LQJOH-HOO DQVFULSWRPLFV HYHDOV PPXQH HVSRQVH … · 2020. 8. 19. · LQJOH-HOO DQVFULSWRPLFV HYHDOV...

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Se T a a1,2*, Me a L. S a fe 3,4*, M a ed S a a 1,2, Ma M e 5, Ca Kee4,5, D a a O d e -R eda6, Ma e Pa e 6, V ad Be e 7, S ee e B a 4,5#, T e d e A e a d 1,8# 1 S c a a d C a a B U , E ea M ec a B Lab a , He de be , Ge a ; 2 C ab a f P D de ee be ee EMBL a d He de be U e , Fac f B c e ce , Ge a ; 3 De a e f I fec D ea e , M ec a V , He de be U e , Ge a ; 4 Re ea c G Ce a a a d a fec , Ge a Ca ce Re ea c Ce e (DKFZ), He de be , Ge a ; 5 De a e f I fec D ea e , V , He de be U e , Ge a ; 6 F C e C e Fac , E ea M ec a B Lab a , He de be , Ge a ; 7 Ge c C e Fac , E ea M ec a B Lab a , He de be , Ge a ; 8 S a Sc f P a ac a d P a ace ca Sc e ce , U e f Ca f a Sa D e , La J a, Ca f a, USA * e e a c b ed e a K : a , a d , e a e e a ce , e-ce a c c ,

e e e #C e d a : S ee e B a , P .D. De a e f I fec D ea e, V He de be U e I Ne e e e Fe d 344 69120 He de be , Ge a P e: +49 (0) 6221 56 7865 E a : .b a @d f .de T e d e A e a d , P D S c a a d C a a B U EMBL Me e f a e 1 69117 He de be , Ge a P e: +49 (0) 6221 387 8690 E a : e d e.a e a d @e b .de

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Ab ac H a e a e e a ce f a a ba e ec f a e , e

ed ed e a a ab e ab d d a c b f eac ce e a e e e a a fec . He e, e de e ed a e e c b e-ce

RNA-Se a d - e RNA a a d a ed a e a a d fec ed a a , a de a e e c . We f d a e fe c e fec a d a a fec a a ce e a d ea e a efe e a fec f fe a ce . I , eac e a e e a ce ea e a a e

ba a e e f e fe - a ed e e a d, a fec , de e a a a a c a e a b e a d c e f e fe - a ed

e e . T e e f d e a e a e a e e , eac ce ea e a a e e e fec . O e e ca be a cab e e a d a d

e , e e a e e a e e f d d a ce e a a e e .

I d c T e a e e e b e f e ab a . I c ed f

a ce e eac e f ec f c f c c b e a (Pe e & A , 2014) . T e a ce e f d e e a e e a e e ab e e e c e , e c - ec e b e ce , e e- d c e e e d c e ce ,

e a c b a e de ec e Pa e ce a d e e ce . D e e c a c a e e e e e e f e , e a e e a ce a e ed e e e f e da . T c a e f- e e a a ed a e c - a a d ed b e

e ce ca ed e c , e e e ed b e a Pa e ce (K e c a & C e e , 2016) . D ffe e a f e e ce e a e a ce ea e e e a N c /W -de e de b f ca a d e e ab e ec e e

ce . Ab e e ce e e e e e c e ce e e ec e e d ffe e a e e e e d c e, b e , f Pa e ce (Sa c e a , 2015;

Ge a & C e e , 2019) . O e e a 10 ea , e a a d a e bee de e ed a d a e e e ed a e be a e de a c e d ffe e a a d f c f e

e a e e (Sa e a , 2011, 2009) .

H a e a e e a ce ( IEC ) a e e ec f e e a a e - a d c e a - e a ed c a e e . T e ac a a f - a e ca ba e f e

defe e a d e e fec (Ma e e a , 2018) . H e e , ed ed e a a ab e ab a a e e e e a e e e c e f

e ce e , a c a d ffe e IEC e c b e e e e e a d c ea a ce f e a fec . T a f ed e ca ed b e c a e e a c a ed

e d c e -ce a c e f e a e a e e . T ead e c e a e e f a a f e e c e , e e a e f a

a e e a e bee add e ed a ed ce e , a de a ed

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https://paperpile.com/c/BTOBCv/WRO41https://paperpile.com/c/BTOBCv/WRO41https://paperpile.com/c/BTOBCv/hlsxNhttps://paperpile.com/c/BTOBCv/hlsxNhttps://paperpile.com/c/BTOBCv/BafRB+Hngsvhttps://paperpile.com/c/BTOBCv/BafRB+Hngsvhttps://paperpile.com/c/BTOBCv/BafRB+Hngsvhttps://paperpile.com/c/BTOBCv/BafRB+Hngsvhttps://paperpile.com/c/BTOBCv/yB2UZ+Wwe8Mhttps://paperpile.com/c/BTOBCv/yB2UZ+Wwe8Mhttps://paperpile.com/c/BTOBCv/yB2UZ+Wwe8Mhttps://paperpile.com/c/BTOBCv/Uxyrihttps://paperpile.com/c/BTOBCv/Uxyrihttps://paperpile.com/c/BTOBCv/Uxyrihttps://doi.org/10.1101/2020.08.19.255893http://creativecommons.org/licenses/by/4.0/

ca ac e d ce e a e e -ce a c e a d e e e a c a ce d ffe e a . I e e , c ea e de ce e a e

ce a e ca e a a fec (Be e e a , 2014; W f & G ff, 2009) . I a ece a e a d e e ce e b c e e f e fe a ed e e (ISG ) a e fe - de e de a e . T ba a e e

f ISG a bee ed be e b e f e e a ce f e ce a fec (W e a , 2018) . U d ffe e a , e ce e e e f e e c ISG a d bec e e e e fe (IFN) (W e a , 2018) . T e e b e a e a , a ea - , e ce a d d ffe e a ed ce e d ffe e a e e f a fec .

W e e c ce - e- ec f c a a a e e e - e e e ce d ffe e a e d ffe e e- ec f c ea e e a . A e e e e e e

ca e e a e de a e a e-ce e d ab e d ec e e e e e f - a e e ac f d ffe e ce e a d f d d a ce

a a .

He e, e e ab ed a e e e a e ce - e- ec f c a a e e a e- e e e . T e e e e a e e-ce RNA e e c a d e

RNA b d a a f e e c - fec ed a e -de ed a d . I de e ab e ce e - ec f c a a e a d , e a e c ea ed a e-ce

RNA-Se a a f a e b e a e e -da e e . A e e a d fec ed b a e e c a (HA V1), e a e

c a ac e ed HA V1- fec f a a IEC e a d de e ed a HA V1 ab e fec a e a ce e a efe e ce f fe a ce . S e ce

a c c a a e ea ed a ce - e- ec f c a c a a e f e e e a e a a d . We f d a b a ead - a e a d d a fec ,

eac e a ce e a e e e f e f e fe - a ed e e c ea a d c a a e e .

