Figure S1 a 24Hpi 48Hpi 72Hpi 96Hpi B 2.0

A SRA_PC9 UNC_PC9 SRA_A427 SRA_H522 SRA_H596 UNC_A427 UNC_H522 UNC_H596 SRA_OE21 SRA_Calu3 UNC_OE21 SRA_Caco2 SRA_H1299 UNC_H1299 SRA_SCC25 UNC_SCC25 SRA_Vero E6 SRA_Vero SRA_KYSE30 SRA_HCC827 UNC_KYSE30 UNC_HCC827 SRA_Detroit562 UNC_Detroit562 WashU_VeroE6

B ACE2 GAPDH ACTB SRA_PC9 UNC_PC9 SRA_PC9 SRA_PC9 UNC_PC9 UNC_PC9 SRA_A427 SRA_H522 SRA_H596 UNC_A427 UNC_H522 UNC_H596 SRA_A427 SRA_A427 SRA_OE21 SRA_H522 SRA_H596 SRA_H522 SRA_H596 SRA_Calu3 UNC_A427 UNC_A427 UNC_OE21 UNC_H522 UNC_H596 UNC_H522 UNC_H596 SRA_OE21 SRA_OE21 SRA_Calu3 SRA_Calu3 UNC_OE21 UNC_OE21 SRA_Caco2 SRA_H1299 UNC_H1299 SRA_Caco2 SRA_Caco2 SRA_H1299 SRA_H1299 SRA_SCC25 UNC_H1299 UNC_H1299 UNC_SCC25 SRA_SCC25 SRA_SCC25 UNC_SCC25 UNC_SCC25 SRA_Vero E6 SRA_Vero SRA_Vero E6 SRA_Vero E6 SRA_Vero SRA_KYSE30 SRA_HCC827 UNC_KYSE30 UNC_HCC827 SRA_KYSE30 SRA_KYSE30 SRA_HCC827 SRA_HCC827 UNC_KYSE30 UNC_KYSE30 UNC_HCC827 UNC_HCC827 SRA_Detroit562 UNC_Detroit562 WashU_VeroE6 SRA_Detroit562 SRA_Detroit562 UNC_Detroit562 UNC_Detroit562 WashU_VeroE6 WashU_VeroE6

Figure S1 A 24hpi 48hpi 72hpi 96hpi B 2.0

1.5 MOI=0.015

1.0

vRNA punctae/cell vRNA 0.5

0.0 0.015 0.06 0.25 1 MOI=0.06 MOI

MOI=0.25

MOI=1

100M

Figure S2 A ACE2 CRISPR KO Polyclonal Populations

H522 H522 H522 Calu-3 ACE2 ACE2 ACE2 ACE2 WT-1 WT-2 KO KO -+-+ -+-+Detection Enzyme

500 bp - Parental band 300 bp - 200 bp - Cleaved bands 44% 51% Cleavage Efficiency

B ACE2 CRISPR KO Monoclonal Populations

cut site ACE2 sgRNA Ref: 5’-AACTTTTCCAGTACTGTAGATGGTGCTCATTGT

ACE2+/+ MC: 5’-AACTTTTCCAGTACTGTAGATGGTGCTCATTGT

ACE2-/- MC-1: 5’-AACTTTTCC-----TGTAGATGGTGCTCATTGT

ACE2-/- MC-2: 5’-AACTTTTCCAGTA-----GATGGTGCTCATTGT

ACE2+/- MC: 5’-AACTTTTCCAGTACTGTAGATGGTGCTCATTGT

Figure S3 Bafilomycin A SGC-AAK1-1 E64D Apilimod Camostat mesylate 10uM 5uM 5uM 0.1uM 50uM *** 150 150 150 150 300 250 100 100 100 100 200 * 150 50 50 ** 50 50 100 ** * ** 50 0 0 0 0 0 RNA copies relative to DMSO (%) H522 H522 H522 H522 H522 ACE2 ACE2 ero E6 ACE2 ero E6 ACE2 ACE2 Vero E6 − Vero E6 − V − V − Vero E6 −

