Lee P, Hruby D E
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
J Virol. 1993 Jul;67(7):4252-63. doi: 10.1128/JVI.67.7.4252-4263.1993.
The three major vaccinia virus (VV) virion proteins (4a, 4b, and 25K) are proteolytically matured from larger precursors (P4a, P4b, and P25K) during virus assembly. Within the precursors, Ala-Gly-X motifs have been noted at the putative processing sites, with cleavage apparently taking place between the Gly and X residues. To identify the sequence and/or structural parameters which are required to define an efficient cleavage site, a trans-processing assay system has been developed by tagging the carboxy terminus of the P25K polypeptide (precursor of 25K) with an octapeptide FLAG epitope, which can be specifically recognized by a monoclonal antibody. By using transient expression assays with cells coinfected with VV, the proteolytic processing of the chimeric gene product (P25K:FLAG) was monitored by immunoblotting procedures. The relationship between the P25K:FLAG precursor and the 25K:FLAG cleavage product was established by pulse-chase experiments. The in vivo cleavage of P25K:FLAG was inhibited by the drug rifampin, implying that the reaction was utilizing the same pathway as authentic VV core proteins. Moreover, the 25K:FLAG protein was found in association with mature virions in accord with the notion that cleavage occurs concomitantly with virion assembly. Site-directed mutagenesis of the Ala-Gly-Ala motif at residues 31 to 33 of the P25K:FLAG precursor to Ile-Asp-Ile blocked production of the 25K:FLAG product. The efficiency of 25K:FLAG production (33.71%) is, however, approximately only half of the production of 25K (63.98%) within VV-infected cells transfected with pL4R:FLAG. One explanation for the lower efficiency of 25K:FLAG production was suggested by the observation in the immunofluorescent-staining experiment that 25K:FLAG-related proteins were not specifically localized to the virus assembly factories (virosomes) within VV-infected cells, although virosome localization was prominent for P25K-related polypeptides. Since VV core protein proteolytic processing is believed to take place during virion maturation, only the P25K:FLAG which was assembled into immature virions could undergo proteolytic maturation. Furthermore during these experiments, a potential cleavage intermediate (25K') of P25K was identified. Amino acid residues 17 to 19 (Ala-Gly-Ser) of the P25K precursor were implicated as the intermediate cleavage site, since no 25K':FLAG product was produced from a mutant precursor in which the sequence was altered to Ile-Asp-Ile. Taken together, these results provide biochemical and genetic evidence to support the hypothesis that the Ala-Gly-X cleavage motif plays a critical role in VV virion protein proteolytic maturation.
三种主要的痘苗病毒（VV）病毒粒子蛋白（4a、4b和25K）在病毒组装过程中从较大的前体（P4a、P4b和P25K）经蛋白水解成熟而来。在前体中，已在假定的加工位点发现丙氨酸 - 甘氨酸 - X基序，切割显然发生在甘氨酸和X残基之间。为了确定定义有效切割位点所需的序列和/或结构参数，通过用八肽FLAG表位标记P25K多肽（25K的前体）的羧基末端开发了一种转加工分析系统，该表位可被单克隆抗体特异性识别。通过对共感染VV的细胞进行瞬时表达分析，通过免疫印迹程序监测嵌合基因产物（P25K:FLAG）的蛋白水解加工。通过脉冲追踪实验确定了P25K:FLAG前体与25K:FLAG切割产物之间的关系。P25K:FLAG的体内切割受到利福平药物的抑制，这意味着该反应与真实的VV核心蛋白利用相同的途径。此外，发现25K:FLAG蛋白与成熟病毒粒子相关，这与切割与病毒粒子组装同时发生的观点一致。将P25K:FLAG前体第31至33位残基的丙氨酸 - 甘氨酸 - 丙氨酸基序定点突变为异亮氨酸 - 天冬氨酸 - 异亮氨酸可阻断25K:FLAG产物的产生。然而，在转染了pL4R:FLAG的VV感染细胞中，25K:FLAG的产生效率（33.71%）大约仅为25K产生效率（63.98%）的一半。免疫荧光染色实验中的观察结果为25K:FLAG产生效率较低提供了一种解释，即尽管P25K相关多肽在病毒体定位方面很突出，但25K:FLAG相关蛋白在VV感染细胞内并未特异性定位于病毒组装工厂（病毒体）。由于认为VV核心蛋白的蛋白水解加工发生在病毒粒子成熟过程中，只有组装到未成熟病毒粒子中的P25K:FLAG才能进行蛋白水解成熟。此外，在这些实验过程中，鉴定出了P25K的一种潜在切割中间体（25K'）。P25K前体的第17至19位氨基酸残基（丙氨酸 - 甘氨酸 - 丝氨酸）被认为是中间切割位点，因为从序列改变为异亮氨酸 - 天冬氨酸 - 异亮氨酸的突变前体中未产生25K':FLAG产物。综上所述，这些结果提供了生化和遗传证据来支持丙氨酸 - 甘氨酸 - X切割基序在VV病毒粒子蛋白水解成熟中起关键作用这一假说。
