Fang H, Pincus S H
Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
J Virol. 1995 Jan;69(1):75-81. doi: 10.1128/JVI.69.1.75-81.1995.
To study the variability of human immunodeficiency virus type 1 (HIV-1), we used immunotoxins to select for variants within a population of H9 cells persistently infected with a molecular clone of HIV-1 designated NL4-3. Chimeric immunotoxin CD4-PE40 (a chimeric fusion protein consisting of the amino-terminal two domains of CD4 and the carboxy-terminal domains of Pseudomonas exotoxin A) was used to select for cells lacking cell surface expression of HIV Env (envelope proteins gp160, gp120, and gp41). The cells described here (A1, A7, C9, and E9) fail to express HIV proteins because they have markedly diminished transcription of the integrated provirus (A1, A7, and E9) or no HIV provirus (C9). Analysis demonstrated that two different cloned variants, A1 and E9, contain the complementary sequence of tRNA(3Lys) (45 bp) inserted 3' to the primer-binding site, following by a 169-bp deletion through the start of the gag gene. No HIV mRNA was detected by Northern (RNA) blotting, but PCR demonstrated the presence of the viral message. These variants were found very infrequently in the unselected H9/NL4-3 cell population, and they contained proviruses distinct from that found in the dominant population. In addition, all of these variants had similar patterns of CD4 surface expression that allowed them to escape reinfection within the tissue culture. The data are discussed with regard to mechanisms and errors of HIV reverse transcription, as well as the evolution of mutants within a population of persistently infected cells.
为研究1型人类免疫缺陷病毒（HIV-1）的变异性，我们使用免疫毒素从持续感染HIV-1分子克隆NL4-3的H9细胞群体中筛选变异体。嵌合免疫毒素CD4-PE40（一种由CD4的氨基末端两个结构域和铜绿假单胞菌外毒素A的羧基末端结构域组成的嵌合融合蛋白）用于筛选缺乏HIV包膜（Env）（包膜蛋白gp160、gp120和gp41）细胞表面表达的细胞。此处描述的细胞（A1、A7、C9和E9）不表达HIV蛋白，因为它们整合的前病毒转录明显减少（A1、A7和E9）或没有HIV前病毒（C9）。分析表明，两个不同的克隆变异体A1和E9在引物结合位点下游3'处含有插入的tRNA(3Lys)互补序列（45 bp），随后是从gag基因起始处开始的169 bp缺失。通过Northern（RNA）印迹未检测到HIV mRNA，但PCR证明存在病毒信息。这些变异体在未筛选的H9/NL4-3细胞群体中很少发现，并且它们含有与优势群体中发现的前病毒不同的前病毒。此外，所有这些变异体具有相似的CD4表面表达模式，使它们能够在组织培养中逃避再次感染。我们将结合HIV逆转录的机制和错误以及持续感染细胞群体中突变体的进化来讨论这些数据。
