Lu H H, Li X, Cuconati A, Wimmer E
Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11794-5222, USA.
J Virol. 1995 Dec;69(12):7445-52. doi: 10.1128/JVI.69.12.7445-7452.1995.
The poliovirus (PV) genome was manipulated by replacing its 2A-encoding sequence with the corresponding sequence of coxsackie B4 virus (CBV4) or human rhinovirus type 2 (HRV2). In vitro translation of the resulting chimeric PV genomes revealed a normal cis-cleavage activity for both heterologous 2A(pro) proteinases in the chimeric PV polyproteins. However, only the genome containing the 2A-encoding sequence of CBV4 (PV/CBV4-2A) yielded viable virus in transfected cells, producing a mixture of large and small plaques on HeLa cell monolayers. The large-plaque variants were found to contain single-amino-acid mutations at a specific site near the C terminus of the CBV4 2A(pro) protein. When the same single-amino-acid mutations were directly introduced into the parental PV/CBV4-2A genome, chimeric viruses with a large-plaque phenotype and a wild-type PV-like growth pattern were obtained upon transfection, an observation demonstrating that these point mutations alone had a drastic effect on the growth of the PV/CBV4 chimeric virus. On the other hand, the chimeric genome containing the 2A-encoding sequence of HRV2 (PV/HRV2-2A) produced a null phenotype in transfected HeLa cells, although low-level replication of this chimeric genome was evident. We conclude that only 2A(pro) of the more closely related enterovirus CBV4 is able to functionally substitute for that of PV in vivo, and a subtle genetic modification of the CBV4 2A(pro) protein results in a drastic improvement in the growth of the chimeric PV/CBV4-2A virus. In addition, this chimeric cDNA approach enabled us to dissect multiple biological functions encoded by the 2A(pro) proteins.
通过用柯萨奇B4病毒（CBV4）或人鼻病毒2型（HRV2）的相应序列替换脊髓灰质炎病毒（PV）基因组的2A编码序列，对PV基因组进行了操作。对所得嵌合PV基因组进行体外翻译，结果显示嵌合PV多聚蛋白中的两种异源2A（pro）蛋白酶均具有正常的顺式切割活性。然而，只有包含CBV4 2A编码序列的基因组（PV/CBV4-2A）在转染细胞中产生了有活力的病毒，在HeLa细胞单层上形成了大小斑块的混合物。发现大斑块变体在CBV4 2A（pro）蛋白C末端附近的特定位点含有单氨基酸突变。当将相同的单氨基酸突变直接引入亲本PV/CBV4-2A基因组时，转染后获得了具有大斑块表型和野生型PV样生长模式的嵌合病毒，这一观察结果表明，仅这些点突变就对PV/CBV4嵌合病毒的生长产生了巨大影响。另一方面，包含HRV2 2A编码序列的嵌合基因组（PV/HRV2-2A）在转染的HeLa细胞中产生了无效表型，尽管该嵌合基因组有明显的低水平复制。我们得出结论，只有亲缘关系更近的肠道病毒CBV4的2A（pro）能够在体内功能性替代PV的2A（pro），并且CBV4 2A（pro）蛋白的细微基因修饰导致嵌合PV/CBV4-2A病毒的生长有显著改善。此外，这种嵌合cDNA方法使我们能够剖析2A（pro）蛋白编码的多种生物学功能。
