Correlation Between EV71 Infection And Stress Granule And RNA Decay Pathway

1.EV71 infection disrupts stress granule formation and translocates RNA binding proteins TDP-43,FUS/TLS and HuR to cytoplasmIn response to viral infection,host cells develop stress granules(SGs)by global inhibition of protein synthesis as a mean of defense or promoting cell survival although viruses have evolved ways to overcome the SG formation.Structural or nonstructural components of some viruses can interact with key components of SGs to divert SG assembly pathways to aid their replication.We found that EV71 infection induced SG formation at an early time postinfection(pi),and the formed SGs were disrupted when viral proteins began expressing.We demonstrated that the SG formation was unrelated to PKR activation and not dependent on eIF2a phosphorylation,the most common form of translational arrest.Instead,the suppression of SG formation by EV71 was caused by the cleavage of eIF4G by viral proteinase.Importantly we identified that HuR,TDP-43,and FUS/TLS,three RNA binding proteins implicated in neurodegenerative diseases and common pathological and genetic determinants of ALS and FTLD,relocalized from nucleus to cytoplasm after EV71 infection and bound to viral genomic ssRNA.We also demonstrated that these proteins aggregated to form cytoplasmic structures distinct from SGs.In addition,We found that TDP-43 plasma levels were significantly elevated in EV71 infected patients with clinic indications of neural injuries.2.Nuclear proteins TIAl and TIAR are translocated to cytoplasm after EV71 infection and facilitate virus replication by interacting with 5’-UTR of viral genome Enterovirus 71 is one of the major causative pathogens of HFMD in children,In particular,EV71 infection can cause severe neurological disorders and was associated with several large-scale outbreaks in Asian Pacific regions.Upon infection,the viral RNA is translated in an IRES-dependent manner to yield a large polyprotein precursor.IRES-dependent translation needs not only the involvement of canonic initiation factors,but also IRES-specific trans-acting factors(ITAFs).In the current study,we found that T-cell-restricted intracellular antigen 1(TIA-1),and TIA-1 related protein(TIAR),two RNA binding proteins(RBPs)shuttle out of nucleus after EV71 infection and localize to the sites of viral replication.Using an RNA pull-down assay,we demonstrated that both of these RBPs bound to the stem-loop I of 5’-UTR of viral genome.Moreover,we found that TIA-1 and TIAR can facilitate EV71 replication by stabilizing the viral genome in host cells through overexpression of TIA-I and TIAR and RNA interference mechanisms.3.miRNA can be recruited into P-body and stress granule,and P-body and its component have an positive effect on EV71 infectionDcpla and GW 182 are two component proteins of P-body.Evidences have shown that both of these two proteins involve in miRNA pathway to regulate intracellular gene expression.In the current study,we found that everexpression of Dcpla and GW 182 can induce to form a larger number of granules in cells,while overexpression of other components of P-body cannot.In addition,we found that overexpressed Dcpla colocalize with endogenous DDX6,another component of P-body.However,we failed to observe colocalization of overexpressed GW 182 and DDX6,indicating the granules induced by overexpressed these two proteins are different aggregates.In addition,we also observed that stress granules can be induced by overexpression of G3BP1.We found that TDP-43 and eIF4G can be recruited into the granules while FUS/TLS cannot.It has been well documented that both P-bodies and stress granules take part in miRNA pathway.Therefore,in the study,we also investigated the correlation between miRNA pathway and these two granules using labeled miRNA-141.The results demonstrated that miRNA can localize into all the granules formed by overexpressed Dcpla,GW182 and G3BP1.In order to further examine the correlation between P-body formation and EV71 infection,we infected cells with EV71 and observed that EV71 infection can promote P-body formation.To investigate influence of P-body on EV71 infection,certain components of P-body were knockdown by RNAi.As a result,we found that knockdown any of the proteins can inhibit EV71 replication,suggesting that P-body and its components have a positive effect on EV71 propagation.Taken together,this part of our study further demonstrated that P-body and stress granule involve in regulation the miRNA pathway,and P-body and its components facilitate EV71 infection.Our result may offer a novel angel of view for design antiviral targets in the future.