Re HA 1 IFN-

T a e e a e e ce e d a ce - e- ec f c a e , e e a ed a e -de ed a d fec ed a a e a

(HA V1). H a a 1 (HA V1) a a -e e ed, e a d RNA c f d d de. HA V1 fec ead a e e a d ca ead dea

c d e a d c ed ad (C e e a , 2017; Wa e & M c e , 2003) . T c f e ab f HA V1 e ca e e a ce , e ed a HA V1 f ca ab e f fec e a f ed IEC , Cac -2 ce , a ca be ee b e c ea e e

be f fec ed ce a d e c ea e e a f a e e c be e e (F .S1A-C). F e fec HA V1, Cac -2 ce ed a c

e e e c a ac e ed b e d c f b e I (IFN 1) a d e III e fe (IFNƛ) (IFN ) (F .S1D). T IFN- ed a ed e e c e a a a

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a e b Cac -2 ce a a e- ea e f ce e e IFN c ed HA V1 fec (F .S1E-F). T c e e e a IFN c

HA V1 fec (Ma e a , 2016; G e a , 2015) . C e e a , e a a e a e a e e a ce e, T84, c de c bed be e

e e (S a fe e a ) , e f d be e fec ab e b HA V1 e e IFN- ed a ed a a e ed b e f b e e I a d III IFN ece (dKO) (F .S1G). T e e e e da a c f e f c f IFN c HA V1

fec a e a e e a ce a d a e e a ce f e a d ffe e ce - e a e fe e e c e ac a fec .

HA 1 -

T e a e e e d ffe e ce e e a e a e e e d a e c a e e b a d c IFN- ed a ed e e, e e ed a

e a a d . H a e a a d a e a ad a ced a ce a e eca a e ce a c e , a a , a d f c f e a a d

e ab c ed e a f e e c fec fea b e e a e (S a fe e a , 2020) . I e a a d e e e a ed f e ce -c a c a ed f

a e e ec (Sa e a , 2011) . T e c a e a d ce a c f e a d e e e f ed b f e ce ce a f ad e e a d c

a d a e f a e a e e a ce e (F .1A a d da a ) (Pe a a e a , 2017) . T e e a d e e ead fec ab e b HA V1 a ca be ee b e de ec f fec ed ce a d eff c e e ca f e HA V1 e e e e (F .1B-D). I fec f a d b HA V1 d ce a c e e e c a ac e ed b e d c f b e I IFN (IFN 1) a d e III e fe (IFN ) (F .1E-F). I e e , e e a f ca e a e e e e a ed b e

a f ed IEC e (F .S1), f e e a ce f a a d e c a ac e - a e e ac . P e- ea e f a d

e e e I e III IFN f ca ed ced HA V1 fec (F .1G). T a a ac f IFN a a HA V1 c e e e e b

a f ed ce e a d a a d (K a e e a , 2019) a d e e -de c bed f c f IFN c e e c fec a e e a e e (Lee & Ba d d e, 2017) .

- NA-

T c a ac e e e e e f e a a d e e c fec a e e-ce e e , e e ed e-ce RNA e e c ( cRNA-Se ) b a 10 Ge c

a f (Z e e a , 2017) . O e e a ea , cRNA-Se e e ed a a a a ac e ea ce e , ea e , a d e a c a a e . Ye , a cRNA-Se a d c a e d e e a a ab e d c a c

ed ed a f e , a e a d e de d ed d ffe e ce be ee e a d a d . I f e e c a e e , a a f e -de ed a d , e

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https://paperpile.com/c/BTOBCv/Kvhrf+S9PEThttps://paperpile.com/c/BTOBCv/Kvhrf+S9PEThttps://paperpile.com/c/BTOBCv/Kvhrf+S9PEThttps://paperpile.com/c/BTOBCv/Kvhrf+S9PEThttps://paperpile.com/c/BTOBCv/Kvhrf+S9PEThttps://paperpile.com/c/BTOBCv/DQbFohttps://paperpile.com/c/BTOBCv/DQbFohttps://paperpile.com/c/BTOBCv/DQbFohttps://paperpile.com/c/BTOBCv/Woqu5https://paperpile.com/c/BTOBCv/Woqu5https://paperpile.com/c/BTOBCv/Woqu5https://paperpile.com/c/BTOBCv/yB2UZhttps://paperpile.com/c/BTOBCv/yB2UZhttps://paperpile.com/c/BTOBCv/yB2UZhttps://paperpile.com/c/BTOBCv/Tph48https://paperpile.com/c/BTOBCv/Tph48https://paperpile.com/c/BTOBCv/Tph48https://paperpile.com/c/BTOBCv/GWqIbhttps://paperpile.com/c/BTOBCv/GWqIbhttps://paperpile.com/c/BTOBCv/GWqIbhttps://paperpile.com/c/BTOBCv/OKn79https://paperpile.com/c/BTOBCv/OKn79https://paperpile.com/c/BTOBCv/698gihttps://paperpile.com/c/BTOBCv/698gihttps://paperpile.com/c/BTOBCv/698gihttps://doi.org/10.1101/2020.08.19.255893http://creativecommons.org/licenses/by/4.0/

e f ed a c e e e cRNA-Se a a f a e b e e a c ea e a ce e-a a ed efe e ce e-ce a a f a e . F , a e b e e e b ec ed cRNA-Se f ed b da a e a a d c e a e ea ed e e e ce f e ce b a (F .2A). We a e de f ed e ce

e e e e ed e b a b f d b a - ec f c a e a d a c e e a e e e f e IEC e . T e a (>87.5%) f e

ce a ed f e b e c e ded IEC e a a f ac (12.4%) c e ded a a d e ce (F .2A). O da a a e a ee e e ece b ed cRNA-Se da a f e b e (Wa e a , 2020) . H e e , a a e e e e de a ed f a ab ce e c a 14 ce e e e ce

e (Wa e a , 2020) c e ec a a f a da ce e e e e ed a d . Add a , da a c a a a e be f ce (8800 ce 6167 ce )

(F .S2A-F) d a e a ca e f f d ce - e- ec f c a e (F .2B a d F .S3A). F a ce e e e e ed cRNA-Se da a e f a e b e , e e ec ed e d ffe e a e e ed e e a a e (F .2B). T e

e d - e a a (S ee e a , 2018) c f ed e e ec ed b f ca f d ffe e a f e ce a d c a ec e a d e e e e c e (ab e f c )

b e ce ( ec e f c ) (F .2C). Eac f e e ea e a e c a ac e ed b ec f c ad e a d a e f e e e e a e d ffe e a a ec e (F .2D).

T e e e e da a de de a ed e-ce a a f e a e e a ce e a d e a c a a e a e .

H -

H a e a d e e b ec ed cRNA-Se f ed b de e e de e f e de ec ed ce e b ce - e- ec f c a e e e f e e b e

(F . 3A). M f e a c a ed a d b e ed ce e b e e e a f d a d e e ce f Pa e a d f ce (F .3A). T c e e

e de c b e ab e ce f e e e a e a d (F e a , 2018) a d e fac a e ce e e e ec f c d ffe e a c be e e

a d (W e a , 2018; R c e a , 2016; D e a , 2020) . C e a f c a -a e a e a c a f e be ee a d a d b e e ea ed a e a c e a

e e ce e e Pea ≥0.75 f e ce a d a -a f ce ; ≥0.6 f e e c e , a e e e c e a d e e c e e ; a d ≥0.5 for b e a d ec e a -a f ce (F .S4). T e c e a f e ce , a -a f ce , a d e e c e a d e c e a (F .S4C-D),

de e e e ec ed d ffe e ce e e e f e e c e e a c e e , f e e ab f a d fa f eca a e e

a c a a .

