Study On Subcellular Localization Of Leader Protein Of Foot-and-Mouth Disease Virus Serotype Asia1 In The BHK-21 Cells And Proteomics Change Caused By Leader Protein

Foot-and-mouth disease virus (FMDV), is a positive stranded RNA virus that belongs to the Aphthovirus genus of the Picornaviridae family. The genome of FMDV is a positive-strand RNA of approximately 8,500 nucleotides. Upon infection, the viral RNA is translated into a polyprotein., The polyprotein yields structural and nonstructural proteins upon cleavage by three virus-encoded proteinases, leader protein (L^pro), 2A, and 3Cpro. L^pro, the first protein to be translated, that is an important virulence factor of FMDV in livestock hosts. L^pro inhibits the induction of transcription of IFN-βmRNA and blocks the host innate immune response. Further studies revealed that L^pro is associated with the apoptosis of host cell.Protein functions are closely related to subcellualr localization. At present,research about subcellular localization of L^pro was performed by immune fluorescence experiment after cell fixation. Actually, lots of research shows that immobilized cell and broken membrane using chemical reagent can induce changes of subcellular localization. Green Fluorescent Protein (GFP) and analogue do not change cellular localization and function of target protein as they are fused with another proein. So, confirming subcellular localization of L^pro in the living cells using fusion with GFP has important significant to understanding function of L^pro. However, so far, Noting is no about precise subcellular localization of L^pro. In this study, the recombinant plasmid pEGFP-L^prowas transfected into BHK-21, then the cells were observed under confocal microscopy. These results show that the L^pro protein localized in BHK-21cytoplasm nucleus. These indicated that L^pro maybe has necleolar localization signals (NLS), that provided reliable data for futher functional nucleolar localization signals identification. At present, research on function L^pro was carried out at morphology and transcriptomic level,but many viruses protein only affect intracellular protein modification and turnover, while influence the gene transcription and mRNA processing. Therefore, studying interaction between virus protein and cellular protein only from the genome or transcriptomic level has certain limitations. To analyse change characteristics of cellular proteomics were caused by L^pro, The recombinant plasmid pCDNA-was transfected into BHK-21, a differentially proteomic analysis was conducted using 2D electrophoresis followed by MALDI-TOF-MS/MS identification. Altered expression of 21 protein spots with at least 1.5-fold quantitative differences in transfection cells, were identified in 2D gels, with 21 of these being characterized by MALDI-TOF-MS/MS, including 10 up-regulated proteins and 11 down-regulated proteins. The altered proteins could be sorted into 6 groups according to their cellular function: cytoskeleton, energy metabolism, RNA editing, protein processing, nucleosome assembling, signal transduction. According to cellular function annotation of differentially expressed proteins, it was speculated that down-regulation expression of cytoskeleton and nucleus related protein might be involved in mechanism of degradation transcription factor and translation initiation factor, Up-regulation of Endophilin-B1 and down-regulation of Peroxiredoxin-2 may be one mechanism of L^pro inducing apoptosis intracellular.