A. Comparison of bootscaning analysis performed on CRF12_BF and CRF12_BF-like showed a similar recombination pattern, with a breakpoint located between α-helix I and II. Reference sequences of subtype B (red), F (black), and A (blue) - out group - were obtained from Los Alamos Database. Plots represent bootstrap values based on 100 re-samplings, supporting branching with reference sequences within a 140 nt window moving in steps of 20 bases.
The structural domains as well as the phosphorylation sites (asterisks) of Vpu are shown in Figure 1B. Underlined residues represent differences between CRF12_BF and CRF-like variant.
C. Diagram depicts the construction step of a chimeric viral genome: vpu coding sequence from a naturally occurring BF intersubtype recombinant variant (named CRF12_BF-like) amplified by PCR from a genomic DNA sample obtained from an infected patient. The use of modified oligonucleotides allowed the introduction of both PacI and Eco47III restriction sites. The purified PCR product was cloned into a commercial vector (pGEM-T) to obtain the pVpuBF plasmid. In parallel, these restriction sites were introduced in pNL4-3 by PCR-based site-directed mutagenesis. After digestion and ligation steps a chimeric variant harboring the Vpu BF sequence into the subtype B genomic background (pNL4-3 VpuBF) was obtained.