Mansky L M, Temin H M
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706, USA.
J Virol. 1995 Aug;69(8):5087-94. doi: 10.1128/JVI.69.8.5087-5094.1995.
The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.
人类免疫缺陷病毒1型（HIV-1）是逆转录病毒科慢病毒属的成员，与其他一些属的逆转录病毒相比，其基因变异水平较高。在无细胞系统中纯化的HIV-1逆转录酶的高错误率为这种高基因变异提供了一种解释。为了测试HIV-1逆转录过程中的体内突变率是否与无细胞研究预测的一样高，因此是否高于其他属逆转录病毒的突变率，我们开发了一种体内检测方法，用于检测HIV-1中的正向突变，使用lacZα肽基因作为突变的报告基因。该系统允许研究在单个复制周期中发生的突变率和类型。我们发现HIV-1的正向突变率为每碱基对每周期3.4×10⁻⁵个突变。碱基置换突变占主导；G到A的转换突变是最常见的碱基置换。HIV-1的体内突变率分别比之前报道的另外两种逆转录病毒——脾坏死病毒和牛白血病病毒的突变率高3倍和7倍。相比之下，我们计算出的HIV-1体内突变率比具有相同靶序列的纯化HIV-1逆转录酶的错误率低约20倍。这一发现表明，HIV-1在体内的逆转录不像无细胞研究中根据纯化逆转录酶的保真度所预测的那样容易出错。我们的数据表明，纯化的HIV-1逆转录酶的保真度可能无法准确反映自然感染中的基因变异水平。