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M in si NA -

ScRNA-Se a e f a d b e a e ce -ce e e e e , e ce e , a d de e e e a e e e . H e e , ac e ca ac f a a a f

e d c e ed f a . T e c e a , e e ed e H P e RNA c e e d f e ed RNA - b d a (Ha a a e a , 2020) . T e d

a f - e e e- ec e de ec f 12 d ffe e a c , f a c de ec ed a e eac d f b d a (F .S5A). T de e e e

be ce e- ec f c a e e e e e ed a d , e a e e ea ed e d ffe e a a a a d de f ed a e e e f eac d d a e a ce e; ee a e F .3B a d e e ded F .S3B. F , e e ec ed 11 e e f e e ed RNA - b d a c e e e e e e e ed a ec f c ce e ad e bac d e e a ce f e e (F .S5B). T e be d ec ed a a

SMOC2, MKI67, CHGB, FCGBP, OLFM4, FABP6 a d LYZ ed d c e e a e a ce b de e ce e ec f c . O e e a d, be

d ec ed a a SLC2A2, BEST4, LGR5 a d SPIB ed e e a a bac d ec f c a (F .S5B). RNA a f 11 ce e a e e e , e a ed

a c a e e f e a d (T a e a , 2017; Na e a , 2019; Se e a , 2018; S e e a , 2019) , a ed a da e e e e f e e e e a d ded a a a e ce a a a c a e ec fe a a ea

a a d (F .3C).

T de f c ce e e e fec ed b HA V1, e de ed a HA V1 RNA- ec f c be a d e f ed e e ed RNA b d a fec ed a d .

(F .3D-I a d F .S5B). I b d a f c fec ed a d e HA V1 RNA- ec f c be e ea ed abe a da e ec f c f e HA V1 be. I HA V1- fec ed a d , a RNA a c - ca ed e e e f MKI67 (F .3D), a a e f fe a c a d e e e ed

ed a c c a -a f ce (F .3B). Add a , a e e e c e ( a e FABP6) e e a f d be fec ed b HA V1 (F .3E). V a RNA a a de ec ed, a e e e e , e - e ce ( a e OLFM4) (F .3F), e e e d c e ce ( a e CHGB) (F .3G), a e a b e ce ( a e FCGBP) (F .3H). T e e e d ca e e e a f HA V1 fec IEC a d a e a ee e a ece e a HA V1 ca fec a e a e e a ce e (K a e e a , 2019) .

HA 1 - -

T add e e e e ce e e e e e a a d d ffe e e d a fec , e e f ed cRNA-Se f HA V1- fec ed a d . S e-ce a c a

a a c f ed a a e a e e a ce e c d be fec ed b HA V1 (F .4A-B). D ffe e a e e e e a a f c - fec ed a d a d a d a

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4 a d 16 - fec HA V1 e ea ed e a f e a a fec , c d e a a ed fec , - f a a e e a d e fe ed a ed e e (F .S6). I e e , d ffe e a e e e e a a e a e

d ffe e ce e e ea ed a ce - e- ec f c a c a e e fec (F .4C-E). T e e c de ca ca a e e e f e de ec ed ce e e.g.

e a f APOA1 e e c e , d e a f OLFM4 e ce a d TA ce , a d e a f CHGA e e e d c e ce (F .4E). M a , a a e ea ed a d ffe e ISG a e e a ed d ffe e ce e a fec

(F .4C-D a d F .S6). T e e f d e a eac ce e e e e a a d a a e a c a a c ba a fec .

T be e e a a e e e e f d d a ce e e e fe - ed a ed e e HA V1 fec (F .1), e e f ed de a ed d ffe e a a a ISG

(F .4F). F e e e c e ea e, 68 f 80 ISG e e f d f ca c a e e e a 4 16 c a ed c a a f ca ISG e a ed a 16 . O e ce e c d e a d TA ce de a ed e ce - e- ec f c c a e a b ea (4 ) a d a e (16 ) e (F .4F).

S , e e ed a f a ce ba ed e ISG f e e ea ed a a a f ce acc d e ce a ea e (F .4G a d F .S7B-C). F e

c e f ce ba ed e ISG f e e ea ed f c e (F .4H a d F .S7E-F) c e 1 be c ed f e a d TA ce a d c e 4 be

c ed f e e c e (F .4I a d F .S7H-I). D ffe e a a a e ea ed d c a e f ISG e e ed HA V1 fec c a e ec f c e ce e e e e ed e ed c e (F .4J a d F .S7K-L). I a , a a a e f c - fec ed

a d a e ea ed a ea e- ec f c c e f ce acc d e ISG e e f e (F .S7A,D,G a d J). E e c - fec ed a d eac c e e e e d ffe e

ba a e e f d c ISG (F .S7J). A e e , e e f d a eac e a ce ea e c a ac e ed b a d c ba a a e f ISG e e a d a

a fec , eac e a ce ea e e d d ffe e b a d c e e e c a ac e ed b ce - e- ec f c a e f ISG e e .

D c I e e ed a e a a d , cRNA-Se , a d e RNA

b d a c a ac e e e e e fec b e e e c HA V1 a a de e a eca a e e ce a c e f e a . U e

RNA b d a a a c e a a fec ce e f a a d, e f d a HA V1 fec a de ec ed ce e b d a a efe e ce f fe a ce . ScRNA-Se c f ed a HA V1 c d fec a e a ce e . I a , e ea ed a eac d d a e a ce ea e ed a d c a c a e e

a fec . We f d ce - e- ec f c a c a a e f ISG e e d ffe e ea e c a ac e ed b e e e f e ISG . I a ,

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a a a e ea ed a e ba a e e f ISG e e - fec ed a d a a ce - e- ec f c. T e e f d e a e e a e e , eac

d d a ce e e a d ffe e a e c ba a fec a d a a d ffe e f c e c ea a ce f e a e .

I e a - a e e ac a d a a e e e c a e . I e e de f ce e c e e a d c e a fec eac ce

e a a c e . He e, e a e de e ed a ec ca e e c b cRNA-Se a d e RNA b d a e c e e e a .

A c a a c a c a e ec f c ce e cRNA-Se da a e e a da ed ce - e- ec f c a e e e . De e e a a ab f e-ce a a e f e a f

e e e b ed ece (S e e a , 2019) , efe e ce a a a a a ab e f e a e e e e a ed . Rece , Wa e a b ed a cRNA-Se

da a e f a e a a f 6167 e e a ce a d 7 ce e a a ed (Wa e a , 2020) . O cRNA-Se da a e f a e b e e e da a

b ed b (Wa e a , 2020) e e c a e ce ( f 8800 6167) a d ce e ce e a a ed (14 7). Ha a c e e e c e a e f ce e a d

d ffe e a ea e a efe e ce a a e ec a a e a d d e b e a c a c a e be ee a d a d e ce a e a

e e e ce f f -d ffe e a ed ce a e e a ead fa e a a . He e, e a e a da ed a e a c a f e ce f e a e e a ce e e a d c e a e e f e f e c e a .

A cRNA-Se d fec -ce a de e c a e a a ca ed ec e a f ce d c a , f a a d

a e ac a . T ead e c e a e cRNA-Se a ed a d a fec ce e (W e e a , 2019; O Nea e a , 2019; C ba Ve a e a ,

2019) a e e e e ce e e e a e ce a e e e e PBMC (Za e a , 2018) e e a ce (Ca e a , 2020) . O e e,

e a e e a a ed e e f a d be e f ed a d c c ded a e ce f a ded e be a cRNA-Se da a f fec ed a d a d

a d ce f ca d ffe e ce (F .S8). O e a , e c ec ed a -da e a e cRNA-Se da a e f a e a a d 16,682 ce e ea

e d e e ed 6000 ce (F e a , 2018) a d 2000 ce (C e e a , 2019) . T a c a a f a a c a a e a e ce e c a c c

e ed TA ce ce a d b e ce a e a f e a b e e e e e fec .

