The induction of Autophagy by 2B protein of Coxsackievirus B3, and identification of the Autophagic motif

Abstract

Objective To identify the 2B protein of coxsackievirus B3 woodruff strain inducing autophagy,and further,identify the motif of 2B protein related to autophagy.Method The eukaryotic recombinant plasmids (pEGFP-2B) expressing the fusion protein of 2B protein and enhanced green fluorescent protein(EGFP) was constructed.The 9 kinds of recombinant plasmids expressing the fusion proteins of brachytmema proteins and EGFP were constructed.The plasmid (pmCherry-LC3) which can express the fusion protein of red fluorescent protein (mCherry) and microtubule-associated protein light chain 3 (LC3) was structured.The expression of mCherry-LC3 in the HeLa cells,tansfected with pmCherry-LC3 and infected with CVB3,was detected by Laser Confocal Scanning Microscope (LCSM) and Western blotting.The status of autophagy in the HeLa cells co-transfected with plasmids pEGFP-2B and pmCherry-LC3 was observed by LCSM,the expression of LC3 was detected by Western blotting.The HeLa cells were transfected with 9 kinds of plasmids expressing the brachytmema fusion proteins,respectively,the expression of fusion proteins was detected by fluorescence microscope.The HeLa cells dealing with 3-methyladenine (3-MA) were co-transfected with plasmids pEGFP-2B or pEGFP-2B106-249 and pmCherry-LC3,and the fluorescins were observed under fluorescence microscope.Results The mCherry-LC3 fusion protein expressed in punctuate pattern around the nucleus in HeLa cells infected by CVB3.The band of LC3-Ⅱ was distinct according to the viral inoculation by Western blotting.The green and red fluorescin expressed in punctuate pattern around the nucleus in HeLa co-transfected with pEGFP-2B and pmCherry-LC3,and the two kinds of the fluorescins were interloped.The distinct band of LC3-Ⅱ was detected by Western blotting.The HeLa cells were transfected with 9 kinds of plasmids expressing brachytmema proteins,respectively,the shortest brachytmema protein which can induce green fluorescence in punctuate pattern around the nucleus was EGFP-2B106-249,while no punctuate patterns of the green and red fluorescin were observed in HeLa cells dealing with 3-MA.The LC3-Ⅱ levels increased according to pEGFP-2B106-249 transfection by Western blotting.Conclusions 2B protein of CVB3 woodruff strain can induce autophagy in HeLa cells ; The motif of 2B protein related to autophagy located on 2B106-249. Key words: Coxsackievirus B3 autophagy; Motif; Fusion protein; Microtubule-associated protein light chain 3