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

“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.

• 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.

Pattern Recognition Theory

Charles Janeway, Yale University

Pattern Recognition Theory-25 years later

IRF3 and Activation by TBK/IKKε

Structural Domains of IRF-3

• Dimerization
• Nuclear localization
• CBP/p300 association
• Transactivation of IRF-3
• target genes
• (IFN, RANTES, IL15)

N DNA Binding C NES

Pro

Regulatory Domain IRF Association Domain

11 10 9 8 7 6 5 4 3 2 1 + + + +

• +
• +

+ + +

• +
• +

+ + +

• SeV

IFN-α Cytoplasmic Nuclear Fraction

I II III

382 - GGASSLENTVDLHISNSHPLSLTSDQYKAYLQD-414

(396/398)(402/404/405) (385/386)

H3 H4 C N H3 H4 N C

Structural Interactions in the C-terminal Region of IRF-3

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

Triggering the Interferon Antiviral Response Through an IKK-Related Pathway

Sonia Sharma,* Benjamin R. tenOever,* Nathalie Grandvaux,* Guo-Ping Zhou, Rongtuan Lin,† John Hiscott†

IKKα

745

Kinase Domain Leucine Zipper Helix- Loop- Helix K44A S176/180E

IKKβ

756

K44A S177/181E

15 15 301 301 453 456 487 486 592 606 643 63 9

27% 52% 61%

IdenDty vs

IKKα IKKε 27% 100% 100% 27% 24%

IKKε

716

33%

K38A

TBK-1

730

31%

K38A

9 300 500 527 578 619 591 632 527 499 9 300

TBK1 IKKε

Sensing of RNA virus infection by RIG-I

• Specificity

Complexity Diversity

RIG-I MAVS CARD domains Helicase domain

Inflammatory Cytokines

TRAF6 TNFa; IL6 NF-κB p65 p50 p50 IκB P IKKα NEMO IKKβ p65 p50

NF-κB sites

IFN Regulation

IRF3 P IRF3 P IRF3 TANK TBK1 IKKε IFNb1; ISGs IRF3 IRF3 P P TRAF3 P P P IRF7 IRF7 P IRF7

IRF sites

P IRF7 P IRF7

Specificity Complexity Diversity

Zevini et al , Trends in Immunol 2017 Chiang et al J. Virol. (2015) Beljanski et al J. Virol. (2015) Olagnier D et al. PLoS Path. (2014) Goulet ML et al. PLoS Path (2013) Sze A et al. Cell Host Microbe (2013) Olagnier & Hiscott J. Nature Immunol. (2012) Belgnaoui S, et al. Cell Host & Microbe (2012) Paz S, et al. Cell Res. (2011). Nakhaei P, et al. PLoS Path. (2009) Goubau D et al. Eur. J. Immunol. (2009) Zhao TJ et al. Nature Immunol. (2007) Romieu R et al. Cancer Res. (2006) tenOever B et al. J. Virol. (2004) Sharma S et al. Science (2003)

The cytosolic RIG-I the initial trigger of the pathway: antiviral immune response

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

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

Diversity: Transcriptome analysis of the host antiviral response to 5’pppRNA

5’pppRNA 24h 956 DEG IFN 6h 99 DEG IFN 24h 146 DEG

37 58 83 778 4 5

6h 6h 6h 24h 24h 24h 5’pppRNA IFNα-2b 100 IU/mL IFNα-2b 1000 IU/mL

Overlapping and unique gene networks

identified in response to 5’pppRNA and IFNα-2b

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

Functional characterization of genes differentially regulated by RIG-I

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

cGAS ISGs Type I IFN IRF 9 STAT1 STAT1 P P Type I IFN IFNAR1 IFNAR2 JAK1 TYK2 STAT1 STAT2 STAT1 STAT2 P P IRF9 STAT1 STAT2 P P IRF9

Antiviral State

TMEM173 is STING – An ER resident sensor of intracellular foreign DNA

IFNb1 Ca2+ IRF3 P IRF3 P P Endoplasmic Reticulum STING TBK1 PLCγ Pi3K AKT Nucleus P IRF3 IRF3 P P ROS TNFa

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

• +

STING expression is transcriptionally regulated by IRF3 & NFκB RelA

5’pppRNA

STING is upregulated by 5’pppRNA in vivo

MAVS TBK1 IKKε IRF3 IRF3 P P p p p 5’ MAVS RIG-I IRF3 IRF3 P P Type I IFN STING STING

Figure 3

RNA

p p p 5’ RNA Legionella HSV-1 EBV Pol III

ds AT-rich DNA ds DNA-RNA ds vDNA ss vRNA Reverse Transcription

Retrovirus

cGAMP

STING +ATP +GTP STING

cGAS IRF3 IRF3 P P

Enveloped RNA Viruses

IRF9 STAT1 STAT2 p p STING Type I IFN 5’pppRNA SENV JEV

Membrane Fusion Process

SENV VSV RIG-I p p p 5’ p p p 5’ RNA

Generation of a therapeutic RIG-I agonist

Poly (I:C)

RNA description Structure Relative activity

Influenza Measles Sendai Rabies Dengue 2

G20/AnG20 VSV WT M2 M3 M4 M5 CL2 CL9 M1 M6 M7 M8

1 10 100 1000

dsRNA modification enhances RIG-I agonist activity and maintains specificity

NS1 ISG56 pSTAT1 B-actin + + + + + CL9

• -

+ + + + + + + + + + + + + VSV WT M5 M8 IAV: RNA :

• Activity of 5’pppRNA agonists is primary

sequence-dependent

• Poly A-rich region is critical for antiviral

activity

• Duplex stability (i.e. GC rich regions) may

inhibit helicase activity of RIG-I, leading to decreased activity

RIG-I TLR3

• M8
• M8
• M8
• M8

siScr siRIG-I siTLR3 siMDA5 STAT1 MDA5 B-actin

10 20 30 40 50 60 C/C DENV M8 C/C DENV M8 C/C DENV M8 % DENV-positive cells siTLR3/MDA5 siRIG-I siScr

control WT M5 M8 poly (I:C)

BIRC3 CCL3 CCL5 CXCL10 DDX58 IFIT1 IFIT2 IFITM1 IFITM2 IFITM3 IFNAR1 IFNAR2 IFNB1 IL1A IL1B IL6 IL8 IL10 IL12A IL28RA IL29 IRF3 IRF7 ISG15 MX1 MX2 SOCS3 STAT1 TANK TLR3 TLR7 TNF CCL4 CD40 CD80 CD83 CD86 4-1BB HLA-DRA CD74 HLA-DQA

Antiviral and inflammatory response Dendritic cell maturation

Both intensity and breadth of immune response are enhanced in M8-treated cells

Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection

RESEARCH ARTICLE

crossmark

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

Specific deletion of Ifnar expression

• n 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.

Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection

RESEARCH ARTICLE

crossmark

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

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

Ø 5’pppRNA activated RIG-I-dependent antiviral and inflammatory response that inhibited a range of RNA viruses in vitro and in vivo. (Goulet et al, 2013; Olagnier et al, 2014) Ø Sequence modification improved RIG-I agonist antiviral activity 10-100 fold, enhanced dendritic cell maturation & T cell priming.

(Chiang et al, 2015)

Conclusions

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)