General concepts of antiviral immunity. source: wikipedia

Transcription

1 General concepts of antiviral immunity source: wikipedia

2 Antiviral defense mechanisms - the birth of immunity Evolution of antiviral defense 4 Principles of innate and adaptive defense mechanisms R/M System - CRISPR/Cas9 - RNAi The emergence of a PRR-based defense mechanism in vertebrates - the innate immune system

3 Who is fighting who? impose Host defend source: cell

4 Antiviral defense impose Host defend source: cell

5 How it all started: defense against foreign genetic material selfish genetic element impose defend Genome

6 Viruses evolved alongside the primordial soup cellular organisms pre cellular replicators Koonin et al. Virology 2015

7 There is no evidence for monophyly of viruses Source: Koonin et al. Virology 2015

8 There are no shared common genes - but there are hallmark genes that are shared between different groups Source: Koonin et al. Virology 2015

9 Viruses - some facts obligate intracellular parasite that needs host ribosomes and mitochondria to propagate viruses are highly abundant (most individuals and species, second most biomass) viruses for every life form viruses are highly diverse in structures and genomes (2 kb to over 2 Mb) high mutability, variability (quasispecies) transmission is the only selection factor in viral evolution (penetration, innate and adaptive immune escape, replication, transport & spread within host, assembly of novel virions) pathogenicity is usually unrelated (or inversely related) to viral transmission (selection pressure is against pathogenicity)

10 Animal viruses

11 What is a systematic approach to classify viruses?

12 The Baltimore classification of animal viruses VI: Single-stranded (+) sense RNA with DNA intermediate in life-cycle II: Single-stranded (+)sense DNA +DNA ± DNA VII: Double-stranded DNA with RNA intermediate +RNA -DNA ± DNA I: Double-stranded DNA +RNA -RNA +RNA ± RNA IV: Single-stranded (+)sense RNA -RNA Translation III: Doublestranded RNA V: Single-stranded (-) sense RNA

13 The Baltimore classification of viruses 1st: Nucleic acid genome and replication strategy I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc) II: Single-stranded (+)sense DNA (Parvoviruses) III: Double-stranded RNA (Reoviruses; Birnaviruses) IV: Single-stranded (+)sense RNA (Picornaviruses; Togaviruses, etc) a) Polycistronic mrna e.g. Picornaviruses; Hepatitis A. Genome RNA = mrna. b) Complex Transcription e.g. Togaviruses. V: Single-stranded (-) sense RNA a) Segmented, e.g. Orthomyxoviruses. b) Non-segmented, e.g. Rhabdoviruses, Paramyxoviruses. VI: Single-stranded (+) sense RNA with DNA intermediate in life-cycle (Retroviruses) VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses) 2nd: capsid structure (icosahedral or helical) envelope (lipid bilayer) or not

14 Replication strategies of (+) and (-) strand RNA viruses Single-stranded (+)sense RNA Single-stranded (-) sense RNA Source: Microbiol. Mol. Biol. Rev. June 2013 vol. 77 no

15 Evolution of antiviral defense mechanisms - nucleic acids are the target Benjamin R. tenoever Cell Host and Microbe 2016

16 Evolution of antiviral defense mechanisms Benjamin R. tenoever Cell Host and Microbe 2016

17 Antiviral defense in prokaryotes source: wikipedia

18 dsdna viruses dominate the prokaryotic world

19 Bacteriophages infect prokaryotes and archaea Source: Nature Reviews Microbiology 8, (May 2010)

20 Restriction nucleases

21 Restriction endonucleases degrade incoming DNA Phage Restriction endonucleases recognize specific DNA sequences 5 -GAATTC-3 3 -CTTAAG-5 Restriction There EcoRI are different Escherichia coli types (Type I- IV), depending on their recognition site and mode of cutting 5'-GAATTC-3' 3'-CTTAAG-5' 5'-G 3'-CTTAA AATTC-3' REase G-5' 5' overhang Goldberg et al. Nat. Rev. Immunology 2015

22 Restriction endonucleases degrade incoming DNA Phage How does this system discriminate self from non-self? 5 -GAATTC-3 3 -CTTAAG-5 Restriction REase 5 -GAATTC-3 3 -CTTAAG-5 Goldberg et al. Nat. Rev. Immunology 2015

23 The restriction modification (R-M) system Phage Methyl transferases mask the recognition site for the restriction enzyme 5 -GAATTC-3 3 -CTTAAG-5 Restriction REase MTase CH3 5 -GAATTC-3 3 -CTTAAG-5 CH3 Goldberg et al. Nat. Rev. Immunology 2015

