Eisenman R N, Mason W S, Linial M
J Virol. 1980 Oct;36(1):62-78. doi: 10.1128/JVI.36.1.62-78.1980.
We have studied the biosynthesis of avian retrovirus proteins related to reverse transcriptase in permissive avian embryonic cells. Analysis of immune precipitates from avian sarcoma virus (ASV)-infected cells demonstrated the presence of the 180,000-dalton gag-pol "read-through" protein (Pr180gag-pol) and a 130,000-dalton polypeptide (Pr130gag-pol). Pr130gag-pol was found, in serological and peptide mapping studies, to consist primarily of sequences related to reverse transcriptase and the gag-encoded protein p15. Pr180gag-pol was found to be phosphorylated, whereas Pr130gag-pol was not. In addition, only Pr180gag-pol but not Pr130gag-pol was susceptible to cleavage with the virion protease p15. Although the structure of Pr130gag-pol would suggest that it is generated by removal of a portion of the gag region from Pr180gag-pol, an analysis of labeling kinetics has failed to demonstrate unequivocally whether Pr130gag-pol is a cleavage product of Pr180gag-pol or a primary translation product. We were repeatedly unable to detect either Pr180gag-pol or Pr130gag-pol in virus particles released from the cell, whereas both beta and alpha subunits were readily observed. Several presumed intermediates between Pr130gag-pol and the beta subunit of reverse transcriptase were also observed in virions. These studies indicate cleavage of polyemrase precursors at the time of virus budding. On the basis of these data, we present a processing scheme for the generation of reverse transcriptase subunits. We have also examined reverse transcriptase biosynthesis in cells producing two mutants that fail to package the enzyme. Previous work showed that integrated proviruses of both mutants are missing DNA sequences in pol: one mutant, PH9 (Mason et al., J. Virol. 30:132-140, 1979), contains a deletion near the 3' end of pol, whereas the other, SE52d (linial et al., Virology 87:130-141, 1978), may have inserted a host cell sequence near the 5' end of pol. Neither mutant synthesized Pr180gag-pol or Pr130gag-pol, but instead produced novel proteins comprised of sequences shared with gag proteins plus a region antigenically related to reverse transcriptase. Both proteins were defective as precursors to reverse transcriptase. Whereas Pr180gag-pol and Pr130gag-pol were precipitated by an antiserum raised against p32 (a virion protein derived from the portion of the beta subunit removed during processing of beta to alpha [Schiff and Grandgenett, J. Virol. 28:279-291, 1978]), the novel protein synthesized by PH9 ws not precipitated. This suggets that the alpha subunit is generated by a COOH-terminal cleavage of the beta subunit.
我们研究了在允许性禽胚胎细胞中与逆转录酶相关的禽逆转录病毒蛋白的生物合成。对感染禽肉瘤病毒（ASV）的细胞的免疫沉淀物分析表明，存在180,000道尔顿的gag-pol“通读”蛋白（Pr180gag-pol）和130,000道尔顿的多肽（Pr130gag-pol）。在血清学和肽图谱研究中发现，Pr130gag-pol主要由与逆转录酶和gag编码蛋白p15相关的序列组成。发现Pr180gag-pol被磷酸化，而Pr130gag-pol未被磷酸化。此外，只有Pr180gag-pol而不是Pr130gag-pol易被病毒体蛋白酶p15切割。尽管Pr130gag-pol的结构表明它是通过从Pr180gag-pol中去除一部分gag区域而产生的，但标记动力学分析未能明确证明Pr130gag-pol是Pr180gag-pol的切割产物还是初级翻译产物。我们反复无法在从细胞释放的病毒颗粒中检测到Pr180gag-pol或Pr130gag-pol，而β和α亚基很容易观察到。在病毒体中也观察到了几种Pr130gag-pol与逆转录酶β亚基之间的假定中间体。这些研究表明在病毒出芽时多聚酶前体发生了切割。基于这些数据，我们提出了一个产生逆转录酶亚基的加工方案。我们还研究了在产生两种不能包装该酶的突变体的细胞中的逆转录酶生物合成。先前的工作表明，两种突变体的整合前病毒在pol中缺失DNA序列：一种突变体PH9（Mason等人，《病毒学杂志》30:132 - 140，1979）在pol的3'末端附近有一个缺失，而另一种突变体SE52d（Linial等人，《病毒学》87:130 - 141，1978）可能在pol的5'末端附近插入了一个宿主细胞序列。两种突变体都不合成Pr180gag-pol或Pr130gag-pol，而是产生了由与gag蛋白共享的序列加上与逆转录酶抗原相关的区域组成的新蛋白。两种蛋白作为逆转录酶的前体都有缺陷。用针对p32（一种病毒体蛋白，源自β亚基在加工成α亚基过程中去除的部分[Schiff和Grandgenett，《病毒学杂志》28:279 - 291，1978]）产生的抗血清沉淀时，Pr180gag-pol和Pr130gag-pol都能被沉淀，而PH9合成的新蛋白不能被沉淀。这表明α亚基是由β亚基的COOH末端切割产生的。
