Mechanism Of Orf Virus-Encoded ORF120 Protein Inhibiting The G3BP1-Mediated Formation Of Stress Granules

Contagious ecthyma,also called “Orf”,is an acute and contact infectious disease caused by Orf virus(ORFV),which mainly infects goats and sheep.Clinically,the disease is mainly characterized by erythema and vesicle on the lips,skin and mucous membrane of infected animals,and pustules,ulcers and verrucous scabs at the later stage of the disease.Currently,the disease has threaten the development of sheep industry.As a zoonotic infection,it also poses a potential threat to public health and safety.ORFV has a large genome and numerous coding proteins.At present,there are still many viral proteins with unknown functions.The previous work of the research team has found that the ORF120 gene coding protein is an important virulence factor and interacts with Ras GTPase activating protein-binding protein 1(G3BP1)in host cells.Since G3BP1 is the core component of stress granules(SGs),we speculate that ORF120 protein is involved in the regulation of SGs formation.In order to determine whether ORF120 protein is involved in the regulation of SGs formation,OFTu cells were infected with the ORFV wild strain(OV-SY17)and ORF120 gene deletion mutant(OV-SY17Δ120)and ORF120 gene rescue strain(OVSY17-RV120)at an MOI of 10,respectively.The immunofluorescence assay results showed that G3BP1 was diffusely distributed in the infected cells of OV-SY17 and OVSY17-RV120 strains,and no specific spot fluorescence of stress particles was observed.In addition,the fluorescence detection results of the SGs marker protein cytotoxic granule-associated RNA binding protein(TIA1)in the cells infected by the above virus strains showed that TIA1 was mainly distributed in the nucleus,and no punctate particles were found in the cytoplasm.The results showed that OV-SY17 could inhibit the formation of SGs.However,at 6 hours after the infection of OV-SY17Δ120 gene deletion mutant,punctate particles formed by G3BP1 aggregation could be observed in the infected cells,and the number of punctate particles gradually increased with the infection time.At the same time,the distribution of TIA1 in cells infected by OVSY17Δ120 gene deletion mutant showed that in the first 6 hours,TIA1 was mainly located in the nucleus,but from 6 hours after infection,TIA1 rapidly migrated to the cytoplasm,and particles formed by TIA1 aggregation could be observed in the cytoplasm,and the co-localization of G3BP1 and TIA1 was clearly observed in the structure of stress particles induced by OV-SY17Δ120 infected.The above results further confirmed that ORF120 protein had an inhibitory effect on the formation of SGs.Next,this study in-depth explored the regulatory relationship between ORF120 protein and important components of SGs,G3BP1 and TIA-1.His-ORF120 and HAG3BP1 or HA-TIA1 fusion plasmids were co-transfected into OFTu cells and He La cells.Western blot was used to detect the expression levels of G3BP1 and TIA-1 in cells overexpressing ORF120 protein.The results showed that ORF120 protein could inhibit the expression of G3BP1 and TIA-1 in a dose dependent manner,while overexpression of G3BP1 or TIA-1,a dose dependent decrease in ORF120 protein was also observed.The results suggest that ORF120 protein may inhibit the formation of SGs by reducing the expression of G3BP1.Further research on the regulatory relationship between ORF120 protein and G3BP1 showed that transfection with His ORF120 plasmid at higher doses could reduce the expression of G3BP1 in cells,suggesting that ORF120 protein may inhibit the formation of SGs by reducing the expression of G3BP1 at higher doses.In order to clarify the regulatory effect of ORF120 protein on the formation of SGs in host cells,this study first stimulated He La cells to produce SGs with the stress particle inducer Arsenite(Ars),and conducted immunofluorescence observation after transfection of His-ORF120 plasmid.The observation results showed that overexpression of ORF120 protein can reduce the number of SGs induced by Ars,and a significant co-localization of ORF120 protein and G3BP1 was observed.The results indicate that G3BP1 may be a target for ORF120 protein to regulate SGs formation.Considering that the accumulation of SGs induced by Ars is passed through eIF2α-G3BP1,so it is speculated that the inhibition of ORF120 protein on SGs formation involves eIF2α-Changes in G3BP1 related proteins.Western blot analysis showed that ORF120 protein can reduce Ars induced eIF2α phosphorylation levels and G3BP1 expression.The above results indicate that ORF120 protein can inhibit SGs formation induced by Ars through eIF2α-G3BP1 pathway.In summary,OV-SY17 cannot induce the formation of SGs in cells.Then,the viral encoded protein(ORF120 protein)involved in inhibiting the formation of SGs in cells was identified.Further,the regulatory relationship between ORF120 protein and the core component G3BP1 protein of SGs,as well as the mechanism of Orf virus-encoded ORF120 protein inhibiting the G3BP1-mediated formation of stress granules was investigated.This study is expected to reveal a new mechanism of immune escape of ORFV and provide a theoretical basis for screening drug targets targeting the replication of poxvirus.