Molecular and genetic determinants of rous sarcoma virus integrase for concerted DNA integration.

Retroviruses have the capability of inserting their DNA genome into the host DNA by the virus-encoded integrase (IN). IN from different retrovirus species possess three similar structural domains even though their amino acid sequences are significantly different (Fig. ​(Fig.1)1) (3, 13). The N-terminal domain (∼50 residues) contains a zinc binding region (6), promotes IN multimerization (46), and is necessary to catalyze 3′ OH processing and strand transfer. The 3′ OH processing activity is responsible for removal of dinucleotides from the catalytic strands on the blunt-ended viral DNA genome. The central or catalytic core domain (∼180 residues) contains a conserved triad of amino acids comprising the D,D(35)E motif (28) that is involved in coordinating divalent metal ions for enzymatic activities (2, 13). The core is also involved in target binding for strand transfer (1, 21, 27). The C-terminal domain (∼50 residues) binds to the viral DNA at ∼7 to 9 bp from the long terminal repeat (LTR) ends (18, 26, 30), binds to DNA in a nonspecific fashion (3), and also appears to be involved in multimerization of IN (25, 30). The reported subunit structure for purified virion IN from avian myeloblastosis virus (AMV) is a dimer (20, 32), while several retrovirus recombinant INs purified from bacteria are observed as monomers, dimers, and tetramers (12, 13, 14, 25). FIG. 1. Structural schematic of RSV IN. (Top) RSV IN (286 amino acids) is divided into three functional domains identified as the N-terminal, catalytic core, and C-terminal regions (13). A hinge region between the core and C-terminal domain is marked as a star ... Models describing potential interactions of IN at the viral DNA ends for concerted integration into the host DNA have been proposed (13, 44). The models are based on several approaches, including complementation of IN mutants (6, 17), crystallographic studies of individual or paired domains of IN (5, 8, 10, 42, 44), cross-linking studies of IN with DNA substrates (18, 19, 22, 26), and protection of viral DNA ends by IN in preintegration complexes (PICs) (4, 7, 9, 33, 43) and in reconstituted nucleoprotein complexes with purified AMV IN with viral DNA substrates (40). The collective theme suggests that IN forms multimers at the viral DNA ends for concerted integration of the two ends into a target, here termed full-site integration. Attempts to compare the mutant retrovirus IN enzymatic activities in vitro to the observed replication and integration capabilities in vivo of retroviruses carrying the same mutations in the IN gene have been difficult for several reasons. First, recombinant wild-type (wt) and mutant IN have either failed to catalyze full-site integration or have done so in a nonefficient manner, thus offering significant insights into only the 3′ OH processing and half-site integration reactions (3, 16, 35). Half-site integration is defined as insertion of one viral DNA end into a target substrate. Second, even though mutations introduced into IN within the context of the viral genome were defective for integration (class I mutants) (16), others produced pleiotropic effects on the replication of retroviruses, indirectly affecting DNA integration in vivo (class II mutants) (3, 16). In order to map the functions associated with the different domains in Rous sarcoma virus (RSV) (Prague A strain) IN for full-site integration, we introduced single amino acid mutations into recombinant IN. RSV IN would be an appropriate candidate for site-directed mutagenesis, since wt recombinant RSV IN has a specific activity for full-site integration similar to that of AMV IN purified from virions (11, 31, 41). The replication capabilities of several RSV IN virus mutants which had appeared to specifically affect viral DNA integration in vivo (Fig. ​(Fig.1)1) (36, 37) have been mapped; other mutants did not affect integration (23, 24). Here, we correlate in vitro activities of recombinant RSV IN mutants for full-site integration to several RSV PrA IN mutants (A48P and P115S) that possess significant integration defects in vivo. The RSV A48P IN mutation would apparently map in a disordered linker region between the N-terminal and the central catalytic domains, as shown with human immunodeficiency virus type 1 (HIV-1) IN (13, 42). A significant integration defect observed with RSV containing the A48P IN mutation in vivo (37) could not be correlated with a defect for full-site integration or other activities by recombinant A48P IN in vitro, suggesting a possible undiscovered pleiotropic effect for integration with RSV A48P IN in vivo. The RSV P115S IN mutation is located in the turn region between α1 and β4 (Fig. ​(Fig.1)1) that is in a structurally equivalent region of HIV-1 IN (5) possessing a similar mutation (P109S) (15, 38). Recombinant HIV-1 P109S IN possesses no 3′ OH processing and strand transfer activities, which is consistent with the inability of the HIV-1 virus containing this mutation to integrate its genome into host DNA. However, in this report, recombinant RSV P115S IN possesses the same full-site integration activity as the wt RSV IN. Furthermore, a positive correlation was observed between the temperature-sensitive defect for integration with the RSV P115S IN mutant in vivo (36) and the thermolabile 3′ OH processing, half-site, and full-site integration activities of recombinant P115S IN in vitro. Wt RSV IN is stable under the same heating conditions at 57°C. Substitutions into the highly conserved W233 of recombinant RSV IN suggest that the C-terminal region is involved in multimerization of IN at the LTR terminal attachment (att) sites, as defined by DNase I footprint protection studies (40).