Role of murine leukemia virus reverse transcriptase deoxyribonucleoside triphosphate-binding site in retroviral replication and in vivo fidelity

Retroviral populations exhibit a high evolutionary potential, giving rise t o extensive genetic variation. Error-prone DNA synthesis catalyzed by rever se transcriptase (RT) generates variation in retroviral populations. Struct ural features within RTs are likely to contribute to the high rate of error s that occur during reverse transcription. We sought to determine whether a mino acids within murine leukemia virus (MLV) RT that contact the deoxyribo nucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P enc apsidating cell line, which expresses the amphotropic MLV envelope and a re troviral vector (pGA-1). pGA-1 expresses the bacterial P-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were de termined. As expected, most substitution mutations of amino acids that dire ctly interact with the cNTP substrate significantly reduced viral titers (> 10,000-fold), indicating that these residues played a critical role in cata lysis and viral replication. However, the D153A and A154S substitutions, wh ich are predicted to affect the interactions with the triphosphate, resulte d in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with th e base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in stati stically significant decreases in fidelity (1.3- to 2.4-fold). These result s suggest that mutations of residues that contact the substrate dNTP can af fect viral replication as well as alter the fidelity of reverse transcripti on.