key: cord-0783521-98qw0kah
authors: Kimura, Izumi; Konno, Yoriyuki; Uriu, Keiya; Hopfensperger, Kristina; Sauter, Daniel; Nakagawa, So; Sato, Kei
title: Sarbecovirus ORF6 proteins hamper the induction of interferon signaling
date: 2021-03-12
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.108916
sha: fd2d34779ade18a764884d11bc6629f9a2162f82
doc_id: 783521
cord_uid: 98qw0kah

The presence of an ORF6 gene distinguishes sarbecoviruses such as SARS-CoV and SARS-CoV-2 from other betacoronaviruses. Here, we show that ORF6 inhibits the induction of innate immune signaling including upregulation of type I IFN upon viral infection, as well as type I and III IFN signaling. Intriguingly, ORF6 proteins from SARS-CoV-2 lineages are more efficient antagonists of innate immunity than their orthologs from SARS-CoV lineages. Mutational analyses identified residues E46 and Q56 as important determinants of the antagonistic activity of SARS-CoV-2 ORF6. Moreover, we show that the anti-innate immune activity of ORF6 depends on its C-terminal region and ORF6 inhibits the nuclear translocation of IRF3. Finally, we identify naturally occurring frameshift/nonsense mutations that result in an inactivating truncation of ORF6 in approximately 0.2% of SARS-CoV-2 isolates. Altogether, our findings suggest that ORF6 contributes to the poor IFN activation observed in COVID-19 patients.

In the present study, we show that an ORF6 gene is commonly encoded in 96 all sarbecoviruses, including SARS-CoV and SARS-CoV-2, while no orthologs are 97

found in other betacoronaviruses such as MERS-CoV, OC43 and MHV. We 98 demonstrate that all Sarbecovirus ORF6 proteins inhibit the induction of IFN-I upon  99 viral infection, as well as antiviral signaling triggered by IFN-I/-III. Intriguingly, the 100 anti-IFN activities of the ORF6 proteins of SARS-CoV-2 lineages are more potent 101 than those of SARS-CoV lineages. We further provide evidence suggesting that the 102 emergence of SARS-CoV-2 variants expressing truncated ORF6 proteins may 103

contribute to the attenuation of viral pathogenicity. 104 J o u r n a l P r e -p r o o f

ORF6 is conserved in the subgenus Sarbecovirus but absent from other 106 betacoronaviruses 107 We first assessed the phylogenetic relationship of betacoronaviruses including 108 SARS-CoV, SARS-CoV-2, MERS-CoV, OC43 and HKU1. The respective viral 109 strains were classified based on their subgenera in the phylogenetic tree of the 110 full-length viral genome ( Figure 1A ; see also Table S1 ) as well as five viral core 111 genes encoding ORF1ab, spike (S), envelope (E), membrane protein (M) and N 112

( Figure 1B) . However, some viral core genes such as E (encoding 75 amino acids in the case of 116 SARS-CoV-2) are relatively short, making it difficult to reliably infer their 117 phylogenetic relationships. Indeed, two viruses belonging to the Sarbecovirus 118 outgroup, BtKY72 and BM48, were separated in the phylogenetic tree of the E gene 119

( Figure 1B) . In contrast, the other six phylogenetic trees showed almost identical 120 relationships among the five subgenera of betacoronaviruses (Figures 1A and 1B) . 121 These results suggest that viral recombination did not occur among 122 betacoronaviruses analyzed, although recombination events can occur among 123 sarbecoviruses. 124 We then compared the genome organizations of the different subgenera. 125 As shown in Figure 1C , the arrangement of the core genes (ORF1ab-S-E-M-N) is 126

conserved. Insertions of additional ORF(s) between ORF1ab and S were detected 127

in Hibecovirus (Hp/Zhejuang2013) and Embecovirus species, while additional 128

ORFs were detected between S and E in all betacoronaviruses ( Figure 1C ). 129

Interestingly, ORF insertions between M and N were observed only in members of 130 the Sarbecovirus and Hibecovirus subgenera ( Figure 1C ). When we compared the 131 sequences of these ORFs, the genes in Sarbecovirus were unalignable with those 132

