Primate lentiviral virion infectivity factors are substrate receptors that assemble with cullin 5–E3 ligase through a HCCH motif to suppress APOBEC3G

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   Fig. 1.

   SIV Vif proteins are BC-box-containing substrate receptors that assemble with Cul5–E3 ligase through a Cul5-box-independent mechanism. (A) Alignment of the SOCS-box motifs of cellular proteins with the putative BC-box motifs of primate lentiviral Vif proteins. Critical residues matching the BC-box consensus are in red; divergent residues are in green. (B) Clustering of primate lentiviruses according to distinct BC-box motifs. Phylogenetic tree based on full-length genome of primate lentiviruses was adapted from the 2002 HIV Sequence Compendium. Red, most divergent viral BC-box; blue, viral BC-box with an Ala at position 6 and a hydrophobic amino acid at position 10; green, viral BC-box with a Cys at position 6. (C) SIV Vif proteins lack a Cul5-box and have significant divergence in the BC-box. (D) Amino acid sequences of consensus BC-box and a comparison with the highly divergent viral BC-box. (E–G) Vif proteins from various primate lentiviruses assemble with Cul5, ElonginB, and ElonginC. Coimmunoprecipitation of Cul5, ElonginB, and ElonginC with HIV-1 and SIV_AGMTanVif (E), SIV_MAC251Vif (F), or SIV_SYK173Vif (G). Cell lysates from SIV_AGMTanVif-HA-transfected or HIV-1 Vif-HA-transfected H9 cells were immunoprecipitated with anti-HA affinity matrix, separated by SDS/PAGE, and then transferred to nitrocellulose membranes and reacted with antibodies against HA, Cul5, ElonginB, or ElonginC. Cell lysates from SIV_MAC251Vif-HA-transfected (F, lane 2) or SIV_SYK173Vif-HA-transfected (G, lane 2) 293T cells were immunoprecipitated with anti-HA affinity matrix, separated by SDS/PAGE, and then transferred to nitrocellulose membranes and reacted with antibodies against HA, Cul5, ElonginB, or ElonginC. (H) SIV_AGMTanVif selectively interacts with Cul5 despite lacking a Cul5-box. 293T cells were cotransfected with HA-tagged Cul1, Cul2, Cul3, or Cul5 and SIV_AGMTanVif-myc. Cell lysates were immunoprecipitated with anti-HA affinity matrix. Immunoprecipitates were analyzed by SDS/PAGE, followed by immunoblotting with antibodies against HA or c-myc. (I) Quantification of SIV_AGMTanVif interaction with Cul5 or Cul2. Results were obtained from three independent experiments.

   
   
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   Fig. 2.

   The SLQ motif in SIV_AGMTanVif acts as a BC-box and is required for Vif function. (A) Reduced interaction of SIV_AGMTanVifAAA with Cul5–ElonginB–ElonginC. Cell lysates from SIV_AGMTanVif-myc- or SIV_AGMTanVifAAA-myc-transfected 293T cells were immunoprecipitated with anti-myc affinity matrix. Immunoprecipitated samples were analyzed by immunoblotting with antibodies against c-myc, Cul5, ElonginB, or ElonginC. (B) SIV_AGMTanVif, but not SIV_AGMTanVifAAA, induces degradation of AGM-A3G. 293T cells were cotransfected with AGM A3G-HA and c-myc-tagged SIV_AGMTanVif, SIV_AGMTanVif-AAA, or control vector as described. AGM-A3G stability was analyzed by SDS/PAGE, followed by immunoblotting with antibodies against HA, c-myc, and ribosomal protein to assure equal loading. (C) SIV_AGMTanVifAAA could not exclude AGM-A3G from released SIV_AGMTan virions. 293T cells were transfected with SIV_AGMTanΔVif and SIV_AGMTanVif or SIV_AGMTanVifAAA expression vector plus AGM-A3G. Comparable amounts of virions were used, as indicated by immunoblotting with SIV-positive monkey sera. The amount of AGM A3G-HA detected in released virions was analyzed by immunoblotting using the anti-HA antibody. (D) SIV_AGMTanVifAAA, unlike WT SIV_AGMTanVif, could not suppress the antiviral activity of AGM-A3G. WT SIV_AGMTan and Vif mutant (SIV_AGMTanΔVif) viruses were generated from transfected 293T cells or 293T plus AGM-A3G, and their infectivity was examined by using MAGI-CCR5 cells. Virus production was normalized by the level of p27. The infectivity of SIV_AGM produced from 293T cells was set as 100%.

