Viral evasion of intracellular innate immune sensing pathways 1. - - PowerPoint PPT Presentation

Viral evasion of intracellular innate immune sensing pathways

• 1. Hiding the viral genome
• 2. Inhibiting interactions with key host

inducers of the IFN response

• 3. Regulating phosphorylation events
• 4. Regulating ubiquitinylation & related pathways
• 5. Cleavage & degradation

6.Transcriptional shut-off 7 . RNA processing & trafficking regulation

• 8. Translational shut-off
• 9. Decoys
• 10. Everything counts

Ten Strategies: Evasion of Induction of Interferons (1)

Ten Strategies: Inhibition of Interferon Signaling (2)

Ten Strategies: Inhibition of Host Gene Expression (3)

Dengue virus immunopathogenesis

• Viral evasion strategies
• Metabolic stress & innate immunity

Epidemiology and pathogenesis of dengue infection

Ø Most common arthropod-borne viral pathogen

• transmitted to humans by Aedes aegypti and

Aedes albopictus Ø 2.5 billion people at risk in tropical regions Ø Estimated 300 million infections per year, with 25,000-50,000 deaths annually Ø Primary infection: often a self limiting acute infection (‘breakbone fever’) Ø Secondary heterologous infection generally lead to more severe immunopathogenic disease with risk of dengue hemorrhagic fever or shock syndrome (DHF/DSS) Ø No effective antiviral agents and no effective vaccine to treat or prevent dengue infection

Dendritic Cells and Dengue Virus

Ø Skin-resident DC and Langerhans cells are the initial targets of infection Ø DC are able to mount a rapid inflammatory and antiviral response to DENV Ø DENV-infected DC: induce an innate immune response to infection, undergo maturation and are subject to viral evasion mechanisms (interference with STAT signaling; viral protease cleavage of STING) Ø Interaction between DENV and DC is crucial for both control of infection and the evolution of disease severity Understanding the mechanisms involved in DENV sensing and the innate immune response to infection is critical for the comprehension of DENV disease evolution and control of infection

Differentiated myeloid cells are the main targets for DENV infection

20 40 60 80 100 20 40 60

% of DENV2+ cells

% of CD14- CD1a+ cells

r2= 0.9829 p<0.0001 n=15 DENV2 SSC-A 79.6% CD1a CD14

• DENV2

Monocyte + DENV2 Mo-DC Day 0 Day 7 *** *** *** **

.

***

mRNA relative expression

MT1A MT2A MT1E MT1G MT1H ERO1L DDIT4 ISG15 OAS2 HERC5 IFIT1 IFIT2 IFIT3 OASL HES4 CCL5

Antioxidant genes Source of ROS DNA-damaged induced gene Antiviral response

12 18 24 6

• 6000
• 4000
• 2000

2000 4000 6000

6000 4000 2000 2000 6000 4000

Up-regulated Down-regulated

Number of DEGs

Time (h) 6 12 18 24

Differential gene expression appears early following DENV2 infection of dendritic cells

Antiviral Response Myeloid Signalling Inflammatory Response Stress Response Apoptosis Diverse Signalling

0 6 12 18 24

Stress-Associated Pathways Are Enriched Early After DENV2 Infection

6H 24H

Is there a link between DENV2-induced oxidative stress response and DENV2-induced antiviral/ inflammatory responses?

DPI

10 20 30 40 50 60 70

mRNA relative expression

DENV2 - + + + + + + +

• + + + + + + +
• + + + + + + +
• + + + + + + +

IFN-β CCL5 IL-1β IDO1

Apocynin Tempol Ebselen PDTC Trolox

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

NADPH oxidase-dependent ROS formation restricts DENV infection and replication

DPI DENV2 DPI -

• +

+ + 0.1 1

% of DENV E+ cells

• +

+ + 0.1 1 DENV2 p= 0.036 p= 0.004 p= 0.044

DENV E FSC-A

uninfected DENV 38.7±3.9 52.1±4.0 1.54±0.3

Viral Titer

• DPI

DENV2 1 10 mRNA relative expression

• 0.1 1

10 0.1 + DPI 10

Time (h)

