Shi C, Mellors J W
Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pennsylvania 15261, USA.
Antimicrob Agents Chemother. 1997 Dec;41(12):2781-5. doi: 10.1128/AAC.41.12.2781.
We have developed a new recombinant retroviral system in which a library of infectious molecular clones of human immunodeficiency virus type 1 (HIV-1) is constructed with reverse transcriptase (RT) genes derived from viral RNA sequences in plasma. HIV-1 RT is amplified from plasma HIV-1 RNA by nested RT-PCR and cloned into a RT-defective HIV-1 proviral vector (xxLAI-np), generating 10(3) to 10(4) recombinant proviral clones from each reaction. The bulk cloning products or individual molecular clones are transfected into MT-2 cells to generate infectious virus. The resultant viruses are assayed for drug susceptibility in CD4+ cell lines to determine either the dominant phenotype of the recombinant virus mixture or the phenotypes of the individual viral clones. DNA sequencing of the cloned RT genes can identify mutations associated with phenotypic resistance of clonal mixtures or individual clones. This method can be used to rapidly detect the in vivo emergence of HIV-1 quasispecies resistant to RT inhibitors.
我们开发了一种新的重组逆转录病毒系统，其中构建了一个人类免疫缺陷病毒1型（HIV-1）感染性分子克隆文库，其逆转录酶（RT）基因来源于血浆中的病毒RNA序列。通过巢式逆转录聚合酶链反应（RT-PCR）从血浆HIV-1 RNA中扩增HIV-1 RT，并将其克隆到一个RT缺陷型HIV-1前病毒载体（xxLAI-np）中，每个反应产生10³至10⁴个重组前病毒克隆。将大量克隆产物或单个分子克隆转染到MT-2细胞中以产生感染性病毒。对所得病毒在CD4⁺细胞系中进行药物敏感性测定，以确定重组病毒混合物的主要表型或单个病毒克隆的表型。对克隆的RT基因进行DNA测序可鉴定与克隆混合物或单个克隆的表型抗性相关的突变。该方法可用于快速检测体内对RT抑制剂耐药的HIV-1准种的出现。
