Figure 4

PFV-1 IN specifically cleaves the conserved palindromic sequence found at LTR-LTR junctions . (A) The LTR-LTR junction in infected cells forms a 20 nucleotide-long bipartite palindrome. The LTR-LTR viral DNAs were PCR-amplified, cloned and sequenced following 5-days infection of BHK-21 cells with wild type virus. The vast majority of sequences (90%) were similar whereas approximately 10% had some divergence of the U3 junction. (B) The LTR-LTR junction is cleaved by recombinant PFV IN. This purified IN was shown to be functional by its 3' processing activity on the blunt-ends of PFV LTR (see lanes 3 and 7, panel C) and its strand transfer activity (not shown). The U5 strand of an oligonucleotide spanning over the WT LTR-LTR palindromic junction was labelled at its 5' extremity, annealed to its U3 complementary strand and incubated in the presence of PFV-1 IN. Products were resolved on a 15% denaturing polyacrylamide gel. A G+A chemical sequencing reaction was run alongside to identify the cleavage site. A specific cleavage immediately downstream of the conserved 5'CA was obtained. The complementary strand was used for the U3 LTR-LTR junction. (C) The cleavage of the LTR-LTR junction by IN is operating on the two strands of the palindrome leading to cohesive digestion fragments (lanes 2 and 6) indistinguishable from the products generated by the classical 3' processing in vitro reaction on the blunt-ended LTRs (lanes 3 and 7). Cleavage products were obtained as for panel B. 3' processing of either U5 or U3 blunt double-stranded LTRs was carried out under similar conditions and products were run alongside to confirm the structure of the palindrome cleavage products. Lanes 2, 3, 6, 7 and 10: 150 nM PFV-1 IN; Lanes 1, 4, 5, 8 and 9: 150 nM IN + 20 mM EDTA. EDTA was used to impair the cation-dependant activity of IN. This digestion is highly specific of the viral palindromic sequence since a mutated palindrome (which sequence is indicated panel D) was not cleaved by IN (lane 10). (D) A palindrome motif is required for cleavage by PFV-1 IN. Cleavage of oligonucleotides with mutations that disrupt the palindrome motif (mutated nucleotides different from the PFV wild-type sequence are marked with an asterisk), and with a scrambled sequence was assessed. Oligonucleotides carrying different palindromes chosen because they correspond to LTR-LTR junctions of other retroviruses such as HIV-1 and MLV were also tested as putative substrates of the PFV-1 IN. Assays were performed under the same conditions as in Fig. 3C. The ability of the IN to cleave the oligonucleotides onto their two strands is indicated in the right column. The vertical arrow indicates the cleavage site of the wild-type PFV LTR-LTR junction. These experiments were found reproducible in four independent assays.