   
   
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   Fig. 3.

   Expression of Cul5 mutants blocks SIV_AGMTanVif function in the presence of AGM-A3G. (A) Cul5 mutants blocked SIV_AGMTan infectivity in human and monkey cells in the presence of AGM-A3G. SIV_AGMTan or Vif mutant viruses were produced from 293T cells, 293T/AGM-A3G cells, COS7, or COS7/AGM-A3G cotransfected with control vector VR1012, Cul5ΔNedd8, or Cul5ΔRbx1. Virus input was normalized by the level of p27. The infectivity of SIV_AGM produced from 293T cells cotransfected with VR1012 was set as 100%. (B) Cul5 mutants blocked SIV_AGMTanVif-induced degradation of AGM-A3G. SIV_AGMTan or Vif mutant viruses were transfected with control vector VR1012, Cul5ΔNedd8, or Cul5ΔRbx1 in 293T/AGM-A3G cells. Cell lysates from transfected cells were prepared and separated by SDS/PAGE, transferred to nitrocellulose membranes, and reacted with an HA tag-specific monoclonal antibody for the detection of AGM-A3G and with antibody to ribosomal P antigens. (C) Cul5 mutants allow incorporation of AGM-A3G into virion even in the presence of SIV_AGMTanVif. The SIV_AGMTan or SIV_AGMTan mutant plus VR1012, Cul5ΔNedd8, or Cul5ΔRbx1 was transfected into 293T/AGM-A3G cells. AGM-A3G levels in viral particles were detected with an HA-specific monoclonal antibody. Comparable amounts of virions were used, as indicated by immunoblotting using SIV-positive monkey sera.

   
   
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   Fig. 4.

   SIV_AGMTanVif interacts with Cul5 through a previously uncharacterized HCCH motif. (A) Alignment of the conserved HCCH motif in SIV/HIV Vif proteins located upstream from the BC-box (SLQ motif). (B) Illustration of HCCH mutants. (C) The HCCH motif is required for interaction with Cul5 but not ElonginB–ElonginC. 293T cells were transfected with WT or mutant c-myc-tagged SIV_AGMTanVif constructs. Equal amounts of cell lysates were immunoprecipitated with anti-c-myc affinity matrix, followed by SDS/PAGE and immunoblotting with antibodies against c-myc, Cul5, ElonginB, and ElonginC. (D) The HCCH motif of SIV_AGMTanVif is required for interaction with Cul5 and has no effect on ElonginB–ElonginC interaction. (E) The HCCH motif is required for degradation of AGM-A3G by SIV_AGMTanVif. 293T/AGM-A3G cells were transfected with c-myc-tagged WT SIV_AGMTanVif or the indicated mutants. Cell lysates were analyzed by SDS/PAGE followed by immunoblotting against c-myc-Vif, HA-A3G, and ribosomal p19. (F) SIV_AGMTanVif HCCH mutants cannot overcome AGM-A3G. SIV_AGMTan or Vif mutant viruses were produced from 293T cells or 293T/AGM-A3G cells. Virus input was normalized by the level of p27. The infectivity of SIV_AGMTan produced from 293T cells cotransfected with VR1012 was set as 100%. (G) Cul5-box from SOCS3 did not restore SIV_AGMVifC117S interaction with Cul5.