key: cord-1015790-hgg33kwz
authors: Konno, Yoriyuki; Kimura, Izumi; Uriu, Keiya; Fukushi, Masaya; Irie, Takashi; Koyanagi, Yoshio; Nakagawa, So; Sato, Kei
title: SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant
date: 2020-05-11
journal: bioRxiv
DOI: 10.1101/2020.05.11.088179
sha: d4559c24f1476fb7aecf70794cfa2c4ddb1926b3
doc_id: 1015790
cord_uid: hgg33kwz

One of the features distinguishing SARS-CoV-2 from its more pathogenic counterpart SARS-CoV is the presence of premature stop codons in its ORF3b gene. Here, we show that SARS-CoV-2 ORF3b is a potent interferon antagonist, suppressing the induction of type I interferon more efficiently than its SARS-CoV ortholog. Phylogenetic analyses and functional assays revealed that SARS-CoV-2-related viruses from bats and pangolins also encode truncated ORF3b gene products with strong anti-interferon activity. Furthermore, analyses of more than 15,000 SARS-CoV-2 sequences identified a natural variant, in which a longer ORF3b reading frame was reconstituted. This variant was isolated from two patients with severe disease and further increased the ability of ORF3b to suppress interferon induction. Thus, our findings not only help to explain the poor interferon response in COVID-19 patients, but also describe a possibility of the emergence of natural SARS-CoV-2 quasispecies with extended ORF3b that may exacerbate COVID-19 symptoms. Highlights ORF3b of SARS-CoV-2 and related bat and pangolin viruses is a potent IFN antagonist SARS-CoV-2 ORF3b suppresses IFN induction more efficiently than SARS-CoV ortholog The anti-IFN activity of ORF3b depends on the length of its C-terminus An ORF3b with increased IFN antagonism was isolated from two severe COVID-19 cases

outbreak has originated from cross-species coronavirus transmission from these 88 mammals to humans, the exact origin remains to be determined (Andersen et al., 89 2020). 90

One prominent feature that distinguishes COVID-19 from SARS in terms of In this study, we therefore aimed to characterize the viral factor(s) determining 97

immune activation upon SARS-CoV-2 infection. We particularly focused on 98 differences in putative viral IFN-I antagonists and revealed that the ORF3b gene 99

products of SARS-CoV-2 and SARS-CoV not only differ considerably in their length, 100 but also in their ability to antagonize type I IFN. Furthermore, we demonstrate that 101 the potent anti-IFN-I activity of SARS-CoV-2 ORF3b is also found in related viruses 102 from bats and pangolins. Mutational analyses revealed that the length of the C-103

terminus determines the efficacy of IFN antagonism by ORF3b. Finally, we describe 104 a natural SARS-CoV-2 variant with further increased ORF3b-mediated anti-IFN-I 105 activity that emerged during the current COVID-19 pandemic. 106

To determine virological differences between SARS-CoV-2 and SARS-CoV, we set 109 out to compare the sequences of diverse Sarbecoviruses. Consistent with recent 110

reports (Lam et ORF3b length was remarkably different between SARS-CoV-2 and SARS-CoV 124 (Figure 1B) , we hypothesized that the antagonistic activity of ORF3b against IFN-I 125 differs between these two viruses. To test this hypothesis, we monitored human 126 IFNB1 promoter activity in the presence of ORF3b of SARS-CoV-2 (Wuhan-Hu-1) 127

and SARS-CoV (Tor2) using a luciferase reporter assay. The influenza A virus (IAV) 128 non-structural protein 1 (NS1) served as positive control (Garcia-Sastre et al., 1998; 129 Krug et al., 2003) . As shown in Figure 1C , all three viral proteins dose-dependently 130 suppressed the activation of the IFNB1 promoter upon Sendai virus (SeV) infection. 131

Notably, the antagonistic activity of SARS-CoV-2 ORF3b was slightly, but 132 significantly higher than that of SARS-CoV ORF3b (Figure 1C, bottom) . Thus, our 133 data demonstrate that SARS-CoV-2 ORF3b is a potent inhibitor for human IFN-I 134 activation, even though it only comprises 22 amino acids. 135 136 SARS-CoV-2-related ORF3b proteins from bat and pangolin viruses suppress 137 IFN-I activation on average more efficiently than their SARS-CoV counterparts 138