in Hibecovirus, suggesting that these ORFs emerged independently after the 133 divergence of these subgenera. Of note, we found that ORF6 is highly conserved in 134 sarbecoviruses including SARS-CoV and SARS-CoV-2, but is absent from other 135 betacoronaviruses ( Figure 1C) (Figure 2A ) was similar to that of the 144 full-length viral genome (Figure 1A) , suggesting that recombination events 145

involving the ORF6 gene have not occurred among sarbecoviruses. For our 146 phenotypic analyses, we generated expression plasmids for ORF6 from 147 SARS-CoV-2 (Wuhan-Hu-1) as well as SARS-CoV-2-related viruses from bats 148 (RmYN02, RaTG13 and ZXC21) and a pangolin (P4L). We also included ORF6 149 from SARS-CoV (Tor2), SARS-CoV-related viruses from bats (Rs4231, Rm1 and 150 HKU3-2), and the two bat sarbecoviruses that are phylogenetically located at the 151 outgroup of SARS-CoV-2 and SARS-CoV (BtKY72 and BM48). Western blotting 152 revealed that the expression levels of ORF6 proteins of the SARS-CoV-2 lineage 153 are lower than those of the SARS-CoV lineage and the two outgroup viruses 154 ( Figure 2B ). 155 We then monitored human IFNB1 promoter activity in the presence of 156 ORF6 using a luciferase reporter assay. the SARS-CoV-2 lineage were more potent inhibitors than those of the SARS-CoV 162 lineage ( Figure 2C, top) , despite their lower expression levels ( Figure 2B ). Next, 163

we analyzed the ORF6 proteins for their ability to inhibit signaling triggered by type I 164 IFN (IFN-α) and type III IFN (IFN-λ3). In agreement with a previous study (Kochs et  165 al Figure S1A ). 181 To verify the immuno-suppressive activity of ORF6 in different 182 experimental systems, the expression levels of endogenous IFNB1, IFNL1, and 183

three ISGs (IFI44L, BST2 and PARP9), were measured after SeV infection ( Figure  184 2D) or IFN-α treatment ( Figure 2E) . The ORF6 proteins of SARS-CoV-2 and 185 SARS-CoV significantly suppressed the upregulation of these genes ( Figures 2D  186  and 2E) . The suppressive effect mediated by SARS-CoV-2 ORF6 was significantly 187

higher compared to its SARS-CoV counterpart (Figures 2D and 2E ). the inhibitory activities of SARS-CoV-2 ORF6 were significantly stronger than those 206 of SARS-CoV ORF6 (Figure 2 ). To determine the residue(s) that are responsible 207

for this difference, we aligned and compared the ORF6 amino acid sequences 208 ( Figure S2A ). As shown in Figure 3A , we found ten amino acids whose chemical 209

properties are different between ORF6 proteins of the SARS-CoV lineage (n=241) 210

and those of the SARS-CoV-2 lineage (n=57, 648) , and these ten residues are 211 highly conserved in each lineage. Furthermore, the ORF6 proteins of the 212 SARS-CoV lineage harbor two additional amino acids (i.e., Y and P) at the 213 C-terminus compared to those of the SARS-CoV-2 lineage. Mutational analysis 214 ( Figure 3B ) revealed that substitution E46K attenuates the anti-IFN activity of 215 SARS-CoV-2 ORF6, while Q56E has the opposite effect ( Figure 3C and S2B) . To 216 verify the effect of residues 46 and 56 on ORF6-mediated anti-IFN activity, we 217 J o u r n a l P r e -p r o o f introduced the respective reverse mutations into SARS-CoV ORF6 ( Figure 3D) the importance of the C-terminal region of ORF6 for its biological activity, we 234 generated a series of ORF6 mutants, in which we deleted the C-terminal region or 235 changed a stretch of positively charged residues to alanines ( Figure 3F ). With the 236 exception of the ∆C1 mutant of SARS-CoV-2 (Wuhan-Hu-1), all mutants were 237 expressed at levels similar to wild type (WT) ORF6 ( Figure 3G ). Luciferase reporter 238 assays revealed that deletion of the C-terminal region (∆C2) or the substitution of 239 acidic residues to alanines (Ala) completely abrogated the anti-IFN effects of 240 SARS-CoV-2 ORF6 (Figures 3H and S2D) . Similarly, the anti-IFN activities of the 241 ORF6 proteins of SARS-CoV (Tor2) and two outgroups (BtKY72 and BM48) were 242 partially attenuated by mutations of the C-terminal region, although they still 243 retained some of their anti-IFN activity (Figures 3H and S2D ). These findings 244

