Inter-subtype comparison of Nef sequences in the context of known Nef functional motifs. Panel A : Consensus A, B, C and D Nef sequences were determined using patient-derived clones from this study and are shown aligned to HXB2. Amino acid residues/motifs previously associated with Nef-mediated modulation of CD4 downregulation (magenta), HLA-I downregulation (cyan) or both (purple) are indicated. Insertions were observed at codons 23, 24, 25, 63, 64, and 65 in greater than 10% of clones for at least one subtype (indicated by asterisks). Panel B : The position of residues/motifs associated with Nef CD4 downregulation (magenta), HLA-I downregulation (cyan), or both functions (purple) are highlighted on a structure model of the Nef protein (composite crystal structure kindly provided by Art F. Y. Poon, ). Panel C: The position of amino acids whose sequence varied between consensus B and C Nef sequences from this study are shown using a structure model of Nef. Differences between consensus sequences (32 residues) are highlighted in red and majority amino acids whose frequency varied by greater than 15% between B and C (additional 29 residues) are indicated in orange.