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transfer (Figure 3). While disintegration probably has no physiological significance, it has been useful in
defining aspects of integrase biochemistry.
The three in vitro activities of integrase require divalent metal ions as cofactors. The only two metals
that support these activities are Mn 2+ and Mg 2+. Since quite high metal concentrations must be added to
assays (1–10 mM for optimal activity), it has been presumed that Mg 2+ is the ion used in vivo.
C. Evidence for a Multimer as the Active Unit of Integrase
Several lines of evidence demonstrate that the active unit of integrase is a multimer. It is clear, as an
isolated protein in solution, that integrase forms dimers [6,10–12], and it has been shown by
sedimentation equilibrium studies that Rous sarcoma virus (RSV) integrase exists in reversible
equilibrium between monomeric, dimeric, and tetrameric forms [13]. Protein-protein cross-linking
studies of HIV-1 [14] and RSV [15] integrases confirm the existence of protein dimers and tetramers in
solution, and in vivo, the yeast GAL4 two-hybrid system has demonstrated that HIV-1 integrase can
interact with itself [16].
Complementation studies using mutant proteins in vitro provide compelling evidence that the active
form of integrase must be at least a dimer [14, 17]. This can be inferred from the result that when certain
inactive forms of integrase—generated either by truncation or point mutation—are mixed, robust
activity can be reconstituted. This indicates that different monomers in a multimer are capable of
providing different essential functions in the context of an active complex.
Collectively, these studies suggest that integrase acts as a multimer. This would also seem the most
straight-forward model to explain the observation that viral integration requires two coordinated cutting
and joining reactions on the target DNA during strand transfer. However, physical studies have not yet
addressed what form of integrase actually binds to DNA and carries out the chemical reactions of
integration.
III. Properties of HIV-1 Integrase
A. Domain Structure of Retroviral Integrases
A consistent view of the domain structure of retroviral integrases has emerged by combining the results
from biochemical studies using deletion and site-specific mutants, limited proteolysis experiments, and
sequence comparisons among the family of retroviral integrases. The organization of the domains of
integrase is shown schematically in Figure 4.
The central domain of HIV-1 integrase, consisting approximately of residues 50 to 200, is largely
conserved among retroviral integrases, and forms
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