suggest that the C-terminal region is crucial for efficient anti-IFN activity of ORF6. 245 To address whether ORF6 interacts with RAE1 and NUP98 via its 246 C-terminal region, we performed co-immunoprecipitation (co-IP) experiments. As 247 shown in Figure 3I , Sarbecovirus ORF6 proteins including those of SARS-CoV-2 248 (Wuhan-Hu-1), SARS-CoV (Tor2) and two outgroups (BtKY72 and BM48) bound to 249 both RAE1 and NUP98. In contrast, C-terminally truncated mutants thereof failed to 250 bind these cellular proteins ( Figure 3I ). These observations suggest that RAE1 and 251

NUP98 associate with the anti-IFN-activity exerted by Sarbecovirus ORF6 proteins. 252

In fact, a recent study demonstrated that SARS-CoV-2 ORF6 hampers the nuclear 253 translocation of IRF3 and STAT1 via RAE1 and NUP98 and suggested that 254 overexpression of RAE1 and NUP98 rescues the IFN response in the presence of 255 ORF6 (Miorin et al., 2020) . While our microscopic analyses ( Figure 3J ) confirmed 256 that SARS-CoV-2 ORF6 inhibits the nuclear translocation of IRF3 ( Figure 3K ), 257 overexpression of RAE1 and NUP98 did not rescue nuclear translocation of IRF3 258

( Figures 3J and 3K) , IFN induction or IFN signaling ( Figure 3L ) in the presence of 259

ORF6. Instead, Western blotting revealed that overexpression of RAE1 and NUP98 260 increased the expression level of ORF6 in a dose-dependent manner ( Figure 3M ). 261

Altogether frameshift and/or nonsense mutations ( Figure 4A ; see also February 8, 2020 (GISAID ID: EPI_ISL_451350) ( Figure 4A ). We assessed the 276 frequency of SARS-CoV-2 variants encoding truncated ORF6 for each country, but 277 found no specific deviations on the emergence of the ORF6-truncated SARS-CoV-2 278 at country level (Table S3) . 279 Based on the classification into pangolin lineages 280

(https://github.com/cov-lineages/pangolin) and GISAID clades ( Wales, UK, and classified into Pangolin lineage B.1.5 and GISAID clade G (Table  290 S4). We then obtained 137 SARS-CoV-2 genome sequences that meet the 291 abovementioned criteria (isolated in Wales, UK, Pangolin lineage B.1.5 and GISAID 292 clade G) including the 12 sequences belonging to cluster 41 and conducted a 293 J o u r n a l P r e -p r o o f phylogenetic analysis. As shown in Figure 4B , 11 of the 12 ORF6-truncated 294 SARS-CoV-2 mutants in cluster 41 formed a single clade. This observation 295

suggests that these ORF6-truncated SARS-CoV-2 mutants have sporadically 296 spread via human-to-human transmission. Together with our findings that deletion 297 of the C-terminal region of SARS-CoV-2 ORF6 abolishes its ability to suppress IFN 298 responses (Figure 3H) The observation that ORF6 proteins from SARS-CoV-2 and related viruses 323

in bats and pangolins are on average more active in suppressing IFN responses 324 than their SARS-CoV counterparts is reminiscent of the recently identified IFN 325 antagonist ORF3b. This protein is also more potent in viruses of the SARS-CoV-2 326 lineage than in SARS-CoV and related animal viruses bind RAE1 and NUP98 via their C-terminal region (Figure 3I) , it has been 337 suggested that ORF6 inhibits innate immune signaling by targeting these two host 338

