The Roles Of PI3K/AKT Cell Signal Pathway And Innate Immunity Restriction Factor SAMHD1in PRRSV Infection

Porcine reproductive and respiratory syndrome (PRRS), casued by porcine reproductive andrespiratory syndrome virus (PRRSV), is an acute and highly contagious viral disease and posessignificant economic losses to the pork industry worldwide. In2006, an outbreak of HP-PRRSVdescribed as ‘‘pig high fever disease’’ occurred in China and caused disastrous loses to the farmers. Atpresent, the virus is sustaining evolution and is currently a major concern for the swine industryworldwide. Due to the pathogenesis and immunological mechanism of PRRSV is in a complex manner,PRRSV is particularly difficult to control and the development a safe and effective vaccine for PRRSVhas difficulties. When viruses invade cells, various cellular mechanisms are activated to inhibit viralreplication. Most recently, the studies of PRRSV are focus on the mechanisms that how the virus toevade the recognition and elimination of host innate immune reponse, through the cell signalingpathways or encoded proteins. Phosphatidylinositol-3-kinase (PI3K)/Akt is an important cellularpathway, and has been shown to participate in various replication steps of multiple viruses. The sterilealpha motif and HD domain1(SAMHD1) protein has been identified as a novel innate immunityrestriction factor, function as a triphosphohydrolase that depletes cells of deoxynucleotide triphosphates(dNTPs), inhibits lentiviral complementary DNA (cDNA) synthesis and blocks HIV-1infection inmyeloid-lineage cells. Porcine alveolar macrophages (PAMs) are the target cells of PRRSV, whether thePI3K/Akt pathway is regulated by PRRSV and the relationship of PRRSV and SAMHD1require muchmore attentions and researches.In the study, our data provide new evidences of a novel role for the PI3K/Akt pathway inHP-PRRSV strain HuN4infection. The PRRSV regulates the PI3K/Akt pathway by differentmechanisms in PAMs and MARC-145cells. The PI3K/Akt pathway has a negligible effect on theprolifeation of PRRSV in MARC-145cells, but regulates the formation of virus-induced CPE. Thedownstream targets of Akt, FoxO1and Bad were regulated by PRRSV through the control of thePI3K/Akt pathway to control host-cell survival and the activity of GSK-3was essential for the virusinfection. To select the mutant virus which was conferred the drug resistance, GSK-3inhibitor X wasused to suppress the HuN4-F112virus replication in MARC-145for14passages. After fourteenpassages, the viral titer increased and restored to level of the control without pharmaceutical treatment.Then complete genome of the selected mutant virus which tolerated to the GSK-3inhibitor X wassequenced. Alignment of the complete genome sequence of the parental virus to the mutant virus withGSK-3inhibitor X resistance indicated that there are eighteen nucleotides changes. Six of them led tonon-synonymous mutation, one was found in both Nsp12and GP3, and two in Nsp2and GP4,respectively. The complete genome sequence analysis revealed that some amino acids in Nsp2, Nsp12,GP3and GP4were essential for the interaction with GSK-3individually or coordinately, which wasrequired for the virus replication.To explore the antiviral effect of SAMHD1on PRRSV infection, the porcine SAMHD1complete gene sequence was first cloned from porcine PBMCs by RACE PCR. The whole recombinant porcineSAMHD1protein was acquired by prokaryotic expression. Specific MAbs against porcine SAMHD1were prepared using the purified recombinant porcine SAMHD1as immunogen. In our analysis of thephylogenesis, subcellular localization and tissue distribution, the SAMHD1sequences were clearlyseparated in evolutionary terms and SAMHD1was localized to the nucleus. SAMHD1mRNA wasdetected in all of the swine tissues examined, and higher levels of SAMHD1mRNA were detected inthe tonsil, lung, liver, and lymph node tissues. The western blotting and immunofluorescence analysesshowed that proliferation of HP-PRRSV strain HuN4, was efficiently blocked in MARC-145cellsoverexpressing the porcine SAMHD1and viral titers were significantly reduced, compared to theHuN4-infected control cells. Analysis of the phosphorylation of SAMHD1found that the overexpressedSAMHD1was mainly in nonphosphorylated status. Real-time quantitative RT-PCR analysis showedthat the synthesis of HuN4genome cRNA was inhibited and the transcripts of ISG15and ISG56wasobviously upregulated in MARC-145cells overexpressing SAMHD1. The results suggested thatoverexpresstion of SAMHD1may perform its dNTP triphosphohydrolase functions to inhibit thesynthesis of cRNA and upregulates the expression of the antiviral genes to block the proliferation ofPRRSV.Here, we show that TLR-3and RIG-I/MDA5pathways are involved in SAMHD1expression, thephosphorylation and nucleus translocation of interferon regulation factor3(IRF-3) plays major role inSAMHD1expression. SAMHD1is inducible expression in porcine alveolar macrophages (PAMs) andMARC-145cells by IFN-α. Overexpression of TBK1enhances the SAMHD1promoter luciferaseactivity and upregulates expression. The mutation in the phosphorylation site S172A of TBK1impairedthe inducible expression of SAMHD1. Overexpression of IRF-3enhances the SAMHD1promoterluciferase activity, but only active form of IRF-3can play a role in inducing SAMHD1expression.Degradation of IRF-3expression by RNA interference and inhibition its phosphorylation and nucleartranslocation by BX795reveal that the IFN-α and NDV infection inducible upregulation of SAMHD1expression was significantly impaired. Complementary IRF-3expression, the inducible expression ofSAMHD1was restored restored to level of the control.Our data found that the phosphorylation of IRF-3was significantly blocked in MARC-145cellsinfected with HuN4. Meanwhile, the expression of SAMHD1was also in basic level. In contrast, thephosphorylation of IRF-3and expression of SAMHD1was significantly upregulated in PAMs infectedwith HuN4. The RIG-I/MDA5/TBK1pathway was participated in the activation of IRF-3by HuN4infection. Inhibition of the phosphorylation and nuclear translocation of IRF-3, the inducible expressionof SAMHD1was significantly reduced in PAMs infected with HuN4, and the proliferation of HuN4virus was increased at the early stage of infection. These findings indicate that SAMHD1plays animportant role in blocking HuN4virus proliferation in PAMs. Analysis the phosphorylation ofSAMHD1found that SAMHD1was phosphorylated in MARC-145infected with HuN4virus, but notin PAMs. Immunoprecipitation asssy showed that SAMHD1was phosphorylated by CyclinA2/CDK1complex in MARC-145cells. The activity of CDK1was upregulated by HuN4virus infection. The proliferation of HuN4virus was obviously inhibited by CyclinA2/CDK1inhibitor. Degradation of theexpression of SAMHD1, the proliferation of HuN4in MARC-145cells was not affected. The resultsshowed that the expression and activity of SAMHD1was inhibited in MARC-145cells infected byHuN4virus, via inhibiting the phosphorylation of IRF-3and promoting the activity of CyclinA2/CDK1.In summary，our data showed that the activation of PI3K/Akt is regulated by HP-PRRSV. As aninnate immunity restriction factor, SAMHD1could inhibit the proliferation of HP-PRRSV, and theactivity of SAMHD1was regulated by PRRSV for effective infection. To investigate the regulatorymechanism of SAMHD1by PRRSV makes sense to explore the pathogenic mechanism of infection andfurther highlights the antiviral researches of PRRSV.