6 12 18 24

• 0.1

+ DPI

• 0.1 1 10

1

HMOX-1 mRNA relative expression

• DPI

DENV2 ** **

• ***

SOD2 mRNA relative expression

*

MT2A FTH1P11 TXNRD1 NQO1 EIF2AK3 MT1G FTH1P16 GCLM MT1E NQO2 SOD2 UGCG GCLC FTH1P2 SQSTM1 MT1A FTH1P3 MT1X HMOX1 AKR1C3

Time (h)

DENV infection stimulates an Nrf2-driven antioxidant response

Nrf2 is a master regulator of the oxidative stress response

Mitsuishi Y, Front Oncol, 2012

Ø Nuclear factor-erythroid 2 related factor 2

• bZIP transcription factor
• involved in the redox homeostasis and in the protection to
• xidative stress
• regulates the expression of cytoprotective and antioxidant genes

Si Nrf2

5 10

• -

IFNb * CXCL10

Si Ctrl+DV2 Si Nrf2+DV2

**

• NOXA

Si-Nrf2

mRNA relative expression

+ + - DENV2 DENV2

• +
• Si-control

IFIT1

mRNA relative expression

RSAD2 + Nrf2

10 80 40 60 6 10 6 80 160 120

DDX58

10 20 30 40 50

+ +

• -
• +

2.103 1.103 2.104

Si-Nrf2 Si-control

• -

+ +

10 15 5

+ + BCLX

10 15 5

RIPK1

4 6 2

• +

+ +

mRNA relative expression

** * **

p= 0.031

• +

Events

596 1088 1088 1996 Si Ctrl

ROS + + **

15 20 25 92.0% 88.8%

DENV-E

Si Ctrl Si Ctrl+DV2 Si Nrf2+DV2

DENV2

Nrf2 knockdown deregulates ROS production and antiviral/apoptotic responses in DENV infection

+

Itaconate – suppresses inflammation via induction of the transcription factor Nrf2 through direct binding

• f the Nrf2 repressor Keap1

ITAC & SFN stimulate Nrf2 & anti-oxidant response element Modulation of Nrf2 & KEAP1 inversely control the antiviral response

Conclusions

Ø DENV2 infection generates an NOX-dependent ROS production in mature DC Ø DENV2-induced ROS formation is essential for the activation of the innate immune response and the activation of IRF3/STAT1 and NF-κB signaling in DC Ø DENV2-infected DC undergo NOX-mediated mitochondrial-dependent apoptosis Ø Nrf2 protects cells from stress-associated damages including over-activation of the immune and apoptotic responses

DENV induces NOX- dependent ROS production required for antiviral & apoptotic responses

Olagnier et al Mol Therapy 2017

NRF2 Arginosuccin ate shunt Citrate Malate Fumarate SDH (CII) Succinate mROS HIF-1α IL1-β AlphaKG Glutamate ITACONIC ACID

Inflammation

HMOX-1

HBV HCV HIV Influenza EV71 RSV EBOV DENV

Antibacterial Activity

Interferon pathway

IRG1 Antiviral Activity Metabolic re-programming inhibits antiviral responses via Nrf2

Acknowledgements

VGTI-FL

David Olagnier Cindy Chiang Vladimir Beljanski

Nadine VanMontfoort Kevin Yin Carmen Nichols Zhong He Elias Haddad Lydie Trautmann

McGill University

MarieLine Goulet Julien van Grevenynghe

Samar Bel Hadj Stephanie Oliere Suzanne Paz Rongtuan Lin

Washington University St. Louis

Michael S. Diamond

Mount Sinai School of Medicine

Ana Fernandez-Sesma Adolfo Garcia-Sastre

Leiden University Medical Center

Martijn Van Hemert Irina Albulescu Florine Scholte Marjolein Kikkert Eric Snijder

VGTI-Oregon

Vic Defilippis Dan Streblow

Fox Chase Cancer Center

Sid Balachandran Suraj Peri

Genomics/Bioinformatics VGTI-FL

Mark Cameron Peter Wilkinson Courtney Steel Stephanie Richards Andrew Smith

Acknowledgements

Luciano Castiello Michela Muscolini Alessandra Zevini Enrico Palermo Matteo Ferrari Dominga Lovecchio Fani Souvalidou Shirley Mann

Thank you