Structural Organization of Avian Retrovirus Integrase in Assembled Intasomes Mediating Full-site Integration

Abstract

Retrovirus preintegration complexes (PIC) purified from virus-infected cells are competent for efficient concerted integration of the linear viral DNA ends by integrase (IN) into target DNA (full-site integration). In this report, we have shown that the assembled complexes (intasomes) formed in vitro with linear 3.6-kbp DNA donors possessing 3′-OH-recessed attachment (att) site sequences and avian myeloblastosis virus IN (4 nM) were as competent for full-site integration as isolated retrovirus PIC. The att sites on DNA with 3′-OH-recessed ends were protected by IN in assembled intasomes from DNase I digestion up to ∼20 bp from the terminus. Several DNA donors containing either normal blunt-ended att sites or different end mutations did not allow assembly of complexes that exhibit the ∼20-bp DNase I footprint at 14 °C. At 50 and 100 mM NaCl, the ∼20-bp DNase I footprints were produced with wild type (wt) U3 and gain-of-function att site donors for full-site integration as previously observed at 320 mM NaCl. Although the wt U5 att site donors were fully competent for full-site integration at 37 °C, the ∼20-bp DNase I footprint was not observed under a variety of assembly conditions including low NaCl concentrations at 14 °C. Under suboptimal assembly conditions for intasomes using U3 att DNA, DNase I probing demonstrated an enhanced cleavage site 9 bp from the end of U3 suggesting that a transient structural intasome intermediate was identified. Using a single nucleotide change at position 7 from the end and a series of small size deletions of wt U3 att site sequences, we determined that sequences upstream of the 11th nucleotide position were not required by IN to produce the ∼20-bp DNase I footprint and full-site integration. The results suggest the structural organization of IN at the att sites in reconstituted intasomes was similar to that observed in PIC.