Since the lengths of ORF3b proteins in SARS-CoV-2-related viruses including those 139 from bats and pangolins were on average shorter than those from SARS-CoV and 140 related viruses (Figure 1B) , we next investigated whether they were also generally 141 more efficient in antagonizing IFN-I. A phylogenetic analysis of Sarbecovirus ORF3b 142 genes showed that the evolutionary relationship of Sarbecovirus ORF3b genes was 143 similar to that of the full-length viral genomes (Figures 1A and 2A) . For our 144 functional analyses, we generated expression plasmids for ORF3b from SARS-CoV-145 2-related viruses from bats (RmYN02, RaTG13 and ZXC21) and a pangolin (P4L), 146

as well as SARS-CoV-related viruses from a civet (civet007) and bats (Rs7327, 147

Rs4231, YN2013 and Rm1), representing different lengths of this protein ( Figure  148 2B). As shown in Figure 2C , all four SARS-CoV-2-related ORF3b significantly 149 suppressed human IFN-I activation. In contrast, only two SARS-CoV-related ORF3b 150

proteins, Rs4231 and Rm1, exhibited anti-IFN-I activity at the concentrations tested 151

( Figure 2C) . Intriguingly, these two SARS-CoV-related ORF3b proteins are C-152

terminally truncated and shorter than ORF3b of SARS-CoV Tor2 (Figure 2A) . These 153 findings suggest that the C-terminal region (residues 115-154) may attenuate the 154 anti-IFN-I activity of ORF3b. To test this hypothesis, we generated a C-terminally 155 truncated derivatives of SARS-CoV (Tor2) ORF3b harboring a premature stop codon 156

at position 135. The K135* mutant mimics the ORF3b of Rs4231 ( Figure 2B ).

Reporter assays revealed that this derivative exhibits higher anti-IFN-I activity than 158

wild-type (WT) SARS-CoV ORF3b ( Figure 2D) , demonstrating that the C-terminal 159 region of SARS-CoV ORF3b indeed attenuates its anti-IFN-I activity.

A SARS-CoV ORF3b-like sequence is hidden in the SARS-CoV-2 genome 162

ORF3b of SARS-CoV-2 is shorter than its ortholog in SARS-CoV ( Figures 1B and  163 2A). However, when closely inspecting the nucleotide sequences of these two 164 viruses, we noticed that the SARS-CoV-2 nucleotide sequence downstream of the 165 stop codon of ORF3b shows a high similarity to the SARS-CoV ORF3b gene 166 (nucleotide similarity=79.5%; Figure 3A ). In contrast to SARS-CoV ORF3b, however, 167 SARS-CoV-2 harbors four premature stop codons that result in the expression of a 168 drastically shortened ORF3b protein ( Figure 3A ). Similar patterns were observed in 169 SARS-CoV-2-related viruses from bats and pangolins. Since the ORF3b length is 170

closely associated with its anti-IFN-I activity ( Figures 2C and 2D) , we hypothesized 171 that reversion of the premature stop codons in SARS-CoV-2 ORF3b affects its ability 172

to inhibit human IFN-I. To address this possibility, we generated four SARS-CoV-2 173

ORF3b derivatives, 57*, 79*, 119* and 155*, lacking the respective premature stop 174 codons ( Figure 3B, top) . As shown in Figure 3C , all four derivatives inhibited human 175 IFN-I activation in dose-dependent manners. Consistent with the results obtained 176

with SARS-CoV ORF3b mutants (Figure 2D) , the 155* mutant, comprising the very 177 C-terminal region (positions 119-154), was poorly expressed and exhibited relatively 178 low anti-IFN-I activity ( Figure 3C) . Notably, however, we found that the extended 179

ORF3b derivatives, particularly 57*, 79*, 119*, exhibited higher anti-IFN-I activity 180

compared to WT ORF3b ( Figure 3C ). These findings confirm that the length of 181

