Abstract
Successful integration of viral genome into a host chromosome depends on interaction between viral integrase and its recognition sequences. We have used a reconstituted concerted human immunodeficiency virus, type 1 (HIV-1), integration system to analyze the role of integrase (IN) recognition sequences in formation of the IN-viral DNA complex capable of concerted integration. HIV-1 integrase was presented with substrates that contained all 4 bases at 8 mismatched positions that define the inverted repeat relationship between U3 and U5 long terminal repeats (LTR) termini and at positions 17-19, which are conserved in the termini. Evidence presented indicates that positions 17-20 of the IN recognition sequences are needed for a concerted DNA integration mechanism. All 4 bases were found at each randomized position in sequenced concerted DNA integrants, although in some instances there were preferences for specific bases. These results indicate that integrase tolerates a significant amount of plasticity as to what constitutes an IN recognition sequence. By having several positions randomized, the concerted integrants were examined for statistically significant relationships between selections of bases at different positions. The results of this analysis show not only relationships between different positions within the same LTR end but also between different positions belonging to opposite DNA termini.
Highlights
Integration of viral genome into the host chromosome is essential for stable infection of the retrovirus and occurs in a concerted reaction in which the ends of the viral DNA are brought together and inserted into the acceptor in a coordinated manner (1, 2)
HIV-1 integrase was presented with substrates that contained all 4 bases at 8 mismatched positions that define the inverted repeat relationship between U3 and U5 long terminal repeats (LTR) termini and at positions [17,18,19], which are conserved in the termini
Sequence Selection by IN at Mismatched Positions in the U5 LTR—To examine sequence requirements by HIV-1 IN for the U3 and U5 IN recognition sequences, we constructed a library of mini donor DNA substrates that contained randomized nucleotides at different positions in the IN recognition sequences
Summary
Reagents—[␣-32P]dCTP (3,000 Ci/mmol) was purchased from Amersham Biosciences. Proteinase K (30 units/mg) and glycogen were from Roche Molecular Biochemicals. The U5 N8 and U5 N3GTG oligodeoxyribonucleotides along with U3(WT) were used to prepare HIV-1 donor-concerted DNA integration substrates with randomization in the U5 terminus sequence. Plasmid Constructions and Preparations—Plasmid pHHIV2 was used in this study as a template to amplify donor DNA and is a variation of pBCSKϩ in which a wild type HIV-1 donor DNA PCR product was inserted into pBCSKϩ catalyzed by IN, resulting in the loss of 2 bp from the LTR ends This plasmid was propagated in E. coli MC1061/P3 under the conditions described above. 15 ng (0.15 pmol of ends) of donor DNA was mixed with 50 ng of acceptor DNA (0.02 pmol) and 80 ng of HIV-1 IN (1.25 pmol) in an 8.5-l preincubation reaction mixture containing, at final concentrations, 25 mM MOPS (pH 7.2), 23 mM NaCl, 10 mM dithiothreitol, 5% polyethylene glycol 8000, 10% dimethyl sulfoxide, 0.05% Nonidet P-40, 1% glycerol, 1.6 mM HEPES (pH 8.0), and 3.3 mM EDTA. HIV-1 sequences with the following GenBankTM accession numbers were analyzed for sequence conservation/variability and relationship between bases (Table VIII): AB023804, AB032740, AB032741, AB049811, AB052867, AB052995, AF003887, AF003888, AF004394, AF004885, AF033819, AF042100, AF042101, AF042106, AF049494, AF049495, AF064699, AF069140, AF070521, AF075719, AF084936, AF086817, AF110959, AF110962, AF110963, AF110964, AF110965, AF110966, AF110967, AF110968, AF110969, AF110970, AF110971, AF110972, AF110973, AF110974, AF110975, AF110976, AF110977, AF110978, AF133821, AF164485, AF197338, AF197339, AF197340, AF197341, AF119819, AF119820, AF256205, AF256206, AF256207, AF256208, AF256210, AF256211, AF259954, AF259955, AF286236, AF286365, AF290030, AF321523, AF385934, AF385935, AF385936, AJ006287, AJ237565, AJ245481, AJ271370, AJ271445, AJ288981, AJ288982, AJ291719, AJ302646, AJ302647, D10112, D86068, K02007, K02013, K02083, K03454, K03455, L02317, L20571, L20587, L31963, L39106, M17449, M17451, M19921, M26727, M38429, M62320, M93258, M93259, NC_001802, U12055, U21135, U23487, U34603, U34604, U37270, U43096, U43141, U51189, U54771, U69584, U69585, U39362, U69586, U69587, U69588, U69589, U69592, U88822, X01762, X04415, and Z11530
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