Background: We have demonstrated that the non-nucleoside reverse transcriptase inhibitors (NNRTI-R) mutants V106A and P236L reduce NL4-3 replication fitness compared to K103N, Y181C, and wt. Unexpectedly, none of these mutations affected processivity of DNA polymerization by reverse transcriptases (RTs). Each mutant RT had altered RNase H cleavage under steady-state conditions, when normalized for polymerase specific activity. The less-fit V106A and P236L had significantly reduced RNase H cleavage compared to K103N and Y181C. This suggested that a reduction in the RNase H:polymerase ratio contributes to the reduced replication fitness of NNRTI-R HIV. We further studied the effects of NNRTI-R on polymerization by measuring the kinetics of single nucleotide incorporation under pre-steady state conditions.

Methods: Incorporation of dGTP by wt and mutant RTs was studied using a heteropolymeric DNA/DNA primer/template, in substrate excess. The concentration of catalytically active RTs was used to normalize RT input. Reactions were initiated by rapid mixing in a KinTek quench flow machine, and terminated with EDTA at times ranging from 10ms–5.5s. Substrate and product were electrophoretically resolved and quantitated by PhosphorImaging. Data points were fit to the burst equation using linear regression, to obtain k_ss (rate of product dissociation), which is the rate-limiting step for polymerization. The initial burst rate, which represents the rate of catalysis, was plotted for [dGTP] ranging from 0.625–80mM, and data points were fitted to a hyperbolic equation to obtain k_pol (maximal rate of dGTP incorporation) and K_d (dGTP affinity).

Results: The table below summarizes data for each mutant RT.

RT	kpol (s-1)	Kd (mM)	kpol/Kd (mM-1 s-1)	kss (s-1)
WT	26.3 ±1.28	3.0 ±0.6	8.85	0.160 ±0.018
P236L	46.2 ±1.98	3.3 ±0.6	13.7	0.039 ±0.018
V106A	pending	pending	pending	0.136 ±0.028
K103N	21.3 ±1.61	3.4 ±1.3	6.33	0.279 ±0.062

 

Conclusions: These results indicate that P236L reduces the rate of nucleotide incorporation by slowing product dissociation during polymerization, but that catalysis of nucleotide incorporation and dGTP affinity are not affected. The reduced rate of product dissociation during polymerization may contribute to the replication defect of P236L, but the normal rate of product dissociation by V106A suggests that reductions in RNase H cleavage play a significant role in the reduced replication fitness of NNRTI-R mutants.