Kuhn R J, Tada H, Ypma-Wong M F, Dunn J J, Semler B L, Wimmer E
Department of Microbiology, State University of New York, Stony Brook 11794.
Proc Natl Acad Sci U S A. 1988 Jan;85(2):519-23. doi: 10.1073/pnas.85.2.519.
By following a strategy of genetic analysis of poliovirus, we have constructed a synthetic "mutagenesis cartridge" spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type 1 (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed us to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. We suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).
通过采用脊髓灰质炎病毒的遗传分析策略，我们构建了一个合成的“诱变盒”，它跨越了1型（马奥尼株）基因组感染性cDNA克隆中与基因组相连的病毒蛋白编码区及侧翼切割位点。在感染性克隆中插入新的限制性酶切位点，使我们能够用含有各种突变的短互补合成寡核苷酸对替换野生型序列。已进行了一组突变，在与基因组相连的病毒蛋白区域内产生了甲硫氨酸密码子。所得病毒具有与野生型相似的生长特性。导致负责与RNA连接的酪氨酸残基发生改变的实验产生了无活力的病毒。在一个突变体中，体外检测的蛋白水解加工似乎未受该突变影响。我们认为酪氨酸残基的位置对与基因组相连的病毒蛋白功能很重要。
