Fig 8.

Cyclosporine experiments in ferret cells and CypA alignments. (A to C) Infectious titers of HIV-1 GFP vector were determined in the presence (black columns) and absence (gray columns) of 5 μM CsA for owl monkey kidney (A), Mpf (B), and FtAEpC (C) cells. (D) Immunoblotting for CypA in human and Mustelidae cell lines. Jurkat ppia^−/− cells (14) were used as a negative control. (E) Alignment of the ferret and human CypA amino acid sequences reveals high conservation overall and complete conservation in the central hydrophobic pocket involved in HIV-1 capsid binding. The amino acids involved in HIV-1 capsid binding, which are known from several mutational and high-resolution structural studies (13, 20, 71), are highlighted with boxes. Comparison is made between the human protein reference sequence (top line, accession no. NP_066953) and the predicted amino acid sequence determined from the ferret CypA cDNA isolated from Mpf cell mRNA in the present study (middle line) and a sequence predicted from an M. putorius furo whole-genome shotgun (WGS) sequence contig (bottom line, accession no. AEYP01032892). The dashed arrows at each end indicate the span of the degenerate primers used to obtain the Mpf cell cDNA sequence. Stringent selection pressure for amino acid level conservation is apparent. For example, at the nucleotide sequence level (not shown), there were 49 differences between Mpf cell CypA and human CypA in the coding region of the mRNA (10.1% nonidentity), but virtually all were synonymous. Only three amino acid differences were present, and these were located at both termini (arrowheads indicating the fifth and the final two C-terminal amino acids); the T5I difference (black arrowhead) is uncertain since both threonine and isoleucine were encoded by the degenerate primer, the domestic cat sequence has an isoleucine at this position, and the ferret WGS contig predicts a threonine. Polymporphism is evident in the two ferret sequences; they differed at 10 nt (not shown), but besides T5I there were only two other predicted amino acid differences, A26S and R37H.