Avian Retrovirus U3 and U5 DNA Inverted Repeats

Abstract

The U3 and U5 termini of linear retrovirus DNA contain imperfect inverted repeats that are necessary for the concerted insertion of the termini into the host chromosome by viral integrase. Avian myeloblastosis virus integrase can efficiently insert the termini of retroviruslike DNA donor substrates (480 base pairs) by a concerted mechanism (full-site reaction) into circular target DNA in vitro. The specific activities of virion-derived avian myeloblastosis virus integrase and bacterial recombinant Rous sarcoma virus (Prague A strain) integrase (ϳ50 nM or less) appear similar upon catalyzing the full-site reaction with 3-OH recessed wild type or mutant donor substrates. We examined the role of the three nonsymmetrical nucleotides located at the 5th, 8th, and 12th positions in the U3 and U5 15-base pair inverted repeats for their ability to modify the full-site and simultaneously, the half-site strand transfer reactions. Our data suggest that the nucleotide at the 5th position appears to be responsible for the 3-5-fold preference for wild type U3 ends over wild type U5 ends by integrase for concerted integration. Additional mutations at the 5th or 6th position, or both, of U3 or U5 termini significantly increased (ϳ3 fold) the full-site reactions of mutant donors over wild type donors.Upon retrovirus infection, the viral RNA genome is reverse transcribed into a linear blunt ended DNA genome. The retrovirus U3 and U5 DNA termini contain LTR 1 sequences with short imperfect inverted repeats located at the very end of the blunt ended LTRs (1). In vivo, the inverted repeats are necessary for virally encoded integrase to catalyze the removal of a dinucleotide from the 3Ј-OH termini and the subsequent fullsite integration reaction (2). The full-site reaction involves the concerted insertion of the two recessed LTR DNA termini into the host genome. This reaction also results in the formation of a small size host duplication at the site of insertion whose size is virus-specific (1).In vitro, the mechanisms involved in the recognition of the blunt ended LTR termini by integrase for the 3Ј-OH processing reaction and for half-site strand transfer of the recessed LTR termini into target DNA have been investigated (2-10). The half-site reaction involves the insertion of only one LTR terminus into the DNA target. These in vitro analyses using purified integrase from several retrovirus species have established that the imperfect inverted repeat sequences located at the LTR termini are also necessary for catalysis (1,11,12). Besides the essential CA dinucleotide located 2 nucleotides downstream from the blunt ended viral termini, approximately 5-10 nucleotides internally also play varying roles in the 3Ј-OH processing and half-site strand transfer reactions (2, 13).The full-site integration reaction can be catalyzed efficiently using retrovirus-like donor substrates with integrase purified from virions (14, 15). Study of the specific interactions of integrase with the U3 and U5 LTR termini for the full-site integration reaction i...

Presteady-state Analysis of Avian Sarcoma Virus Integrase

“…However, a qualitative visual inspection of the gels revealed the rapid appearance of U3 joining products within 15 s and a delayed onset of U5 joining products for 120 s even though the formation of both U3 and U5 processing products occurred within the first 5-10 s of the reaction. These results extend previous reports that integrase favors U3 over U5 (14,25) and illustrate the importance of having both U3 and U5 sequences present for the correct assembly of an integrase-LTR productive complex. This suggests that after processing, the joining activity of integrase is ordered such that the U3 end of the viral LTR is inserted prior to the U5 LTR.…”

“…In the course of retroviral replication, the RNA genome is reverse-transcribed into a duplex DNA copy, and as a result of the strand-transfer mechanism of reverse transcriptase, the U5 and U3 sequences become the termini of the DNA genome (11). Purified integrase along with Mn 2ϩ or Mg 2ϩ as a cofactor is sufficient to catalyze both processing and joining reactions in in vitro assays using synthetic oligodeoxynucleotide substrates with DNA sequences derived from the U3 and/or U5 viral LTR ends (12)(13)(14).…”

“…Consequently, products larger than the original substrate in these assays that use a substrate containing only one LTR-end-derived sequence are considered to be products of only halfreactions, as only one viral DNA end is joined to a target site (19). Assays involving substrates designed to resemble the linear retroviral genome, with an LTR end-derived sequence at either terminus, have resulted in Ͼ95% of the joining products coming from the recombination of two separate substrate molecules integrating into a single target molecule (14,20,21). Despite the lack of similarity to the nucleoprotein complex assembled in vivo, the assays involving such trimolecular reactions have been used to suggest the preference of integrase for a U3/U3 combination of ends over that of a U5/U5 combination, with the specificity for the biologically significant U3/U5 arrangement intermediately between the two (14).…”

“…Assays involving substrates designed to resemble the linear retroviral genome, with an LTR end-derived sequence at either terminus, have resulted in Ͼ95% of the joining products coming from the recombination of two separate substrate molecules integrating into a single target molecule (14,20,21). Despite the lack of similarity to the nucleoprotein complex assembled in vivo, the assays involving such trimolecular reactions have been used to suggest the preference of integrase for a U3/U3 combination of ends over that of a U5/U5 combination, with the specificity for the biologically significant U3/U5 arrangement intermediately between the two (14). More recently, Brin and Leis (22), using a reconstituted HIV-1 integration system, demonstrated that concerted DNA integration requires the presence of both U3 and U5 ends in the donor DNA.…”

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

“…The design of the 12-15 deletion mutant allowed the 12th position to remain wt with changes occurring at the 13th position and beyond. Previous results suggested that the nonsymmetrical nucleotides between avian U3 and U5 att sites at positions 8 and 12 did not appear to significantly influence either full-site or half-site strand transfer activities of IN (21). Each att DNA substrate was assembled with IN at several concentrations and the samples were subjected to DNase I probing and strand transfer.…”