Modeling the late steps in HIV-1 retroviral integrase-catalyzed DNA integration

Model oligodeoxyribonucleotide substrates representing viral DNA integratio n intermediates with a gap and a two-nucleotide 5 ' overhang were used to e xamine late steps in human immunodeficiency virus, type 1 (HIV-1) retrovira l integrase (IN)-cataIyzed DNA integration in vitro. HIV-1 or avian myelobl astosis virus reverse transcriptase (RT) were capable of quantitatively fil ling in the gap to create a nicked substrate but did not remove the 5 ' ove rhang. HIV-1 IN also failed to remove the 5 ' overhang with the gapped subs trate. However, with a nicked substrate formed by RT, HIV-1 IN removed the overhang and covalently closed the nick in a disintegration-like reaction. The efficiency of this closure reaction was very low, Such closure was not stimulated by the addition of HMG-(I/Y), suggesting that this protein only acts during the early processing and joining reactions. Addition of Flap en donuclease-1, a nuclease known to remove 5 ' overhangs, abolished the closu re reaction catalyzed by IN, A series of base pair inversions, introduced i nto the HIV-1 U5 long terminal repeat sequence adjacent to and/or including the conserved CA dinucleotide, produced no or only a small. decrease in th e HIV-1 IN-dependent strand closure reaction. These same mutations caused a significant decrease in the efficiency of concerted DNA integration by a m odified donor DNA in vitro, suggesting that recognition of the ends of the long terminal repeat sequence is required only in the early steps of DNA in tegration, Finally, a combination of HIV-1 RT,Flap endonuclease-1, and DNA ligase is capable of quantitatively forming covalently closed DNA with thes e model substrates, These results support the hypothesis that cellular enzy me(s) may catalyze the late steps of retroviral DNA integration.