24 The restriction modification (R-M) system Phage Methyl transferases mask the recognition site for the restriction enzyme 5 -GAATTC-3 3 -CTTAAG-5 Primordial innate defense mechanism against non-self NAs MTase Restriction REase No adaption (stays always the same), no memory Goldberg et al. Nat. Rev. Immunology 2015

25 The CRISPR/Cas system

26 The CRISPR/Cas system provides adaptive immunity to prokaryotes and archaea CRISPR-mediated adaptive immunity occurs in three stages: I. acquisition II. expression III. interference acquisition CRISPR phase adaptation I Transcription of pre-crrnas Phage sequence Spacer elements interference CRISPR Cas phase targeting III crrna processing and Cas complex loading expression phase II Goldberg et al. Nat. Rev. Immunology 2015

27 CRISPR: clustered regularly interspaced short palindromic repeats Cas: CRISPR-associated protein cas genes Leader CRISPR locus Repeat Spacer? Repair and/or recombination enzymes Acquisition Spacer acquisition 4 distinct enzymatic functionalities are required to mediate CRISPR-dependent protection Expression Transcription Pre-crRNA Cas6 or RNase III crrna processing RNase Interference crrnp assembly and surveillance Target degradation van der Oost et al. Nat. Rev. Microbiology 2014

28 Diversity of CRISPR Cas systems Type I-A Type I-E Type II Type III-A Type III-B Cas1 Cas2 Spacer acquisition Cas6 Cas6e RNase III Cas6 Cas6 crrna processing Cascade/I-A crrna Cas6e Cascade/I-E Cas9* Csm complex Cmr complex crrnp assembly and surveillance Cas5, Cas7, Cas8, Csa5 Cas5, Cas7, Cse1, Cse2 tracrrna Csm4, Csm3, Cas10*, Csm2 Cmr3, Cmr4, Cas10*, Cmr5 Cas3* Cas3* Cas9* domains Csm6? Cmr4 subunits? Target degradation DNA interference RNA interference van der Oost et al. Nat. Rev. Microbiology 2014

29 Spacer acquisition Cas1 / Cas2 Leader CRISPR locus protospacer protospacer adjacent motif Processing PAM Phage van der Oost et al. Nat. Rev. Microbiology 2014

30 CRISPR expression (transcription / processing) The CRISPR array is transcribed to produce a pre-crispr RNA (pre-crrna) type I / III type II Leader CRISPR locus RNase III 5ʹ Cas6 3ʹ 5ʹ 3ʹ 3ʹ pre-crrna Repeat Spacer tracrrna crrna 5ʹ crrna 3ʹ 3ʹ 5ʹ tracrrna CRISPR RNA (crrna) transactivating crrna (tracrrna) guide RNA guide RNA van der Oost et al. Nat. Rev. Microbiology 2014

31 CRISPR interference Target scanning tracrrna Cas9 PAM recognition Seed base pairing Complete base pairing and conformational change Activation of nuclease domains Target degradation by non-cas DNases type II systems van der Oost et al. Nat. Rev. Microbiology 2014

32 CRISPR: clustered regularly interspaced short palindromic repeats Cas: CRISPR-associated protein cas genes Leader CRISPR locus Repeat Spacer? Repair and/or recombination enzymes Acquisition Spacer acquisition Adaptive (remembers previous infection) Expression Transcription Pre-crRNA Cas6 or RNase III crrna processing RNase Interference crrnp assembly and surveillance Target degradation van der Oost et al. Nat. Rev. Microbiology 2014

33 Antiviral defense in eukaryotes

34 (+)RNA viruses dominate the eukaryotic world

35 Replication strategies of (+) strand RNA viruses Single-stranded (+)sense RNA Source: Microbiol. Mol. Biol. Rev. June 2013 vol. 77 no

36 Replication strategies of (+) strand RNA viruses Single-stranded (+)sense RNA dsrna as a replication intermediate during virus replication Long dsrna does not exist in the cytoplasm under normal conditions Source: Microbiol. Mol. Biol. Rev. June 2013 vol. 77 no

37 RNA interference (RNAi) as a defense mechanism against viruses

38 Cosupression in plants chalcone synthase (CHS) is the key enzyme in flavonoid synthesis CHS-transgenic plants should make more CHS and as such more pigment + p35s-chsa Source: The Plant Cell, Vol. 2, , April 1990

39 Cosuppression in plants is triggered by dsrna Source: Nature Reviews Molecular Cell Biology 15, (2014)

40 During cosuppression small RNA molecules are formed that are complementary to the transgene 5 tomato lines transgenic with tomato 1-aminocyclopropane-1-carboxylate oxidase (ACO) T1.1 T1.2 T5.1 T5.2 T5.3 Source: Science 29 Oct 1999: Vol. 286, Issue 5441