A cRNA-Se de a c e e e e f a c a a a d e e a a e ce e f ce e , b e a d d ffe e a ea e , e f d RNA b d a be c c a a a a a a de a a

f a ab e e e f a e e e . U b d a f e a RNA e e e a a e a ac e a e f a ab ce e a d fec . F ,

ca be e e e a d ec f c f ca a c a ed f e ce ce

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https://paperpile.com/c/BTOBCv/88dx9https://paperpile.com/c/BTOBCv/88dx9https://paperpile.com/c/BTOBCv/88dx9https://paperpile.com/c/BTOBCv/Z7WSmhttps://paperpile.com/c/BTOBCv/Z7WSmhttps://paperpile.com/c/BTOBCv/Z7WSmhttps://paperpile.com/c/BTOBCv/Z7WSmhttps://paperpile.com/c/BTOBCv/Z7WSmhttps://paperpile.com/c/BTOBCv/Z7WSmhttps://paperpile.com/c/BTOBCv/tyWjB+tVCRS+bpyBrhttps://paperpile.com/c/BTOBCv/tyWjB+tVCRS+bpyBrhttps://paperpile.com/c/BTOBCv/tyWjB+tVCRS+bpyBrhttps://paperpile.com/c/BTOBCv/tyWjB+tVCRS+bpyBrhttps://paperpile.com/c/BTOBCv/tyWjB+tVCRS+bpyBrhttps://paperpile.com/c/BTOBCv/tyWjB+tVCRS+bpyBrhttps://paperpile.com/c/BTOBCv/tyWjB+tVCRS+bpyBrhttps://paperpile.com/c/BTOBCv/tyWjB+tVCRS+bpyBrhttps://paperpile.com/c/BTOBCv/qDnxmhttps://paperpile.com/c/BTOBCv/qDnxmhttps://paperpile.com/c/BTOBCv/qDnxmhttps://paperpile.com/c/BTOBCv/PNZOmhttps://paperpile.com/c/BTOBCv/PNZOmhttps://paperpile.com/c/BTOBCv/PNZOmhttps://paperpile.com/c/BTOBCv/pmZ04https://paperpile.com/c/BTOBCv/pmZ04https://paperpile.com/c/BTOBCv/pmZ04https://paperpile.com/c/BTOBCv/MJ1FAhttps://paperpile.com/c/BTOBCv/MJ1FAhttps://paperpile.com/c/BTOBCv/MJ1FAhttps://doi.org/10.1101/2020.08.19.255893http://creativecommons.org/licenses/by/4.0/

a d a e e e a fa a e a e ca e e e ec f c a b d a a ab e. Sec d, a ac a ed b a e a a f c e a cRNA-Se f e ce ce c c c f e ed b e a a ab f a b d e f e d ffe e ce e a e . M a , cRNA-Se a d RNA b d a

e e f ce - e-a a ead f e ad a a e c a ed c e e cRNA-Se de ec ce e a b d e . A ed c e a be ee

ab da ce f RNA ec e a d c a e e ca ead d c e a c e f e ce e def a d/ c e d ce - e- a e be ee e a c a d e e e . L ca a RNA e e ce - e- ec f c a e e e e e e . He e, e e f a ca f e (>10 c a e ) RNA

b d a fec ed a d e ea e d e e ed de ec f e ce e (T a e a , 2017; Na e a , 2019; Se e a , 2018; S e e a , 2019) .

U de a d e e c a e e a d e e a e e c ba a fec a bec e a f e d f e e d e e c ec c ac f a

a e e (B a e a , 2018) . I e fe a d e a c a ed e fe - a ed e e a e e c b e e f - e defe ce a a e . A e d ffe e

e f IFN , e III IFN ( .e. IFN𝛌) (K e e a , 2003; S e a d e a , 2003) a bee be e e a a c e c e e c e (Pe a a e a , 2017,

2018; P e a , 2011; N ce e a , 2015) . O cRNA-Se a a c f a HA V1 fec ed ce e e f IFN𝛌 a d ISG . O e c a , e I IFN (IFNB1) a de ec ed cRNA-Se da a, c f e e III IFN efe e ce f IEC

e e a fec (Ma a e a , 2015) .

S e ce a e bee be e a a fec c a ed d ffe e a ed ce . I a bee a a e e ce a e ef ac IFN , e e e a ba a e e f ISG c a e e b e f e c e a ce a fec (W e a ,

2018) . I e e , d ffe e e e ce a d c b e f ISG e e a e . D ffe e a f a e b c e ce e d -, e - a d ec de a

a e ee d ffe e ea e a e a d ffe e ba a e e f ISG (W e a , 2018) . He e, b e a a d , e c d a a a a -d ffe e a e a

e a ea e a d e e ce d a d c ba a e e e e f ISG . T a c b e a b c ea a a a a e e c a ce - e- ec f c a e .

T e e f e a a d d -e e c a e e ac a e e (D a & C e e , 2017) . B a c a a a f a fec e a a d ,

c a e f - e e c a ed a e a -de ed ce e , a e d ce a ca IFN- ed a ed a a e e (K a e e a , 2019; La e e a ,

2020) . ScRNA-Se f b a a d a ed - fec ed ce e e ea ed a a e e e e f b a e ca a d e a c a e e f ce

a fec (C be e a , 2015; R e e a ; W e e a , 2019) . He e, e e e f cRNA-Se d f a fec f a d . O da a a IFN- ed a ed

