Guillon C, Oriol G, Gruters R A
UMR103 CNRS/bioMérieux, ENS Lyon, France.
J Virol Methods. 1997 Jul;66(2):237-46. doi: 10.1016/s0166-0934(97)00061-x.
During the course of infection, human immunodeficiency virus type 1 (HIV-1) displays wide genotypic and phenotypic differences. Construction of chimeric viruses is useful to determine the genotypic basis that underlies phenotypic variations, but the procedure is time-consuming. Previously, it has been shown that co-transfection of truncated hemi-genomic HIV-1 proviral DNA can lead to generation of full-length infectious virus. In the study of HIV phenotypes, using this technique, it is important to determine whether recombination between the two hemigenomes occurs without mutations. After co-transfection, progeny recombinant viruses replicated at the same rate as the control. We purified progeny viruses from culture supernatants and determined mutations at the recombination site. It appeared that correct in vivo ligation depended on the purity of DNA and the restriction site used. It also appeared that some of the mutations observed affect replication, as progeny viruses bearing one of these mutations disappeared during in vitro cultures, whereas other mutants did not. Although this technique is widely applied to generate chimeric viruses, the results should be evaluated with care, since mutations influencing the phenotype of the progeny viruses may have been introduced.
在感染过程中，1型人类免疫缺陷病毒（HIV-1）表现出广泛的基因型和表型差异。构建嵌合病毒有助于确定表型变异背后的基因型基础，但该过程耗时。此前已表明，共转染截短的半基因组HIV-1前病毒DNA可导致产生全长感染性病毒。在HIV表型研究中，使用该技术时，确定两个半基因组之间是否发生无突变的重组很重要。共转染后，子代重组病毒的复制速度与对照相同。我们从培养上清液中纯化子代病毒，并确定重组位点的突变。似乎体内正确连接取决于DNA的纯度和所用的限制酶切位点。还似乎观察到的一些突变影响复制，因为携带这些突变之一的子代病毒在体外培养过程中消失，而其他突变体则没有。尽管该技术广泛应用于产生嵌合病毒，但由于可能已引入影响子代病毒表型的突变，因此应谨慎评估结果。
