Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism

Abstract

HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3' end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3'-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri- or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (Ki approximately 5 microM), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3' nucleotide from the primer to the pyrophosphate moiety in the nucleoside di- or triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

Figure 1

Nucleotide-dependent primer modification by HIV-1 RT. [³²P]ddAMP-terminated L32 primer (5 nM), annealed to WL50 template, was incubated with excess HIV-1 RT (200 nM) at 37°C, and the products were separated by electrophoresis through a 20% denaturing polyacrylamide gel. The arrows indicate specific, nucleotide-dependent products formed during the primer modification reaction. The braces indicate ddATP, which migrates as multiple bands. “x” indicates an unidentified labeled product that was independent of added nucleotide. Panels show labeled products formed under the following conditions: (A) fifteen-min incubation in the presence of 3.2 mM of thermostable pyrophosphatase-treated (+PPase) or untreated (−PPase) dNTP, pyrophosphate (PPi), or NTP. A partial sequence of the primer and template is shown. ∗, radioactive ³²P. (B) Five-minute incubation with 3.2 mM PPase-treated GTP and indicated concentrations of PPase-treated dTTP. (C) Fifteen-minute incubation with 800 μM PPase-treated NTP, dNTP, or ddNTP, as indicated. (D) Five-minute incubation with 3.2 mM PPase-treated NTP, NDP, or NMP, as indicated. Lanes: 15, 5-min incubation with 3.2 mM PPi; 16, [³²P]ddATP as a reference. (E) Five-minute incubation with indicated amounts of PPase-treated GTP.

Figure 2

Properties of labeled products synthesized by HIV-1 RT (A) Products formed after incubation for 5 min at 37°C of [³²P]ddAMP-terminated L32-primer annealed to WL50 template in the presence of HIV-1 RT and 3.2 mM PPase-treated ATP (lane 1) or GTP (lane 3). Labeled dinucleoside polyphosphates Ap₄ddA (lane 2) or Gp₄ddA (lane 4) were synthesized from [³²P]ddATP by firefly luciferase (FL). Bands labeled ddATP and “x” are identified in the legend to Fig. 1. The ³²P-labeled substrate is indicated at the bottom of the panel by an asterisk. (B) Digestion of labeled products with CIP or SVPD. Lanes: 1, 4, and 7, products formed after incubation of [³²P]ddAMP-terminated L32 primer annealed to WL50 template with GTP and HIV-1 RT; 2, 5, and 8, ³²P-labeled Gp₄ddA synthesized by firefly luciferase; 3, 6, and 9, control DNA, ddAMP-terminated, 5′-³²P-labeled L32 primer annealed to unlabeled WL50 template. Portions of each reaction mixture were untreated (lanes 1–3) or digested with CIP (lanes 4–6) or SVPD (lanes 7–9). The expected product of complete SVPD digestion of the 5′-labeled control DNA is [³²P]dCMP. (C) Structure of Gp₄ddA. ∗, radioactive ³²P.

Figure 3

Primer rescue by HIV-1 RT. 5′-³²P-labeled, ddAMP-terminated L32 primer annealed to WL50 template was incubated with excess HIV-1 RT and the indicated concentrations of PPase-treated ATP (lanes 1–8) or GTP (lanes 9–16) at 37°C for 10 min. After heat inactivation of the RT, the primer was extended by incubation with exonuclease-free Klenow polymerase and dNTPs for 10 min at 37°C. The products were separated by electrophoresis through a 20% denaturing polyacrylamide gel. The original L32-ddAMP primer is indicated as primer and extended primer products are indicated as ext. primer.

Figure 4

Gp₄ddA synthesis activity of HIV-1 RT, AMV RT, and M-MuLV RT. (A) Products formed after incubation of 5 nM [³²P]ddAMP-terminated L32 primer annealed to WL50 template, 3.2 mM GTP, and no enzyme for 1h at 37°C (lane1); the indicated amounts of HIV-1 RT for 3 min at 37°C (lanes 2–4); AMV RT (Boehringer Mannheim) for 1h at 42°C (lanes 5–7); or M-MuLV RT (GIBCO/BRL) for 1h at 37°C (lanes 8–10). (B) Products formed by the reaction described in A with 200 nM HIV-1 RT and the indicated concentrations of Cl-TIBO for 15 min at 37°C (lanes 1–8), or with 700 nM M-MuLV RT and the indicated concentrations of Cl-TIBO for 1 h at 37°C (lanes 9–16). The highest concentration of Cl-TIBO contained 20% dimethyl sulfoxide, the solvent for this compound. Lanes 1 and 9 (0*) show products formed in the absence of Cl-TIBO but in the presence of 20% dimethyl sulfoxide.

Figure 5

Schematic representation of removal of chain-terminating nucleotide from the primer terminus through either pyrophosphorolysis (Left) or dinucleoside polyphosphate synthesis (Right).