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Cristina Cantale

The Viral Integrases


The Integrase Protein


The sequence

The IN primary structure has been deeply examined; only to mention some of the approaches, secondary structure prediction methods (Lin T. et al., 89) and multiple alignment procedures in conjunction with point and deletion mutageneses and partial proteolysis have been used in a concerted effort with the aim of elucidating the reaction mechanism of viral integration up to the molecular level.
Multiple sequences alignments have been carried out, comparing portions of IN sequences from different sources. Integrases from retroviruses and their analog proteins from retrotransposones and some families of bacterial Insertion Elements (IS) share distinctive aspects, beside a very low general similarity (Johnson M.S. et al., 86).There is a pattern of AAs that has been considered as an integrase fingerprint, because it is highly conserved among all these proteins. The motif is located at the inner part of the sequence and it is called DD(35)E motif (Fayet O. et al., 90 - Kulkosky J. et al., 92).
A point mutation of these AAs eliminates the strand transfer reaction, as largely demonstrated; this suggests that they are part of the catalytic core.
Another characteristic motif is found at the N terminus of integrases from retroviruses and retrotransposones, consisting of HHCC motif, resembling a zinc-finger motif, which is often involved in DNA interactions.
Partial proteolysis has been another powerful instrument to clarify the functional organization of IN, together with site-directed mutagenesis. A very large spectrum of techniques have been used and experimental set-ups have been developed, including epitope mapping and monoclonal antibodies (Nielsen B.M. et al., 96)

Two main hypotheses have been advanced about functional organization of IN. The first scenario takes into account monomers including one active site and one DNA-binding domain, arranged into a tetramer. In the second one the same single active site is flancked by two different DNA binding domains, one for viral DNA and the other for target DNA, leading to a dimeric system.
The different domains in IN proteins analyzed for understanding the different aspects of the integration reaction are:

The functional specialization of these three domains has been derived mainly from in vitro experiments, but in vivo tests are also needed, to examine aspects which are present and possibly fundamental in the in vivo systems, which are more complex and not entirely simulated in the in vitro ones.
Furthermore it is really important to underline that the results obtained by in vitro assays are deeply dependent on the assay details. Metals and salts presence, their concentration, protein concentration, ionic strength, temperature and any other experimental parameter may play an essential role in conditioning the final results.
This fact increases the importance of similar results obtained from different groups, but it also recommends a great caution towards in drawing general conclusions.

N-TERMINAL DOMAIN

The H-X3-H-X20-30C-X2-C motif at N-terminus was the first motif observed by comparison between IN sequences from different sources (Johnson et al., 86).
As this motif resembles known metal binding Zn finger domain, which is a characteristic element of a variety of DNA-binding proteins, it was at first supposed that this region was involved in DNA recognition and correct positioning.
Moreover, mutants with different deletions at the N-terminal region were still able to bind DNA and even to accomplish a detectable DNA disintegration reaction in vitro, demostrating the inconsistence of this first hypothesis (Khan E. et al., 90 - Engelman A. and Craige R., 92 - Vincent K. et al., 93 - Vink C. et al., 93).
However the same assays demonstrated that N-terminus integrity was necessary for processing and transfer reactions, suggesting that its funtionality could correlate with the site-specific cleavage activity.
Similar conclusions were drawn by in vivo tests carried out using Mo-MuLV IN mutants (Roth M.J. et al., 90). Point mutations at the conserved cysteines or histidines of HIV-1 IN and MLV IN are not completely desruptive for catalytic activity in vitro, while they abolish infectivity in vivo.
Generally speaking, there is an aspect of the in vivo assays that has to be considered, adding further complexity. The IN protein, beside its specific functions regarding the overall viral DNA transfer, is involved in all the other steps of the life cycle. It is part of the gag-pol polyprotein (whose correct folding permits following proteolysis), of the PIC and of the final mature virion. IN has a multitude of interactions inside these structures, which are not known and which can be influenced by a IN mutation, playing also a role in the overall life cycle and thus affecting the results of an in vivo assay. Mutations of IN are reported which affect gag proteins or which are lethal for the virus at differents stages (Ansari-Lari et al., 95 - Shin C. et al., 94).
An hypothesis of tertiary structure of HHCC motif was formulated by spectroscopy, using a 55-AAs peptide simulating (1-55)HIV-1 IN in a Zn2+ complex (Burke C.J. ae al., 92), so demostrating that this motif can fold indipendently and it is able to bind Zn2+.
Recently, it has been proposed that this domain can promote higher order multimerization of integrase dimers, fundamental for the stable formation of a complex between the IN protein and viral DNA (Ellison V. et al., 95). This reaction requires a divalent cation (mainly Mn2+, but it was demonstrated that also Mg2+ is efficient, the results depending on the assay conditions (Engelman a. and Craige R., 1995)). Zinc-binding domain from other proteins are reported to play a role in protein-protein interaction.
Despite of the amount of work carried out to define the role of the N-terminus, a model able to explain all the different and often disagreeing observations is not yet available, also because the different results are affected by the specific reaction conditions used.

