Functional Analysis On 2A And Its ’Cleavage’ Proteins Of Duck Hepatitis A Virus

Duck hepatitis A virus（DHAV）,which mainly infects ducklings,causes duck virus hepatitis with a mortality rate over 95%.2A is the first non-structural protein in DHAV genome,the researches against 2A would help to further understand the pathogenesis of DHAV.DHAV has three tandem 2A proteins,named 2A1,2A2 and 2A3 in sequence.The main contents and results of the analysis on the function of 2A protein are as follows:1 DHAV 2A1 peptide mediates ribosomal‘skipping’function2A1 is the first non-structural protein in DHAV genome.Its C-terminal amino acids and the first amino acid‘P’of 2A2 form a‘-Gx Ex NPGP-’motif together.Based on this motif and the G/P site,the following researches were carried out:（1）The ribosomal‘skipping’function mediated by 2A1:overlapping PCR technology was used to amplify the EGFP-2A1-RFP fragment（2A1 contains the first amino acid‘P’of 2A2）,then this fragment was cloned into the pc DNA3.1（+）vector to obtain the p EGFP-2A1-RFP plasmid.Then the plasmid was transfected into duck embryo fibroblast（DEF）and its expression was tested by western blot（WB）.The results showed that three kinds of products were detected,which is the EGFP-2A1 protein,RFP protein and EGFP-2A1-RFP fusion protein.This indicates that the DHAV 2A1 peptide mediates the ribosomal‘skipping’function,and G/P is the functional site.（2）the length of N-terminus of 2A1 peptide affects its ribosomal‘skipping’efficiency:the N-terminus of 2A1 was extended to VP1（the structural protein of DHAV）sequence,or truncated to varying length of C-terminus,then overlapping P CR technology was used to link EGFP upstream and RFP downstream by the diffe rent（VP1）+2A1 sequences.Then the overlapping EGFP-（VP1）+2A1-RFP fragments were cloned into pc DAN3.1（+）vector and the completion of ribosomal‘skipping’f or each plasmid was tested.The results showed that the N-terminal extension of 2A1 improved the ribosomal‘skipping’efficiency.When the N-terminus was extende d towards VP1 by 10 aa（5’-leucine-alanine-phenylalanine-glutamicacid-leucine-asparti cacid-leucine-glutamicacid-isoleucine-glutamicacid-3’）,the ribosomes could completely‘skip’and only EGFP-（VP1）-2A1 and RFP proteins were detected.In contrast,the shortening of N-terminus decreased the efficiency of ribosomal‘skipping’.When 2A1 is shortened to only contain 10 amino acids of the C-terminus,2A1 didn’t play the ribosomal‘skipping’function and only the EGFP-2A1-RFP fusion protein was detected.This indicates that the length of N-terminus of 2A1 peptide is important f or the efficiency of ribosomal‘skipping’function.（3）time phase when the ribosomal‘skipping’function occurs:western blot assay was performed to test the expression of p EGFP-12-RFP（VP1+2A1=12 aa）plasmid at different time points and Image J2x software was used to quantify the expression for each protein product.The results showed that the proteins were fully expressed at 24 h already.The expressed fusion protein couldn’t be proteolyzed and the expression of independent upstream or downstream proteins didn’t increase with time passing.It indicates that the independent expression of upstream and downstream proteins is not due to post-translational proteolysis.At the same time,RT-q PCR assay was performed to test m RNA levels of EGFP,peptide with‘skipping’site and RFP.The results showed that the m RNA levels reached the highest at 24 h,then they declined.However,the decrease of transcriptional level didn’t affect the protein expression level.This indicates that the independent expression of upstream and downstream proteins mediated by 2A1 was not due to the abnormal RNA transcription.（4）the expression imbalance between upstream protein and downstream protein flanked by 2A1:The expression level of upstream protein is 16-118 times higher than that of downstream protein through WB analysis.In order to explore whether this phenomenon is caused by the upstream EGFP reporter gene,the upstream EGFP was replaced by EYFP reporter gene and p EYFP-2A1-RFP was constructed.The results showed that the expression level of upstream EYFP-2A1 protein was still 128 times higher than that of downstream RFP protein.Since EYFP is derived from EGFP,the order of EGFP and RFP was reversed and the p RFP-2A1-EGFP plasmid was constructed in order to further verify.The results showed that the expression level of upstream RFP-2A1 protein was still 2 times higher than that of downstream EGFP protein.This study indicates that the expression imbalance is due to the specific property of 2A1.