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Cristina Cantale

The Integrase Protein

Integration mechanism

Since then, a large amount of work has been carried out using in vitro systems and mainly HIV IN purified protein (but also INs from ASLV, RSV (Rous Sarcoma virus), MoMULV (Moloney Murine leukemia virus) and very recently HTLV-II (Human T-cell Leukemia virus type II)), with the aim of clarifying the integration mechanism.
It was demonstrated that IN is able to carry out three different reactions:

• cleavage of the two last nucleotides and formation of recessed 5' AC (OH) 3' (processing reaction)
• nucleophilic attack of 5' CA(OH) 3' end on a phosphodiester bond of DNA target and DNA strand transfer reaction
• reversal of DNA strand transfer reaction, called disintegration (up to now this reaction has been found just using HIV IN and ASV; due to this capacity of promoting and turning over catalytically both a direct and a reverse reaction, HIV and ASV INs protein are enzymes (Chow S.A. et al., 92 - Bujacz G. et al., 95)).

The overall reaction carried out by IN is a transesterification, produced by a nucleophilic attack on an activated phosphodiester bond performed by water or by the recessed 5'CA(OH)3' hydroxyl group end.
It appears that the same catalytic core domain is involved in both processing and the DNA transfer.
Both reactions proceed by a one step mechanism, as demonstrated using a known chirality substrate (phosphorothioate), without the formation of covalent protein-DNA intermediate (Engelman A. et al., 91)
These reactions do not need any external energy but a divalent cation (Mg²⁺ or Mn²⁺) is necessary for the reaction to proceed. Beside the hypotheses of involvement of divalent cations in active core mechanism, a novel in vitro assay using immobilized LTR oligonucleotides (Hazuda et al., 94-1) suggests that the requirement for Mn²⁺ is correlated with the formation of the oligomeric structure of IN in solution (Wolfe et al., 96). This is considered the very first step of the overall integration reaction, that is the assembly of a stable complex between integrase and viral DNA (Ellison V. and Brown P.O., 94 - Ellison V. et al., 95 - Vink C. et al., 94).
As two sterically and temporally coordinated reactions (one at each end of viral DNA) are required for integration of viral DNA, IN has to be at least a dimer, carrying the double strand viral DNA.
Staggered cleavage of the host DNA should involve another dimer (at least), suggesting that IN works as a tetramer (at least). Complementation experiments using IN mutants lacking different portions support this hypothesis (van Gent et al., 93 - Jones C.S. et al., 92 - Engelman A. et al.,93).
The use of mutants has proved also that there are different domains of IN which play different roles (transesterification, multimerization, DNA recognition).Furthermore it has permitted the identification of the AAs that are fundamental for IN activities, confirming the results obtained from the sequence analysis of various retroviral INs.
To complete the picture, it should be emphasized that, even if the in vitro experiments have the great worth to have clarified many aspects of IN behaviour, they are not able to simulate entirely the in vivo system.
The same mechanism of integration is only partially reproduced.
The in vitro system lacks the aspects of concerted two ends strand transfer reactions and just one strand is processed and joined to the DNA target, with the final product having a typical Y form.

Along with the actual mechanism, the aspects connected with viral DNA and host DNA recognition characteristics need to be deepened.
As previously reported, the specificity of viral DNA for LTR sequences is not so high and there are reported examples of IN proteins able to react with an oligonucleotide simulating LTR from different retroviruses (e.g. MoMULV IN with HIV LTR ends in an aspecific fashion, but not the reverse (Vink C. et al., 91-2)). Nucleotides next to the subterminal CA have been reported to be involved, namely the subterminal 6 to 8 nucleotides (Reicin A.S. et al., 95). The prevailing idea is that the specificity is not mainly connected with sequence but with other aspects of LTR viral DNA.
Hovewer it has been underlined that in the in vivo systems IN and the viral DNA are not free in cytoplasm, but both are part of an ordered complex, the PreIntegration Complex (PIC). PICs are so stable assemblages that they can be extracted from cytoplasm of infected cells retainig their activities.
Consequentely, it has been proposed that IN doesn't need such a large sequence specificity to recognize its substrate and only a short repeat CA, highly conserved, is essential for right positioning and catalysis (van Gent et al., 91 - Hazuda et al., 94-2) , together with a subterminal portion interacting with the HHCC region of IN (Vincent K.A. et al., 93).
The main aspects promoting IN attack on host DNA for strand transfer reaction are not still completely understood.
It seems that in vivo the site of attack is strongly influenced by chromatin. There are some preferences, like regions complexed with transcription factors (Kassavetis et al., 89) or by histones (Morse et al., 92) or DNaseI sensitive sites. Probably there is some sequence bias, too.
Some in vitro experiments were carried out using more and more complex target DNA structures; a particularly efficient integration into nucleosomal DNA (Pryciak P.M. and Varmus E.H., 92) and in the most severely deformed and kinked DNA regions within the nucleosomal core (Pruss et al., 94), was observed.
It has been proposed that this is due to the bending of DNA in these regions, which may activate integration (Muller H.P. et al., 94).The bending promotes a DNA conformation (which is favourable for integration), widening the minor and/or major groove(s) on the exposed face of the DNA helix. There are also other parameters that can be influenced by DNA bending, like affinity for Mn ²⁺; also transfer reaction might require local denaturation of DNA, easier in a bent region.
In any case, the in vivo system is very complex: specific interations with host proteins have to be taken into account, following what observed for retrotransposone Ty3 (Chalker D.L. and Sandmeyer S.B., 92), together with the subnuclear localization of viral PIC; the host cell state during integration could play a role too. Such interactions have been also proposed to explain the capacity of retroviral DNA to protect itself from the autointegration process (Lee M.S. and Craige R., 94).

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Last updated 25th Oct '96