HIV-1 integrase interaction with U3 and U5 terminal sequences in vitro defined using substrates with random sequences

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.

Fig. 1. Mismatched positions in HIV-1 LTR ends and sites of randomization

Twenty base pairs of the IN recognition sequences derived from the U3 and U5 LTR ends are shown. Bases that differ between the two LTR ends are in bold.

Fig. 2. Integration of donors with randomized mismatched positions in U3 and/or U5 LTR

Integration reactions were reconstituted as described under “Experimental Procedures.” A, gel electrophoresis analysis of integration products from reactions with wild type (wt) donor (lane 1), donor containing randomized positions 5, 6, 9, 11, 13, 14, 16, and 20 in U5 LTR end (U5N8) (lane 2), donor with U3 LTR end randomized at the same positions (U3N8) (lane 3), or donor with both IN recognition sequences containing randomized mismatched positions (N8-N8) (lane 4). B, summary of the percentage of RFII-like products shown in A (closed bars) compared with wild type and the total number of colonies containing two-ended integrants after integration reaction products introduced into bacteria (open bars). Integration efficiency of wild type mini donor DNA was set as 100%. The data shown are an average of two separate experiments with the standard deviation between experiments of 0.5–2%. C, percent of integrants derived from Supplemental Material to Tables I–III formed by a concerted mechanism involving two ends of the same donor DNA.

Fig. 3. Relationships between randomized positions in donor DNA-HIV-1 IN complex

Donor contained IN recognition sequence with randomized mismatched positions at one end and wild type U3 (A) or wild type U5 IN recognition sequence at the other end (B). Data were derived from Supplemental Material to Table I(i) for A and from Supplemental Material to Table II(ii) for B. χ² test was used to reveal statistically significant relationships between positions.

Fig. 4. Example of relationship between positions in U5 LTR end

A, relationship of base content at position 13 with base selection at position 16. B, relationship of base content at position 16 with base selection at position 13. DNA integration reactions were carried out with IN, HMG-(I/Y), acceptor DNA, and a mini donor DNA containing randomized bases at mismatched positions in U5 LTR end, whereas U3 LTR end contained wild type terminal U3 sequence. Frequencies of particular base selection at position 16 (A) and position 13 (B) are shown by height of correspondent bars.

Fig. 5. Randomization of positions 17–19 in U3 and/or U5 LTR

Concerted DNA integration reactions were carried out with IN, HMG-(I/Y), acceptor DNA, and a wild type (wt) mini donor DNA, donor DNA in which positions 17–19 were randomized in U5 (U5N3GTG), U3 LTR (U3N3GTG), and in both (N3N3GTG). A, total number of colonies containing two-ended integrants. B, percent of integrants derived from Supplemental Material to Tables V–VII formed by a concerted mechanism involving two ends of the same donor DNA.