a a e e b a d d a e a ea e c b e

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https://paperpile.com/c/BTOBCv/Cdksb+1Fw5S+z7Iq9+88dx9https://paperpile.com/c/BTOBCv/Cdksb+1Fw5S+z7Iq9+88dx9https://paperpile.com/c/BTOBCv/Cdksb+1Fw5S+z7Iq9+88dx9https://paperpile.com/c/BTOBCv/Cdksb+1Fw5S+z7Iq9+88dx9https://paperpile.com/c/BTOBCv/Cdksb+1Fw5S+z7Iq9+88dx9https://paperpile.com/c/BTOBCv/Cdksb+1Fw5S+z7Iq9+88dx9https://paperpile.com/c/BTOBCv/Cdksb+1Fw5S+z7Iq9+88dx9https://paperpile.com/c/BTOBCv/Cdksb+1Fw5S+z7Iq9+88dx9https://paperpile.com/c/BTOBCv/Cdksb+1Fw5S+z7Iq9+88dx9https://paperpile.com/c/BTOBCv/a1y9Ihttps://paperpile.com/c/BTOBCv/a1y9Ihttps://paperpile.com/c/BTOBCv/a1y9Ihttps://paperpile.com/c/BTOBCv/fRRH8+KY7Nhhttps://paperpile.com/c/BTOBCv/fRRH8+KY7Nhhttps://paperpile.com/c/BTOBCv/fRRH8+KY7Nhhttps://paperpile.com/c/BTOBCv/fRRH8+KY7Nhhttps://paperpile.com/c/BTOBCv/fRRH8+KY7Nhhttps://paperpile.com/c/BTOBCv/Tph48+YJr9G+igNfO+tIrX4https://paperpile.com/c/BTOBCv/Tph48+YJr9G+igNfO+tIrX4https://paperpile.com/c/BTOBCv/Tph48+YJr9G+igNfO+tIrX4https://paperpile.com/c/BTOBCv/Tph48+YJr9G+igNfO+tIrX4https://paperpile.com/c/BTOBCv/Tph48+YJr9G+igNfO+tIrX4https://paperpile.com/c/BTOBCv/Tph48+YJr9G+igNfO+tIrX4https://paperpile.com/c/BTOBCv/Tph48+YJr9G+igNfO+tIrX4https://paperpile.com/c/BTOBCv/Tph48+YJr9G+igNfO+tIrX4https://paperpile.com/c/BTOBCv/Bl2lphttps://paperpile.com/c/BTOBCv/Bl2lphttps://paperpile.com/c/BTOBCv/Bl2lphttps://paperpile.com/c/BTOBCv/xh7zxhttps://paperpile.com/c/BTOBCv/xh7zxhttps://paperpile.com/c/BTOBCv/xh7zxhttps://paperpile.com/c/BTOBCv/xh7zxhttps://paperpile.com/c/BTOBCv/xh7zxhttps://paperpile.com/c/BTOBCv/xh7zxhttps://paperpile.com/c/BTOBCv/xh7zxhttps://paperpile.com/c/BTOBCv/pOUbchttps://paperpile.com/c/BTOBCv/GWqIb+o3NIAhttps://paperpile.com/c/BTOBCv/GWqIb+o3NIAhttps://paperpile.com/c/BTOBCv/GWqIb+o3NIAhttps://paperpile.com/c/BTOBCv/GWqIb+o3NIAhttps://paperpile.com/c/BTOBCv/GWqIb+o3NIAhttps://paperpile.com/c/BTOBCv/GWqIb+o3NIAhttps://paperpile.com/c/BTOBCv/vFG6s+1pJE5+tyWjBhttps://paperpile.com/c/BTOBCv/vFG6s+1pJE5+tyWjBhttps://paperpile.com/c/BTOBCv/vFG6s+1pJE5+tyWjBhttps://paperpile.com/c/BTOBCv/vFG6s+1pJE5+tyWjBhttps://paperpile.com/c/BTOBCv/vFG6s+1pJE5+tyWjBhttps://paperpile.com/c/BTOBCv/vFG6s+1pJE5+tyWjBhttps://paperpile.com/c/BTOBCv/vFG6s+1pJE5+tyWjBhttps://doi.org/10.1101/2020.08.19.255893http://creativecommons.org/licenses/by/4.0/

e e a e e b ac a e a c a a c a ac e ed b ce - e- ec f c b e f ISG (F .4 a d S7). I e e , e a b e ed a

e e e e f e a c a e e fec ce f e a e ea e. H e e a d a , c e ce ba ed e ISG f e e ea a e d ffe e ce f e ISG e e be ee e a ce ea e e a e

e e e e be ee ce f e a e ea e (F .S7). T ce - e- ec f c f e e f ISG b e ed f e e e c e ea e, e ce , a -a f ce , a d

b e ce c f e e a f b e ee ed e- e e d ec e ce e- ec f c e e e - a e e ac . F a b ca f

e , f d e a d d a ce e e e a e e e e d ffe e f c d a e e a d c b e d ffe e e

e e e a d e e a c ea a ce f e a e .

We a c a e e e f e a e e-ce RNA e e c a d RNA a f a d be de a cab e e a d de a d a RNA (A) a e e e a e a ac d a a e e a d ab e de

e e e e a d ce - e- ec f c a c a e a fec .

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Ma e a a d Me d C T84 a c ca c a ce (ATCC CCL-248) e e a a ed a 50:50 e f D becc d f ed Ea e ed (DMEM) a d F12 (G bC ) e e ed 10% fe a b e e a d 1% e c / e c (G bc ). Cac -2 a c ec a ade ca c a (ATCC HTB-37) e e a a ed D becc d f ed Ea e ed (DMEM) (G bC ) e e ed 10% fe a b e e a d 1% e c / e c (G bc ). H a A 1 (HA V1) a a d f f S ac Sc -C e , S . J de C d e ' Re ea c H a , TN, USA. HA V1 a a f ed Cac -2 ce a d

-c a e a a e e ed f fec e e e . H H a e a ece ed f c a d a e e e ec f e U e H a He de be . T d a ca ed acc da ce e ec e da f

e U e a He de be e f ed c e f a b ec acc da ce e Dec a a f He . A a e e e ece ed a d a a ed a a ed a e . T e c a a ed b e E c c f e U e H a He de be de e c S-443/2017. S e ce c a c e e

a ed f 2 M EDTA d c a f e a e f 1 a 4 C. C e e a d a ed ce c d PBS. F ac e c ed c e e f e ed 70 M f e a d

e f ac e e b e ed de a c c e. F ac c a e e be f c e e ed a d a a . T e e a a a e ed, a d c e e e- e ded Ma e . C e e a a ed a d a a ed ba a a d d ffe e a

c e ed a ( ee Tab e 1).

C d F a c ce a B Ad DMEM/F12 +G aMAX +HEPES +P/S

W 3A 50% b e B27 1:50 N2 1:100 N-ace -c e e 1 M R- d 10% b e N 100 / L EGF 50 / L Ga 10 M N c a de 10 M A83-01 500 M Sb202190 10 M

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D M Ad DMEM/F12 +G aMAX +HEPES +P/S

B27 1:50 N2 1:100 N-ace -c e e 1 M R- d 5% b e N 50 / L EGF 50 / L Ga 10 M A83-01 500 M Sb202190 10 M

1. O . A ZO-1 (Sa a C B ec ) a ed a 1/100 f a ; P a d -AF647 (M ec a P be ) a ed a 1/200 f a ; La (Abca ) a ed a 1/100 f a , e c a a a E-cad e (BD T a d c ), a d abb c a a -M c -2 (Sa a C B ec ) e e ed a 1:500 f

f e ce ce a d HA 1 ca d a b d (Abca ) a ed a 1/300 f a . Sec da a b d e e e c a ed AF488 (M ec a P be ),

AF568 (M ec a P be ), a d AF647 (M ec a P be ) d ec ed a a e a a ce.

NA , DNA, PC RNA a a e ed f ce N ce S RNA e ac (Mac e -Na e ) a e

a fac e ' c . cDNA a ade SCRIPT e e e a c a e (B Rad) f 250 f a RNA a e a fac e ' c . -RT-PCR a e f ed Ta SYBR ee (B Rad) a e a fac e c , HPRT1 a ed a

a e e . P e ed:

Ge e Na e S ec e F e Re e

IFN 2/3 H a ccaca a ccca caa a a a a ca

IFNb1 H a cc ca acca c a c ca cca cca

HPRT1 H a cc c c a a a a ac ca cc cca aa

HA V1 A cca c caca aa a caac c c a cca c cac c

I

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Ce eeded BIDI a b 8- e c a be de . A d ca ed e - fec , ce e e f ed 4% a af a de de (PFA) f 20 a e e a e (RT). Ce

e e a ed a d e eab ed 0.5% T -X f 15 a RT. P a a b d e e e d ed a e-b ffe ed a e (PBS) a d c ba ed f 1 a RT. Ce e e a ed 1X PBS ee e a d c ba ed ec da a b d e a d DAPI f 45 a RT. Ce e e a ed 1X PBS ee e a d a a ed PBS. Ce e e a ed b e f e ce ce a N Ec e T -S (N ). O a d e e f ed 2% a af a de de (PFA) f 20 a e e a e (RT). Ce e e a ed a d e eab ed 0.5% T -X f 15 a RT. P a a b d e

e e d ed a e-b ffe ed a e (PBS) a d c ba ed f 1 a RT. A b d a e ed a d a e e e a ed 1X PBS ee e a d c ba ed ec da

a b d e f 45 a RT. A b d a e ed a d a e e e a ed 1X PBS ee e a d e a e f a . O a d e e a ed a e ed d c

c f ca c c e (N , Pe E e ) 60 (1.2 e ca a e e, P a A , N ) a e e b ec e a d a EMCCD ca e a (Ha a a C9100-23B).

