嗜神經(jīng)病毒躲避宿主先天性免疫反應(yīng)的機(jī)制及應(yīng)用趙凌老師

1、 嗜神經(jīng)病毒躲避宿主先天性免疫反應(yīng)的機(jī)制及對策趙趙 凌凌 教授教授華中農(nóng)業(yè)大學(xué)華中農(nóng)業(yè)大學(xué)10-15-2013Rabies virus180nm x 75nmRobert Hurt-USCRabies pathogenesis4Rabies infection and innate immunityWang et al. Journal of Virology, 20055Zhao et al. Journal of Virology, 2009重組狂犬病病毒構(gòu)建重組狂犬病病毒構(gòu)建6Expression of MIP-1MIP-1 attenuated RABV pathogenicity,

2、while expression of RANTES or IP-10 increased RABV pathogencityZhao et al. Journal of Virology, 20097Control rHEP HEP-MIP1a a HEP-RANTES HEP-IP10D3D6D9 HE staining of mouse brainsZhao et al. Journal of Virology, 2009Expression of MIP-1 MIP-1 enhances VNA production and protectionZhao et al. Journal

3、of Virology, 2010Zhao et al. Journal of Virology, 2010外周過量表達(dá)外周過量表達(dá)MIP-1會會吸引更多的樹突狀細(xì)胞和吸引更多的樹突狀細(xì)胞和B細(xì)胞細(xì)胞 Antigenic GroupVirusHostDiseaseI229ENL63humanhumanrespiratory infectionrespiratory infection, croupTGEVpigrespiratory and enteric infectionCCVdogenteric infectionFECVcatenteric infectionFIPVcatrespir

4、atory,enteric, hepatitis and neurological infectionIIOC43humanRespiratory and possibly enteric infectionSARS-CoVHKU1humanhumanRespiratoryRespiratoryMHVmouseRespiratory, enteric, neurologic infectionHEVpigRespiratory, enteric, neurologic infectionBCVcowEnteric infectionTCVturkeyRespiratory and enteri

5、c infection IIIIBVchickenRespiratory and enteric infection, hepatitisCoronaviruses cause diseases in humans and domestic animalsAdapted from Holmes and Lai, Fields VirologyWhy MHV? MHV produces a broad spectrum of disease in the mouse -pneumonia (MHV-1) -hepatitis (MHV-A59) -encephalitis (MHV-A59/JH

6、M) -demylination (MHV-A59) It provides excellent small animal models for hepatitis, for SARS, and for multiple sclerosisPart I: MHV ns2 interferes type I interferon responses Mutation of ns2 confers attenuation of hepatitis but not CNS disease IC 500PFUIH 500PFU Roth-Cross, J.K. et al, JVI, 2009, 83

7、(8):3743-3753.brainliverNs2 is an organ specific virulence factor 1a1b2a4 5a5bHESEMINMHV 2H phosphodiesterase; 2 predicted catalytic His-x-Thr/Ser motifs (Mazumder et a., 2002; Snijder et al.,2003)ns2(Mazumbder et al., 2002Snijder et al.,2003; Roth-Cross, 2009)Mutation of ns2 confers attenuation of

8、replication in macrophages and microglia but not in other cell types Zhao, L. et al, JVI, 2011.Oct; 85(19):10058-10068. MOI 1MOI 0.01 ns2 mutants recover the ability to replicate efficiently in macrophages and microglia from IFNAR knockout miceMOI 1 Zhao, L. et al, JVI, 2011.Oct; 85(19):10058-10068.

