For retroviral DNA to direct production of progeny virions it must become covalently integrated into the host cell chromosome (reviewed in Coffin et al. 1997; Hansen et al. 1998). Analyses of mutants have identified the viral integrase coding region (part of the retroviral pol gene) as essential for the integration process (Donehower 1988; Donehower and Varmus 1984; Panganiban and Temin 1984; Quinn and Grandgenett 1988; Schwartzberg et al. 1984). Also essential are regions at the ends of the viral long terminal repeats (LTRs) that serve as recognition sites for integrase protein (Colicelli and Goff 1985, 1988; Panganiban and Temin 1983).
The viral genomic RNA is reverse transcribed to form a linear double-stranded DNA molecule, the precursor to the integrated provirus (Brown et al. 1987, 1989; Fujiwara and Mizuuchi 1988). The provirus is colinear with unintegrated linear viral DNA (Dhar et al. 1980; Hughes et al. 1978) but differs from the reverse transcription product in that it is missing two bases from each end (Hughes et al. 1981). Flanking the integrated HIV provirus are direct repeats of the cellular DNA that are 5 base pairs in length (Vincent et al. 1990). This duplication of cellular sequences flanking the viral DNA is generated as a consequence of the integration mechanism (Coffin et al., 1997).
Linear viral DNA is found in a complex with proteins in the cytoplasm of infected cells. These complexes (termed "preintegration complexes", PICs) can be isolated and have been shown to mediate integration of viral DNA into target DNA in vitro (Bowerman et al. 1989; Brown et al. 1987; Ellison et al. 1990; Farnet and Haseltine 1990, 1991).
The development of in vitro assays with purified integrase has allowed its enzymatic functions to be elucidated. The provirus is formed by two reactions catalyzed by the viral integrase: terminal cleavage and strand transfer. Studies with purified integrase have shown that it is sufficient for both 3' end cleavage (Bushman and Craigie 1991; Craigie et al. 1990; Katzman et al. 1989; Sherman and Fyfe 1990) and joining of the viral DNA to the cellular chromosome or naked target DNA (Bushman et al. 1990; Craigie et al. 1990; Katz et al. 1990). HIV integrase catalyze the removal of two bases from the 3' end of each viral DNA strand, leaving recessed 3' hydroxyl groups (Brown et al. 1989; Fujiwara and Mizuuchi 1988; Roth et al. 1989; Sherman and Fyfe 1990). This terminal cleavage reaction is required for proper integration. It may allow the virus to create a standard end from viral DNA termini that can be heterogeneous due to the terminal transferase activity of reverse transcriptase (Miller et al. 1997; Patel and Preston 1994). In addition, the terminal cleavage step is coupled to the formation of a stable integrase-DNA complex (Ellison and Brown 1994; Vink et al. 1994). Following terminal cleavage, a recessed hydroxyl is exposed that immediately follows a CA dinucleotide. More internal LTR sites are also important for integration (Balakrishnan and Jonsson 1997; Bushman and Craigie 1990; Leavitt et al. 1992). After end processing, integrase catalyzes the covalent attachment of hydroxyl groups at the viral DNA termini to protruding 5' phosphoryl ends of the host cell DNA (Brown et al. 1987; Brown et al. 1989; Fujiwara and Mizuuchi 1988). The DNA cleavage and joining reactions involved in integration are shown in the figure below. Both the viral DNA 3' end cleavage and strand transfer reactions are mediated by single-step transesterification chemistry as shown by stereochemical analysis of reaction products (Engelman et al. 1991). Biochemical analysis of purified integrase revealed that it requires a divalent metal - either Mg2+ or Mn2+ - to carry out reactions with model substrates, that probably mediate the reaction chemistry (Bushman and Craigie 1991; Craigie et al. 1990; Katzman et al. 1989; Sherman and Fyfe 1990; Gao et al. 2004).