ELI A S e a a e e c ec ed a 16 - fec . S e a a e e e d ed. IFN 2/3 a e a a ed e IFN 2/3 DIY ELISA (PBL I e fe ce) a e

a fac e c . O O a d e e ed d ffe e a ed a (Tab e 1) ee da fec . A e

e f fec , ed a e ed a d a d e e e e ded c d PBS e e a e ce f Ma e . T a f fec f b e a ca a d ba a e a de , a d e e d ed a 27G eed e 10 e . M c fec ce e e c ba ed ed a a d fec ed a e e e c ba ed e a a c a .

I fec e e a ed ceed f e . Ce e e e d a d a e ed. Ce e e a ed e e DMEM/F12 a d e e e ded

d ffe e a ed a f e c e f e e e e .

NA- Ob a ed f e a ea b e e e e PBS a 4 C ce e

e de c bed e d (S e e a , 2019) . T e b a e a a c a ece c . T e d c a ed e a e c ba ed d c a (HBSS

Ca/M -F ee, 10 M EDTA, 100 U/ e c , 100 / L e - c , 10 M HEPES, a d 2% FCS) 200 L f 0.5M EDTA added a e e f d c a . T e a

c ba ed d c a f 15 a 37 C a . Ce e e e c ba ed ce f 10 a d e a e 15-20 e . A e c c c e a a e ea ed

a c ad bee e ea ed a d e ef e e a e ece f e e e e ed. T e c e e e d a 500 f 5 . T e e a a a e ed a d c e e a ed e e c d PBS. C e e e d e ed e ce b c ba

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T LE f 5-10 a 37 C c c c e a a ed e ce e e e e . Ce e e e a 500 f 5 a d a ed e e PBS. A ed

b d ce c a a a e ed b a 3 c ba ce ACK b ffe (T e ). Ce e e e a 500 f 5 a d a ed e e PBS. S e a a a e ed a d e ce e e a e e ded PBS e e ed 0.04% BSA a d a ed a 40- ce a e a d ed d ec f e ce RNASe . F a d a e , c ed a d a e ed af e 0 ( c ), 4 a d 16

- fec e e a ed c d PBS e e e ce Ma e a d c ba ed T LE E e f 25 a 37 C. W e c c c e a a e ea ed a ce ad eac ed a

e ce a e, e e e e e ded DMEM/F12 a d a 500 f 5 . S e a a a e ed a d e ce e e a e e ded PBS e e ed 0.04% BSA a d a ed a 40- ce a e . Re ce e e e e e

a ed DAPI (BD B c e ce , 1:1000) f dead ce abe a d FACS ed (A aF , BD B c e ce ) ed d ec f e-ce RNA e .

- NA- S e-ce e e e aded e 10 C c e e 10 Ge c S e Ce 3 L b a K 2 (10 Ge c ) acc d e a fac e

c . I a ce a d bead, e e e e e a ed, f ed b e e e a c , cDNA a f ca , f a e a , a d a ada f ed b a e de PCR. Re b a e e e a c ec ed b Q b a d B a a e , ed

a d e e ced Ne Se 500 (I a; - de, a ed-e d 26 75 b ). Tec ca a da (F e S8) a a e f ed a a d f f e

a d ca be d e affec e e e a a c c f e. P - NA Ra e e c da a a ce ed e Ce Ra e f a e ( e 3.1.0). Read e e a ed a c efe e ce e e c ea ed e efe e ce a e e (GRC 38) a d a a (NC_001943.1). T e e e ec a de f e (UMI) c

a ce e e ed R ( e 3.6.2) a d ce ed e R ac a e Se a ( e 3.1.3) (S a e a , 2019) L - a ce e e e ed, ba ed e f c e a. F , e e ed a e ce a e f c d a e e ead . F e a d da a, a e ce c d a ead > 10% e e e c ded. F e e da a, a e ce c d a ead > 20% e e e ed. Sec d, e ed e acce ab e

be f de ec ed e e . F b e f a e , ce 5000 de ec ed e e e e d ca ded. T e e a da a e e f e ce ed Se a . T acc f

d ffe e ce e e c de ac ce , UMI c e e a ed a d ca ed e a ed e a e b a e e a a f e ac a e c a f (Hafe e e &

Sa a, 2019) . Af e a d, e b e a d a d a e e e e a ed de e de e e ba c a d e e e a a ab effec . I e a a e f ed e

ca ca c e a a a (B e e a , 2018) a d a ea e e b a a (S a

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https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/dapihttps://paperpile.com/c/BTOBCv/YOjIlhttps://paperpile.com/c/BTOBCv/YOjIlhttps://paperpile.com/c/BTOBCv/YOjIlhttps://paperpile.com/c/BTOBCv/UUb6Nhttps://paperpile.com/c/BTOBCv/UUb6Nhttps://paperpile.com/c/BTOBCv/s9qJvhttps://paperpile.com/c/BTOBCv/s9qJvhttps://paperpile.com/c/BTOBCv/s9qJvhttps://paperpile.com/c/BTOBCv/YOjIl+dLlLIhttps://doi.org/10.1101/2020.08.19.255893http://creativecommons.org/licenses/by/4.0/

e a , 2019; Ha e d e a , 2018) . T e e c ec ed c e e ed f a a a d c e d ea a a a d - e a ed c f a a a e c a . C We e f ed c a c e a a (PCA) 3000 a ab e e e (ba ed a e a e e e a d d e f eac e e). T e 30 c a c e e e ed

c c a a ed ea e e b (SNN) a a d d a -ba ed c e e L a a a e f ed. F a , U f a f d a a a d ec (UMAP) a a a ca c a ed 30 e b f e ca a a

f e a f d c e. Ma e e e f e e ce e e e de f ed b c a e e e f eac e e a e a a e e f e ce e ece e e a c a ac e c (ROC) e . T e a a e c e e c a f a ce e, e a e e e e ec ed a e e e c a f ca e def ed b e AUC (a ea de e

ROC c e). T e e a e a ca ca a e f e a ce e e ed a a e eac f e c e f e e a e . F e a d , T e Se a abe a fe

e (B e e a , 2018) a ed a e ce e f e e e a d' ce . Bef e a d, e a d a ce e e f e ed a d e e a ce e e ed f e e efe e ce. A a f e a d ce e a e a a c a ed

e e ed c e a d e de f ed a e e e . T e c a be ee e a ed ce e f e e a d e a d a e f ed b ca c a e Pea

c e a be ee e a e a a ed e e e e f eac ce e.

