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

Abstract

Retrovirus preintegration complexes (PIC) in virus-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 3Ј-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 5Ј 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 fullsite integration was initiated by isolating PIC from virus-infected cells (8). Purified HIV-1 and murine leukemia virus (MLV) PIC are capable of incorporating ϳ15 to 50% of their viral DNA into an exogenously supplied DNA target as fullsite 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 ϳ95% 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-toautointegration 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 3Ј-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 3Ј-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

Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

“…IN (pNY clone) was expressed in Escherichia coli BL21(DE3) cells and purified to near homogeneity (41). Concentrations of HIV-1 IN were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using RSV IN as a standard (12), which are very similar to the concentration of HIV-1 IN determined using the molar extinction coefficient of 50,460 M Ϫ1 cm Ϫ1 at 280 nm (24).…”

“…Integration assay. The integration assay was performed as described previously (41). Briefly, IN was assembled with the donor substrate in presence of 20 mM HEPES (pH 7.0), 5 mM dithiothreitol, 10 mM MgCl 2 , 25 M ZnCl 2 , 100 mM NaCl, and 10% polyethylene glycol (6,000 Da) at 14°C for 15 min.…”

“…Substrates with blunt ends are favored over 3Ј-OH recessed ends by HIV-1 IN to produce FS products (28). The concerted insertion of viral DNA ends into a target results in the correct host site duplication at the site of insertion (29,37,41,42,45). Another intermediate in the integration pathway has been uncovered in addition to the SC assembled with purified virion or recombinant avian retrovirus IN (5,45).…”

Integrase, LEDGF/p75 and HIV replication

“…2. This essential aspect of the in vivo reaction has typically been difficult to recapitulate in vitro with the purified enzyme, although recent adjustments to assay parameters have boosted efficiency [155,156]. The effect of p75 differed sharply between different recombinant lentiviral IN proteins: its addition enhanced full-site integration for EIAV IN while HIV IN was instead directed virtually exclusively to catalyze uncoupled (half-site) integration in which only one viral DNA end is joined to one strand of target DNA [100].…”

Biochemical functions of integrase-binding domain of lens epithelium-derived growth factor

“…Then the full-site (FS) integration occurs when a pair of SC integrates into both strands of target DNA at the opposite sites ( Figure 2A). Besides these, the donor DNA can form other products by binding each other (dimer) or Y-shape Sinha & Grandgenett 2005). To form the SC, different roles were envisioned to describe.…”