Interactions between HIV-1 reverse transcriptase and the downstream template strand in stable complexes with primer-template

Abstract

Background: Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) forms stable ternary complexes in which RT is bound tightly at fixed positions on the primer-template (P/T). We have probed downstream interactions between RT and the template strand in the complex containing the incoming dNTP (+1 dNTP*RT*P/T complex) and in the complex containing the pyrophosphate analog, foscarnet (foscarnet*RT*P/T complex).

Methods and results: UV-induced cross-linking between RT and the DNA template strand was most efficient when a bromodeoxyuridine residue was placed in the +2 position (the first template position downstream from the incoming dNTP). Furthermore, formation of the +1 dNTP*RT*P/T complex on a biotin-containing template inhibited binding of streptavidin when biotin was in the +2 position on the template but not when the biotin was in the +3 position. Streptavidin pre-bound to a biotin residue in the template caused RT to stall two to three nucleotides upstream from the biotin residue. The downstream border of the complex formed by the stalled RT was mapped by digestion with exonuclease RecJ(F). UV-induced cross-linking of the complex formed by the pyrophosphate analog, foscarnet, with RT and P/T occurred preferentially with bromodeoxyuridine in the +1 position on the template in keeping with the location of RT one base upstream in the foscarnet*RT*P/T complex (i.e., in the pre-translocation position).

Conclusions: For +1 dNTP*RT*P/T and foscarnet*RT*P/T stable complexes, tight interactions were observed between RT and the first unpaired template nucleotide following the bound dNTP or the primer terminus, respectively.

Figure 1. DNA primer-templates.

(A) Primer-templates used for photo-cross-linking studies. “B” indicates a BrdU residue and * shows the position of an α-³²P-labeled dC residue in the template. Dashes indicate that the sequence is the same as the line above. Subscript “_H” indicates a dideoxynucleotide residue introduced at the 3′ end of an oligodeoxynucleotide primer. (B) Primer-templates used for SA binding and SA-biotin barrier experiments. “Bio” indicates the position of the internal Biotin-ON structure (shown in panel (C)). WL50 has the same sequence as WL50-Bio39 with the Biotin-ON structure replaced by T.

Figure 2. Photo-cross-linking of HIV-1 RT to template containing two adjacent BrdU residues.

(A) Stable complexes were formed by incubating 40 nM HIV-1 RT, 9 nM P/T (the indicated primer annealed to CBB template (Figure 1)), and 100 µM of the +1 dNTP (+1 complex); or 200 nM HIV-1 RT, 9 nM P/T, and 3.2 mM foscarnet (PFA complex) for 10 min at 37°C followed by cooling in ice. Heparin was added and the mixture was kept in ice and exposed to UV light as described in the text. Samples were analyzed by SDS-PAGE. Positions of MW markers are indicated at the left of each panel. (B) Complexes were prepared and analyzed as in (A) except that 5 nM of each P/T was mixed with 200 nM RT only or with RT plus 100 µM of each of the indicated dNTPs or RT plus 3.2 mM foscarnet. “+1” indicates the dNTP complementary to the first unpaired template nucleotide following the primer terminus. (C) Portions of the CBB template (T) sequence and selected primer (P) sequences are shown. “B” indicates BrdU. Subscript “_H” denotes a dideoxynucleotide residue. The experiment in (A) was repeated with heterodimer RT obtained from Worthington Biochemical Corp. with similar results.

Figure 3. Photo-cross-linking of HIV-1 RT to P/Ts containing a single BrdU residue.

Complexes in (A) were formed with 400 nM HIV-1 RT and 5 nM CTB template annealed to each of the indicated chain-terminated primers (Figure 1). Complexes in (B) were formed with 800 nM HIV-1 RT and 20 nM CBT template annealed to each of the chain-terminated primers. For both panels the reaction mixtures contained100 µM of the next complementary dNTP (+1 complex) or 3.2 mM foscarnet (PFA complex). Complexes were treated with heparin, exposed to UV light and analyzed by SDS-PAGE as in Figure 2. Positions of MW markers are shown at the left of each panel. Portions of the template and selected primer sequences are shown at the bottom of each panel.

Figure 4. Effect of preformed RT stable ternary complexes on SA binding to a template biotin residue.

Five nM 5′-³²P-labeled P/T L35-ddA/WL50-Bio39 (A,C) or L36-ddA/WL50-Bio39 (B,D) were incubated without or with 100 nM RT in the absence of ligands or with 0.8, 3.2 or 6.4 mM dNTP. In A and B, SA (50 nM) was added for 5 min at 37°C, and 0.6 µM biotin was added to bind excess SA. Then RT was dissociated with SDS and urea, and SA-biotin-DNA complexes were separated from free DNA by electrophoresis on nondenaturing gels. For C and D, complexes formed with dNTP were transferred to ice, treated with heparin to dissociate RT•P/T binary complexes and fractionated by nondenaturing gel electrophoresis. Arrows to the right of each panel indicate positions of free DNA (P/T) and SA-biotin-DNA complexes (A,B) or dNTP•RT•P/T ternary complexes (C,D). A portion of each P/T sequence is shown. “Bio” indicates the biotin-ON linkage. Subscript “_H” denotes a dideoxyribonucleotide residue.

Figure 5. Stalling of primer extension by HIV-1 RT due to a biotin residue placed at a specific position in the template in the absence or presence of SA.

(Left panel) Five nM 5′-[³²P]-L20 primer annealed to WL50-Bio39 template was extended with 200 nM RT and various concentrations of dNTP (indicated above the lanes) and 6 µM biotin for 45 min at 37°C. (Right panel) 5′-[³²P]-L20/WL50-Bio39 P/T was first incubated for 2-3 min with 100 nM SA at 37°C and then RT, dNTPs and biotin were added and primer extension was carried out as above. Labeled products were fractionated on 20% polyacrylamide under denaturing conditions. Arrows indicate 50 nt (full length of template), 39 nt (primer extended to the position of the biotin residue), and 37 nt (primary stop site for primer extension in the presence of SA.). Diagrams at the bottom of the figure show the direction of primer extension (arrows) and the likely position of RT (box) after stalling on a template containing the biotin residue (triangle) in the absence (left) or presence (right) of SA (shown as a tetramer of circles).

Figure 6. Products of template digestion by RecJ_F exonuclease in a P/T containing a bound SA molecule in the absence or presence of HIV-1 RT and dNTPs.

(Left panel) Five nM L32 primer was annealed to 3′-[³²P]ddAMP-labeled WL50-Bio39 template and incubated with 50 nM SA at 37°C for 5 min followed by incubation with RecJ_F (1 U per µl of reaction mixture) at 37°C for the indicated times. (Right panel) L32 primer/3′-[³²P]ddAMP-WL50-Bio39 template was incubated with SA as described above. Then, 12.5 nM HIV-1 RT and 100 µM each of the four dNTPs were added and incubated at 37°C for 10 min followed by digestion with RecJ_F as above. Digestion products were fractionated on 20% polyacrylamide under denaturing conditions. Arrows at the right of the panels indicate 51 nt (full-length labeled template) and major pause sites for RecJ_F digestion at 45 nt and 43 nt. A digestion product stopping at the biotin residue would be 40 nt, designated 40(Bio). Diagrams at the bottom show the position of RecJ_F (filled crescent) after digestion reaches a pause site in the absence (left) or presence (right) of RT (box) and dNTPs. Arrows indicate direction of RecJ_F digestion. SA is shown as a tetramer of circles and biotin as a triangle.