Crawford S, Goff S P
J Virol. 1985 Mar;53(3):899-907. doi: 10.1128/JVI.53.3.899-907.1985.
Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation.
通过对克隆的前病毒DNA进行限制性酶切位点定向诱变，产生了莫洛尼鼠白血病病毒pol基因5'端的缺失突变。DNA序列分析表明，其中一个这样的缺失完全定位在pol基因的5'端，不影响编码逆转录酶的区域，并保留了突变下游的翻译阅读框。在携带突变基因组的细胞系中，主要的病毒前体多蛋白（Pr65gag、Pr200gag-pol和gPr80env）以野生型水平合成。这些细胞系组装并向培养基中释放野生型水平的病毒粒子。在突变病毒粒子中，Pr65gag和Pr200gag-pol前体向成熟蛋白的切割完全受阻。令人惊讶的是，这些病毒粒子含有高水平的活性逆转录酶；对突变病毒粒子合成的内源性逆转录产物的检测显示，负链强终止DNA的量正常，表明RNA基因组被包装，并且在去污剂通透的病毒粒子中的逆转录没有受到显著损害。gPr80env加工成gP70env和P15E不受突变影响，但未观察到P15E切割成P12E。突变粒子的感染性很差；分析表明感染在早期被阻断。这些数据与以下观点一致：pol基因的5'端编码一种蛋白酶，直接负责将Pr65gag，可能还有Pr200gag-pol加工成病毒粒子的结构蛋白。看来，病毒粒子的出芽和释放不需要gag基因产物的切割，并且pol基因产物完全加工成逆转录酶的成熟形式对其功能激活也不是必需的。
