Schatz G, Pichova I, Vogt V M
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
J Virol. 1997 Jan;71(1):444-50. doi: 10.1128/JVI.71.1.444-450.1997.
In retroviruses, the viral protease (PR) is released as a mature protein by cleavage of Gag, Gag-Pro, or Gag-Pro-Pol precursor polypeptides. In avian sarcoma and leukemia viruses (ASLV), PR forms the C-terminal domain of Gag. Based on the properties of a mutation (cs22) in the cleavage site between the upstream NC domain and the PR domain, the proteolytic liberation of PR previously was inferred to be essential for processing of Gag and Pol proteins. To study this process in more detail, we have analyzed the effects that several mutations at the NC-PR cleavage site have on proteolytic processing in virus-like particles expressed in COS and quail cells. Mutant Gag proteins carrying the same mutations also were synthesized in vitro and tested for processing with purified PR. In both types of studies, N-terminal sequencing of the liberated PR domain was carried out to exactly identify the site of cleavage. Finally, synthetic peptides corresponding to the mutant proteins were assessed for the ability to act as substrates for PR. The results were all consistent and led to the following conclusions. (i) In vivo, if normal processing between NC and PR is prevented by mutations, limited cleavage occurs at a previously unrecognized alternative site three amino acids downstream, i.e., in PR. This N-terminally truncated PR is inactive as an enzyme, as inferred from the global processing defect in cs22 and a similar mutant. (ii) In Gag proteins translated in vitro, purified PR cleaves this alternative site as rapidly as it does the wild-type site. (iii) Contrary to previously accepted rules describing retroviral cleavage sites, an isoleucine residue placed at the P1 position of the NC-PR cleavage site does not hinder normal processing. (iv) A proline residue placed at the P2 position in this cleavage site blocks normal processing.
在逆转录病毒中，病毒蛋白酶（PR）通过切割Gag、Gag-Pro或Gag-Pro-Pol前体多肽以成熟蛋白的形式释放。在禽肉瘤和白血病病毒（ASLV）中，PR形成Gag的C末端结构域。基于上游NC结构域和PR结构域之间切割位点的一个突变（cs22）的特性，先前推断PR的蛋白水解释放对于Gag和Pol蛋白的加工至关重要。为了更详细地研究这一过程，我们分析了NC-PR切割位点的几个突变对在COS和鹌鹑细胞中表达的病毒样颗粒中蛋白水解加工的影响。携带相同突变的突变型Gag蛋白也在体外合成，并与纯化的PR一起进行加工测试。在这两种类型的研究中，对释放的PR结构域进行N端测序以准确鉴定切割位点。最后，评估与突变蛋白相对应的合成肽作为PR底物的能力。结果均一致，并得出以下结论。（i）在体内，如果突变阻止了NC和PR之间的正常加工，则在下游三个氨基酸处一个先前未被识别的替代位点（即PR中）会发生有限的切割。从cs22和一个类似突变体中的整体加工缺陷推断，这种N端截短的PR作为酶是无活性的。（ii）在体外翻译的Gag蛋白中，纯化的PR切割这个替代位点的速度与切割野生型位点的速度一样快。（iii）与先前描述逆转录病毒切割位点的规则相反，位于NC-PR切割位点P1位置的异亮氨酸残基并不妨碍正常加工。（iv）在这个切割位点的P2位置放置一个脯氨酸残基会阻止正常加工。