THE CATALYTIC CORE

It has been demonstrated by deletion mutants that the shortest sequence of HIV-1 IN still able to accomplish disintegration reaction is mapped at IN50-186. Therefore this region, which is also the most resistant to proteolytic cleavage, has to contain the catalytic core (Engelman A. and Craige R., 92) - Bushman F.D. et al., 93).
Key AAs forming the catalytic triad are D-64, D-116 and E-152 (in HIV-1).
Even a conservative mutation of one of these AAs eliminates all detectable activities of integration and viral replication both in vitro and in vivo (Kulkosky J. et al., 92).
The role of these AAs is supposed to be the coordination of a divalent metal cofactor, in analogy to the behaviour of other enzymes catalyzing phosphoryl transfer reactions (KulkoskyJ. et al., 92)
Other AAs adjacent to DDE motif (like W61, T66, V75, S81, T115, S123 and I135) are well conserved among retroviruses and the mutation of each of them can be detrimental.
Beside containing the AAs involved in the catalytic core, it is suggested that the central core region is also involved in other functions.
It is reported that D116 is also involved in stable binding of IN to its viral DNA, but the opinions about the role of the central core in DNA binding, both unspecific and specific, are divergent, due to discrepancy in results (Hazuda D. et al , 94-2 - Vink C. et al., 94.
Recently, results have been reported about chimeric INs. Swapping the N terminal domain in HFV with HIV resulted in a chimeric IN having 3' processing activities with HFV LTR, indicating that central domain is crucial for substrate recognition (Pahl A. and Flugel R.M., 95). The same result is suggested by other chimeras obtained swapping Visna with HIV-1 (Katzman M. and Sudol M., 95).
Further results suggest that this region is involved into dimerization. A potential leucine zipper domain motif has been identified, mapped at 151-168 in HIV-1 IN (Lin T. et al ., 91) and it has been pointed as the dimerization domain. In this region K159, R166 and E152 are highly conserved residues.
Recently the tertiary structure of HIV-1 and ASV INs core domain has been solved.

C-TERMINAL DOMAIN

While studying the N-domain connection to DNA, the C-teminal domain was discovered to be the one specifically involved into DNA recognition. The same experiments previously referred, (e.g. Khan E. et al. 90) showed that IN deletion mutants at the C-terminal domain were no longer able to bind DNA.
It is unkown which characteristic motif is connected to this function. The C-terminal domain is considered to be the least conserved, with less distinctive aspects between the IN sequences.
Mutagenesis, complementation and other assays have mapped, at the 200-270 region, a not-specific DNA binding function, which does not require divalent metal ion (Engelman et al., 94). So these region can be involved in the interaction with the target DNA.
Deletion mutans at different levels have been tested in vitro to better define the binding region. An interesting result has been reported about a single point mutation, W235A, having no effect in vitro, but totally blocking the provirus capacity to replicate in vivo. W235 was the only AA reported to be highly conserved in this region (Johnson M.S. et al.,86). W235 has been consequently proposed to be a key component of some local structure involved in the target DNA interactions (Cannon P.M. et al.,94).
A more recent analysis of HIV-1 IN C-terminal domain has been carried out, comparing retroviruses from different sub-families (Cannon P.M. et al.,96). Three conserved regions among all retroviruses (except HFV) were evidenced and designated as L, C and N. Region L is only conserved in the lentiviruses; region C and N are conserved in all retroviruses, altought the consensus sequences differ between lenti- and non lenti-viruses. C and N are encompassed by that part of C-terminal region which is considered essential for DNA binding activity, that is HIV-IN 213-266 (Engelman A. et al., 94) . W235 is near the beginning of the C region. Some mutants selected on the basis of the previous observations, were tested using in vivo assays. The results confirm that W235 and K186, whose mutation produce block of infectivity at a step beyond reverse transcription and migration into the nucleus, can be involved in target DNA binding.
The complete meaning of these observation has to be investigated. The presence of these sequence features, characteristic of lentiviruses, could be correlated with aspects of their life cycle that are distinct from other retroviruses, as the ability to enter into the nucleus of host cell that are not in mitosis.

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Last updated 25th Oct '96