Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, United States.
Virus Res. 2011 Jan;155(1):91-7. doi: 10.1016/j.virusres.2010.09.004. Epub 2010 Sep 16.
Foot-and-mouth disease virus (FMDV) initiates translation from two in-frame AUG codons producing two forms of the leader (L) proteinase, Lab (starting at the first AUG) and Lb (starting at second AUG). In a previous study, we have demonstrated that a cDNA-derived mutant FMDV (A24-L1123) containing a 57-nucleotide transposon (tn) insertion between the two AUG initiation codons (inter-AUG region) was completely attenuated in cattle, suggesting that this region is involved in viral pathogenesis. To investigate the potential role of the Lab protein in attenuation, we have introduced two epitope tags (Flag: DYKDDDK and HA: YPYDVPDYA) or a small tetracysteine motif (tc: CCGPCC) into the pA24-L1123 infectious DNA clone. Mutant viruses with a small plaque phenotype similar to the parental A24-L1123 were recovered after transfection of constructs encoding the Flag tag and the tc motif. However, expression of the Flag- or tc-tagged Lab protein was abolished or greatly diminished in these viruses. Interestingly, the A24-L1123/Flag virus acquired an extra base in the inter-AUG region that resulted in new AUG codons in-frame with the second AUG, and produced a larger Lb protein. This N terminal extension of the Lb protein in mutant A24-L1123/Flag did not affect virus viability or L functions in cell culture.
口蹄疫病毒（FMDV）从两个框架内 AUG 密码子起始翻译，产生两种形式的先导（L）蛋白酶，Lab（从第一个 AUG 开始）和 Lb（从第二个 AUG 开始）。在之前的研究中，我们已经证明，一种 cDNA 衍生的突变型 FMDV（A24-L1123）在两个 AUG 起始密码子（AUG 间隔区）之间含有一个 57 个核苷酸的转座子（tn）插入，在牛中完全减毒，表明该区域参与病毒发病机制。为了研究 Lab 蛋白在减毒中的潜在作用，我们在 pA24-L1123 感染性 DNA 克隆中引入了两个表位标签（Flag：DYKDDDK 和 HA：YPYDVPDYA）或一个小四肽基序（tc：CCGPCC）。在转染编码 Flag 标签和 tc 基序的构建体后，回收了具有类似于亲本 A24-L1123 的小斑表型的突变病毒。然而，在这些病毒中，Flag 或 tc 标记的 Lab 蛋白的表达被废除或大大减少。有趣的是，A24-L1123/Flag 病毒在 AUG 间隔区获得了一个额外的碱基，导致与第二个 AUG 框内的新 AUG 密码子，并产生了更大的 Lb 蛋白。突变型 A24-L1123/Flag 中的 Lb 蛋白的 N 端延伸不会影响病毒活力或 L 在细胞培养中的功能。
