Structural organization of avian retrovirus integrase in assembled intasomes mediating full-site integration.
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 approximately 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 approximately 20-bp DNase I footprint at 14 degrees C. At 50 and 100 mm NaCl, the approximately 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 degrees C, the approximately 20-bp DNase I footprint was not observed under a variety of assembly conditions including low NaCl concentrations at 14 degrees 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 approximately 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.
Institute for Molecular Virology, Saint Louis University Health Sciences Center, St. Louis, Missouri 63110, USA.
Ajaykumar
A
Vora
Sibes
S
Bera
Duane
D
Grandgenett
D001287:Attachment Sites, Microbiological; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D019426:Integrases / Q000378:metabolism / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism; D030101:Orthoreovirus, Avian / Q000201:enzymology* / Q000502:physiology; D012190:Retroviridae / Q000201:enzymology / Q000502:physiology; D012965:Sodium Chloride / Q000494:pharmacology; D016662:Virus Integration
D012965:Sodium Chloride; D004247:DNA; D019426:Integrases; D008274:Magnesium