Effects Of GBP1 And GBP2 Proteins On The Proliferation Of Porcine Rotavirus

Rotavirus(RV)belongs to the family Reoviridae and the genus Rotavirus.It is a segmented double-stranded ribonucleotide(ds RNA)virus.Its structure is icosahedral symmetry,no envelope,and its capsid is wrapped with 11 ds RNA segments.Porcine rotavirus(PoRV)can cause acute enteritis in young piglets and is infectious,which has caused serious harm to the development of the breeding industry.Rotavirus(RV)is a genus of rotavirus in the family Eutheroviridae,and is a segmented double-stranded ribonucleotide(ds RNA)virus with an icosahedral symmetric structure and no capsule,and its capsid is wrapped with 11 segments of ds RNA.porcine rotavirus(PoRV)can cause acute enteritis in young piglets,and is highly infectious,causing serious harm to the development of the farming industry.It is highly infectious and poses a serious hazard to the farming industry.Guanylate-binding protein 1(GBP1)and guanylate binding protein 2(GBP2)belong to the family of guanylate-binding proteins(GBPs),which are interferon(IFN)-inducible GTPases The GBPs family has a GTPase structural domain with multiple functional sites on its structural domain.Several papers have reported that their activities are related to antiviral effects,and their main roles are resistance to protozoan infection,inhibition of bacterial proliferation,antiviral replication,antitumor effects,and promotion of inflammatory response formation,playing an important role in anti-pathogenic microbial responses.Based on this,in-depth analysis of the interaction mechanism between PoRV and GBP1 and GBP2 proteins and study of the effect of GBP1 and GBP2 on PoRV replication can help to understand the molecular mechanism of PoRV replication and explore the unknown functions of GBP1 and 2 proteins,providing important evidence for candidate disease resistance genes.1.Effect of GBP1 and GBP2 on PoRV proliferationInoculation of PoRV in MA104 cells produced stimulation of elevated expression of GBP1 and GBP2.p CDNA3.1-GBP1-full-length-HIS(1773bp)and p CDNA3.1(+)-GBP2(47aa-592aa)-HIS(1635bp),p CDNA3.1(+)-GBP2(131aa-592aa)-HIS(1383bp)eukaryotic expression recombinant plasmids.After transfection of the plasmids into cells,the intracellular localization of GBP1 and GBP2 was determined by indirect immunofluorescence,Western blot,fluorescent quantitative PCR,and TCID50,and the effects of GBP1 and GBP2 on the replication effect of PoRV were verified on three types of cells,MA104,293 T,and IPEC-J2,to investigate the effects of GBP1 and GBP2 on PoRV replication process.The results showed that GBP1 and GBP2 were localized in the cytoplasm,and all three cells verified that GBP1 and GBP2 could inhibit PoRV replication,and that GBP1 and GBP2 exerted effects on the replication phase of the PoRV replication process,thus inhibiting the replication of the virus.2.Interaction of GBP1 and GBP2 with PoRV viral proteinsPoRV viral proteins consist of structural proteins(VP1-VP4,VP6,VP7)and non-structural proteins(NSP1-NSP5).The expression of multiple viral proteins on 293 T cells was verified by Western blot,and multiple viral proteins of GBP1,GBP2 and PoRV were co-transfected in MA104 cells.Intracellular co-localization and protein interactions were verified by laser confocalization,and the screened PoRV proteins were cotransfected with GBP1 and GBP2,and the existence of interactions between GBP1 and GBP2 and VP2 proteins was verified by CoIP method.3.Exploration of the mechanism of anti-PoRV of GBP1 and GBP2According to the GTPase structural regions that are unique to the family of GBPs that play functions,corresponding to the sites of protein sequences and amino acid sequences,and other different structural regions are divided into three parts,N-terminal,intermediate helix part,and C-terminal.the N-terminal has a globular GTPase structural domain with the function of hydrolysis and binding GTP,which is the G region.The middle helix part acts as a linker and is the M region.the C-terminus has a GTPase effector structural domain and is the E region.The full-length GBP1(1-590aa)and GBP2(1-592aa)proteins were designed to be truncated as GBP1-G(1-280aa),GBP1-M(281-361aa),GBP1-E(362-560aa),GBP1-ME(281-560aa),GBP2-G(1-280aa),and GBP2-M(281-464aa),GBP2-E(465-557aa),and GBP2-ME(281-592aa),each with four segments,were constructed as truncated recombinant plasmids,and it was determined that the truncated bodies were expressed intracellularly and localized in the cytoplasm,and that the G region in the GBP1 and GBP2 truncated bodies produced an inhibitory effect on PoRV replication.Using a pan-GTPase enzyme inhibitor,the inhibitory effect of PoRV replication was lost in GBP1 and GBP2 after inhibition of GTPase enzyme activity.Since the GTPase enzyme active region corresponds to the position of the truncated body G region,it indicates that the inhibition of PoRV replication by GBP1 and GBP2 requires the participation of the GTPase enzyme active region to function.To investigate the antiviral pathway of GBP1 and GBP2,three different interferon promoters IFN-α,IFN-β,IFN-λ1 and three different cytokine NF-кB,TNF-α,IL-6 promoters were selected and cotransfected into 293 T cells with GBP1,GBP2 and p RL-TK,and then the dual luciferase assay was performed to investigate the GBP1 and GBP2 antiviral pathway.The results showed that both GBP1 and GBP2 could stimulate the promoter activity level to increase,and GBP1 and GBP2 might exert antiviral effects through activating interferon pathway and inflammatory pathway.