Screening Of P-body Molecules Related To PRRSV Proliferation And The Effect Of APOBEC3F/3G On PRRSV Proliferation

PRRS,commonly known as "Porcine blue ear disease",is a highly contagious disease caused by PRRSV.It is characterized by immunosuppression,virus persistence and secondary infection,which is one of the important diseases affecting the development of the global pig industry.P-Body is an m RNA-protein subcellular complex that does not have an envelope structure in the cytoplasm.It mainly monitors,quality controls,and degrades m RNA.The P-Body participates in the completion of important cellular activities such as gene silencing,translation inhibition and innate immune process,with a functional diversity.APOBEC3F/3G is a member APOBECs family,located in P-Body,with cytidine deaminase activity,and is an important host limiting factor,which were reported to inhibit the proliferation of a variety of viruses.At present,the effect of PRRSV proliferation process on the dynamic change of P-Body component molecules and whether APOBEC3F/3G has inhibitory effect on PRRSV proliferation weren’t reported.Therefore,this study takes PRRSV,P-Body,and APOBEC3 F / 3G as the research objects.To explore the effect of the PRRSV proliferation process on the dynamic changes of P-Body component molecules,whether APOBEC3 F has an inhibitory effect on PRRSV proliferation,and whether IFNγ can inhibit the proliferation of PRRSV by increasing APOBEC3 G overexpression,which were broaden the thinking of the relationship between PRRSV and host antiviral innate immunity and provide theoretical basis for the development of new antiviral protein drugs.The main research contents are as follows:1.Screening of P-Body module molecules related to PRRSV proliferation: Based on the gene sequence of P-Body module published in Gene Bank database,42 pairs of P-Body module protein-specific primers were designed,and q-PCR method were used to analyze the dynamic changes of the transcription level of P-Body module protein in Marc145 cells infected with PRRSV 24 h.The m RNA levels of APOBEC3 F,APOBEC3G,Ago2,ccr4-not,DCP1 a,DCP1b,DCP2,EDC3,EDC4,GW182,HNRNPU,LSM2,LSM3,LSM4,LSM6,DHX9,MOV10,PUM1,RAP55,SMG7,STAU2,SYNCRIP,and UPF3 A were down-regulated.The m RNA levels of DDX6,LSM1 and PUM2 were up-regulated.The m RNA levels of DDX3 X,ILF3,LSM5,LSM7,SMG1,SMG5,SMG6,Ro52,TNRC6 B,TNRC6C,TTP,TUT4,UPF1,UPF2,UPF3 B,and XRN1 weren’t changed.The results showed that the changes in the transcription level of most components of P-Body were affected by PRRSV infection.2.APOBEC3 F / 3G Gene Cloning and Bioinformatics Analysis: The CDS regions of APOBEC3F(m A3F)and APOBEC3G(m A3G)genes were amplified from Marc145 cells of monkey origin,and the CDS regions of APOBEC3F(p A3F)genes was amplified from peripheral mononuclear cells of the Sutai pigs origin,respectively,and then cloned and sequenced.Online biological software were used to analyze the biological information of A3F/3G gene from different host sources.The results showed that the m A3 F and m A3 G CDS were 1122 bp and1152bp,respectively,and the p A3 F CDS was 1257 bp,which were all in line with the expected size;the m A3 F gene had the highest nucleotide and amino acid sequence similarity to macaca leonina(Gen Bank accession number: KX583651),96.5% and 93.0%,respectively;p A3 F has the highest nucleotide and amino acid sequence similarity to sus scrofa(Gen Bank accession number: EU871586),99.0%and 98.8%,respectively;while the nucleotide and amino acid sequence homology between m A3 F and p A3 F is only 56.2%,36.9%,there are species differences in A3 F gene;m A3 F and p A3 F genes cannot exist stably in organism,but m A3 G gene can exist stably.The m A3 F,p A3 F,and m A3 G proteins all have the Pfam APOBEC＿N CD1,Pfam APOBEC＿C CD2 domains unique to members of the APOBECs family.The secondary and tertiary structures of m A3 F,p A3 F,and m A3 G proteins mainly include random coils,α-helices,and β-sheets,without transmembrane structure and signal peptide,and with multiple glycosylation and phosphorylation modification sites.3.The effect of APOBEC3 F on the proliferation of PRRSV: Construction of eukaryotic recombinant expression plasmids pc DNA 3.1-Flag-m A3 F,pc DNA3.1-Flag-p A3 F,and transfection of pc DNA 3.1-Flag-m A3 F,pc DNA 3.1-Flag-p A3 F at different doses in Marc145 cells.PRRSV was infected 24 h later and samples were collected 36 h later.IFA method was used to detect the expression and localization of Flag-m A3 F and p A3 F in cells;q-PCR and Western blot methods were used to detect the effect of overexpression of m A3 F and p A3 F on PRRSV proliferation.Different doses of A3 F interference fragments were transfected into Marc145 cells.PRRSV was infected 24 h later and samples were collected 36 h later.q-PCR and Western blot methods were used to detect the effect of interference with endogenous A3 F on PRRSV proliferation.The results showed that m A3 F protein was mainly located in the cytoplasm and p A3 F was located in the nucleus and cytoplasm.After overexpression of m A3 F or p A3 F,the N gene transcription level and translation level of PRRSV were significantly down-regulated,and after the endogenous m A3 F,the N gene transcription level and translation level of PRRSV were significantly up-regulated.The results showed that m A3 F and p A3 F can significantly inhibit the proliferation of PRRSV,and showed a dose-dependent effect.4.Effect of IFNγ on the anti-PRRSV effect of APOBEC3G: Construction of eukaryotic recombinant expression plasmids pc DNA 3.1-Flag-m A3 G,and transfection of pc DNA 3.1-Flag-m A3 G at different doses in Marc145 cells,PRRSV was infected 24 h later and samples were collected 36 h later.IFA method was used to detect the expression and localization of Flag-m A3 G in cells.q-PCR and Western blot methods were used to detect the effect of overexpression of m A3 G on PRRSV proliferation.Four groups were set: empty vector,IFNγ,pc DNA 3.1-Flag-m A3 G,pc DNA 3.1-Flag-m A3 G + IFNγ,all were transfected into Marc145 cells respectively,treated with IFNγ after 24 h,PRRSV was infected 24 h later and samples were collected 36 h later.q-PCR and Western blot were used to detect whether IFNγ could increase m A3 G overexpression to affect the proliferation of PRRSV,and depended on the JAK-STAT signaling pathway.The results showed that the m A3 G protein was mainly located in the nucleus and cytoplasm.After overexpression of m A3 G,the N gene transcription level and translation level of PRRSV were significantly down-regulated.The JAK1,JAK2,and SATA1 m RNA levels in the IFNγ + pc DNA3.1-m A3 G treatment group were significantly up-regulated,and the m RNA levels of IFNGR1 and IFNGR2 weren’t significantly changed in m RNA levels.There weren’t significantly changed in JAK1,JAK2,SATA1 m RNA levels in pc DNA3.1-m A3 G treatment group,and the m RNA levels of IFNGR1 and IFNGR2 were significantly up-regulated.Compared with the pc DNA3.1-m A3 G treatment group,the IFNγ + pc DNA3.1-m A3 G treatment group’s m A3 G gene transcription level and translation level were significantly up-regulated,and the PRRSV N gene transcription level and translation level were significantly down-regulated.The results showed that m A3 G can significantly inhibit the proliferation of PRRSV,and showed a dose-dependent effect.IFNγpromoted A3 G overexpression to inhibit PRRSV proliferation,and may depend on the JAK-STAT signaling pathway.