factors (Miorin et al., 2020) . In contrast to this recent study (Miorin et al., 2020) , 339 however, overexpression of RAE1 and NUP98 did not rescue the IFN response in 340 the presence of ORF6 in our hands. Intriguingly, we found that the expression 341 levels of ORF6 are increased upon expression of RAE1 and NUP98 (Figure 3M) Incidentally, in the Dox-inducible ORF6 expression system in A549 cells, 364 differences in the ability of SARS-CoV-2 ORF6 and SARS-CoV ORF6 to suppress 365 the upregulation of IFNB1 appeared to disappear ( Figure 2G ). Differences between 366

HEK293 cells and A549 cells may be explained by at least two possibilities: First, 367 the expression levels of ORF6 upon Dox stimulation in A549 cells are lower than 368 those achieved by transient transfection of HEK293 cells ( Figure S1B) . Second, the 369 induction of IFNB1 by SeV infection in A549 cells (~1,500 fold) is dramatically 370 higher than that in HEK293 cells (~50-100 fold) (Figures 2D and 2G) . Thus, the 371 relative antagonistic activity of ORF6 may be lower in A549 cells compared to 372 HEK293 cells. 373

By analyzing more than 67,000 SARS-CoV-2 sequences, we found that 374

variants lacking the C-terminal region of ORF6 due to frameshift and/or nonsense 375 mutations emerged more than 50 times during the current COVID-19 pandemic 376 (Figure 4 and Table S4 ). In contrast, truncated ORF6 genes have not been 377 detected in SARS-CoV-2-related viruses isolated from animals so far. By analyzing 378 the ORF6 sequences from a variety of sarbecoviruses belonging to the SARS-CoV 379

lineage, however, we also found three SARS-CoV-related viruses isolated from two 380 bats (GenBank accession numbers: MK211374 and KJ473816) and a palm civet 381 (GenBank accession number: FJ959407) harboring truncated ORF6 sequences (44, 382 50 and 44 amino acids, respectively) due to frameshift mutations ( Figure S3) . 383 Furthermore, we detected a human SARS-CoV, strain TWJ (GenBank accession 384 number: AP006558) that encodes a shortened ORF6 protein due to a frameshift 385 mutation ( Figure S3) Since the C-terminal region of SARS-CoV-2 ORF6 is essential to elicit its 392

anti-IFN activity, SARS-CoV-2 variants expressing C-terminally truncated ORF6 393 most likely lost an IFN antagonist. Although the frequency of SARS-CoV-2 isolates 394 with C-terminally truncated ORF6 is low (~0.2%), our phylogenetic analyses provide 395 strong evidence for human-to-human transmission of these viruses ( Figure 4B ).

Since ORF6 is a potent IFN antagonist, the emergence of SARS-CoV-2 ORF6 397 frameshift mutants may contribute to the attenuation of viral pathogenicity. However, 398

the relative contribution of ORF6 to disease severity is hard to assess at this point 399 since most of the viral sequences currently deposited in GISAID are derived from 400 symptomatic patients (mostly severe cases ORF3b is a potent interferon antagonist whose activity is increased by a naturally 562 occurring elongation variant. Cell Rep 32, 108185. 563

Konno, Y., Nagaoka, S., Kimura, I., Takahashi See also Table S1 . All viral genome sequences used in this study and the respective GenBank or 904 GISAID (https://www.gisaid.org) accession numbers are summarized in Table S1 . 905

