Pathway Induced by RNA-Virus

beta-Glucan

RNA virus Peptidoglycan 16 2021

2 2 0 =2 00 00 CLEC7A NANA NANANA NA NANA

32 1010 0 00 3 00 SYK iE-DAP MDP 00 00

dsRNA 3649 41 16 1010 NANA =

0 0000 16 1010

NANA NA 0

156 133112

======

==0 == NA NANA 00 00

======00 00

= 2 16 00 0 = = NOD1 NOD2 0 4 3 IFIH1 00 00 00 00 14 1611 DDX58 == == 115 9080 00 00 0000 00 0000 2 4 43 8 118 == == 221 310212 58 6354

= 00 =0 3 = == 11 140

== == 16 1010

147 143118 0 = 00 == == = CARD9 = = 00 = = PPIP5K1 00 00

== 3 RIPK2

77 7071 = 82 10578

== MALT1 CARD9 == ==

122 8666 = = 00 =0 2 119 199101 11 148 217 151162 BCL10 == == 2 3

99 8272

== == = 2 2 = =

== TRAF3 == ==

199 159163 = == = IKBKG 87 8787 TANK CXCL8 =

93 5440 16 1010 181 188137 0

2 00 00 IKKα IKKβ

= 293 286258 80 8756 00 00 =

======TRAF6

277 220220 2 3 = == 00 0 34 6876

== 73 5443 10 1715 == == = MKK

= 3 = 3 =

73 3022 ======3 == 212 263253 87 8469

======227 179207 TBK1 IKKε == == 183 168142 3166 29342411 = == = 2 =2 3 DDX3X 36 5343 IκB = MAP3K1 NFKB1 133 8866

= = == 773 258197 = MAPK8 p38 = == 3 2 RELA ERK 1498 14261651 ======P 80 6154 = ==

== = IRF3 47 4643 = = 209 146154

== =

= = FOS 262 147104 = 905 278127 NFKB1

62 9658 2 FOS 383 1815 = 9 = 2 0 038

P P 0 7 00 JUN

= RELA == =

090 00 JUN = = IRF3 IRF3 00 00 3 == 206 217 243 02 = 500 288455 15 1620

MAPK8 IRF3 AP1 NF-κB NF-κB AP1 MAPK8 MAPK9

123 133121 235 345261 = =

== = 2 = 2 2

= = = Type I IFNs Pro-inﬂammatory cytokines 2 2 2 2 ======327 267268 166 185151 IκB MKK IκBα IκBβ IκBε Bcl-3 MKK1 MKK2 MKK3 MKK4 MKK5 MKK6 MKK7

125 8284 73 3022 180 3921 94 6448 308 362283 432 298243 95 12794 169 127127 97 6161 70 6471 117 8157 = = = =

2 == = 3 == 9 = 2 2 = == 2 2 3 ======4 3 == =

= =

======6 ======

= = =

======NANA ======2 = = = = = 2 2 2 2 2 3 = 2 ======NANANA = = 2 NANANA 78 6977 36 5343 43 5941 36 5951 683 722759 498 514499 NA NANA 156 195182 35 4749 12 2319 70 7487 p38 ERK p38α p38β p38γ p38σ MAPK3 MAPK1 MAPK6 MAPK4 MAPK7 MAPK15

214 210178 16 1010 128 9271 16 1510 91 9076 1192 1252992 727 559509 16 1010 47 5332 16 1010 ======

== 00 0 00 == == 00 0 = = = 3 00 00 == 00 0 00 = = = =

00 00 == 00 0 ======0000 = 00 00

= = =

== == NA NA == 0000 == == = 00 00 NA NA

======NA NA NA 3 2 3 3 00 00 00 NANANA NA NA NA 259 239228 NA NA NA 12 2217 3 68 37 4235 93 7790 803 873719 168 118157 45 5345 NA NA NA

