Viral evasion of intracellular innate immune sensing pathways Course - PowerPoint PPT Presentation

Viral evasion of intracellular innate immune sensing pathways Course in Virology Erasmus University Medical Center May 30, 2018

Pattern Recognition Theory - a general theory of innate immune recognition (pattern recognition theory) - activation of the adaptive immune response is controlled by the more ancient innate immune system. “I contend that the immune system has evolved specifically to recognize and respond to infectious microorganisms, and that this involves recognition not only of specific [proteins], but also of certain characteristics or patterns common on infectious agents but absent from the host. Charles Janeway, Yale University

Pattern Recognition Theory-25 years later

IRF3 and Activation by TBK/IKK ε

Structural Domains of IRF-3 Regulatory Domain DNA Binding NES Pro N C IRF Association Domain 382 - GGA SS LENTVDLHI S N S HPL S L TS DQYKAYLQD- 414 (385/386) (396/398)(402/404/405) Cytoplasmic Nuclear Fraction - Dimerization SeV - + + - - + + - - + + - Nuclear localization - + + - + + + - + + + IFN- α - CBP/p300 association - Transactivation of IRF-3 III II I - target genes - (IFN, RANTES, IL15) 1 2 3 4 5 6 7 8 9 10 11

Structural Interactions in the C-terminal Region of IRF-3 C N N H4 H3 H3 H4 C The ribbon diagrams illustrate the interactions between the IAD of IRF-3 and the IBiD region of CBP. Left: Intramolecular interactions between the IAD of IRF-3 (in green) and the flanking autoinhibitory structures (in red). Phosphorylation sites are in yellow. Right: Intermolecular interactions between the IAD of IRF-3 (in green) and the IBiD region of CBP (in blue). Qin et al, 2003, 2005; Takahashi et al, 2003

IdenDty vs Helix- Leucine Loop- Triggering the Interferon IKK α IKK ε Zipper Kinase Domain Helix IKK α 100% 27% Antiviral Response Through an 15 K44A S176/180E 301 453 487 592 63 745 9 IKK-Related Pathway 24% IKK β 52% 15 K44A S177/181E 301 456 486 606 643 756 Sonia Sharma,* Benjamin R. tenOever,* Nathalie Grandvaux,* Guo-Ping Zhou, Rongtuan Lin, † John Hiscott † 33% IKK ε 27% 100% 9 K38A 300 500 527 578 619 716 31 % TBK-1 27% 61% 9 300 499 527 591 632 730 K38A TBK1 IKK ε

Sensing of RNA virus infection by RIG-I � � Specificity � Complexity � Diversity

The cytosolic RIG-I the initial trigger of the pathway: antiviral immune response CARD domains RIG-I Specificity Zevini et al , Trends in Immunol 2017 Helicase MAVS Chiang et al J. Virol. (2015) domain Beljanski et al J. Virol. (2015) TRAF6 TRAF3 Olagnier D et al. PLoS Path. (2014) Goulet ML et al. PLoS Path (2013) Sze A et al. Cell Host Microbe (2013) NEMO TANK Complexity IKKα IKKβ TBK1 IKKε Olagnier & Hiscott J. Nature Immunol. (2012) P Belgnaoui S, et al. Cell Host & Microbe (2012) IκB Paz S, et al. Cell Res. (2011). P P Nakhaei P, et al. PLoS Path. (2009) IRF3 IRF7 p50 P P Goubau D et al. Eur. J. Immunol. (2009) IRF3 IRF7 p65 p50 Zhao TJ et al. Nature Immunol. (2007) Diversity IRF3 IRF7 Romieu R et al. Cancer Res. (2006) NF- κ B P P tenOever B et al. J. Virol. (2004) TNFa; IL6 IFNb1; ISGs Sharma S et al. Science (2003) P P IRF3 p50 IRF7 p65 IRF3 IRF7 P NF-κB sites P IRF sites Inflammatory Cytokines IFN Regulation

C-terminal Structure of RIG-I with the 5’ppp binding pocket

Diversity: Transcriptome analysis of the host antiviral response to 5’pppRNA CCL5 IFIT3 CCL3L1 HLA-F IL28A CXCL10 PRIC285 PLEKHA4 IFI27 IFI6 IFITM1 CCL4L1 IL29 IL6 IDO1 CX3CL1 HSPA6 CCL3 HLA-H IFIT2 IFIH1 TNFAIP3 IFIT1 OLR1 GBP4 MX1 IFITM3 IFI44 ISG15 RARRES3 HSPA7 IFNB1 OAS2 DDX58 CCL3L3 IL8 RSAD2 IL28B OASL SAMD9 HLA-B ISG20 PMAIP1 C15orf48 HERC5 DDIT3 LCN2 CFB GBP1 TOP2A CDKN3 RFC5 CES1 PBK KIAA0101 TIMELESS MCM7 CCNB2 AKR1B10 TP53I3 PDCL3 GINS2 BOP1 DLGAP5 FASN AKR1B15 CXCR7 KCNF1 HTRA1 ALDH3A1 MCM6 RUVBL2 FOS CDC45 PRKCA CDC20 PSAT1 CDCA3 ACO1 AURKB TYMS MALL CCNB1 CDK1 RBM14 BTBD11 MCM4 GPX2 CCDC34 KIF20A SCD NEK2 SGK1 HJURP EIF4B CCNA2 SNRNP25 TK1 MCM5

