Recent study has shown that the C-terminal portion of 3A (amino acids (aa) 81–153) is not essential for foot-and-mouth disease virus replication in cell culture, however, the complete C-terminal portion (aa 77–153) of 3A is highly variable and prone to occur deletions and mutations, therefore, we presume that this region plays a very limited role and probablely is completely nonessential for virus viability. In this study, to identify the largest non-essential region of the C-terminal portion in 3A for FMDV viability, several deletions containing aa 80–153, 77–153 and 76–153 of 3A protein were introduced into an FMDV full-length infectious cDNA clone pOFS by the overlapping extension PCR. Additionally, to explore the importance of the highly conserved residue 76 L of 3A for the FMDV of Cathay topotype, two mutants containing 3A L76I and 3A L76V were generated based on the 3A deletion mutant by point mutation. We also introduced the enhanced green fluorescent protein (eGFP) into one of the 3A deletion mutants by the extension PCR to investigate the genetic flexibility of 3A to express foreign genes. All linearized full plasmids were transfected into BSR/T7 cells to rescue infectious foot-and-mouth disease viruses. The rescused viruses were analyzed by RT-PCR, nucleotide sequencing, immunofluorescence assay and western blot and were characterized by plaque assays and one-step growth kinetics. The results demonstrated that the deletion of aa 80–153 and aa 77–153 and the substitutions of 3A L76I and 3A L76V did not affect the production of infectious virus, while the fusion of the eGFP gene to the C-terminus of 3A resulted in nonviable FMDV. Our results firstly reported that the aa 77–153 rather than aa 81–153 of 3A protein was dispensable for FMDV replication in cell culture. This study is of great significance for development of FMD marker vaccine and foreign gene expression in the future.

中文翻译：

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鉴定口蹄疫病毒3A蛋白C端部分最大的非必需区域，以便在细胞培养中复制。

最近的研究表明，3A的C末端部分（氨基酸（aa）81–153）对于细胞培养中的口蹄疫病毒复制不是必不可少的，但是，完整的C末端部分（aa 77– 3A的153）具有很高的可变性，并且易于发生缺失和突变，因此，我们推测该区域的作用非常有限，可能对于病毒的生存力完全不重要。在这项研究中，为了确定FMDV活力中3A中C末端部分的最大非必需区域，将3A氨基酸80–153、77–153和76–153的几个缺失引入了FMDV全长重叠延伸PCR扩增感染性cDNA克隆pOFS。此外，为了探究高度保守的3A残基76 L对国泰拓扑型FMDV的重要性，基于点突变的3A缺失突变体产生了两个包含3A L76I和3A L76V的突变体。我们还通过扩展PCR将增强的绿色荧光蛋白（eGFP）引入了3A缺失突变体之一，以研究3A表达外源基因的遗传灵活性。将所有线性化的完整质粒转染到BSR / T7细胞中，以挽救传染性口蹄疫病毒。通过RT-PCR，核苷酸测序，免疫荧光测定和western印迹分析了所回收的病毒，并通过噬斑测定和一步生长动力学对其进行了表征。结果表明，aa 80–153和aa 77–153的缺失以及3A L76I和3A L76V的取代不影响感染性病毒的产生，而eGFP基因与3A C端的融合导致FMDV不可行。我们的结果首次报道，3A蛋白的aa 77–153而不是aa 81–153是细胞培养中FMDV复制所必需的。该研究对FMD标记疫苗的开发和今后外源基因的表达具有重要意义。