzed by immunoblotting using antibodies against HA for the detection of hAPO3G and Ribosomal P19. (E) Evaluation of Vif function. Virus input was normalized by the level of p24. The infectivity of HXB2ΔVif plus wild-type (WT) Vif produced from 293/h-APO3G cells was set as 100%. The results are the average of three independent experiments. (F) 293 cells were transfected with pVif-myc, pVifL163S-myc, or pVifL169S-myc. Equal amounts of cell extract were immunoprecipitated with anti-myc-tag antibody and analyzed by immunoblotting using antibodies against Vif and Cul5. (G) Reduced degradation of hAPO3G by VifL163S or VifL169S. (H) In vitro interaction between ElonginC and Vif or VifΔSLQ. Recombinant GST-ElonginC (lane 1) was incubated with recombinant MBP-Vif (lane 2) or MBP-VifΔSLQ (lane 3) bound to the amylose column. (Top) Bound MBP-Vif (lane 4) or MBP-VifΔSLQ (lane 5) proteins were detected by staining with Coomassie blue. (Bottom) Coprecipitated GST-ElonginC (lanes 4,5) was detected by anti-ElonginC antibody.
Figure 4.
Figure 4.
The N-terminal region of Cul5 (including helix 2) is critical for binding to ElonginC and is required to form a complex with HIV-1 Vif. (A) Interaction of ElonginC, ElonginB, and Rbx1 with Cul5, Cul5ΔN1, or Cul5ΔN2. (B) HIV-1 Vif is predicted to form a complex with Cul5 that interacts with ElonginC and ElonginB but not Cul5ΔN1 or Cul5ΔN2. (C) Lack of interaction between HIV-1 Vif and Cul5ΔN1 or Cul5ΔN2. (D) Interaction of HIV-1 Vif, ElonginC, and ElonginB with Cul5, Cul5ΔRbx, or Cul5ΔNedd8.
Figure 5.
Figure 5.
HIV-1 Vif forms a stable complex with Cul5-ElonginB-ElonginC but not Cul2-ElonginB-ElonginC. (A) Comparable expression of Cul1-HA, Cul2-HA, Cul3-HA, and Cul5-HA as well as Vif-myc, ElonginB, and ElonginC in transfected 293 cells. (B) HIV-1 Vif was coimmunoprecipitated with Cul5-HA but not other cullins. (C) Cul5 but not other cullins was coimmunoprecipitated with HIV-1 Vif. (D) Cul5 was coimmunoprecipitated with wild-type (WT) Vif but not C114S or C133S mutant Vif. (E) Coimmunoprecipitation of h-APO3G with wild-type (WT; lanes 1,4), mutant C114S (lanes 2,5), and mutant C133S Vif (lanes 3,6). (F) Degradation of hAPO3G by wild-type but not C114S or C133S mutant Vif.
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References

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