41 Cosuppression is a mode of post transcriptional gene silencing (PTGS) Double stranded RNA is the mediator of PTGS or and small RNAs complementary to the respective target mrna are formed

42 PTGS has been described in different organisms Fungi Plants Nematodes Quelling Quelling Cosuppression Cosupression PTGS PTGS Genetic Interference interference

43 Genetic interference in C. elegans is triggered by dsrna of the same sequence as the target mrna GFP-reporter strain PD4251 Progeny of animals injected with a control RNA stranded (ds)-unc22a) or a dsrna homologous to (double- GFP Genetic interference = RNA interference Source: Nature 391, (19 February 1998)

44 RNAi can be observed in vitro by adding long dsrna to a cell lysate (Drosophila cell line) The dsrna is degraded into smaller fragments of nt via an ATP-consuming process

45 A 21 mer dsrna molecule is the active RNAi component sirna sense 5 P 3 OH antisense Source: Genes Dev Jan 15; 15(2) 19 nt 2 nt

46 RNAi works just as well in vertebrates HeLa cells transfected with synthetic sirna duplexes targeting Lamin A/C Source: Nature 411, (2001)

47 sirnas slice their target mrna

48 sirna-mediated PTGS is not the only mechanism mirna mirna-mediated regulation results in repression of translation

49 dsrna is processed by Dicer into Small interfering RNA (sirna) Source: Nature Reviews Genetics 10, (February 2009)

50 sirnas are loaded into the RNA-induced Silencing Complex (RISC) Drosophila: Dicer generates sirnas R2D2 helps to load the RISC The passenger strand is cleaved HEN1 2!-O-methylates the guide strand Source: Nature Reviews Genetics 10, (February 2009)

51 sirnas are loaded into the RNA-induced Silencing Complex (RISC) RISC targets RNA of the same sequence as the initial dsrna substrate Source: Nature Reviews Genetics 10, (February 2009)

52 In Drosophila there are three major RNAi effector functions that employ distinct RISC complexes Source: Nature Reviews Genetics 10, (February 2009)

53 In the mammalian system, mirna mediated regulation is the predominant RNAi mechanism Source: Nature Reviews Genetics 16, (2015)

54 Major differences in RNAi pathways between different domains of life Arabidopsis (Plants) C. elegans (Nematodes) Drosophila (Insects) Human (Mammals) Dicer Products Ago/Piwi proteins mirnas, sirnas mirnas, pirnas sirnas mirnas, pirnas, sirnas mirnas, pirnas (sirnas?) RdRP yes yes no no Spreading* yes yes no* no*

55 Flock house virus (FHV) is a positive strand RNA virus that infects insects Nodaviridae 4.5 kb bipartite (+)RNA genome Source:

56 RNAi defective flies cannot fight FHV infection Source: J. Virol. December 2011 vol. 85 no. 24

57 RNAi defective flies cannot fight FHV infection Source: J. Virol. December 2011 vol. 85 no. 24

58 FHV encodes for a powerful inhibitor of RNAi B2 Source: Nature Reviews Genetics 10, (February 2009)

59 B2 proteins block RNAi in other domains of life Plant that expresses GFP mrna that is targeted by its own sirnas; additional coexpression of FHV encoded or related B2 proteins Source: Science May 17;296(5571)

60 RNAi works just as well in vertebrates HeLa cells transfected with sirna duplexes targeting Lamin A/C Source: Nature 411, (2001)

61 but only 21nt sirna molecules work! Longer dsrna evokes antiviral responses. And there is no role for RNAi in antiviral defense in vertebrates

62 Evolution of pattern recognition and the interferon system

63 Viral nucleic acids are still the trigger of the innate immune response dsrna receptor signal transduction Transcription antiviral effector functions

64 Virus-infected cells produce a protein factor that interferes with viral replication = interferons Type I interferons

65 The Type I Interferon system Type I IFN IFNAR1 IFNAR2 JAK1 TYK2 STAT2 STAT1 Type I IFN ISGF3 IRF9 cytokine ISRE Nucleus antiviral immunity

66 The Type I Interferon system 14 IFN-a subtypes Type I IFN 1 IFN-b 1 IFN-! 1 IFN-" 1 IFN-# IFNAR1 IFNAR2 TYK2 JAK1 STAT1 STAT2

67 The Type I Interferon system - effects ISGF3 Antigen presentation, MHC1 expression... Inibition of protein translation Degradation of (viral) RNAs ISRE Inhibition of proliferation IFN-stimulated genes (ISGs) antiviral immunity

68 The Type I Interferon system - cell autonomous effects Source: Nature Reviews Immunology 12, (May 2012)

69 The IFN feedback loop IFNβ IFNAR1 IFNAR2 IRF3 IRF3 IRF7 IFNβ Promoter IFNβ IRF3 IRF7 IFNα Promoter IFNα IRF3 Nucleus IFNα

