Middle East Respiratory Syndrome Coronavirus (MERS-CoV) ORF5 IRES Activity Study

Research background and purposeMiddle East Respiratory Syndrome(MERS)is a zoonotic disease caused by Middle East respiratory syndrome coronavirus(MERS-cov),which can cause severe acute respiratory symptoms in humans.Similar to SARS and SARA-CoV 2,MERS belongs to the genus β-coronavirus.Highly pathogenic respiratory infectious diseases.In our previous study,we found a deletion variant in the ORF5-E protein of the MERS-cov virus sequence in the throat swab samples of imported MERS patients from Korea.Further study found that the cloned ORF5 gene was difficult to express in cells.EGFP expression can be observed after the fusion of ORF5(containing stop codon)and EGFP,suggesting that ORF5 gene may contain a function similar to the internal ribosome entry site(IRES),but its mechanism needs to be further studied.Therefore,in this study,the function of MRES ORF5 protein was studied,the recombinant expression vector of ORF5 was used to screen the key sites affecting the secondary structure through the prediction model,and the secondary structure of RNA was changed to study the relationship between the secondary structure of ORF5 and the functional activity of IRES.Method1、The eukaryotic expression vector of ORF5-EGFP fusion protein was constructed.After frameshift mutation and termination mutation in some sites of ORF5 protein,EGFP fusion protein could be expressed,suggesting that ORF5 protein has the function of initiating the translation of related proteins.2、Using the software RNA Fold to predict the secondary structure of ORF5 RNA,there were three important stem-loop structures,namely stem-loop Ⅰ(amino acid sequence from 124th to 127th position),stem-loop Ⅱ(amino acid sequence from 155th to 157th position)and stem-loop Ⅲ(amino acid sequence from 184th to 188th position).3、The synonymous code mutation of ORF5 RNA changed the stem and loop of the secondary structure of ORF5,and the IRES activity of ORF5 was significantly changed by luciferase detection.4、The constructed MERS ORF5-E-Renilla-EGFP expression vector was used to change the secondary structure of ORF5 RNA by site-directed mutagenesis.Dual luciferase assay and Western Blot were used to detect the expression of the target protein.Results1、The pEGFP-ORF5 eukaryotic expression vector was constructed,and the fusion protein could be expressed after frameshift and termination of its site,indicating that ORF5 has a different mechanism from the conventional eukaryotic translation expression mechanism.2、The software RNA Fold was used to predict the secondary structure of ORF5 RNA.There were three important stem ring structures,which were stem ring Ⅰ,Ⅱ,Ⅲ.3、Mutagenesis of the stem-loop of the secondary structure of ORF5 RNA significantly altered IRES activity.4、In the mock virus model pEGFP-5ERI vector,after changing the secondary structure of RNA at the corresponding site,the fusion expression of E protein and Renilla protein was significantly reduced by dual luciferase assay.Conclusions1、ORF5 protein plays a role in mediating viral protein translation,similar to the internal ribosome entry site of IRES.2、The stem-loop structure of MERS ORF5 affects IRES activity.3、The stem-loop Istructure(amino acid sequence from 124th to 127th position)located on the sequence of ORF5 may be an important part of its IRES activity.