Clathrin Facilitates the Morphogenesis of Retrovirus Particles
Figure 6
Effects of mutations in the DLL motif in SIVmac p6 on replication and particle morphogenesis.
(A) Sequence of SIV p6 highlighting motifs that were mutated to specifically block recruitment of Tsg101 (PTAP), ALIX (PY) or clathrin (DLL). (B) Mapping of the ALIX-binding site on SIVmac p6. WT or mutant SIVmac Gag expression plasmids were co-transfected with a plasmid expressing HA-tagged ALIX. Cell lysates and pelleted VLPs were subjected to Western blot analysis with anti-CA and anti-HA antibodies. (C and D) Effects of DLL- mutation on SIVmac particle yield and infectiousness. WT and DLL-mutant SIVmac proviral plasmids were transfected into 293T cells. Cell lysates and pelleted VLPs were subjected to Western blot analysis with anti-CA antibody (C) and infectious virion release was quantitated using the TZM-bl assay (D). (E) Effects of DLL- mutation on SIVmac replication. C8166 cells were inoculated with equal amounts (as determined by infectious virus measurement) of WT and D21A/D51A mutant SIVmac virus. Supernatant samples were collected at 2–3 day intervals thereafter and the amount of infectious SIVmac contained therein was measured using the TZM-bl assay. (F) Effects of combined PTAP, PY and DLL-motif mutation on SIVmac Gag VLP yield. 293T cells were transfected with plasmids expressing WT or the indicated mutant SIVmac Gag proteins. Cell lysates and pelleted VLPs were subjected to Western blot analysis with anti-CA antibodies. (G) Effects of ALIX overexpression on SIVmac VLP yield. 293T cells were transfected with an empty vector or an ALIX expression plasmid along with plasmids expressing codon-optimized versions of the WT or the indicated mutant SIVmac Gag proteins. Cell lysates and pelleted VLPs were subjected to Western blot analysis with anti-CA antibodies. (H) Effects of AP180C overexpression on SIVmac VLP yield. 293T cells were transfected with an empty vector or increasing amounts (0, 0.5, 1, or 1.5 ug) of an AP180C expression plasmid along with plasmids expressing PTAP mutant or WT SIVmac Gag proteins. Cell lysates and pelleted VLPs were subjected to Western blot analysis with anti-CA antibodies. (I) Scanning EM images of 293T cells transfected with plasmids expressing codon optimized WT or the indicated mutant SIVmac Gag-IRES-eGFP cassettes. Cells were selected for imaging based on similar levels of GFP fluorescence. Additional examples are shown in Figure S7. (J) Scanning EM images of 293T cells transfected with plasmids expressing codon optimized SIVmac Gag-IRES-eGFP cassettes, in the presence of AP180C. Cells were selected for imaging based on similar levels of GFP fluorescence. Additional examples are shown in Figure S8.