The Primary Research On Molecular Mechanisms Of Duck A Virus Modulating Stress Granules Formation In Host Cells

DHAV(Duck hepatitis A virus)is a common virus that severely endanger the ducking industry of our country.SG(stress granule)is a condensate membraneless structure which forms under unusual physiological or pathological conditions such as oxidative,heat shock,virus infection.And SG generally forms with cell translation arrested.The previous study of our lab indicates that DHAV-1 infection could induce SG formation in a eukaryotic initiation factor 2α-dependent manner in DEF(Duck embryo fibroblast)cells.To better understand the mechanisms of DHAV-1-induced SG formation,we studied how DHAV-1modulated the SG formation in host cells,and findings are presented below:1.Determining the ability of DHAV-1 mediated SG formation and SG disassemblyDEF cells were infected with DHAV-1 CH strain at MOI of 0.01 and performed as follow:(1)The samples were collected at 0,4,8,12 and 24 hpi.Then we observed the SG formation status using indirect immunofluorescence assay and selecting G3BP1 as SG marker.The results showed that no observable SG formation was observed during 0 to 8hpi.The formation of SG was first detected at 12 hpi then SG vanished at 24 hpi.(2)Inducing SG formation in DEF cells by arsenate sodium at 0 hpi,4 hpi,8 hpi,12 hpi and24 hpi,and SG formation was successfully induced at 0 hpi to 12 hpi but failed at 24 hpi.This result indicates that DHAV-1 could induce SG formation in host cells and were also able to restrain the ability of SG formation in host cells.2.DHAV-1 3C could antagonize SG formation in host cells by down-regulating G3BP1 levelAfter infecting DEF cells with DHAV-1 at MOI of 0.01 for 24 h,the G3BP1 level of infected cells were lower than that of uninfected cells.And with the hours after infection increasing,the G3BP1 level continuing to drop.At the same time,the level of another SG marker,TIA-1 in DEF cells were also examined.There was no detectable change of TIA-1level.These results indicates that DHAV-1 could down regulate G3BP1 level in DEF cells but not TIA-1 level.The eukaryotic expression plasmids of nonstructural proteins of DHAV-1 and empty vector were transfected into DEF cells.Then cells were induced by arsenate.And the location of the nonstructural proteins and G3BP1 was observed.The results demonstrated that 3C protein could colocalize with G3BP1 in cells and the cells expressing 3C protein could not form SG.These results illustrated that DHAV-1 3C protein could confine the SG formation induced by arsenate in cells.By transient transfection,we found the protein level of G3BP1 in was lower in cells expressing 3C protein than compare group that cells transfected with empty vector.Then we transfected the eukaryotic expression plasmids of G3BP1 and wild-type or mutant 3C proteins.However,the mutant 3C protein without protease activity was still able to down-regulate G3BP1 level,which indicated that DHAV-1 3C protein could decrease G3BP1 level in cells in a protease activity-independent manner.3.G3BP1 could inhibit DHAV-1 propagation in DEF cellsTo figure out the impact of G3BP1 on DHAV-1 propagation,we infected the G3BP1-overexpression DEF cells with DHAV-1 CH strain(MOI of 0.01)and detected viral RNA copy numbers at 24 hpi using RT-qPCR.Compared to control group,the viral copy number of DHAV-1 were reduced in G3BP1-overexpression cells.The viral copy number of DHAV-1 was obviously increased in G3BP1-konckdown cells compared to control group that transfected with shNC plasmid.These results demonstrated that G3BP1 could confine DHAV-1 propagation in host cells.Taken together,these results indicated that DHAV-1 infection could induce SG formation in cells firstly and then antagonize SG assembly by down-regulating G3BP1 level with its 3C protein in a protease activity-independent manner.Therefore,DHAV-1could modulate the SG formation in host cells and promote DHAV-1 propagation.