We aligned the viral genomes and amino acid sequences of ORF1ab, S, E, M, N 906

and ORF6 using the L-INS-i program of MAFFT version 7.453 (Katoh and Standley,  907 2013). We then constructed phylogenetic trees using the full-length genomes 908 (Figure 1A) , ORF1ab, S, E, M, N genes ( Figure 1B) and ORF6 gene (Figure 2A) . 909 We Sarbecovirus ORF6s (the accession numbers and sequences are listed in Table  925 S1) were synthesized by a gene synthesis service (Fasmac). The ORF6 derivatives 926

were generated by PCR using PrimeSTAR GXL DNA polymerase (Takara), the 927 synthesized ORFs as templates, and the primers listed in Table S5 . The obtained 928 DNA fragments were inserted into pCAGGS via XhoI-BglII. To construct the 929 Dox-inducible expression plasmids for HA-tagged ORF6, pLVX-TetOne-Puro 930 (Takara, cat# 631849) was used as a backbone. The HA-ORF6 sequences were 931 generated by PCR using PrimeSTAR GXL DNA polymerase (Takara), the 932 synthesized ORF6s as templates, and the primers listed in Table S5 . The obtained 933 DNA fragments were digested with EcoRI and BglII, and were inserted into the 934

EcoRI-BamHI site of pLVX-TetOne-Puro. To construct a Flag-tagged RAE1 935 expression plasmid, pcDNA3.1 (Thermo Fisher Scientific) was used as a backbone. 936

The Flag-tagged RAE1 sequence was generated by PCR using PrimeSTAR GXL 937 DNA polymerase (Takara), human cDNA, which was synthesized using 938 HEK293-derived mRNA as the template, and the primers listed in Table S5 . The 939

obtained DNA fragments were digested with EcoRV and NotI, and inserted into the 940

EcoRV-NotI site of pcDNA3.1. The HA-tagged NUP98 expression plasmid was 941 described in a previous study (Ebina et al., 2004) . Nucleotide sequences were 942

Emergence of SARS-CoV-2 607 through recombination and strong purifying selection

Extensive diversity of coronaviruses 610 in bats from China

Growth in suckling-mouse 612 brain of "IBV-like" viruses from patients with upper respiratory tract disease

SARS-CoV-2 Orf6 hijacks 616 Nup98 to block STAT nuclear import and antagonize interferon signaling

Genome evolution of SARS-CoV-2 and its 619 virological characteristics

HIV-1 622 competition experiments in humanized mice show that APOBEC3H imposes 623 selective pressure and promotes virus adaptation

Efficient selection for 625 high-expression transfectants with a novel eukaryotic vector

Coronaviridae: the viruses and their 627 replication

RAE1 is 630 a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the 631 nuclear pore complex through multiple domains

Identification of a severe acute respiratory syndrome coronavirus-like virus in a 635 leaf-nosed bat in Nigeria

A dynamic nomenclature proposal for SARS-CoV-2 638 lineages to assist genomic epidemiology

Structural and functional 640 analysis of the interaction between the nucleoporin Nup98 and the mRNA export 641 factor Rae1

A plasmid 644 DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for 645 COVID-19 research

Coronaviruses: structure and 647 genome expression

Prevalence and genetic diversity of 650 coronaviruses in bats from China

Rapid reconstruction of 653 SARS-CoV-2 using a synthetic genomics platform

Establishment of a reverse 656 genetics system for SARS-CoV-2 using circular polymerase extension reaction

Functional mutations in spike 660 glycoprotein of Zaire ebolavirus associated with an increase in infection efficiency

Genomic characterization of a newly discovered coronavirus associated with acute 665 respiratory distress syndrome in humans

Discovery and genetic analysis of novel coronaviruses in least 668 horseshoe bats in southwestern China

SARS-CoV infection in a restaurant from palm civet

Summary of probable SARS cases with onset of illness from 1 674

Middle East respiratory syndrome coronavirus (MERS-CoV

Coronavirus disease

Characterization and complete 682 genome sequence of a novel coronavirus, coronavirus HKU1, from patients with 683 pneumonia

Comparative analysis of twelve genomes of 686 three novel group 2c and group 2d coronaviruses reveals unique group and 687 subgroup features

Deciphering the bat virome catalog to better understand the 690 ecological diversity of bat viruses and the bat origin of emerging infectious diseases

ORF8-related genetic evidence for Chinese horseshoe bats as the source of human 694 severe acute respiratory syndrome coronavirus

