Molecular mechanisms by which human immunodeficiency virus type 1 integrase stimulates the early steps of reverse transcription

Abstract

Reverse transcriptase (RT) and integrase (IN) are two essential enzymes that play a critical role in synthesis and integration of the retroviral cDNA, respectively. For human immunodeficiency virus type 1 (HIV-1), RT and IN physically interact and certain mutations and deletions of IN result in viruses defective in early steps of reverse transcription. However, the mechanism by which IN affects reverse transcription is not understood. We used a cell-free reverse transcription assay with different primers and compositions of deoxynucleoside triphosphates to differentially monitor the effect of IN on the initiation and elongation modes of reverse transcription. During the initiation mode, addition of IN stimulated RT-catalyzed reverse transcription by fourfold. The stimulation was specific to IN and could not be detected when the full-length IN was replaced with truncated IN derivatives. The IN-stimulated initiation was also restricted to the template-primer complex formed using tRNA(3)(Lys) or short RNA oligonucleotides as the primer and not those formed using DNA oligonucleotides as the primer. Addition of IN also produced a threefold stimulation during the elongation mode, which was not primer dependent. The stimulation of both initiation and elongation by IN was retained in the presence of an RT trap. Furthermore, IN had no effect on steps at or before template-primer annealing, including packaging of viral genomic RNA and tRNA(3)(Lys). Taken together, our results showed that IN acts at early steps of reverse transcription by increasing the processivity of RT and suppressing the formation of the pause products.

FIG. 1.

Effects of RT concentration on the initiation mode of reverse transcription. (A) Schematic representation of the in vitro extension assay used to study the initiation mode of reverse transcription. Human tRNA₃^Lys primers (thin lines with looped 5′ ends; 25 nM) were heat annealed to the PBS of the 957-nt HIV-1 RNA template (thick line; 25 nM), and the template-primer complex was then incubated with purified RT heterodimers. The initiation was started by adding 1 μM dGTP, dTTP, and [α-³²P]dCTP (asterisks) and allowed to proceed at 37°C for 30 min. The extension products labeled +1, +3, and +5 represent the corresponding numbers of nucleotides added to the 3′ end of the primer. (B) Formation of initiation products as a function of RT concentration. The initiation mode of reverse transcription was assayed under conditions described above in the presence of 0 to 540 nM RT. The upper panel shows the separation of the ³²P-labeled products on a sodium dodecyl sulfate-polyacrylamide gel, with the identities of the extended products indicated to the right. The lower panel shows a bar graph of the formation of the +5 product expressed as a percentage of the total extension product. Values are means ± standard errors of the means of three independent experiments.

FIG. 2.

IN stimulates the initiation mode of reverse transcription catalyzed by RT. (A) Effect of IN on initiation. The initiation reaction was carried out in the presence of 20 nM RT alone (lane 1) or 20 nM RT plus 20 to 400 nM of IN (lanes 2 to 5) as described in Materials and Methods. (B) Effect of IN on +1 product formation. Primers were limited to a 1-nucleotide extension by adding only [α-³²P]dCTP to initiate reverse transcription. The reaction was carried out with 20 nM RT alone (left) or RT plus a 10-fold molar excess of IN (200 nM) and monitored at the indicated times for the synthesis of the +1 product. (C) Specificity of IN stimulation on +5 product formation during initiation. The initiation reaction with 20 nM RT was carried out in the absence of added protein (lane 1) or in the presence of a 10-fold molar excess of IN (lane 2), BSA (lane 3), T4 g32 (lane 4), or NC (lane 5). (D) Mapping of the IN domains required for stimulating the initiation mode of reverse transcription. RT (20 nM) was incubated without (lane 1) or with (lane 2) 200 nM of full-length IN or the truncated derivatives of IN: N-terminally truncated IN (lane 3; residues 50 to 288), C-terminally truncated IN (lane 4; residues 1 to 212), core domain only (lane 5; residues 50 to 212), and C-terminal domain only (lane 6; residues 213 to 288). In all panels, the numbers on the right denote the identities of the extension products.

FIG. 3.

Effect of different primers on the initiation mode stimulated by IN. (A) Initiation using an 18-nt RNA (R18) as a primer in the absence of IN. The template-primer complex was formed by annealing 25 nM R18 with the 957-nt RNA template (25 nM), and initiation was conducted in the presence of various concentrations of RT (0 to 165 nM). (B) Effect of IN on the initiation mode using complexes formed with R18 as the primer. The initiation reaction was carried out by adding 20 nM RT and various concentrations of IN (0 to 200 nM) as indicated. (C) Effect of IN on the initiation mode using complexes formed with an 18-nt DNA (D18) as the primer. The initiation reaction was performed in the presence of 20 nM RT, with or without 200 nM IN, and monitored at 1 and 3 min after the start of the reaction. In all panels, the numbers on the right have the same meaning as in Fig. 1.

FIG. 4.