9、 Type 1 interferon induction and signaling pathwaysIFN-IFNTYK2JAK1PSTAT1P STAT1 orSTAT2IFNAR1IFNAR2ISREIRF9PSTAT1PSTAT2 ISGF3PSTAT2IRF-9ISGs:OAS, MxA, ISG15, ISG54,MDA5TLR3TLR7/8TLR9dsRNACpGssRNATIRTBK-1IKK-IRF-3IFN-NF-BATF-2RIG-ICBPPMDA5TIRTIRTRIFMyD88MyD88ISREIFN-L2AstroBMMIFNAR-/- BMMns2 mutants

10、are not defective in induction of IFN- / / mRNABoth wt RA59 and ns2 mutants induce minimal amounts of IFN-, mRNA in L2 cells and astrocytes wt RA59 and ns2 mutants induce similar levels of IFN-, mRNA in BMM from both B6 and IFNAR-/- mice Zhao, L. et al, JVI, 2011.Oct; 85(19):10058-10068. BMMBMMMicro

11、glians2 mutants are more sensitive to the antiviral effects of IFN-IFN-/ / than wt A59 in macrophages and microgliabut not in other cell typesL2AstroViral replication and IFN sensitivity in the hepatocytesNo IFNIFNIn vivo macrophage depletionLiposomes, encapsulating the Clodronate molecules (squares

12、), are ingested by macrophages via endocytosis. After fusion with lysosomes (L) containing phospholipases (arrowheads), the latter disrupt the bilayers of the liposomes. The more concentric bilayers are disrupted, the greater is the Clodronate release within the cell. The cells are killed by Clodron

13、ate through apoptosis. Liposome ClodronateA59ns2-H126RA59ns2-H126RPBSLiposome ClodronateH&E stainingN protein stainingns2 mutants replicate and induce hepatitis in macrophage depleted mice500 fold vs 10 fold Model: Kupffer cells provide a barrier to the liver parenchyma to viruses=rA59LSECKCHepatocy

14、tesinusoidparenchyma=ns2-H126APart I: conclusionsl ns2 is an organ specific virulence factor and antagonizes IFN signalingl ns2 is required for replication in macrophages; depletion of macrophages in vivo promotes ns2 mutant virus replicationl we suggest that MHV has to replicate in Kupffer cells in

15、 the liver sinusoids in order to reach the liver parenchyma and induce hepatitis Type 1 interferon induction and signaling pathwaysIFN-IFNTYK2JAK1PSTAT1P STAT1 orSTAT2IFNAR1IFNAR2ISREIRF9PSTAT1PSTAT2 ISGF3PSTAT2IRF-9ISGs:OAS, MxA, ISG15, ISG54,MDA5TLR3TLR7/8TLR9dsRNACpGssRNATIRTBK-1IKK-IRF-3IFN-NF-B

16、ATF-2RIG-ICBPPMDA5TIRTIRTRIFMyD88MyD88ISREIFN-pCAGGS-IFNNS2a-IFNSV5V-IFNNDV-bioassay in Vero cellspCAGGS-no IFNTansfection of pCAGGSor pCAGGS-ns2 or pCAGGS-SV5Vin Vero cellsTreatment with or w/o1000U/ml for 16hInfection of NDV-GFP24h12hGFP ISG screening in KO BMMISG15IFIT1IFIT2PKRRNase LPart II: MHV

17、 ns2 antagonizes OAS-RNase L pathwayInterferon signaling modelViral dsRNAMDA5*, RIG-I*IFNAntiviral ISGsOAS*2-5ARNase LCellular and viral RNASmall RNAs2-PDEns2?Main pathwayOAS-RNase L pathwayOAS= 2,5-oliogoadenylate synthetase2-PDE= 2 phosphodiesterase2-5A=2,5oligoadenylateWild type A59 replication i

18、n BMM was not affected by the OAS-RNase L systemDefective replication of ns2 mutant is restored in RNase L-/- BMMBut not in PKR-/- BMMZhao, L et al. Cell Host & Microbe, 2012, (11) 607616.ns2 expression in 293T cellspCAGGS-ns2pCAGGSpCAGGS-ns2-H126RZhao, L et al. Cell Host & Microbe, 2012, (11) 60761

19、6.ns2 prevents rRNA cleavage and 2-5A production in BMMZhao, L et al. Cell Host & Microbe, 2012, (11) 607616.Overexpression of ns2 in 293T cells prevents rRNA cleavage and 2-5A production induced by poly I:CZhao, L et al. Cell Host & Microbe, 2012, (11) 607616.Ns2 cleaves 2-5A into ATP and AMPZhao, L et al. C