Figure 6

Schematic of reverse transcription and template switching from the 115A to the 89D RNA templates. A 350 nt RNA sequence from position 7150 to 7500 (HXB2 numbering) of virus 115A, 120A, 89D, 122D, and 126D was submitted to Mfold server at MacFarlane-Burnett for RNA structure prediction based on the Zucher algorithms. The RNA structures for the five different templates varied considerably but one stem-loop or hairpin was consistently present around nt 7301 to 7339 and was termed the V4 stem-loop. Only 115A and 89D RNA structures between nt 7296 and 7421 are presented in this figure. Shifting the 350 nt RNA template 50 nt upstream or downstream did not affect the structure of this predicted V4 stem-loop. This RNA structure also contains the strong pause site identified in Figure 4B (blue dumbbell). The structure of virus 115A RNA was examined by sliding a 50 nt window from nt 7276 to 7126 on the 3' end and from nt 7235 to 7075 on the 5'end. To mimic the double stranded DNA/RNA duplex generated by (-) strand DNA synthesis by reverse transcriptase, a 20 nt duplex was added into the submitted sequence at the 3' end (designated as twenty "X's" in the sequence). The progression of RT on the 115-A RNA template for a 50, 100, and 150 nt extension is illustrated. The promiscuous template switching event to the 89-D template, near V4 stem-loop is also depicted. The "red" nucleotides represent the mismatched sequences between 115-A and 89-D templates. Recombination sites were identified and mapped to the sequences between these base mismatches. The "green stars" represent the recombination breakpoints identified in 16 recombinant clones from the reconstituted in vitro assay in the absence of NC (Figure 4B) and "pink squares", those in 18 recombinant clones from the reconstituted in vitro assay in the presence of NC (Figure 4B). The "blue circles" represent the breakpoints from five clones of the single cycle infection assay, i.e. identified in the C3-V4 region).