Palmenberg A C, Parks G D, Hall D J, Ingraham R H, Seng T W, Pallai P V
Institute for Molecular Virology, University of Wisconsin, Madison 53706.
Virology. 1992 Oct;190(2):754-62. doi: 10.1016/0042-6822(92)90913-a.
The primary 2A/2B cleavage within cardiovirus polyprotein was examined by construction of cDNA plasmids which linked fragments from the P2 region of encephalomyocarditis virus (EMCV) and Mengovirus genomes to the EMCV 5' nontranslated region. When RNA transcripts from these clones were tested in reticulocyte extracts, the synthesized proteins were cotranslationally processed at the 2A/2B site. No viral segments outside of the P2 region were required for this activity. Engineered deletions which removed the amino-terminal two-thirds of protein 2A or the carboxyl half of protein 2B had no effect on this scission, nor did insertions into a Ser-Ala-Phe sequence (SAF) within 2B, which is conserved in most cardio- and aphthoviruses. In contrast, mutations which disrupted a conserved Asn-Pro-Gly-Pro (NPGP) sequence abolished primary scission. Precursors thus inactivated were unable to serve as substrate when simultaneously expressed with active (wild-type) 2AB sequences. Microsequencing placed the EMCV primary cleavage site between the Gly/Pro pair within the NPGP sequence. It was also determined that endogenous viral protease 3C is the previously unidentified agent responsible for cardiovirus 1D/2A scission, a cleavage that is part of the primary processing reaction in poliovirus.
通过构建cDNA质粒来研究心病毒多聚蛋白内的主要2A/2B切割，这些质粒将脑心肌炎病毒（EMCV）和 Mengovirus 基因组P2区域的片段与EMCV 5'非翻译区域连接起来。当在网织红细胞提取物中测试这些克隆的RNA转录本时，合成的蛋白质在2A/2B位点进行共翻译加工。此活性不需要P2区域以外的病毒片段。去除蛋白质2A氨基末端三分之二或蛋白质2B羧基一半的工程缺失对这种切割没有影响，在2B内插入Ser-Ala-Phe序列（SAF）也没有影响，该序列在大多数心病毒和口蹄疫病毒中是保守的。相比之下，破坏保守的Asn-Pro-Gly-Pro（NPGP）序列的突变消除了主要切割。当与活性（野生型）2AB序列同时表达时，如此失活的前体不能作为底物。微量测序将EMCV主要切割位点定位在NPGP序列内的Gly/Pro对之间。还确定内源性病毒蛋白酶3C是以前未鉴定出的负责心病毒1D/2A切割的因子，这种切割是脊髓灰质炎病毒初级加工反应的一部分。