P T ec c b e ce ea e f e-ce e e e e da a. T e e a e a b e c de e ce e f c (S ee e a , 2018) ec ed a e. T e

UMAP e bedd a e ed a f e d e a a b (S ee e a , 2018) . S e ce a ed a a a c e a d E e c e a d G b e a e e ec ed e d c e . Af e e d e e a de e ed a d ce e e de ed e e

c a c e . T e e e a f ca c a e e e d e e e de e ed b f a e e a ed add e de (GAM) f eac e e a d e e e

a a e c eff c e e ea e a e e a , e T adeSe ac a e (Va de Be e e a , 2020) . S f ca c a e e e e ed a ea a ea a ( . 1.0.10), eac c e e e a ce de ed e e d e f e e

ea e.

D T de f e c a e e e ac c d . D ffe e a e e e e e

e f ed MAST (F a e a , 2015) , c f a d e de e e e f eac e e, c e e f e e e de ec a d ea e e f e e e

e e e e . T ed ce e e f e fe e ce b e a d a d ce b a , e a a e de e e f f eac ce ea e a d c a be ee c , 4 , a d 16

- fec e e e f ed. Fa e d c e a e (FDR) a ca c a ed b e Be a -H c be e d (Be a & H c be , 1995) a d f ca e e e e e a

e FDR f e a 0.05. S b e e , I e fe e a ed e e a f ca

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https://paperpile.com/c/BTOBCv/YOjIl+dLlLIhttps://paperpile.com/c/BTOBCv/YOjIl+dLlLIhttps://paperpile.com/c/BTOBCv/YOjIl+dLlLIhttps://paperpile.com/c/BTOBCv/YOjIl+dLlLIhttps://paperpile.com/c/BTOBCv/s9qJvhttps://paperpile.com/c/BTOBCv/s9qJvhttps://paperpile.com/c/BTOBCv/s9qJvhttps://paperpile.com/c/BTOBCv/MCvYnhttps://paperpile.com/c/BTOBCv/MCvYnhttps://paperpile.com/c/BTOBCv/MCvYnhttps://paperpile.com/c/BTOBCv/MCvYnhttps://paperpile.com/c/BTOBCv/MCvYnhttps://paperpile.com/c/BTOBCv/MCvYnhttps://paperpile.com/c/BTOBCv/MCvYnhttps://paperpile.com/c/BTOBCv/fUBNThttps://paperpile.com/c/BTOBCv/fUBNThttps://paperpile.com/c/BTOBCv/fUBNThttps://paperpile.com/c/BTOBCv/fUBNThttps://paperpile.com/c/BTOBCv/eETF4https://paperpile.com/c/BTOBCv/eETF4https://paperpile.com/c/BTOBCv/eETF4https://paperpile.com/c/BTOBCv/7lR5ahttps://doi.org/10.1101/2020.08.19.255893http://creativecommons.org/licenses/by/4.0/

c a ed ac e c d (FDR < 0.05) e e ed ca c a e PCA, e ed a -ba ed c e , UMAP a a a d ea a . Ge e e RNA e e f d be d ffe e a e e ed e e b ec ed a e e e e e e e a a a e

E c R ac a e R (K e e a , 2016) M FI H O a d e e fec ed, a e ed af e 16 , a d e bedded OCT. Sec f 10 a c a c a (Le ca) a d ed a -80 e. H P e (RNA c e) a e f ed f e a fac e ' c . T e RNA c e H P e A a e a e a d

e a e d f b d a (ISH) a e a e 12 d ffe e RNA a e e ce a e ed de . B ef , ec e e f ed 4%

a af a de de, de d a ed 50%, 70%, 100% e a , e ea ed ea e. A e H P e be e e b d ed a d a f ed e e . P be e e de ed f e e

de f ed a ce e a e a d/ c b a ed b e a e. T e be ed e e: Ge e a e R e f e a e Ca a be C a e

SLC2A2 Ma e e e c e NPRP-0000689 T1

SMOC2 S e ce 522921 T2

MKI67 Ce c c e 591771 T3

CHGB E e e d c e ce 567681 T4

FCGBP G b e ce NPRP-0000691 T5

OLFM4 S e - a a f ce

311041 T6

BEST4 Be -M ce 481501 T7

FABP6 E e c e ea e 478701 T8

LGR5 S e ce 311021 T9

SPIB M ce 551431 T10

LYZ Pa e ce 421441 T11

HA V 1 A NPR-0001571 T12 T e de ec a e f ed e a e f 4 a e . Af e a , ce c e e e c e a ed DAPI, a d a e e e ed P L G d A fade M a . I a a e f ed e ca e a N DS-Q 2 (N I e )

e P a F 40 b ec e (N I e ) ed e N T -E e ed c c e (N I e ) b -f e d a d f e ce ce (DAPI, GFP, C 3, C 5, a d

C 7 c a e ). T e c c e a c ed e N NIS E e e f a e. Af e eac d, f e e e c ea ed a d a e ed e e d f e f e de ec ced e . A a e f a d f a e e e e e ed

eac e e e a e 15 e a e H P e a e e a f a e (ACD B ).

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F e b e a d c a ad e e e e f ed F (Sc de e a , 2012).

Da a a d C de A a ab T e da a e e e a ed d d a e c e e ce be b ed A a E e .

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https://paperpile.com/c/BTOBCv/6BluZhttps://paperpile.com/c/BTOBCv/6BluZhttps://paperpile.com/c/BTOBCv/6BluZhttps://doi.org/10.1101/2020.08.19.255893http://creativecommons.org/licenses/by/4.0/

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Be e J-P, S a TJ, Ye GW & S ec DH (2014) H a c e a fec f a e b c e ce -de ed e e a e ce e c ed a e e a e b ead

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B e A, H ff a P, S be P, Pa a e E & Sa a R (2018) I e a e-ce a c c da a ac d ffe e c d , ec e , a d ec e . Na . B ec . 36: 411 420

Ca Y, G Z, Va a a P, D a d E, L P, McD e P, O d a -M a e J, S a e AK, F be RW, Wa JP & Ga be M (2020) S e-ce a a f e a a ce e ea - a d a c f e a fec ed ad . Ge c : 957

C e J, La BT, A d N, G e SM, Ha d C, W d-B e C & J HP (2019) S e-ce a c e a a de f e d c ce e a d c e a a a a c

a d de . Sc . Re . 9: 1 12

C be M, Ga R, Ge e R, C e a JM & Sa R (2015) S e-Ce A a f RNA V I fec Ide f e M e Ge e ca D e e V a Ge e S e I fec U . Ce H M c be 18: 424 432

C e V, Me VA, Ka EA, Ha e V, J C & Sc -C e S (2017) A B a d Pa e e . A Re V 4: 327 348

C ba Ve a J, S J, L YT, D e c J, Ke R & B e CB (2019) A c a e f f e a A e ce e e e e e e e e e e a e a a e e fec . b R : 858373 A a ab e a : :// .b . /c e /10.1101/858373 2.ab ac [Acce ed J e 4, 2020]

D S, S Y, B KF, Pa J, C JY, Re L, Fe N, Ya a a LL, S c e -Tac ba L, W e JE, Me ED, M ac DM, A e a MR, K CJ, B c e EC & G ee be HB (2020) Re c Ac d a d L S a P e D ffe e a f H a I e a M Ce . Ga e e g A a ab e a : ://d .d . /10.1053/ . a .2020.03.053

D a D & C e e H (2017) O a d c e e d - a e e ac . C . O . I . 48: 15 22

F a G, McDa d A, Ya a M, De J, Ge V, S a e AK, S c e CK, M e HW, J a a McE a M, P c M, L e PS & G a d R (2015) MAST: a f e b e a ca f a e f a e a c a c a e a d c a ac e e e e e e-ce RNA e e c da a. Ge e B g 16: A a ab e a : ://d .d . /10.1186/ 13059-015-0844-5

F M, Ma a M, T K, Ta a A, M a Y, N S, S S & Sa T (2018) H a I e a O a d Ma a Se f-Re e a Ca ac a d Ce a D e N c e-I ed C e C d . Ce S e Ce 23: 787 793.e6

Ge a H & C e e H (2019) Ta e f e c : e e a e ce . Na . Re .