Overlapping and unique gene networks identified in response to 5’pppRNA and IFNα-2b IFNα-2b IFNα-2b 100 IU/mL 1000 IU/mL 5’pppRNA 6h 24h 6h 24h 6h 24h 5’pppRNA 24h IFN 6h 956 DEG 99 DEG 4 37 778 58 5 83 IFN 24h 146 DEG

Functional characterization of genes differentially regulated by RIG-I 24h Inflammasome Pattern Recognition Receptor Signaling NF- κ B and adaptive immunity TGF- β signal Myc Signaling FOS Regulation Chemokines Ubiquitin signaling Chemokines NF- κ B Regulation Fos Metabolism Regulators of cell Apoptosis cycle progression Pro-inflammatory cytokines Anapahase regulators Molecular Chaperones Type I and III IFNs Chromatin Separation Hypoxia pathway STATs signaling Cell Cycle Regulation Transcriptional Regulation Chaperones and Heat Shock Response 6h IRFs signal STATs signal

Cross-talk between the RNA (RIG-I) and DNA (cGAS-STING) sensing pathways

TMEM173 is STING – An ER resident sensor of intracellular foreign DNA Type I IFN IFNAR1 IFNAR2 PLC γ ROS JAK1 Ca 2+ TYK2 Pi3K Endoplasmic Reticulum P P AKT STAT1 STAT1 cGAS STAT2 P STING IRF9 STAT2 Type I IFN TBK1 IRF9 P P STAT1 IRF3 STAT2 IRF3 P P P P TNFa IFNb1 IRF3 Antiviral IRF3 ISGs P IRF STAT1 P State 9 STAT1 Nucleus P

STING is upregulated by SeV infection through the RIG-I - MAVS pathway

STING expression is transcriptionally regulated by IRF3 & NF κ B RelA - + 5’pppRNA

STING is upregulated by 5’pppRNA in vivo

Figure 3 Legionella 5’pppRNA Enveloped SENV HSV-1 SENV Retrovirus RNA Viruses VSV EBV JEV Membrane Fusion ds AT-rich DNA Process ss vRNA Transcription Reverse Pol III ds DNA-RNA p p p 5’ ds vDNA p p p 5’ RNA RNA cGAS RNA RIG-I +ATP +GTP MAVS p p p cGAMP RIG-I p p p 5’ TBK1 IKKε MAVS STING 5’ STING STING STING P IRF3 IRF3 P Type I IFN STING Type I IFN P P p IRF3 p IRF3 STAT1 STAT2 IRF3 IRF3 IRF9 P P

Generation of a therapeutic RIG-I agonist

dsRNA modification enhances RIG-I agonist activity and maintains specificity poly (I:C) RNA Relative activity control Structure description 1 10 100 1000 WT • 60 Activity of 5’pppRNA agonists is primary M5 M8 % DENV-positive cells Influenza 50 sequence-dependent p p p BIRC3 Measles CCL3 p p p 40 CCL5 Poly A-rich region is critical for antiviral • CXCL10 30 Sendai DDX58 p p p activity IFIT1 20 Antiviral and inflammatory response Rabies IFIT2 p p p IFITM1 • Duplex stability (i.e. GC rich regions) may 10 IFITM2 Dengue 2 IFITM3 inhibit helicase activity of RIG-I, leading 0 IFNAR1 G20/AnG20 IFNAR2 C/C DENV M8 C/C DENV M8 C/C DENV M8 p p p to decreased activity IFNB1 IL1A VSV WT p p p IL1B siScr siRIG-I siTLR3/MDA5 IL6 IL8 M1 p p p IL10 IL12A M2 IL28RA p p p IL29 VSV WT M5 M8 CL9 IRF3 siScr siRIG-I siTLR3 siMDA5 M3 IRF7 p p p ISG15 MX1 M4 - M8 - M8 - M8 - M8 IAV: + + + + + + + + + + + + + + + + + + p p p MX2 SOCS3 - - RNA : M5 STAT1 RIG-I p p p TANK NS1 TLR3 M6 p p p TLR7 TLR3 TNF CCL4 M7 p p p pSTAT1 Dendritic cell CD40 maturation MDA5 CD80 CD83 M8 CD86 ISG56 p p p 4-1BB STAT1 HLA-DRA CL2 CD74 B-actin HLA-DQA CL9 B-actin Both intensity and breadth of immune Poly (I:C) response are enhanced in M8-treated cells

crossmark RESEARCH ARTICLE Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection Models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type I IFN signaling have been used extensively. However, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Pinto et al MBio 2015

crossmark RESEARCH ARTICLE Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection Specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre Ifnarflox/flox) resulted in enhanced DENV replication in vivo . The administration of subneutralizing amounts of cross-reactive anti-DENV antibody to LysM Cre Ifnarf/f mice prior to infection with DENV2 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, and thrombocytopenia.

Pre-exposure therapy with M8 controls DENV2 & DENV3 infection and disease in LysM Cre+ ifnar f/f mice

Post-exposure therapy with M8 controls DENV2 & DENV3 infection and disease in LysM Cre+ ifnar f/f mice

Conclusions 5 ’ pppRNA activated RIG-I-dependent antiviral and inflammatory Ø response that inhibited a range of RNA viruses in vitro and in vivo. (Goulet et al, 2013; Olagnier et al, 2014) Sequence modification improved RIG-I agonist antiviral activity Ø 10-100 fold, enhanced dendritic cell maturation & T cell priming. (Chiang et al, 2015) Ø Therapeutic immunostimulation of the RIG-I pathway diminished the symptoms of severe Dengue virus infection in a new murine model of dengue immunopathogenesis. (Pinto et al, 2015)