Hsu M, Inouye P, Rezende L, Richard N, Li Z, Prasad V R, Wainberg M A
Department of Microbiology and Immunology, McGill University, Montreal, Canada.
Nucleic Acids Res. 1997 Nov 15;25(22):4532-6. doi: 10.1093/nar/25.22.4532.
Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) has low fidelity compared with RTs of other retroviruses and cellular DNA polymerases. We and others have previously found that the fidelity of DNA-dependent DNA polymerization (DDDP) of M184V-mutated HIV-1 RT is significantly higher than that of wild-type RT. Viruses containing the M184V substitution are highly resistant to (-)-2'-dideoxy-3'-thiacytidine (3TC) in vitro and in patients treated with 3TC monotherapy. It was of interest to determine the fidelity of RNA-dependent DNA polymerization (RDDP) of M184V RT compared with wild-type because this step occurs first in reverse transcription; errors made during this step may be copied in subsequent polymerization steps. Using an in vitro mispaired primer extension assay, M184V-mutated RT exhibited 3-49-fold decreased frequency of mispair extension compared with wild-type RT. Fidelity differences between M184V and wild-type RT were most marked in extension of A:G (49-fold) and A:C (16-fold) mispairs, with only a marginal (3-fold) decrease in the extension of A:A mispairs. RT containing a methionine to isoleucine (M184I) mutation showed only slight increases in RDDP fidelity compared with wild-type, ranging from 1.5- to 6-fold increases. Of the three RTs tested, wild-type RT was the most error-prone, with mispair extension frequencies ranging from 6.674 x 10(-1) to 7.454 x10(-2).
与其他逆转录病毒的逆转录酶（RT）以及细胞DNA聚合酶相比，人类免疫缺陷病毒1型（HIV-1）的逆转录酶保真度较低。我们和其他人之前发现，M184V突变的HIV-1 RT的依赖DNA的DNA聚合（DDDP）保真度显著高于野生型RT。含有M184V替代的病毒在体外以及接受3TC单一疗法治疗的患者中对（-）-2'-脱氧-3'-硫代胞苷（3TC）具有高度抗性。确定M184V RT与野生型相比的依赖RNA的DNA聚合（RDDP）保真度很有意义，因为这一步骤在逆转录中首先发生；这一步骤中产生的错误可能会在随后的聚合步骤中被复制。使用体外错配引物延伸试验，与野生型RT相比，M184V突变的RT错配延伸频率降低了3至49倍。M184V和野生型RT之间的保真度差异在A:G（49倍）和A:C（16倍）错配的延伸中最为明显，而A:A错配的延伸仅略有下降（3倍）。含有甲硫氨酸到异亮氨酸（M184I）突变的RT与野生型相比，RDDP保真度仅略有增加，增加幅度在1.5至6倍之间。在测试的三种RT中，野生型RT最容易出错，错配延伸频率范围为6.674×10⁻¹至7.454×10⁻²。
