Halvas E K, Svarovskaia E S, Pathak V K
Mary Babb Randolph Cancer Center and Department of Biochemistry, West Virginia University, Morgantown, West Virginia 26506, USA.
J Virol. 2000 Nov;74(22):10349-58. doi: 10.1128/jvi.74.22.10349-10358.2000.
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 beta-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.
逆转录病毒群体具有很高的进化潜力，会产生广泛的基因变异。逆转录酶（RT）催化的易出错DNA合成导致逆转录病毒群体出现变异。逆转录酶中的结构特征可能导致逆转录过程中出现高错误率。我们试图确定小鼠白血病病毒（MLV）逆转录酶中与脱氧核苷三磷酸（dNTP）底物接触的氨基酸对于逆转录在体内的保真度是否重要。我们利用了先前描述的ANGIE P包装细胞系，该细胞系表达双嗜性MLV包膜和逆转录病毒载体（pGA-1）。pGA-1表达细菌β-半乳糖苷酶基因（lacZ），该基因作为突变的报告基因。对可能与dNTP底物相互作用的残基进行了广泛的诱变，并确定了这些突变对逆转录保真度的影响。正如预期的那样，与dNTP底物直接相互作用的氨基酸的大多数替代突变显著降低了病毒滴度（>10000倍），表明这些残基在催化和病毒复制中起关键作用。然而，预计会影响与三磷酸相互作用的D153A和A154S替代导致突变率在统计学上显著增加。此外，可能影响与碱基和核糖相互作用的保守替代F155W使突变率增加了2.8倍。dNTP结合位点附近残基的替代也导致保真度在统计学上显著降低（1.3至2.4倍）。这些结果表明，与底物dNTP接触的残基突变会影响病毒复制，并改变逆转录的保真度。