ORF3b determines its ability to suppress an IFN-I response. Furthermore, they show 182 IFNβ reporter assays revealed that the "Ecuador variant" ORF3b exhibits 199

significantly higher anti-IFN-I activity than the parental SARS-CoV-2 ORF3b ( Figure  200 3D). These findings show that a naturally occurring SARS-CoV-2 variant, expressing 201

an elongated ORF3b protein with enhanced anti-IFN activity, has already emerged 202

during the current SARS-CoV-2 pandemic. 203

Here, we demonstrate that SARS-CoV-2 ORF3b is a potent antagonist of human 205 IFN-I activation. On average, ORF3b proteins from SARS-CoV-2 and related bat and 206 pangolin viruses were more active than their SARS-CoV counterparts. Notably, a 207 recent study has revealed that the antibodies recognizing ORF3b are highly 208

detectable it is tempting to speculate that atypical symptoms and poor IFN-I responses in 218 SARS-CoV-2 infection may be attributed to the potent anti IFN-I activity of its ORF3b. 219

Like SARS-CoV-2 ORF3b, its orthologs in SARS-CoV-2-related viruses 220 from bats and pangolins efficiently antagonize IFN-I and are generally truncated due 221

to the presence of several premature stop codons. In contrast, the anti-IFN activity 222 of ORF3b proteins encoded by some SARS-CoV-related viruses is attenuated, most 223 likely due to an elongated C-terminus. We hypothesized that the ORF3b length ORF3b gene. 243 We further show that a SARS-CoV ORF3b-like sequence is still present in 244

the SARS-CoV-2 genome, but is interrupted by premature stop codons. We 245 demonstrate that a partial extension of SARS-CoV-2 ORF3b by reverting stop 246

codons increases its inhibitory activity against human IFN-I. Full reversion of all stop 247

codons, however, resulted in an ORF3b protein with poor anti-IFN activity. This is in 248

line with the phenotypic difference between SARS-CoV-2 and SARS-CoV ORF3b 249

proteins and suggests that the very C-terminus of ORF3b impairs its immune 250 evasion activity. 251

Intriguingly, we also identified a naturally occurring SARS- All viral genome sequences used in this study and the respective GenBank or 275 GISAID (https://www.gisaid.org) accession numbers are summarized in Table S1 . 276 We Table S2 ) and the cryptic SARS-291

CoV ORF3b-like sequence in SARS-CoV-2 [Wuhan-Hu-1 (accession no. 292 NC_045512.2), nucleotides 25814-26281) was synthesized by a gene synthesis 293 service (Fasmac). The ORF3b derivatives were generated by PCR using 294

PrimeSTAR GXL DNA polymerase (Takara), the synthesized ORFs as templates, 295 and the primers listed in Hu, B., Zeng, L.P., Yang, X.L., Ge, X.Y., Zhang, W., Li, B., Xie, J.Z., Shen, X.R., The full-length sequences (~30,000 bp) of SARS-CoV-2 (Wuhan-Hu-1 as a 536

representative), SARS-CoV-2-related viruses from bats (n=4) and pangolins (n=4), 537

SARS-CoV (n=190), SARS-CoV-related viruses from civets (n=3) and bats (n=54), 538

and outgroup viruses (n=2; BM48-31 and BtKY72) were analyzed. Accession 539 number, strain name, and host of each virus are indicated for each branch. Note that 540 the branches including SARS-CoV (n=190) and SARS-CoV-related viruses from 541 civets (n=3) were collapsed for better visualization. The uncollapsed tree is shown 542

in Figure S1 , and the sequences used are summarized in and one representative result out of X independent experiments is shown. The band 559

of each viral protein is indicated by a white arrowhead. kDa, kilodalton. In the 560 luciferase assay, the value of the SeV-infected empty vector-transfected cells was 561 set to 100%. The average of three independent experiments with SEM is shown, 562

and statistically significant differences (P < 0.05) compared to the SeV-infected 563 empty vector-transfected cells (#) and the same amount of the SARS-CoV-2 ORF3b-564

transfected cells (*) are shown. E, empty vector. 565

See also Figure S1 and Table S1 . 566 were cotransfected with a plasmid expressing one of 11 HA-tagged Sarbecovirus 580

ORF3b proteins (summarized in B; 100 ng) and p125Luc (500 ng). 24 h post 581 transfection, SeV was inoculated at MOI 10. 24 h post infection, cells were harvested 582

for Western blotting (top) and luciferase assay (bottom). Note that the amino acid 583 sequences of ZXC21 and ZC45 are identical.