70 IFNAR1-deficient mice are hypersusceptible to virus infection Mice infected with VSV i.v. Source: Science Jun 24;264(5167)

71 IFNAR Nucleic acids trigger antiviral immunity DNA RNA IRF3 IRF3 Isaacs et al. Foreign nucleic acids as the stimulus to make interferon. Lancet 2, (1963). IFNβ Promoter IFNβ IRF7 IFNα Promoter

72 What receptors detect viral nucleic acids and how are they distinguished from self-nucleic acids?

73 Nucleic acid sensing TLRs (toll like receptors) Long dsrna RNA TLR3 TRIF TLR7,8,9 DNA

74 TLR3 detects long dsrna LRR TIR 10 in the human system Single-pass type I membrane protein LRR domain faces lumen Source: Science Apr 18;320(5874):379-81

75 But... TLR3 TRIF TLR7,8,9 cytosolic recognition of replicating viruses is still functional

76 RIG-I initiates IRF3 phosphorylation via TBK1 IKKε TBK1 2x CARD RIG-I RNA Helicase domain IRF3 IRF3 ATF2/c-Jun IRF3/7 NF-kB PRDIV PRDIII-I PRDII IFNβ Promoter

77 RIG-I detects certain types of RNA viruses Ifna4 Ifnb RIG-I Type I IFN L929 cells transfected with RNAi vectors and then stimulated with NDV Source: Nat Immunol Jul;5(7)

78 RIG-I utilizes the mitochondrial outer membrane to signal CARD RIG-I RNA Helicase domain - IFN-β promoter stimulator protein 1 (IPS1) - mitochondrial antiviral signalling protein (MAVS) - virus-induced signalling adaptor (VISA) - CARD adaptor inducing IFN-β (CARDIF) - MAVS TBK1 IRF3 IRF3

79 There are two more family members of the RIG-I like helicase family CARD RIG-I CARD MDA5 LGP2

80 What are the ligands that are sensed by RIG-I or MDA5? Japanese encephalitis virus (JEV) Encephalomyocarditis Virus (EMCV) RIG-I: negative strand ssrna viruses MDA5: positive strand ssrna viruses dsrna viruses poly(i):poly(c) Source: Nature 441, (4 May 2006)

81 Replication strategies of (+) and (-) strand RNA viruses Single-stranded (+)sense RNA Single-stranded (-) sense RNA Source: Microbiol. Mol. Biol. Rev. June 2013 vol. 77 no

82 There are large amounts of dsrna during (+) ssrna and dsrna virus infection Control EMCV SARS-CoV ReoV Source: J Virol May;80(10):

83 ... but not during (-) ssrna infection La Crosse virus Influenza Source: J Virol May;80(10):

84 MDA5 forms long filaments along long dsrna Source: Curr Opin Virol Jun;12:20-5

85 What about RIG-I? How does it sense negative strand RNA viruses?

86 (-) strand RNA viruses harbor unmodified 5 triphosphate RNA molecules Source: Nature Immunology 12, (2011)

87 RIG-I detects unmodified 5 triphosphate RNA molecules + CIAP Source: Science Nov 10;314(5801)

88 The CTD of RIG-I binds to the 5 triphosphate moiety of dsrna Source: RNA Dec; 18(12)

89 Different modes of RNA recognition by RIG-I and MDA5 RIG-I dsrna > 20 bp optimal ligand: 5 -(p)pp moiety blunt end conformation unmodified nucleosides MDA5 dsrna > 1-2 kbp

90 The RIG-I CARD tetramer nucleates the formation of MAVS filaments Source: Nature Reviews Immunology 14, (2014)

91 TLRs 7,8,9 Viruses are detected by their nucleic acid genomes... RIG-I like receptors MDA5 RIG-I MAVS TBKI / IKKe IκB ATF-2 c-jun Mitochondrion IRF3 p65 p50

92 TLRs 7,8,9... or their transcriptional, replicative activity. RIG-I like receptors MDA5 5' triphosphate dsrna ppp RIG-I MAVS TBKI / IKKe IκB ATF-2 c-jun Mitochondrion IRF3 p65 p50 Ubiquitous Essential

93 Summary Viruses - classification according to their replication strategy Antiviral defense strategies rely on the recognition of non-self genomes, replicative or transcriptional activity Innate vs. adaptive mechanisms Non-self NAs are either a blue print for defense mechanisms or they are recognized just as such Distinct strategies evolved in all three domains of life

94 Evolution of antiviral defense mechanisms - nucleic acids are the target Benjamin R. tenoever Cell Host and Microbe 2016

95 Evolution of antiviral defense mechanisms Benjamin R. tenoever Cell Host and Microbe 2016