Pathway induced by RNA-virus. Background colors code for a part of a pathway, used in Pathway for general immune response. Modiﬁed pathway from Kawai et. al.[1] and InvivoGen. The activation of the interferon and the NFκB pathways are essential antiviral defense mechanisms (for a review see [2]). Thus, organisms have evolved mechanisms to activate these pathways in response to viral infections. Double- stranded viral RNA is recognized by DExD/H box cytoplasmic RNA helicases such as the retinoic acid-inducible gene I(DDX58 ) and the melanoma diﬀerentiation-associated gene 5 (IFIH1 ). These interact with the adaptor proteins interferon-β promoter stimulator 1 (PPIP5K1 ), eventually converging on the interferon regulatory factors 3 (IRF3 ) and 7 (IRF7 ) that ultimately activate the transcription of interferon-stimulated genes in the nucleus [3]. Similarly,

1 the virus infection also activates the NFκB pathway. Not surprisingly, viruses have evolved mechanisms to evade the host immune responses. EBOV was shown to block IRF3 activation [4], the mechanism being dependent on the viral protein VP35. Interestingly, although the VP35 proteins of both MARV and EBOV can inhibit DDX58 , EBOV VP35 can antagonize activation of DDX58 like receptors by both double-stranded RNA (dsRNA) as well as dsRNA blunt ends, whereas MARV VP35 does not antagonize induction by dsRNA blunt ends [5]. The innate immune receptors IFIH1 and DDX58 , which play a major role in antiviral defense, are not significant differentially expressed, over all time point. The mitochondrial antiviral-signaling proteins IPSI /MAVS, which associates with ligand-bound IFIH1 and DDX58, are up-regulated 7 h after infection with EBOV in HuH7 cells. This expression decreases 23 h p.i.. This regulation pattern is also seen for downstream molecules like the ATP-dependent RNA helicase DDX3X and serine/threonine protein kinase TBK1 . All of them are involved in activation of the interferon regulator IRF3 , which together with IRF7, NFκB and AP1 transcription factors can activate transcription of a manifold of interferon stimulated genes. IRF3 is up-regulated in EBOV-infected HuH7 cells as well. In contrast, molecules of this signaling cascade are equally expressed or down-regulated in bat cells infected with EBOV or MARV. PPIP5K1 /MAVS can also activate the E3 ubiquitin protein ligase TRAF6 , which leads to the activation of the kinase MAP3K1 . MAP3K1 in turn stimulates transcription of FOS. Expression of FOS and Jun, which form the transcription factor AP1 , are highly up-regulated 23 h after EBOV infection. IRF3 , AP1 and NFκB control transcription of many different interferons and IFN stimulated genes. At least some of these transcription factors increase in expression 23 hours p.i. with EBOV. However IFNs are mainly not expressed in cells used in this study, STab. 9. The transmembrane receptor CLEC7A, the associated kinase SYK which mediates signal transduction and the adapter protein CARD9 are not or only very slightly expressed in HuH7 and bat cells. This pathway seems not to play a key role for response to EBOV or MARV in HuH7 or bat. Expression of NOD1 , which confers responsiveness to bacterial lipopolysaccharides, increases slightly in bat cells 23 h p.i. with EBOV and MARV, while expression in HuH7 did not change. NOD2, which also recognizes bacterial peptidoglycans, is not expressed in bat and HuH7 cells. NOD1 recruits the serine/threonine kinase RIPK2 . This leads to phosphorylation and thus activation of IKKβ by IKBKG resulting in the release of the inhibitor complex IκB from NFKB1 and RELA. These form the transcription factor NFκB. Although the expression of CLEC7A, CARD9 , MALT1 , Bcl-10 , NOD1 /2, and RIPK2 are not significantly changed during filovirus infection, we see drastic changes for NFKB1 , RELA, IκB, MKK2,3,6, FOS and Jun.

References

[1] Kawai, T. & Akira, S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34, 637–650 (2011).

[2] Hiscott, J., Nguyen, T. L., Arguello, M., Nakhaei, P. & Paz, S. Manipulation of the nuclear factor-kappaB pathway and the innate immune response by viruses. Oncogene 25, 6844–6867 (2006). [3] Klenk, H. D. & Feldmann, H. Ebola and Marburg Viruses (Taylor and Francis, 2003). [4] Basler, C. F. et al. The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3. J. Virol. 77, 7945–7956 (2003). [5] Ramanan, P. et al. Structural basis for Marburg virus VP35-mediated immune evasion mechanisms. Proc. Natl. Acad. Sci. U.S.A. 109, 20661–20666 (2012).