(D) Anti-IFN-I activity of C-terminally truncated SARS-CoV ORF3b. HEK293T cells 585

were cotransfected with two different amounts of plasmids expressing HA-tagged 586 SARS-CoV ORF3b WT and K135* (50 and 100 ng) and p125Luc (500 ng). 24 h post 587 transfection, SeV was inoculated at MOI 10. 24 h post infection, the cells were 588 harvested for Western blotting (top) and luciferase assay (bottom). 589

For Western blotting, the input of cell lysate was normalized to TUBA. One 590

representative blot out of three independent experiments is shown. In the luciferase 591 assay, the value of the SeV-infected empty vector-transfected cells was set to 100%. 592

The average of three independent experiments with SEM is shown, and statistically 593 significant differences (P < 0.05) compared to the SeV-infected empty vector-594 transfected cells (#) and the same amount of either SARS-CoV-2 ORF3b (Wuhan-595

Hu-1)-transfected cells (*, C) or SARS-CoV ORF3b WT-transfected cells (*, D) are 596

shown. E, empty vector. 597

Ecoepidemiology and complete genome 462 comparison of different strains of severe acute respiratory syndrome

Rhinolophus bat coronavirus in China reveal bats as a reservoir for acute, self-464 limiting infection that allows recombination events

Severe acute respiratory syndrome 467 coronavirus-like virus in Chinese horseshoe bats

Early transmission dynamics in Wuhan, China, of 471 novel coronavirus-infected pneumonia

Bats are natural reservoirs of SARS-like coronaviruses

Extensive diversity of coronaviruses 477 in bats from China

Correction: 480 HIV-1 competition experiments in humanized mice show that APOBEC3H imposes 481 selective pressure and promotes virus adaptation

Efficient selection for high-483 expression transfectants with a novel eukaryotic vector

Prevalence and genetic diversity of 486 coronaviruses in bats from China

Functional mutations in spike 489 glycoprotein of Zaire ebolavirus associated with an increase in infection efficiency

Estimates of 493 the severity of coronavirus disease 2019: a model-based analysis

Discovery and genetic analysis of novel coronaviruses in least horseshoe 496 bats in southwestern China

SARS-CoV infection in a restaurant from palm civet

Enhanced anti-IFN-I upon reconstitution of the cryptic SARS-CoV-2 599 ORF3b 600 (A) Schemes illustrating the genomic regions encoding ORF2, ORF3a, ORF3b and 601 ORF4 of SARS-CoV-2 and SARS-CoV. Open squares with dotted red lines indicate 602 a cryptic ORF3b reading frame in SARS-CoV-2 that is similar to SARS-CoV ORF3b. 603 Asterisks indicate stop codons in the ORF3b frame. 604 (B) SARS-CoV-2 ORF3b derivatives characterized in this study

CoV-2 ORF3b as well as four derivatives with mutated stop codons (57*, 79*, 119* 606 and 155*) are shown. Asterisks indicate the stop codons in the original ORF3b frame. 607 (Bottom) A natural ORF3b variant detected in two sequences deposited in GISAID 608 (accession IDs: EPI_ISL_422564 and EPI_ISL_422565

Anti-IFN-I activity different SARS-CoV-2 ORF3b derivatives. HEK293T cells 611 were cotransfected with two different amounts of plasmids expressing the indicated 612 HA-tagged SARS-CoV-2 ORF3b derivatives (WT, 57*, 79*, 119* and 155*

24 h post transfection, SeV was inoculated at MOI 614 10. 24 h post infection, the cells were harvested for Western blotting (top) and 615 luciferase assay (bottom)

Ecuador variant" ORF3b. HEK293T cells were 617 cotransfected with two different amounts of plasmids expressing HA-tagged 618 "Ecuador variant" ORF3b or parental SARS-CoV-2 ORF3b (50 and 100 ng) and 619 p125Luc (500 ng). 24 h post transfection

For Western blotting, the input of cell lysate was normalized to TUBA. One 623 representative blot out of three independent experiments is shown. A highly exposed 624 blot visualizing the band of the 155* mutant is shown in Figure S2. kDa, kilodalton

SeV-infected empty vector-transfected cells 626 was set to 100%. The average of three independent experiments with SEM is shown, 627 and statistically significant differences (P < 0.05) compared to the SeV-infected 628 empty vector-transfected cells (#) and the same amount of the SARS-CoV-2 ORF3b 629 WT

See also Figure S2