Ji J, Hoffmann J S, Loeb L
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology SM-30, University of Washington, Seattle 98195.
Nucleic Acids Res. 1994 Jan 11;22(1):47-52. doi: 10.1093/nar/22.1.47.
The unusually high frequency of misincorporation by HIV-1 reverse transcriptase (HIV RT) is likely to be the major factor in the rapid accumulation of viral mutations in AIDS, especially in the env gene. To investigate the ability of HIV RT to copy the env gene, we subcloned an HIV env gene fragment into a single-stranded DNA vector and measured the progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosis virus, DNA polymerase-alpha or T7 DNA polymerase, pauses specifically at poly-deoxyadenosine stretches within the env gene. The frequency of bypassing the polyadenosine stretches by HIV RT is enhanced by increasing the ratio of enzyme to template. We measured the fidelity of DNA synthesis within a segment of the hypervariable region 1 of the env gene (V-1) containing a poly-deoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning and sequencing the copied fragments. We found that 27% of the errors identified in V-1 sequence were frameshift mutations opposite the poly-adenosine tract, a site where strong pausing was observed. Pausing of HIV RT at the polyadenosine tract could be enhanced by either distamycin A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experiments in which HIV RT catalyzes gap filling synthesis within the lacZ gene in double-stranded circular M13mp2 DNA. These combined results suggest that the enhanced mutation frequency may be due to increased pausing at netropsin-modified polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift mutagenesis induced by HIV RT and yield predominantly non-viable virus.
人类免疫缺陷病毒1型逆转录酶（HIV RT）错配掺入的异常高频率很可能是艾滋病中病毒突变快速积累的主要因素，尤其是在env基因中。为了研究HIV RT复制env基因的能力，我们将一个HIV env基因片段亚克隆到一个单链DNA载体中，并测量了HIV RT的合成进程。我们观察到，HIV RT，而非禽成髓细胞瘤病毒的RT、DNA聚合酶α或T7 DNA聚合酶，会在env基因内的聚脱氧腺苷序列处特异性停顿。通过增加酶与模板的比例，HIV RT绕过聚腺苷酸序列的频率会提高。我们通过让HIV RT反复复制一段包含聚脱氧腺苷序列的env基因高变区1（V-1）内的DNA片段，然后对复制片段进行克隆和测序，来测量该片段内DNA合成的保真度。我们发现，在V-1序列中鉴定出的错误中有27%是与聚腺苷酸序列相对的移码突变，而聚腺苷酸序列处是观察到强烈停顿的位点。HIV RT在聚腺苷酸序列处的停顿可被放线菌素A或纺锤菌素增强，这两种都是富含（A-T）的小沟结合肽。此外，在HIV RT催化双链环状M13mp2 DNA中lacZ基因内的缺口填充合成的实验中，纺锤菌素会增加移码突变的频率。这些综合结果表明，突变频率的增加可能是由于在纺锤菌素修饰的聚腺苷酸序列处停顿增加所致。因此，纺锤菌素及相关的A-T结合化学物质可能会选择性增强HIV RT诱导的移码诱变，并主要产生无活力的病毒。