H522 H522 H522 H522 H522

Figure S4 ECT2-KIF23-RACGAP1 complex p27-cyclinE-CDK2 complex CDC2-CCNA2-CDK2 complex RasGAP-AURKA-survivin complex actomyosin contractile ring assembly G1/S transition of mitotic cell cycle cell cycle mitotic cell cycle spindle midzone G-protein coupled receptor signaling pathway

KIF23 CCNE1 CDK1 RASA1 RACGAP1 CDKN1B CDK2 BIRC5

CCNA2 AURKA ECT2 CDK2

AuroraB-AuroraC-INCENP complex RasGAP-AURKB-survivin complex BRIC5-AURKB-INCEN-EVI5 complex CRM1-Survivin-AuroraB mitotic complex protein folding and stabilization mitotic cell cycle cytokinesis mitotic cell cycle G-protein coupled receptor signaling pathway barrier septum assembly nuclear transport

AURKB RASA1 INCENP AURKB INCENP AURKB BIRC5 AURKB

BIRC5

AURKA BIRC5 EVI5 XPO1

Chromosomal passenger complex BRCA1-IRIS-pre-replication complex MIS18 complex GINS complex mitotic cell cycle DNA replication centromere complex assembly Pre-replicative complex assembly spindle assembly regulation of CENP-A containing nucleosome assembly GINS2 AURKB GMNN MIS18BP1 OIP5 GINS4 CDCA8 BRCA1

INCENP ORC1 GINS3

BIRC5 CDC6 MIS18A GINS1

Chromatin assembly complex (CAF-1) BRD7-EZH2-SUZ12 complex BRCC complex KNL1-MIS12-NDC80-SPC24-ZWINT complex DNA replication DNA repair mitotic cell cycle checkpoint mitotic S phase BRD7 cell cycle checkpoint chromosome organization EZH2 protein ubiquitination SPC24 NDC80 CHAF1A BRCA2 CHAF1B BRCA1 KNL1

SUZ12 BRCC3 ZWINT

RAD51 MIS12 RBBP4 BABAM2

BARD1-BRCA1-CSTF64 complex NDC80 kinetochore complex E3 ubiquitin ligase - UbcH5c complex BRCA1 B complex RNA 3'-end processing mitotic cell cycle protein ubiquitination DNA recombination chromosome segregation DNA damage response CSTF2 BARD1 NDC80 UBE2D3 BACH1 BARD1 BARD1 NUF2

SPC24 BRCA1

SPC25 BRCA1 BRCA1

BRCA1-BARD1-POLR2A complex BRAC1-BARD1-UbcH7c complex APP-APBB1-LRP1 complex Interferon-stimulated gene factor regulation of RNA biosynthetic process DNA damage response protein processing (proteolytic) transcription complex 3 regulation of transcription UBE2L3 APBB1 STAT2 POLR2A BARD1 BARD1 LRP1 IRF9

ORF7b

BRCA1 STAT1 BRCA1 APP

DTX3L-PARP9-STAT1 complex Defense response to viruses SARS-CoV-2 protein Protein ubiquitination Non-differentially expressed PARP9 DTX3L Undetected

Figure S5 STAT1 siRNA knockdown efficiency

RIG−I MDA5 LGP−2 MAVS

1.0 1.0 1.0 1.0 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0 0 0 0 24 96 120 24 96 120 24 96 120 24 96 120

TLR3 TLR7 TLR9 TRIF MyD88

1.0 1.0 1.0 1.0 1.0 0.8 0.8 0.8 0.8 0.8

Relative copies (siRNA / siNT) * * * 0.6 ND x2 ND x2 0.6 ND x1 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0.2 0 0 0 0 0 24 96 120 24 96 120 24 96 120 24 96 120 24 96 120 Hours post−infection

Figure S6