Deoxynucleoside triphosphate incorporation mechanism of foamy virus (FV) reverse transcriptase: implications for cell tropism of FV

Abstract

Here, we investigated the pre-steady-state deoxynucleoside triphosphate (dNTP) incorporation kinetics of primate foamy virus (PFV) reverse transcriptase (RT) in comparison with those of HIV-1 and MuLV RTs. PFV RT displayed a drastic reduction in primer extension at low dNTP concentrations where HIV-1 RT remains highly active, indicating a low dNTP binding affinity in the case of PFV RT. Indeed, kinetic analysis showed that, as observed with MuLV RT, PFV RT exhibits approximately 10 to 80 times lower dNTP binding affinity than HIV-1 RT. These three RTs, however, show similar catalytic activities. In conclusion, PFV RT displays mechanistic distinctions in comparison to HIV-1 RT and shares close similarity to MuLV RT.

FIG. 1.

dNTP concentration-dependent reverse transcription activity of RT proteins. (A) Schematic illustration of primer extension reaction by RT. A 5′-end, ³²P-labeled, 23-mer T primer (P, 5′-CCGAATTCCCGCTAGCAATATTC-3′) annealed the 38-mer RNA template (T, 5′-GCUUGGCUGCAGAAUAUUGCUAGCGGGAAUUCGGCGCG-3′; template/primer ratio, 2.5:1) was extended by RTs of HIV-1 (B), MuLV (C), and PFV (D), showing approximately 25, 60, and 75% of primer extension (F), respectively, with 250 μM dNTPs (first lane) at 37°C for 5 min as previously described (12), and the reactions were repeated with decreasing dNTP concentrations (125, 50, 25,10, 5, 1, 0.2, 0.1, and 0.05 μM). The dNTP concentrations found in dividing cells (D) (1∼5 μM) and macrophages (M) (0.05 μM) are marked at the bottom of the figure. All three RT proteins used here were fused to the N-terminal His tag and purified from a bacterial overexpression system as described previously (7). PFV RT was purified from pET28a, containing the PFV PR-RT gene provided by Stephen Hughes (1). F, 38-nucleotide-long, fully extended product; P, 23-mer, unextended primer.

FIG. 2.

Active-site determination and dNTP titration of PFV RT. (A) Pre-steady- and steady-state kinetics of PFV RT incorporating dTTP onto the ³²P-labeled, 23-mer T primer annealed to the 38-mer template used in Fig. 1 were analyzed as previously described (12). Reactions were carried out at the indicated times by mixing together a solution of RT (100 nM protein concentration) prebound to T/P (300 nM) and a second solution with 800 μM dTTP under rapid quench conditions. The data were fit into the burst equation (equation 1), product concentration = A[1 − exp(−k_obst) + k_sst] (5, 6), which provides a measure of the active concentration of RT (Amp), the observed rate constant for the burst phase (k_obs), and the rate constant for the linear phase (k_ss) for PFV RT. The pre-steady-state rates of dTTP incorporation onto T/P (k_obs) for PFV RT were 71.5 ± 14 s⁻¹, and their rates during the steady state were 0.51 s⁻¹. (B) Pre-steady-state T and G titration by PFV RT. The ³²P labeled, 23-mer dTTP and dGTP primers (12) annealed to the 38-mer template (50 nM) were extended with excess RT (200 nM active-site concentration) for single round of dTTP and dGTP incorporation at 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, and 150 μM concentrations. These data were used for the determination of K_d and k_pol values of PFV RT, using equation 2, k_obsd = k_pol(dNTP concentration)/(K_d + dNTP concentration) (5, 6), as summarized in Table 1.