Evasion of type I interferon by SARS-CoV-2

Isolation of SARS-CoV-2-related coronavirus from Malayan 700 pangolins

Engineering SARS-CoV-2 using a reverse genetic system

An infectious cDNA clone 706 of SARS-CoV-2

Human-specific 709 adaptations in Vpu conferring anti-tetherin activity are critical for efficient early 710 HIV-1 replication in vivo

Rescue of SARS-CoV-2 from 713 a single bacterial artificial chromosome

A single amino acid substitution within the paramyxovirus Sendai virus 716 nucleoprotein is a critical determinant for production of interferon-beta-inducing 717 copyback-type defective interfering genomes

Feline immunodeficiency 720 virus evolutionarily acquires two proteins, Vif and protease, capable of antagonizing 721 feline APOBEC3

Intraspecies diversity of SARS-like coronaviruses in 724

Rhinolophus sinicus and its implications for the origin of SARS coronaviruses in 725 humans

SARS-CoV-2 nsp13, nsp14, nsp15 and orf6 728 function as potent interferon antagonists

Isolation of a novel coronavirus from a man with pneumonia in Saudi 731

A novel bat coronavirus closely related to 734 SARS-CoV-2 contains natural insertions at the S1/S2 cleavage site of the spike 735 protein

A pneumonia outbreak associated with a new 738 coronavirus of probable bat origin

Comparison of the anti-IFN activity of Sarbecovirus ORF6 and ORF3b. The 778 anti-IFN activities of ORF6 (100 ng; x-axis) and ORF3b (100 ng

the input of the cell lysate was normalized to TUBA, 781 and one representative result out of three independent experiments is shown. In (B), 782 three different doses of IAV NS1 were used as controls on each membrane. kDa, 783 kilodalton. For the luciferase assay (C), the value was normalized to the 784 unstimulated, empty vector-transfected cells

the expression of the target gene was 787 normalized to GAPDH, and the fold change to the value of 0 h is shown

mean values of 789 three independent experiments with SEM are shown

793 the GFP-expressing cells upon Dox stimulation

See also Figure S1

A) Comparison of ORF6 residues that are different between the SARS-CoV-2 and 800 SARS-CoV lineage. Numbers in parentheses indicate the total number of 801 sequences analyzed. The comparison of all ORF6 residues is shown in Figure 802 S2A. 803 (B-E) Anti-IFN activity of SARS-CoV-2 ORF6 mutants (B and C) and SARS-CoV 804 ORF6 mutants (D and E). HEK293 cells were cotransfected with plasmids 805 expressing HA-tagged ORF6 variants and p125Luc. 24 h post transfection, cells 806 were infected with SeV (MOI 10). 24 h post infection

Scheme illustrating the ORF6 C-terminal mutants generated. 809 (G and H) Anti-IFN activity of Sarbecovirus ORF6 mutants. HEK293 cells were 810 cotransfected with plasmids expressing HA-tagged ORF6 variants and p125Luc. 24 811 h post transfection, cells were infected with SeV (MOI 10). 24 h post infection

(I) ORF6 interaction with RAE1 and NUP98. HEK293 cells were transfected with 814 plasmids expressing HA-tagged ORF6 WT or the respective ∆C mutant (∆C2 for 815 cluster 41 is shown in Figure S3. GISAID ID and sampling date

See also Figure S3 and Tables S2-S4

DNA sequencing service (Fasmac), and the sequence data were 943 analyzed by Sequencher version 5.1 software (Gene Codes Corporation)

IFN treatment and SeV Infection 946 HEK293 cells were transfected using PEI Max (Polysciences) according to the 947 manufacturer's protocol. For immunofluorescence staining, HEK293T cells were 948 transfected using calcium phosphate as previously described

For Western blotting, cells (in 12 well) were cotransfected with the pCAGGS-based 950

