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 to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-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 determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the 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 statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.

逆转录病毒种群展现出极高的进化潜能，可产生广泛的遗传变异。由逆转录酶（reverse transcriptase, RT）催化的易错DNA合成，是逆转录病毒种群产生遗传变异的重要来源。逆转录酶内部的结构特征，可能是逆转录过程中出现高错误率的关键因素。本研究旨在明确：鼠白血病病毒（murine leukemia virus, MLV）逆转录酶中，与脱氧核糖核苷三磷酸（deoxyribonucleoside triphosphate, dNTP）底物结合的氨基酸残基，是否对逆转录的体内保真度具有重要作用。我们采用了此前已报道的ANGIE P包装细胞系，该细胞系可表达嗜两性鼠白血病病毒包膜以及逆转录病毒载体pGA-1。pGA-1携带细菌β-半乳糖苷酶基因（lacZ），该基因可作为突变检测的报告基因。我们对可能与dNTP底物存在相互作用的残基进行了大规模诱变，并测定了这些突变对逆转录保真度的影响。正如预期，绝大多数直接与dNTP底物结合的氨基酸残基的替换突变，均显著降低了病毒滴度（降幅超过10000倍），表明这些残基在催化过程与病毒复制中发挥关键作用。然而，被预测会影响三磷酸基团相互作用的D153A与A154S替换突变，导致突变率出现统计学意义上的显著升高。此外，可能影响碱基与核糖相互作用的保守替换F155W，使突变率提升了2.8倍。dNTP结合位点附近残基的替换突变，同样导致保真度出现统计学意义上的显著下降（降幅为1.3~2.4倍）。上述结果表明，与底物dNTP结合的残基发生突变，既可影响病毒复制过程，也可改变逆转录的保真度。