Cuconati A, Xiang W, Lahser F, Pfister T, Wimmer E
Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, 11794, USA.
J Virol. 1998 Feb;72(2):1297-307. doi: 10.1128/JVI.72.2.1297-1307.1998.
The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2A(pro), 2BC, 2C, and 2B. Specifically, 2A(pro) can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. transdominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341-4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome.
酵母双杂交系统用于梳理1型脊髓灰质炎病毒（Mahoney株）P2非结构裂解产物之间所有可检测到的相互作用。已获得证据表明2A蛋白酶（2A(pro)）、2BC、2C和2B之间存在特异性关联。具体而言，2A(pro)可与自身相互作用，2BC及其裂解产物（2B和2C）能以所有可能的组合相互作用，但2C/2C组合除外。通过谷胱甘肽S-转移酶下拉试验在体外证实了检测到的相互作用，该试验使我们能够检测到2C/2C关联。对2B的反式显性负突变体（K. Johnson和P. J. Sarnow，《病毒学杂志》65:4341 - 4349，1991年）进行了检测，发现其与野生型2B仍保持相互作用，这可能反映了病毒RNA复制中2B多聚化的必要性。通过筛选诱变文库寻找失去结合野生型2B能力的2B变体，进一步研究了2B的多聚化。筛选鉴定出中央疏水区域内的两个非保守错义突变，以及涉及C末端同源相互作用的截短和移码突变。将错义突变引入病毒基因组赋予了准感染表型，这一观察结果强烈表明病毒基因组复制需要2B/2B相互作用。