2006) and the 959 pCAGGS-based HA-tagged expression plasmid (10, 30 or 50 ng for Figures 2C 960 and S1D; 10 ng for Figures 3C, 3E, S2B and S2C; 10 or 30 ng for Figures 3H and 961 S2D; 30 ng for Figure S1A). The amounts of transfected plasmids were normalized 962 to 100 ng per well. For the compensation assay (Figures 3L and 3M), cells (in 12 963 well) were cotransfected with the pCAGGS-based SARS-CoV-2 ORF6 expression 964 plasmid (100 ng) and 100, 200 or 400 ng of Flag-tagged RAE1 expression plasmid 965 and 100, 200 or 400 ng of HA-tagged NUP98 expression plasmid (kindly provided 966 by Dr. Yoshio Koyanagi). The amounts of transfected plasmids were normalized to 967 1,000 ng per well. To induce ORF6-HA expression in A549 cells (described above), 968 they were treated with 1 µg/ml Dox (Takara)

Selleck Chemicals) (solved with DMSO) was added at 24 h post 974 transfection. For co-IP, HEK293 cells were transfected with pCAGGS

HA-tagged ORF6 expression plasmids (20 µg, Figure 3I) were transfected into 976 HEK293 cells (in 10-cm dishes) as described above

Briefly, 50 µl cell lysate was applied to a 96-well plate (Nunc), and the 982 firefly luciferase activity was measured using a PicaGene BrillianStar-LT luciferase 983 assay system (Toyo-b-net), and the input for the luciferase assay was normalized 984 by using a CellTiter-Glo 2.0 assay kit (Promega) following the manufacturers' 985 instructions

Transfected cells were lysed with 1x SDS sample buffer (62.5 mM Tris-HCl

50 mM NaCl, 1 mM MgCl 2 , 50 µM ZnCl 2 , 10% 992 glycerol, 1% Triton X-100) containing a protease inhibitor cocktail (Roche)

Briefly, cells were harvested at 48 h post transfection and lysed with lysis 1000 buffer (0.1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA 1001 and 10 mM Tris-HCl

Real-time RT-PCR was performed as previously described

Briefly, cellular RNA was extracted using QIAamp RNA blood 1008 mini kit (Qiagen) and then treated with RNase-Free DNase Set (Qiagen). cDNA was 1009 synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific) 1010 and random primer (Thermo Fisher Scientific). Real-time RT-PCR was performed 1011 using a Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) and the 1012 primers listed in Key Resources Table. For real-time RT-PCR, a CFX Connect 1013 Real-Time PCR Detection System

SARS-CoV-2 ORF6 expression vector either in 1018 combination with RAE1 and NUP98 expression vectors or an empty vector. 16-24 h 1019 post transfection, cells were infected with SeV or left untreated. Subsequently, cells 1020 were fixed for 20 min at room temperature with 4% paraformaldehyde and 1021 permeabilized using PBS containing 0.5% Triton-X 100 and 5% FCS for 20 min at 1022 room temperature. IRF3 was detected using a unconjugated primary antibody

Nuclei were visualized by DAPI staining. Cells 1025 were mounted in Mowiol mounting medium (Cold Spring Harbor Protocols) and 1026 analyzed using confocal microscopy

Using these 1044 sequences, we generated a multiple sequence alignment using FFT-NS-2 program 1045 in MAFFT software version 7.467. We then constructed a maximum 1046 likelihood-based phylogenetic tree using RAxML-NG version 1.0.0 with GTR model 1047 that was chosen based on AIC values using ModelTest-NG version 0.1.5. We 1048 applied a 1,000-time bootstrapping test. 1049 1050 QUANTIFICATION AND STATISTICAL ANALYSIS 1051 Data analyses were performed using Prism 7 (GraphPad Software). The data are 1052 presented as averages ± SEM

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies HRP-conjugated anti-HA Roche Cat# 12013819001

HRP-conjugated anti-Flag Sigma-Aldrich Cat# A8592

Abcam Cat# ab124783

TUBA) Sigma-Aldrich Cat# T9026

HRP-conjugated anti-rabbit IgG Cell Signaling Technology Cat# 7074S

antagonist and is unique to sarbecoviruses including SARS-CoV-2. Although the antagonistic activity of SARS-CoV-2 ORF6 depends on its C-terminal region, approximately 0.2% of all SARS-CoV-2 genomes isolated during the current COVID-19 pandemic harbor premature stop codons in ORF6.