Mechanistic Understanding of an Altered Fidelity Simian Immunodeficiency Virus Reverse Transcriptase Mutation, V148I, Identified in a Pig-tailed Macaque*

We have recently reported that the reverse transcriptase (RT) of SIVMNE 170 (170), which is a representative viral clone of the late symptomatic phase of infection with the parental strain, SIVMNE CL8 (CL8), has a largely increased fidelity, compared with the CL8 RT. In the present study, we analyzed the mechanistic alterations of the high fidelity 170 RT variant. First, we found that among several 170 RT mutations, only one, V148I, is solely responsible for the fidelity increase over the CL8 RT. This V148I mutation lies near the Gln-151 residue that we recently found is important to the low fidelity of RT and the binding of incoming dNTPs. Second, we compared dNTP binding affinity (Kd) and catalysis (kpol) of the CL8 RT and the CL8-V148I RT using pre-steady state kinetic analysis. In this experiment, the high fidelity CL8-V148I RT has largely decreased binding to both correct and incorrect dNTP without altering kpol. The fidelity increase imparted by the V148I mutation is likely because of the major reduction seen in RT binding to dNTPs. This parallels our findings with the Q151N mutant. Third, site-directed mutagenesis targeting amino acid residue 148 has revealed that a valine amino acid at this position is essential to RT infidelity. Based on these findings, we discuss possible structural impacts of residue 148 (and mutations at this site) on the interaction of RT with incoming dNTPs and infer how alterations in these properties may relate to viral replication and fitness.