Abstract
BackgroundRecent 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.MethodsIn 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.ResultsThe 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.ConclusionsOur 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.
Highlights
Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, including domesticated ruminants and pigs, as well as a large number of wildlife animals
The aa 77–153 of 3A protein was dispensable for FMD virus (FMDV) replication in cell culture and the residue 76 is essential for FMDV viability To determine whether the remarkably variable region of 3A plays little role for the production of infectious FMDV, three full-length mutant clones were created by over-lapping PCR fragments flanked by unique restriction enzymes
These results revealed that the aa 77–153 rather than the aa 81–153 of 3A was dispensable for FMDV replication in cell culture and the residue 76 is essential for completion of FMDV life cycle
Summary
Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, including domesticated ruminants and pigs, as well as a large number of wildlife animals. 3A protein The C-terminal deletion mutants of FMDV 3A protein were constructed based on the full length clone pOFS. Six PCR primers (Table 1) were used to amplify three overlapping cDNA fragments containing the corresponding deletion regions as previously described [21]. Four PCR primers (Table 1) were used to amplify two overlapping cDNA fragments and two additional clones pOFS/3A76I-153 and pOFS/3A76V-153 containing the deletion of aa 77–153 in 3A and the substitutions of 3A L76I and 3A L76V were generated as above described. 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, the complete C-terminal portion (aa 77–153) of 3A is highly variable and prone to occur deletions and mutations, we presume that this region plays a very limited role and probablely is completely nonessential for virus viability
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