Fujinaga K, Nakamura Y, Zhong Q, Nakaya T, Ikuta K
Section of Serology, Hokkaido University, Sapporo, Japan.
Biochem Biophys Res Commun. 1996 Dec 4;229(1):96-101. doi: 10.1006/bbrc.1996.1763.
We prepared a series of nef mutant HIV-1 with a frameshift mutation at the Xho I site by up to 50 serial transfers into MT-4 cells. Here, we studied revertants. Immunofluorescence using an anti-Nef monoclonal antibody revealed that cells first became Nef antigen-positive at the 23rd passage. The percentage of Nef antigen-positive cells gradually increased and reached almost 100% by the 27th passage. The sequence of the provirus in the cells supported the generation of a revertant. This revertant mutated at the site immediately after the initially introduced frameshift mutation. This resulted in the substitution of only three amino acids and the insertion of two, which restored the proline-rich domain, a conserved region believed essential to viral replication, at the middle of Nef. Thus, the growth dominance of the revertant virus, compared with the original nef mutant, was directly demonstrated in vitro using serial passages consisting of mixed HIV-1 populations in a single cell.
我们通过在MT-4细胞中进行多达50次连续传代，制备了一系列在Xho I位点具有移码突变的nef突变型HIV-1。在此，我们研究了回复突变体。使用抗Nef单克隆抗体的免疫荧光显示，细胞在第23代时首次变为Nef抗原阳性。Nef抗原阳性细胞的百分比逐渐增加，到第27代时几乎达到100%。细胞中前病毒的序列支持回复突变体的产生。这种回复突变体在最初引入的移码突变后的位点发生了突变。这仅导致三个氨基酸的替换和两个氨基酸的插入，从而在Nef中间恢复了富含脯氨酸的结构域，这是一个被认为对病毒复制至关重要的保守区域。因此，在体外使用由单个细胞中的混合HIV-1群体组成的连续传代，直接证明了回复突变病毒相对于原始nef突变体的生长优势。
