Granowitz C, Berkowitz R D, Goff S P
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Virus Res. 1996 Mar;41(1):25-42. doi: 10.1016/0168-1702(95)01278-8.
Five premature termination mutations and five missense mutations were introduced into the portion of cloned Moloney murine leukemia virus (M-MuLV) DNA encoding the Env cytoplasmic domain. All of the mutant DNAs gave rise to replication-competent virus after transfection of NIH/3T3 cells, but several of the mutant DNAs scored as replication-defective when introduced into Rat2 cells. Cell lines stably expressing the mutant DNAs all released virion particles, and in all but one case infectious virus were generated. These viable mutants were all found to have reverted to the wild-type sequence. To generate fully mutant virus stocks, the mutant DNAs were introduced transiently into COS cells, which are resistant to infection with MuLV, thus prohibiting reversion by error-prone mechanisms involving reverse transcription. Virions harvested from the COS cells were confirmed as mutant by analyzing both virion proteins and the viral DNA they generated, and were then tested for infectivity in NIH/3T3 cells. The mutant viruses were infectious, but still rapidly gave rise to revertants. We conclude that the mutations within the cytoplasmic domain do not provide an absolute block to virus replication, but that the mutants replicate more slowly than the wild-type and quickly give rise to revertants with selective advantage for replication.
将五个提前终止突变和五个错义突变引入克隆的莫洛尼鼠白血病病毒（M-MuLV）DNA编码Env胞质结构域的部分。在转染NIH/3T3细胞后，所有突变DNA都产生了具有复制能力的病毒，但当将其中一些突变DNA引入Rat2细胞时，有几个被判定为复制缺陷型。稳定表达突变DNA的细胞系均释放出病毒粒子，除一种情况外，均产生了感染性病毒。发现这些存活的突变体均已回复为野生型序列。为了产生完全突变的病毒株，将突变DNA瞬时引入对MuLV感染具有抗性的COS细胞中，从而通过涉及逆转录的易出错机制阻止回复。通过分析病毒粒子蛋白及其产生的病毒DNA，确认从COS细胞收获的病毒粒子为突变体，然后在NIH/3T3细胞中测试其感染性。突变病毒具有感染性，但仍迅速产生回复体。我们得出结论，胞质结构域内的突变不会对病毒复制产生绝对阻碍，但突变体的复制速度比野生型慢，并迅速产生具有复制选择优势的回复体。
