Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells.

Integration of the retrovirus linear DNA genome into host chromosomes is essential for replication of human immunodeficiency virus type 1 (HIV-1). Preintegration complexes (PIC) are formed in the cytoplasm after reverse transcription of the viral RNA. Integrase (IN) in the PIC catalyzes the excision of two nucleotides adjacent to the phylogenetically conserved CA dinucleotide from the 3′-OH blunt ends of the viral DNA (7). After nuclear transport of the PIC, the two viral DNA ends are inserted by IN into the host genome in a concerted fashion, here termed “full-site integration” (8, 25, 44). The unpaired dinucleotides at the 5′ ends of the inserted viral DNA are removed, and the single-stranded gaps are repaired by a host cell DNA repair pathway (35). This process results in a short duplication of cell DNA ranging in size from 4 to 6 bp depending on the retrovirus species. Significant progress in our understanding of retrovirus full-site integration was initiated by isolating PIC from virus-infected cells (8). Purified HIV-1 and murine leukemia virus (MLV) PIC are capable of incorporating ∼15 to 50% of their viral DNA into an exogenously supplied DNA target as full-site integration products after 45 to 90 min of incubation at 37°C (6, 11, 13, 21, 39, 48). One report using HIV-1 PIC showed an ∼95% incorporation of viral DNA into a target substrate where incorporation was essentially over after 45 min of incubation at 37°C (21). Cellular factors like barrier-to-autointegration factor (BAF) appear to play a role in maintaining stable MLV and HIV-1 PIC structures, thus promoting full-site integration by preventing integration of the viral DNA into itself, termed “autointegration” (12, 33, 48, 55). Simplified integration assays using model linear retrovirus DNA with terminal U5 and U3 long terminal repeat (LTR) sequences, purified recombinant IN, and a DNA target were also developed (9, 16, 32, 45). These studies established that IN was the only viral protein necessary for both 3′-OH processing and DNA strand transfer activities. Further progress in reconstituting the HIV-1 full-site integration process was made using IN derived from nonionic detergent lysates of virus particles (10, 22, 23). The 3′-OH recessed ends containing attachment (att) site sequences from two different viral donors (480 bp) were integrated by IN in a concerted manner into a circular DNA target (bimolecular reaction) (Fig. ​(Fig.1).1). In a typical reaction at 37°C for 30 min, 4 to 8% of the input donor DNA was incorporated into full-site products by these lysates. The fidelity of the HIV-1 5-bp host site duplications was ∼70%. Subsequently improvements in purified recombinant HIV-1 (46) and simian immunodeficiency virus (24) IN provided an ∼2-fold improved efficiency for producing full-site integration products over HIV-1 lysates. Recombinant HIV-1 IN using blunt-ended DNA donor substrates (∼300 bp) with added HMGA1 is also capable of performing unimolecular full-site integration reactions where the two att ends from the same DNA donor are inserted into the DNA target (4, 29, 30). However, the primary products of the above integration reactions had one donor end either inserted into the DNA target (half-site integration) or into other donors. FIG. 1. Schematic representation of the 4.1-kbp linear DNA substrate, assembly conditions, and strand transfer analyses. The 4.1-kbp donor is depicted (top) with restriction sites and the 3′-OH recessed wt U5 and U3 termini. The donor contained a 420-bp ... In this study, recombinant HIV-1 IN purified at pH 6.8 in the presence of 10 mM MgSO4 has the capacity to catalyze full-site integration with high efficiency and minimal synthesis of half-site integration products. A 4.1-kbp linear donor possessing 3′-OH recessed U5 (67 bp) and U3 (378 bp) LTR termini produced by NdeI digestion was employed. With this donor, we significantly lowered the number of DNA donor ends by a factor of ∼10 as deduced from earlier studies (22, 23, 46). The concentrations of IN were also lowered proportionally, thus decreasing the frequency of unwanted half-site integration reactions. The full-site integration reaction efficiency was routinely in the 30 to 40% range with 5 to 10 nM IN for 120 min at 37°C. No cellular or viral factors were added. The fidelity of the 5-bp host site duplications was ∼70% with coupled U5 and U3 donor ends. The rest of the sequenced clones contained repetitive small size bp deletions mirroring the same results obtained with HIV-1 lysates (22, 23). Other results suggest that IN must be kept in a sufficiently reduced state upon storage (−70°C) to maintain full-site integration activity and to avoid donor-donor integration reactions. In summary, recombinant HIV-1 IN under low protein concentrations without the requirement for organic solvents present possesses significant capacity for full-site integration.