Ji J, Loeb L A
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
Virology. 1994 Mar;199(2):323-30. doi: 10.1006/viro.1994.1130.
The unusually high mutation frequency exhibited by the human immunodeficiency virus (HIV) is a major impediment to developing effective vaccines against the virus and to designing analogs that inhibit viral replication. To investigate the molecular basis of HIV hypermutability, we established cell-free assays to measure the fidelity of HIV-1 reverse transcriptase (RT) in copying either DNA or both RNA and DNA templates that contain the hypervariable region 1 of the HIV-1 env gene (V-1). The fidelity of DNA synthesis was measured by repetitively copying the envelope gene (V-1) DNA by HIV-1 RT, followed by cloning and sequencing these newly synthesized DNA products. We found that the error rate of HIV RT copying either RNA or DNA of the env V-1 region is about one misincorporation per 5 kb polymerized. This rate is similar to that found with the M13mp2 forward mutation assay using the lacZ alpha gene as a template. This similarity suggests that the HIV env hypervariable sequence is not inherently hypermutable. The high error rate of HIV RT suggests that misincorporation by this enzyme is a major source of mutations throughout the viral genome and a determinant for rapid viral evolution. The spectrum of mutations produced by HIV RT in vitro partially correlates with the spectrum of HIV mutations observed in AIDS patients. The differences between these spectra highlight the contribution of phenotypic selection during HIV-1 infection. The overall uniformity of misincorporation of HIV-1 RT further suggests an alternative anti-HIV strategy based on increasing viral mutagenesis by nucleotide analogs.
人类免疫缺陷病毒（HIV）呈现出的异常高突变频率，是研发针对该病毒的有效疫苗以及设计抑制病毒复制类似物的主要障碍。为了探究HIV高变异性的分子基础，我们建立了无细胞测定法，以测量HIV-1逆转录酶（RT）在复制包含HIV-1 env基因高变区1（V-1）的DNA或RNA与DNA模板时的保真度。通过让HIV-1 RT反复复制包膜基因（V-1）DNA，然后对这些新合成的DNA产物进行克隆和测序，来测量DNA合成的保真度。我们发现，HIV RT复制env V-1区域的RNA或DNA时的错误率约为每聚合5 kb发生一次错配。这个速率与使用lacZα基因作为模板的M13mp2正向突变测定法所发现的速率相似。这种相似性表明，HIV env高变序列并非天生就具有高变异性。HIV RT的高错误率表明，该酶的错配是整个病毒基因组突变的主要来源，也是病毒快速进化的一个决定因素。HIV RT在体外产生的突变谱与艾滋病患者中观察到的HIV突变谱部分相关。这些谱之间的差异突出了HIV-1感染期间表型选择的作用。HIV-1 RT错配的总体一致性进一步提示了一种基于通过核苷酸类似物增加病毒诱变的抗HIV替代策略。