（5）the relationship between the 2A1-mediated‘cleavage’and the integrity of encoding m RNA:the results of plasmids transcription in vitro and in transfected cells showed that:the encoding m RNAs were all intact;the m RNA levels of EGFP,peptide with‘skipping’site and RFP were the same.This study indicated that the protein‘cleavage’mediated by 2A1 was not caused by RNA breakage or abnormal abnormality,and the levels of‘cleavage’proteins are independent of the levels of RNA transcriptions.（6）the relationship between 2A1-mediated ribosomal‘skipping’function and cell species:in order to explore whether 2A1 can only function in duck-derived cells,p EGFP-2A1-RFP was expressed in chicken-derived DF-1 cells and mouse-derived BHK21cells.Results showed that the EGFP-2A1 and RFP proteins were detected in these two kinds of cells.This study indicates that the ribosomal‘skipping’function mediated by 2A1is independent of cell species.2 key amino acids of the ribosomal‘skipping’function mediated by DHAV 2A1Each amino acid in‘-Gx Ex NPGP-’motif of p EGFP-2A1-RFP was scanning mutated into Alanine（A）by point mutagenesis kit and the expression of each mutant was tested.Label the motif as‘-G¹⁴x E¹⁶x N¹⁸P¹⁹G²⁰P²¹-’according to the order of each amino acid in2A1 genome.Since N¹⁸ couldn’t be mutated to A at once,it was firstly mutated to a synonymous N’,and then mutated into A.Results showed that G¹⁴ and E¹⁶ only attenuated the ribosomal‘skipping’efficiency.Although large amounts of fusion proteins were detected,there were still a lot of independent proteins detected.The alanine mutation of N¹⁸,P¹⁹,G²⁰ and P²¹ made the complete loss of ribosomal‘skipping’function,and only fusion proteins were detected.3 Interaction between DHAV 2A3 protein and viral 5’UTR（1）The prokaryotic expression plasmid p ET28a-2A3 was constructed and expressed in E.coli Rosetta expression bacteria.2A3 protein,whose molecular weight is about 15k Da,expressed in the forms of inclusion body and soluble protein both and can be purified with Ni²⁺agarose beads to get high-purity 2A3 protein.（2）The 5’UTR of DHAV was transcribed and purified in vitro.（3）The purified 2A3 protein and the in vitro transcribed5’UTR were used to perform the electrophoresis mobility shift assay（EMSA）on horizontal agarose gel electrophoresis,and the result showed that 2A3 protein changed the migration speed of 5’UTR in horizontal agarose gel electrophoresis.4 2A,2A2 and 2A3 proteins affect the activity of succinate dehydrogenase in host cell2A,2A2 and 2A3 proteins were overexpressed in DEF cells separately:（1）A570values from MTT experiment showed that 2A,2A2 and 2A3 could promote the activity of succinate dehydrogenase in host cells（2A increased by 0.150;2A2 increased by 0.336;2A3 increased by 0.356）;（2）In the early stage（24 h）of overexpression,RT-q PCR experiment was performed to detect the m RNA levels of multiple cell cycle genes（CCNE1!CCNE2!CUL1!SKP1!SKP2!RBX1 and CCNT1）in DEF.Results showed that 2A could improve these m RNA levels by 3.4-43.9 times,2A2 could improve them by3.9-8.8 times and 2A3 could improve them by 3.4-9.4 times respectively;（3）The phosphorylation level of Akt protein in the PI-3K/Akt signaling of DEF cells was tested by WB after overexpressing 2A,2A2 and 2A3.Results showed that 2A precursor could promote the phosphorylation level of Akt protein（gray value was 1.7-2.5 times higher than that of p CAGGS panel）,2A2 and 2A3 proteins couldn’t promote the phosphorylation level of Akt protein;（4）Results of RT-q PCR experiment to detect DHAV replication showed that there were no statistical significance on viral copies between the DHAV infected cells transfected with 2A,2A2 and 2A3（The viral copies of DHAV are 6.2×10⁷ copies/μl,7.0×10⁷copies/μl,and 7.5×10⁷ copies/μl respectively）and that transfected with empty p CAGGS（7.4×10⁷ copies/μl）.In conclusion,DHAV 2A1 peptide mediates the ribosomal‘skipping’function and this function occurs during translation.G/P is the‘skipping’site.The N-terminal length of2A1 has an important impact on the ribosomal‘skipping’function.The co-expression levels between upstream and downstream proteins mediated by 2A1 are different.The ribosomal‘skipping’function mediated by 2A1 doesn’t rely on the cell species.Every amino acid in‘-Gx Ex NPGP-’motif can affect the ribosomal‘skipping’efficiency.Among them,N¹⁸,P¹⁹,G²⁰ and P²¹ are the key amino acids.DHAV 2A3 protein can bind to viral5’UTR,indicating that DHAV 2A3 protein is involved in the 5’UTR-mediated functions.2A,2A2 and 2A3 proteins can promote the activity of succinate dehydrogenase in host cells.Overexpression of these three proteins can up-regulate the m RNA levels of host cell cycle genes.2A protein can improve the phosphorylation level of Akt protein in PI-3K/Akt signaling.These phenomena,which lead to some metabolic abnormalities of host cells,might be one the of associated factors of cytopathic effect caused by DHAV infection.