A DELETION MUTATION IN THE 5' PART OF THE POL GENE OF MOLONEY MURINE LEUKEMIA-VIRUS BLOCKS PROTEOLYTIC PROCESSING OF THE GAG AND POL POLYPROTEINS
A DELETION MUTATION IN THE 5' PART OF THE POL GENE OF MOLONEY MURINE LEUKEMIA-VIRUS BLOCKS PROTEOLYTIC PROCESSING OF THE GAG AND POL POLYPROTEINS
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DOI：
10.1128/jvi.53.3.899-907.1985
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发表时间：
1985-01-01
影响因子：
5.4
通讯作者：
GOFF, SP
中科院分区：
文献类型：
--
作者：
CRAWFORD, S;GOFF, SP
通过克隆的前病毒DNA的限制性酶定点诱变，在Moloney鼠白血病病毒的pol基因的5“部分产生缺失突变。DNA序列分析表明，一个这样的缺失完全位于pol基因的5“部分，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。主要的病毒前体多聚蛋白（Pr 65 gag-pol和gPr 80 env）在携带突变基因组的细胞系中以野生型水平合成。这些细胞系组装并将野生型水平的病毒粒子颗粒释放到培养基中。Pr 65 gag和Pr 200 gag-pol前体到成熟蛋白的切割在突变体病毒体中被完全阻断。这些病毒体含有高水平的活性逆转录酶;对突变病毒体合成的内源性逆转录产物的检查显示负链强终止DNA的量正常，表明RNA基因组被包装，并且在洗涤剂渗透的病毒体中的逆转录没有显著受损。gPr 80 env到gP 70 env和P15 E的加工不受突变的影响，但未观察到P15 E到P12 E的切割。突变体颗粒的感染性差;分析表明，感染在早期阶段被阻断。这些数据与pol基因的5“部分编码直接负责将Pr 65 gag和可能的Pr 200 gag-pol加工成结构病毒体蛋白的蛋白酶的想法一致。似乎gag基因产物的切割对于病毒体的出芽和释放不是必需的，并且pol基因产物完全加工成成熟形式的逆转录酶对于其功能活化不是必需的。
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-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. 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 infeciton 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.