1、嗜神經(jīng)病毒躲避宿主先天性免疫反應(yīng)的機制及對策,趙 凌 教授 華中農(nóng)業(yè)大學(xué) 10-15-2013,Rabies virus,180nm x 75nm,Robert Hurt-USC,Rabies pathogenesis,Patients die of circulatory insufficiency, cardiac arrest and respiratory failure.,4,Rabies infection and innate immunity,Wang et al. Journal of Virology, 2005,5,Zhao et al. Journal of Virol

2、ogy, 2009,重組狂犬病病毒構(gòu)建,6,Expression of MIP-1 attenuated RABV pathogenicity, while expression of RANTES or IP-10 increased RABV pathogencity,Zhao et al. Journal of Virology, 2009,7,Control rHEP HEP-MIP1a HEP-RANTES HEP-IP10,D3 D6 D9,HE staining of mouse brains,Zhao et al. Journal of Virology, 2009,Expre

3、ssion of MIP-1 enhances VNA production and protection,Zhao et al. Journal of Virology, 2010,Zhao et al. Journal of Virology, 2010,外周過量表達MIP-1會吸引更多的樹突狀細胞和B細胞,Coronaviruses cause diseases in humans and domestic animals,Adapted from Holmes and Lai, Fields Virology,Why MHV?,MHV produces a broad spectrum

4、 of disease in the mouse -pneumonia (MHV-1) -hepatitis (MHV-A59) -encephalitis (MHV-A59/JHM) -demylination (MHV-A59) It provides excellent small animal models for hepatitis, for SARS, and for multiple sclerosis,Part I: MHV ns2 interferes type I interferon responses,Mutation of ns2 confers attenuatio

5、n of hepatitis but not CNS disease,IC 500PFU,IH 500PFU,Roth-Cross, J.K. et al, JVI, 2009, 83(8):3743-3753.,brain,liver,Ns2 is an organ specific virulence factor,2H phosphodiesterase; 2 predicted catalytic His-x-Thr/Ser motifs (Mazumder et a., 2002; Snijder et al.,2003),(Mazumbder et al., 2002 Snijde

6、r et al.,2003; Roth-Cross, 2009),Mutation of ns2 confers attenuation of replication in macrophages and microglia but not in other cell types,Zhao, L. et al, JVI, 2011.Oct; 85(19):10058-10068.,MOI 1,MOI 0.01,ns2 mutants recover the ability to replicate efficiently in macrophages and microglia from IF

7、NAR knockout mice,MOI 1,Zhao, L. et al, JVI, 2011.Oct; 85(19):10058-10068.,Type 1 interferon induction and signaling pathways,IFN-,L2,Astro,BMM,IFNAR-/- BMM,ns2 mutants are not defective in induction of IFN-/ mRNA,Both wt RA59 and ns2 mutants induce minimal amounts of IFN-, mRNA in L2 cells and astr

8、ocytes,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.,BMM,BMM,Microglia,ns2 mutants are more sensitive to the antiviral effects of IFN-/ than wt A59 in macrophages and microglia,Viral replication an

9、d IFN sensitivity in the hepatocytes,No IFN,IFN,In vivo macrophage depletion,Liposomes, encapsulating the Clodronate molecules (squares), are ingested by macrophages via endocytosis. After fusion with lysosomes (L) containing phospholipases (arrowheads), the latter disrupt the bilayers of the liposo

10、mes. The more concentric bilayers are disrupted, the greater is the Clodronate release within the cell. The cells are killed by Clodronate through apoptosis.,ns2 mutants replicate and induce hepatitis in macrophage depleted mice,500 fold vs 10 fold,Model: Kupffer cells provide a barrier to the liver

11、 parenchyma to viruses,=rA59,LSEC,KC,Hepatocyte,sinusoid,parenchyma,=ns2-H126A,Part I: conclusions,ns2 is an organ specific virulence factor and antagonizes IFN signaling ns2 is required for replication in macrophages; depletion of macrophages in vivo promotes ns2 mutant virus replication we suggest

12、 that MHV has to replicate in Kupffer cells in the liver sinusoids in order to reach the liver parenchyma and induce hepatitis,Type 1 interferon induction and signaling pathways,IFN-,pCAGGS-IFN,NS2a-IFN,SV5V-IFN,NDV-bioassay in Vero cells,pCAGGS-no IFN,Tansfection of pCAGGS or pCAGGS-ns2 or pCAGGS-S

13、V5V in Vero cells,Treatment with or w/o 1000U/ml for 16h,Infection of NDV-GFP,24h,12h,GFP,ISG screening in KO BMM,ISG15,IFIT1,IFIT2,PKR,RNase L,Part II: MHV ns2 antagonizes OAS-RNase L pathway,Interferon signaling model,Viral dsRNA,MDA5*, RIG-I*,IFN,Antiviral ISGs,OAS*,2-5A,RNase L,Cellular and vira

14、l RNA,Small RNAs,2-PDE,ns2?,Main pathway,OAS-RNase L pathway,OAS= 2,5-oliogoadenylate synthetase 2-PDE= 2 phosphodiesterase 2-5A=2,5oligoadenylate,Wild type A59 replication in BMM was not affected by the OAS-RNase L system,Defective replication of ns2 mutant is restored in RNase L-/- BMM,But not in

15、PKR-/- BMM,Zhao, L et al. Cell Host & Microbe, 2012, (11) 607616.,ns2 expression in 293T cells,pCAGGS-ns2,pCAGGS,pCAGGS-ns2- H126R,Zhao, L et al. Cell Host & Microbe, 2012, (11) 607616.,ns2 prevents rRNA cleavage and 2-5A production in BMM,Zhao, L et al. Cell Host & Microbe, 2012, (11) 607616.,Overe

16、xpression of ns2 in 293T cells prevents rRNA cleavage and 2-5A production induced by poly I:C,Zhao, L et al. Cell Host & Microbe, 2012, (11) 607616.,Ns2 cleaves 2-5A into ATP and AMP,Zhao, L et al. Cell Host & Microbe, 2012, (11) 607616.,Ns2 enhances virus replication in the liver,Zhao, L et al. Cell Host & Microbe, 2012, (11) 607616.,Ns2 facilitates the induction of viral hepatitis,Zhao, L et al. Cell Host & Microbe, 2012, (11) 607616.,Mouse hepatitis virus ns2 protein inhibits the IFN-induced OAS-RNase L pathway ns2 reduces the intracellular level of