Effect Of Interaction Between 5’ UTR And 3D Of Enterovirus 71 On Virus Replication

Hand-foot-and-mouth disease(HFMD)is an acute infectious disease caused by an enterovirus.It mostly occurs in children under the age of 5.It is transmitted through close contact and the digestive tract.It typically presents as herpes and ulcers in the mucous membranes of hands,feet,and mouth.Among them,HFMD caused by enterovirus 71(EV71)has more serious neurological symptoms.There is no specific antiviral drug at present,and there is a lack of research on the combined effect of multiple structures in the virus.The EV71 genome contains only one open reading frame(ORF),with a 5’ untranslated region(UTR)and a 3’ UTR on both sides.The ORF encodes 4 structural proteins(VP1-VP4)and 7 nonstructural proteins(2A-2C and 3A-3D).Among them,5’UTR has a complex secondary structure,which can fold into multiple specific spatial structures,and affects the synthesis of viral genomic RNA and the translation of viral protein through a combination with host cell protein factors.The EV71 3D protein is an RNA-dependent RNA polymerase(RdRp),which is mainly responsible for the extension of RNA strands in the process of virus replication.In addition,studies have shown that 5’UTR and the 3D protein can interact with the same factors regulating virus replication in various ways to regulate replication.However,whether 5’ UTR interacts with the 3D protein in regulating virus replication has not been clarified.Based on the infectious cDNA cloning of EV71 SDLY107 and 3D mutant strains,four 5’UTR single point mutant strains and eight combined mutant viruses were constructed.The replication ability and cytopathic effect of each mutant strain were studied,and the interaction between 5’UTR and the 3D protein was explored.The results showed that 5’UTR interacts with 3D protein-regulated autophagy through the AKT-mTOR pathway,and affects virus replication.The research results described the interaction between 5’UTR and the 3D protein in the EV71 virus by experiments for the first time,deepened the understanding of the virus genome,and provided a theoretical basis for the mechanism of EV71 and the development of antiviral drugs.Objective1.The 5’UTR mutant virus was constructed to explore the effect of 5’UTR on the ability of the virus replication and to cause cell-damaging.2.Based on the infectious cDNA cloning of two 3D mutant strains,the combined mutant viruses of 5’UTR and the 3D protein were constructed to explore the interaction mechanism between them.3.To investigate the effect of AKT-mTOR on virus replication.Methods1.Using site-directed mutation and reverse genetics technology,the 5’UTR single point mutant strain was constructed and rescued with plasmid pMD19T-SDLY107-EGFP as a template.At the same time,two 3D mutant plasmids were used as templates to construct the combined mutant strains of 5 ’ UTR and 3D protein.The virus titer was verified and determined by sequencing.2.Detected and compared the level of replication between the mutant virus and the parent virus,and detected the ability of the virus to cause cell-damaging by CCK8 experiment,then studied the interaction between 5’ UTR and 3D protein,and screened the interacting virus.3.The interacting strains and parental strains were infected with the same MOI,the changes in autophagy-related protein content were detected,and the effect of the interaction between 5’UTR and 3D protein on autophagy was analyzed.At the same time,the host cells were treated with AKT inhibitors to explore the role of the AKT-mTOR pathway.Results1.Four 5,UTR mutants,EGFP-EV71(nt88C/T),EGFP-EV71(nt90-102-3 C),EGFP-EV71(nt157G/A),and EGFP-EV71(nt574T/A),were successfully constructed with the infectious cDNA of virulent strain as a template,and eight combined mutants were successfully constructed with the infectious cDNA of the 3D mutant strain as the templates:EGFP-EV71(S37N-nt88C/T),EGFP-EV71(S37N-nt90-102-3C),EGFP-EV71(S37N-nt157G/A),EGFP-EV71(S37N-nt574T/A),EGFP-EV71(S37N-nt157G/A),EGFP-EV71(R142K-nt88C/T),EGFP-EV71(R142K-nt90-102-3C),EGFP-EV71(R142K-nt88C/T),EGFP-EV71(R142K-nt157G/A).and EGFP-EV71(R142K-nt574T/A).After identification,12 mutant strains were successfully saved.2.Through the detection of the replication ability of each mutant and parent strain,it was found that among the four 5’UTR single point mutant strains,except for EGFP-EV71(nt90-102-3C),the replication level was lower than EV71 SDLY107,while the replication level of the combined strain showed different characteristics.A total of five mutant strains with a combined effect were screened:EGFP-EV71(S37N-nt88C/T),EGFP-EV71(S37N-nt574T/A),EGFP-EV71(R142K-nt574T/A),EGFP-EV71(S37N-nt574T/A),EGFP-EV71(R142K-nt88C/T),and EGFP-EV71(R142K-nt157G/A).3.Western blot results showed that the high replicative strain significantly promoted the accumulation of autophagosomes in host cells and hindered the degradation of autophagosomes and lysosomes.However,the low replication strain had a low ability to regulate the autophagy of host cells.In addition,the high replicative strain also significantly inhibited the phosphorylation of AKT and mTORConclusions1.The mutations of nt88C/T,nt90-102-3C,nt157G/A,and nt574T/A of EV71 5’UTR affected the replication ability and cell-damaging ability of the virus.2.EV71 5’UTR interacted with the 3D protein.The combined mutation of S37N and nt88C/T,S37N and nt574T/A,and R142K and nt574T/A induced incomplete autophagy of host cells and promoted virus replication by inhibiting the autophagy pathway Akt-mTOR.The combined mutation of R142K and nt88C/T,and R142K and nt157G/A significantly reduced the inhibitory effect of EV71 on the Akt-mTOR pathway and reduced the replication ability of the virus.