Research On The Activation To IFN-? Of ITIM Motif In PRRSV GP6 Protein

Porcine Reproductive and Respiratory Syndrome(PRRS)is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV)which causes serious reproductive failure in pregnant sows and severe respiratory distress in piglets and growing pigs.The genome size of PRRSV is 15.4kb,containing 10 open reading frames(ORFs).They are ORF1 a,ORF1b,ORF2 a,ORF2b and ORF3~ORF7 in order from 5'end to 3'end.ORF1 a and ORF1 b are non-structural protiens that participates in encoding RNA polymerase and replicase.ORF3~ORF7 encod the structural protiens of the virus: GP2,GP3,GP4,GP5(E protein),GP6(M protein)and GP7(N protein).Analysis of the sequence of the GP6 protein,we find that 24 to 29 amino acid residues containing an immunoreceptor tyrosine inhibitory motif(ITIM).The ITIM motif is an inhibitory sequence containing a conserved structure L / I / VXYXXL / V(X is an amino acid).It is phosphorylated by the Src family of protein tyrosine kinases and can recruit inositol polyphosphate phosphatase(SHIP-1),protein tyrosine phosphatases SHP-1 and SHP-2,etc.These phosphatases containing the SH2 region have catalytic activity,which regulate the conduction of intracellular signaling pathways.This study desighed to investigate the signal transduction mechanism of the ITIM motif in PRRSV GP6 protein.First,we designed a pair of primers with appropriate restriction sites according to the GP6 gene sequence of Genbank.The GP6 fragment was amplified from the PRRSV named VR2332 and cloned into eukaryotic expression vector pc DNA3.0.The recombinant expression vector pc DNA3.0-GP6 was constructed.We used the overlap extension PCR,designed a pair of primers with complementary ends,and the two fragments deleted from the ITIM motif were ligated together to construct the recombinant expression vector pc DNA3.0-GP6?ITIM.After sequencing,the homology of the target gene was more than 99%,and the two recombinant plasmids were constructed successfully.Then,pc DNA3.0-GP6,pc DNA3.0-GP6?ITIM and empty vector pc DNA3.0 were transfected into Marc-145 cells,and the Marc-145 cells blank control were setted.The m RNA levels of TRIF,My D88,TRAF6,IRF3,IRF7,NF-?B,IFN-? and other key molecules were detected by real-time PCR.The results showed that the ITIM motif in GP6 protein could upregulate the m RNA transcription levels of TRIF,IRF3,IRF7,NF-?B and IFN-? and downregulate the m RNA transcription levels of My D88 and TRAF6 in Marc-145 cells,which indicated that the ITIM motif might regulate TRIF mediated signaling rather than My D88.The ITIM motif was able to further upregulate the m RNA transcription levels of TRIF,IRF3 and IRF7,and the upregulation of NF-?B and IFN-? had a certain time limit by Poly(I:C)'s stimulation.Thus,we hypothesized that the ITIM motif in GP6 protein might activate the production of IFN-? by regulating the TLR3-mediated TRIF-dependent signaling pathway.In order to further clarify the immunoregulatory mechanism of ITIM motifs,we used RNA interference technique to downregulate the target genes in Marc-145 cells.pc DNA3.0-GP6 and pc DNA3.0-GP6?ITIM were transfected into Marc-145 cells which were downregulated the target genes,and then,the m RNA levels of TRIF,IRF3,IRF7,NF-?B,IFN-? and other key molecules were detected by real-time PCR.The results showed that silencing of SHP-1 gene in Marc-145 cells,ITIM motif in the GP6 protein could significantly downregulated the m RNA transcription levels of TRIF,IRF3,IRF7,IFN-? from 12 h,and the change of NF-?B was not obvious.After the silencing of SHP-2 gene,the regulation rules of ITIM motif to TRIF,IRF-3,NF-?B and IFN-? increased first,then decreased,and then stabilized,and the m RNA level of IRF-7 was continuously upregulated to 48 h.Our results suggested that SHP-1 played a key role in the activation to IFN-? of ITIM motif in PRRSV GP6 protein,and the relationship between ITIM motif and phosphatase SHP-2 need further exploration.