Residues A188 and H188 are components of the F₈₅F₈₉A₁₈₈R₁₉₁ and F₈₅F₈₉H₁₈₈H₁₉₁ alternative Pak2-activating structural motifs. The indicated amino acid substitutions (top) were introduced into SF2 Nef by PCR-based mutagenesis and cloned into pcDNA3.1 and the resultant plasmids transfected into 293T cells. Lysates were immunoprecipitated with anti-Nef antiserum. (Middle) Immunocomplexes were subjected to an in vitro kinase assay as previously described (4, 14, 33, 47). (Bottom) Western blot analysis of Nef proteins present in cell lysates. Activation (n-fold) over that of SF2 Nef is indicated for one representative experiment out of three. Ctrl, control (empty-vector-transfected cells).

Analysis of subtype C Nef function. (A) HIV-1 subtype C Nef isolates from Botswana were subcloned into pcDNA3.1 as previously described (33). A plasmid expressing HA-tagged Pak2 was cotransfected into 293T cells with each of the Nef-expressing plasmids, the lysates were immunoprecipitated with anti-HA antiserum, and the immunocomplexes were subjected to an in vitro kinase assay (top) as previously described (4, 33). Western blot analysis for Nef and HA-tagged Pak2 expression is shown in the middle and lower panels, respectively. Activation (n-fold) over that of SF2 Nef is indicated. Results for one representative experiment out of three are shown. (B) HIV-1 subtype C Nef isolates were analyzed for cell surface CD4 and MHC class I levels as described for Fig. 2 (33). The percentage of total cells for each quadrant is shown in each lower-right corner. Ctrl, control (empty-vector-transduced cells); 74, AF443074; 93, AF443093; 97, AF443097; 98, AF443098; 110, AF443110. Results for one representative experiment out of two are shown. (C) HIV-1 subtype C Nef isolates were analyzed for the ability to upregulate CD74 expression essentially as described previously (40, 43). Dark solid line, cells transfected with Nef expression plasmids; dashed line, cells transfected with the empty vector pCGCG. The NA7 primary Nef isolate described in reference 23 was used as a control. Results for one representative experiment out of two are shown. (D) Subtype C Nef amino acid sequences from patient isolates were aligned using ClustalW (http://www.ebi.ac.uk/clustalw) and compared against a subtype C consensus sequence (31). Note the five-amino-acid insertion in AF443074 Nef (R₂₆KTEP₃₀) and the E66 deletion in AF443093 Nef, which explain their different electrophoretic mobilities.

Subtype B Nef isolates harboring alternative PASMs activate Pak2. (A) The amino acid compositions of chemically synthesized primary subtype B Nef isolates (1R3 and L588) or SF2 Nef-derived mutants derived by PCR-based mutagenesis are indicated (top). Nef genes were cloned and transfected as described for Fig. 1. Lysates were immunoprecipitated with anti-Nef antiserum. (Middle) Immunocomplexes were subjected to an in vitro kinase assay as previously described (4, 14, 33, 47). (Bottom) Western blot analysis of Nef proteins present in cell lysates. Asterisks denote synthetic Nef isolates 1R3 (AF120749) and L588 (AF082375). Ctrl, control (empty-vector-transfected cells). Activation (n-fold) over that of SF2 Nef is indicated for one representative experiment out of two. (B) HIV-1 subtype B Nef isolates or SF2 Nef mutants were cloned into pLXSN before transduction of CEM cells and staining for analysis of cell surface CD4 and MHC class I levels as previously described (33). The percentage of total cells for each quadrant is shown in each lower-right corner. Results for one representative experiment out of two are shown. (C) Patient isolate (1R3 and L588) Nef amino acid sequences were aligned using ClustalW (http://www.ebi.ac.uk/clustalw) and compared to the SF2 sequence and a subtype B consensus sequence (33). Only the residues differing from those in the consensus sequence are shown. Gaps or deletions are represented by dashes. Note the four-amino-acid duplication in SF2 Nef (R₂₂AEP₂₅) and the five-amino-acid deletion in isolate 1R3 (K₁₅₂VEEA₁₅₆), which explain their different electrophoretic mobilities.

Continued.