Stimulation by IN of the elongation mode of the RT-catalyzed reverse transcription and the effect of primers on IN stimulation. (A) Effect of RT concentration on the elongation mode of reverse transcription. The 957-nt HIV-1 RNA template (25 nM) was heat annealed with 25 nM human tRNA₃^Lys primer, and the resulting template-primer complex was incubated with various concentrations of RT (0 to 540 nM), 200 μM dNTPs, and 10 μCi of [α-³²P]dCTP. The filled arrowhead indicates the position of the fully extended product, which is 259 nt when tRNA is used as the primer. The numbers to the left are the lengths in nucleotides of the DNA size markers. (B) Effect of IN on the elongation reaction using tRNA₃^Lys as the primer. The elongation was monitored after mixing 20 nM RT in the presence of various concentrations of IN as indicated. The fully extended product was identical to that of panel A. (C) Effect of IN on the elongation reaction using R18 as the primer. (D) Effect of IN on the elongation reaction using D18 as the primer. In panels C and D, the reaction was identical to that of panel B except that either R18 or D18 was used to form the primer-template complex with the 957-nt HIV-1 RNA template. The open arrowheads indicate the positions of the fully extended product, which is 192 nt when R18 or D18 is used as the primer.

FIG. 5.

IN has no effect on the annealing of the tRNA primer to the HIV-1 RNA template. (A) Efficiency of primer annealing measured by the initiation assay. RNA templates (25 nM) and tRNA₃^Lys primers (25 nM) were mixed and subjected to heat annealing (HA) or incubation with 1.5 μM of BSA, NC, or IN at 37°C for 1 h. The efficiency of annealing was assessed by measuring the synthesis of initiation products after the addition of 0.15 μM RT and 1 μM dGTP, dTTP, and [α-³²P]dCTP. The reaction was allowed to proceed for 1, 3, or 30 min. The identities of the initiation products are indicated by the numbers to the right and have the same meaning as in Fig. 1. (B) Formation of the template-primer complex. ³²P-labeled tRNA₃^Lys primers and RNA templates were annealed by heat treatment (lane 2) or incubated without added protein (lane 1) or with 30 pmol of BSA (lane 3), IN (lane 4), or NC (lane 5) at 37°C for 1 h. Primer-template complexes were recovered, resuspended in loading buffer, and separated on nondenaturing 8% polyacrylamide gels at 4°C. Free tRNA₃^Lys primer (*P) and annealed RNA-tRNA₃^Lys complexes (T/*P) are as indicated on the right. (C) Initiation using template-primer complexes isolated from cell-free virus particles. RNA was isolated from wild-type (NL4-3) or IN-negative virions (NLΔIN), and equal amounts (500 ng) of total nucleic acids were used as the source of primer-template complexes in the initiation assay described in Materials and Methods. Heat-annealed RNA template-tRNA₃^Lys complexes were subjected to the same viral RNA extraction condition and served as a positive control (HA). The initiation reaction was carried out in the presence of 1 μM dGTP, dTTP, and [α-³²P]dCTP. Because of the small quantity of the virion-derived primer-template complex, the concentration of RT used was increased to 165 nM. The numbers on the right have the same meaning as in panel A.

FIG. 6.

IN enhances the processivity of RT. (A) Effect of IN on initiation in the presence of an RT trap. Heat-annealed RNA template-tRNA₃^Lys complexes were mixed with 20 nM RT alone (lanes 2 and 4) or 20 nM RT plus 200 nM IN (lanes 3 and 5) at 37°C for 10 min, and the initiation reaction was started by adding 1 μM dGTP, dTTP, and [α-³²P]dCTP. The processivity of RT was assessed by measuring the synthesis of the fully extended initiation product (+5) in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 125 nM RT trap. The numbers to the right have the same meaning as in Fig. 1. (B) Effect of IN on elongation in the presence of an RT trap. The reaction condition was identical to that of panel A except that 200 μM dNTPs were added to start the reaction. The fully extended 259-nt elongation product and the lengths in nucleotides of the DNA size markers are indicated to the right. In both panels, lane 1 represents the control for the trap assay, in which the RT trap was added to the reaction mixture before RT.

FIG. 7.

The RT noninteracting IN mutant (IN-FC) does not stimulate RT-catalyzed reverse transcription. (A) IN-FC does not bind RT. Two microliters containing 20 pmol of WT IN, NC, or IN-FC was spotted onto a nitrocellulose membrane. The dried membrane was subjected to UV cross-linking for 30 s, then blocked in HBB buffer (25 mM HEPES [pH 7.8], 10 mM ZnCl₂, 5 mM MgCl₂, and 25 mM NaCl) plus 5% milk at ambient temperature for 4 h. After being washed three times with HBB buffer, the membrane was incubated overnight at 4°C with shaking in the presence of 0.1 μM RT in HBB buffer, 1% milk, and 0.05% NP-40 (bottom). As a negative control, an identical membrane was incubated under the same conditions without RT (top panel). The membrane was then washed three times with HBB buffer and probed with the mouse anti-RT monoclonal antibody at 1:500 dilution at 37°C for 4 h. A chemiluminescence substrate kit (SuperSignal; Pierce) was used for detection according to the manufacturer's instructions. (B) Effect of IN-FC on initiation. Heat-annealed RNA template-tRNA₃^Lys complexes were mixed with 20 nM RT either alone (lane 1) or in the presence of 200 nM IN (lane 2) or 200 nM IN-FC (lane 3) at 37°C for 10 min, and the initiation reaction was started by adding 1 μM dGTP, dTTP, and [α-³²P]dCTP. The numbers to the right have the same meaning as in Fig. 1. (C) Effect of IN-FC on elongation. The reaction condition was identical to that of panel B except that 200 μM dNTPs were added to start the reaction. The fully extended 259-nt elongation product and the lengths in nucleotides of the DNA size markers are indicated to the right of the panel.