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F e

F 1. I HA 1 . A . C - ec f a a d e e a a ed f e e e ce f e e c e (E-cad), G b e ce (M c-2) a d c (ZO-1) b d ec f e ce ce. N c e a e

a ed DAPI. B. H a e a a d e e c ba ed ed a ( c ) fec ed HA V1. 16 a d e e f e , c - ec ed, a d HA V1 fec ed

ce e e a ed b d ec f e ce ce (HA V1 ( ed), c e (DAPI, b e). C . H a e a a d e e c ba ed ed a ( c ) fec ed HA V1. 16

a d e e f ed a d e e e ce f HA V1 fec ed ce ( ee ) e e a ed b d ec f e ce ce. A ca a d ba a e a e b a e e e a ed f ac

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( a e a) a d La ( ed) e ec e . N c e a e a ed DAPI (b e). D . H a e a a d e e fec ed HA V1. A d ca ed e - fec e c ea e

a c be a de e ed b -RT-PCR. E. Sa e a D b e d c f e I (IFN 1) a d III (IFN ) IFN e e e a a ed. F. H a e a a d e e c ba ed

ed a ( c ) fec ed HA V1. 24 e e e ce f IFN e ed a a e ed b ELISA. D ed e d ca e de ec f e a a . G. H a e a

a d e e e- ea ed f 24 2000IU/ L f IFN 1 300 / L f IFN 1-3. I e fe e e a a ed d e c e f fec a d e a f HA V1 c

be a a a ed 24 b -RT-PCR. A-G T ee b ca e ca e e e e f ed f eac e e e . Re e e a e f e ce ce a e a e . E ba d ca e

e a da d de a . S a c a e f a ed - e .

F 2. - . A . U f a f d a a a d ec (UMAP) e bedd f e-ce RNA-Se da a f a e b e c ed b e ce e ( =8,800 ce ). B. D f e ee a e e e f eac ce

e. T e d e e e e e e ce a e f ce e e e e e; e c e e e e a e a e e e ac e ce e. F .S3A a e e ded f e a e

e e . A ce - e-def a e e e a e ded Tab e S2. C . P ed c ed e d - e a ec e f e b e a d e e c e ea e ec ed e UMAP e bedd . D .

Hea a c a e e e e e e e a e d e f e e e c e a d b e ea e . De d a e ef f e ea a d ca e e e f e a c ca

c e f e e e . C ed ba ab e e ea a d ca e e e f ce de ed a e e d e, c ed acc d A.

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F 3. - in si NA

- . A . UMAP f cRNA-Se da a f a e -de ed a d ( =16,682 ce ); d c e d ce a e c ed b e ce e. B. D

f e - ee a e e e f eac ce e. T e d e e e e e e ce a e f ce e e e e e; e c e e e a e a e e e ac e ce e. F .S3B

a e e ded f e a e e e . A ce - e-def a e e e a e ded Tab e S3. C . Re e e a e a e e RNA b d a f a e

e e a a d. DAPI b e. D-I . V a a f ce - e a e f e a e a ce e , e-ce e e e e f e ce - e a e e

UMAP ( ef ) a d e RNA b d a ; ed c e d a RNA a d a ee c e d ce e a e ( ).

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F 4. C - - I G HA 1

. A . UMAP e bedd f a e -de ed a d HA V- fec ed ce (ce e e a a c e e de ec ed cRNA e ). B. Pe ce a e f ce fec ed a de ec ed eac ce ea e a d e. C-D. V ca f e e a a e d ffe e a e e ed c e a e 16 , e

a ca f ca ce (- 10 ad ed - a e) 2 f d c a e ( c /16 ) f e e ce (C) a d e e c e ea e (D). E. D f ea e- ec f c e e c a e

HA V1 fec f c , 4 a d 16 a d .. F. Sa e a E b f ISG. T e d e e e e e e ce a e f ce e e e e e; e c e e e e a e a e e e ac e ea e. T a e a d a e e e f ca c a e e (FDR < 0.05) M c 4 . a d M c 16 . e ec e G. UMAP e bedd f cRNA-Se da a f a e -de ed a d ba ed e f ca c a ISG f ce a 16 . H. U e ed c e f e UMAP da a f G. I . D b f ce ea e a d e e c e f H. J . Hea a f d ffe e a e e ed ISG ac e c e f H.

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S e e a F e

F 1. I HA 1 . A . Cac -2 ce e e fec ed HA V1. A d ca ed e , e a a ed b

d ec f e ce ce f HA V1 ( ed). N c e e e a ed DAPI (b e). B. Q a f ca f e be f HA V1 fec ed ce f A. C. Sa e a A, e ce e ca f HA V1 a a e ed b e e e c be e e -RT-PCR.

D . Sa e a A, e ce a e c a e e d c f e I (IFNb1) a d III (IFN ) IFN e e e a a ed. E. Cac -2 ce e e e- ea ed f 24 2000IU/ L f IFNb1

300 / L f IFN 1-3. I e fe e e a a ed d e c e f fec a d Ha V1 fec ed ce e e a ed d ec f e ce ce a d e be f HAS V1 fec ed ce a a f ed. F. Sa e a E, e ce e ca f HA V1 a a e ed b -RT-PCR f e e e c be . G. Cac -2 ce , T84 d- e a d T84 ce ac b e e I a d e III IFN ece (dKO) e e fec ed HA V1. 24 HA V1 e e e ca a e a a ed b -RT-PCR f e e e c be . A-G T ee b ca e ca e e e e f ed f eac e e e . Re e e a e

f e ce ce a e a e . E ba d ca e e a da d de a . S a c a e f a ed - e . F 2. G - NA- Da a f . A-F. e a d G-L . a d . U f a f d a a a d ec (UMAP) e bedd f e-ce RNA-Se da a f a e b e c ed b a e A. a d G. , N be f UMI c B. a d H. a d e e C. a d I. S a c f e

e-ce RNA- e da a e af e a f e a e a a V f e e a e, N be f ce D. a d J., a UMI c E. a d K. a d de ec ed e e F. a d L.

F 3. C . V f e - e a e

e e f eac ce e e a e e A. b e a d a d B. fea e e e de f ed e ece e e a c a ac e c (ROC) e . T e e ec ed a e

e e c a f ca e def ed b e AUC (a ea de e ROC c e) . T e c e e e e e ce e. A e e a e e ed ab e 2 a d 3.

F 4. C I I

. Hea a Pea c e a be ee e a e a e e e f e e e e ac a ed ce e be ee A. I e b e e e f B. O a d e e e C. I e b e c a ed a d a d D. I e b e c a ed

a d e 500 a ab e e e . F 5. A H . Re e e a e FISH a e f a 12 de ec ed e e f c ec f e a a d . S ed b b d a d

27




a d c ed b e c a e ed. F 6. L - E C M 4 16 . . V ca

f e e a a e d ffe e a e e ed ce e e e a e e e , e a ca f ca ce (- 10 ad ed - a e) 2 f d c a e a d e e

e c e a a e f f ca c a e e (FDR

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