An Amino Acid in the Central Catalytic Domain of Three Retroviral Integrases That Affects Target Site Selection in Nonviral DNA

Integration of a DNA copy of the retroviral genome into cellular DNA is a critical step in the retrovirus life cycle and in the pathogenesis of retrovirus infections. Formation of an integrated provirus requires that the viral integrase act on its two DNA substrates with different levels of specificity. When preparing the viral DNA for integration, two nucleotides that follow conserved CA bases at the 3′ ends of each DNA strand are removed by integrase; this site-specific endonuclease reaction is referred to as processing. In contrast, integrase can insert the processed viral DNA ends into almost any site in cellular DNA; this second endonuclease reaction is referred to as DNA joining or strand transfer. Both of these actions can be modeled in vitro by using purified integrase and oligonucleotides that represent the viral DNA ends (Fig. ​(Fig.1A1A and B) (8, 17, 20). Although any accessible site in nonviral DNA can be used as the target for viral DNA insertion, preferences are noted in vitro and in vivo (7, 19, 32, 33, 39, 43). Characteristics of cellular DNA that affect the susceptibility of target sites were reviewed recently (6, 16, 19). For example, integration preferentially occurs into phosphodiester bonds at areas of DNA distortion on the outside of DNA bends; DNA sequence might play an additional minor role in susceptibility. Understanding how integrase recognizes these features of cellular DNA and identifying the part or parts of integrase responsible for any selectivity in choosing target sites are important for modeling integration, for developing methods of targeted gene delivery that are based on retroviral integration, and for designing a new class of antiretroviral agents that interfere with these enzyme-substrate interactions. FIG. 1. Integrase assays. The names of the assays are shown above the horizontal arrows, and the key aspects of the readouts (e.g., the position of the radiolabel or the pairing of PCR primers) are shown below the arrows. The CA bases near the 3′ ends ... Studies using chimeric integrases involving human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus, and visna virus indicate that the central domain of integrase plays a major role in selecting the target sites for insertion of viral DNA ends. This conclusion was based on results obtained with the standard oligonucleotide joining assay (Fig. ​(Fig.1B)1B) (24) as well as with a PCR-based assay that monitors insertion of viral DNA ends into a longer plasmid DNA target (Fig. ​(Fig.1C)1C) (2, 12, 25, 38). Moreover, the central domain of integrase was solely responsible for the selection of nonviral target sites when chimeric integrases used exogenous alcohols, rather than processed viral DNA ends, as the nucleophilic donor for nicking nonviral DNA (Fig. ​(Fig.1D)1D) (22, 24, 25). In fact, the isolated central fragment of HIV-1 integrase (from residues 50 to 186) exhibited the same target site preferences in this nonspecific alcoholysis assay, which has many similarities to the joining reaction (25), as did the full-length 288-amino-acid HIV-1 protein. Thus, this region of approximately 140 amino acids is capable of binding and positioning nonviral DNA for nucleophilic attack. Within the central domain of integrase, many amino acids can be replaced without affecting target site preferences (2, 13). However, we recently identified residue 119, the second amino acid in the α2 helix in the central domain of HIV-1 integrase, as strongly affecting the choice of nonviral target DNA sites (13). This residue was identified by a novel approach that involved screening a large set of patient-derived HIV-1 integrase variants for alterations in nonviral target site selection, comparing the sequences of proteins that exhibited similar target site preferences, and using this information to guide mutagenesis of a laboratory HIV-1 integrase (13). In fact, HIV-1 integrases with any of five different amino acids (Ser, Thr, Gly, Ala, or Lys) at position 119 exhibited five different patterns of target site selection in nonviral DNA. To test the hypothesis that these results are generalizable to other retroviral integrases, we have now assessed the role of the analogous protein residue in the integrases from a nonprimate lentivirus (visna virus) and a more distantly related alpharetrovirus (Rous sarcoma virus [RSV], formerly classified in the Oncovirinae retrovirus subfamily). Because the preferred sites of viral DNA insertion can differ depending on whether Mn2+ or Mg2+ is present during reactions (13), it was important that each of these divalent metal cations be used for these analyses. To introduce amino acid substitutions into integrase, we used the QuikChange site-directed mutagenesis system (Stratagene, La Jolla, Calif.) with pQE-30 plasmids (Qiagen, Chatsworth, Calif.) that encoded the wild-type integrases, as described previously (13, 41). The entire integrase-coding region for all proteins was confirmed by DNA sequencing, and proteins were purified from M15[pREP4] bacteria (Qiagen) (13). The purified proteins were tested under conditions known to optimize activity for visna virus and RSV integrase, including the use of oligonucleotides derived from the U3 end of viral DNA (21, 22, 42). Conditions for the standard oligonucleotide-based assays (Fig. 1A, B, and D) were as described previously (13) but included either 10 mM Mn2+ or 5 mM Mg2+. In the case of visna virus integrase and its derivatives, reactions that included Mg2+ were supplemented with 30% dimethyl sulfoxide (DMSO) because of our previous demonstration that this maneuver enhances the Mg2+-dependent activity of visna virus integrase (31). For the plasmid insertion assays (Fig. ​(Fig.1C)1C) (26, 34), double-stranded 30/32-mers representing the preprocessed U3 end of viral DNA were incubated under the same reaction conditions but in the presence of 0.5 μg of φX174 DNA (Invitrogen, Carlsbad, Calif.) that had been linearized with SstII. The insertion reactions were stopped by fivefold dilution into 40 μl of 10 mM Tris-HCl (pH 8.0)-1 mM EDTA, and 7 μl was transferred to a tube for PCR. PCR was conducted in 25-μl reaction mixtures that contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.2 or 0.4 mM each deoxynucleoside triphosphate, 2 mM MgCl2, 1 U of Taq polymerase (Fisher, Pittsburgh, Pa.), and 15 pmol each of primer P1, which matched the viral donor DNA strand (for visna virus, 5′CAGGGTAGGCATTTGTTCTCTGTCCTGACA3′; for RSV, 5′AAGACTACAAGAGTATTGCATAAGACTACA3′), and primer P2, which was derived from φX174 (5′GGCGACCATTCAAAGGATAAACAT3′). The reaction mixtures underwent 35 cycles at 95°C for 45 s and 65°C for 3 min, with a final extension at 72°C for 10 min. Subsequently, 3 μl of each PCR was transferred to a 10-ul nested runoff reaction that contained 0.24 pmol of 5′ 32P-labeled φX174 primer P3 (5′GGCAGTCGGGAGGGTAGTCGG3′) and 1 U of Taq polymerase under the same buffer conditions as for PCR but for one cycle of 95°C for 2.5 min, 55°C for 4 min, and 72°C for 20 min. All reactions were analyzed by autoradiography after electrophoresis on denaturing polyacrylamide gels (20% gels for the processing, joining, and alcoholysis assays and 6% gels for the PCR-based insertion assay).