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SARS-CoV-2 genomic surveillance identifies
naturally occurring truncation of ORF7a that limits
immune suppression
Graphical abstract
Highlights
dORF7a mutations are found in SARS-CoV-2 genomes
isolated from around the globe
dThe ORF7aD115 isolate displays a replication defect
dAn ORF7a mutation limits viral suppression of the interferon
response
Authors
Artem Nemudryi, Anna Nemudraia,
Tanner Wiegand, ..., Diane Bimczok,
Mark A. Jutila, Blake Wiedenheft
Correspondence
bwiedenheft@gmail.com
In brief
Nemudryi et al. use global SARS-CoV-2
phylogenomics to identify mutations that
frequently occur in the C-terminal end of
ORF7a accessory protein. They isolate
one of these mutant viruses from a patient
sample and demonstrate that ORF7a
truncation affects viral mechanisms
responsible for suppressing host immune
response.
Nemudryi et al., 2021, Cell Reports 35, 109197
June 1, 2021 ª2021 The Author(s).
https://doi.org/10.1016/j.celrep.2021.109197 ll
Report
SARS-CoV-2 genomic surveillance identifies
naturally occurring truncation of ORF7a
that limits immune suppression
Artem Nemudryi,
1,3,5
Anna Nemudraia,
1,5
Tanner Wiegand,
1,6
Joseph Nichols,
1,6
Deann T. Snyder,
1
Jodi F. Hedges,
1
Calvin Cicha,
1
Helen Lee,
1
Karl K. Vanderwood,
2
Diane Bimczok,
1
Mark A. Jutila,
1
and Blake Wiedenheft
1,4,7,
*
1
Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA
2
Gallatin City-County Health Department, Bozeman, MT 59715, USA
3
Twitter: @artemnemudryi
4
Twitter: @WiedenheftLab
5
These authors contributed equally
6
These authors contributed equally
7
Lead contact
*Correspondence: bwiedenheft@gmail.com
https://doi.org/10.1016/j.celrep.2021.109197
SUMMARY
Over 950,000 whole-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
have been determined for viruses isolated from around the world. These sequences are critical for under-
standing the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations
frequently occur in the C-terminal end of ORF7a. We isolate one of these mutant viruses from a patient sam-
ple and use viral challenge experiments to link this isolate (ORF7a
D115
) to a growth defect. ORF7a is impli-
cated in immune modulation, and we show that the C-terminal truncation negates anti-immune activities
of the protein, which results in elevated type I interferon response to the viral infection. Collectively, this
work indicates that ORF7a mutations occur frequently, and that these changes affect viral mechanisms
responsible for suppressing the immune response.
INTRODUCTION
The spillover of severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) into the human population has resulted in a global
pandemic of coronavirus disease 2019 (COVID-19) with more
than 2.8 million deaths worldwide (https://coronavirus.jhu.edu/)
(Andersen et al., 2020). Although much of the research on this vi-
rus is focused on the Spike protein, recent reports demonstrate
that accessory proteins of SARS-CoV-2 might be involved in
COVID-19 pathogenesis by modulating antiviral host responses
(Young et al., 2020;Zhang et al., 2020). SARS-CoV-2 uses mul-
tiple strategies to evade host immunity. Accessory proteins
ORF3b, ORF6, and ORF7a antagonize various steps of type I
interferon (IFN-I) production and signaling, while the proposed
function of ORF8 is downregulating antigen presentation (Konno
et al., 2020;Lei et al., 2020;Miorin et al., 2020;Tan et al., 2020;
Xia et al., 2020). To occlude signal transmission from IFN recep-
tors, ORF7a subverts phosphorylation of STAT2, suppressing
transcriptional activation of antiviral IFN-stimulated genes
(ISGs) that can otherwise restrict viral replication (Martin-Sancho
et al., 2020;Xia et al., 2020). Although the proposed function for
ORF7a is intracellular, antibodies against ORF7a are elevated in
the serum of COVID-19 patients (Hachim et al., 2020). Work that
is currently under review indicates that recombinant ORF7a pro-
tein interacts with CD14
+
monocytes and triggers expression of
pro-inflammatory cytokines, including interleukin-6 (IL-6) and tu-
mor necrosis factor alpha (TNF-a)(Zhou et al., 2021). However, it
is unclear if these interactions happen in COVID-19 patients.
Together, these data suggest that ORF7a plays a dual role in
SARS-CoV-2 infection by modulating both the IFN and inflam-
matory responses.
Here, we show that C-terminal mutations in ORF7a occur
frequently in samples isolated from patients around the globe.
These mutations are not derived from a single lineage, and
they do not persist over time. Using samples collected from in-
fected patients, we have isolated a virus containing a deletion
mutation in ORF7a that truncates the C-terminal half of the pro-
tein. In vitro viral challenge experiments demonstrate that this
mutation results in a replication defect and obviates viral sup-
pression of the immune response. Collectively, these data sug-
gest that ORF7a truncations are defective in suppressing the
host immune response, which may explain why these mutations
quickly disappear in the immunocompetent population.
RESULTS
SARS-CoV-2 genomic surveillance identifies
truncations of ORF7a
Genome sequencing has been used to track the rise and spread
of new SARS-CoV-2 lineages over the course of the pandemic
Cell Reports 35, 109197, June 1, 2021 ª2021 The Author(s). 1
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Nemudryi et al., SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits
immune suppression, Cell Reports (2021), https://doi.org/10.1016/j.celrep.2021.109197
ll
OPEN ACCESS
(https://www.gisaid.org/)(Bedford et al., 2020;Korber et al.,
2020). As part of this effort, we sequenced SARS-CoV-2 ge-
nomes isolated from patients in Bozeman, Montana (Figure 1A;
Table S1). In total, we determined 55 whole-genome sequences
of SARS-CoV-2 viruses isolated from patients between April and
July 2020 using an amplicon-based Nanopore protocol from the
ARTIC network (https://artic.network/)(Quick et al., 2017;Tyson
et al., 2020). To place the local outbreak in the context of the
global SARS-CoV-2 evolutionary trends, we aligned 55 whole-
genome sequences isolated from patients in Bozeman to
4,235 genomes subsampled (10 genomes per month per coun-
try) from 181,003 SARS-CoV-2 genomes downloaded from the
GISAID. The subsampling protocol removes redundancy and
bias introduced by uneven distribution of the global SARS-
CoV-2 sequencing effort. The resulting alignment was used to
build a phylogenetic tree (Figure 1B). Of the 55 SARS-CoV-2 ge-
nomes determined from patients in Bozeman, only 1 was derived
from the WA1 lineage (lineage A in Figure 1B) that was intro-
duced to Washington state from Wuhan, China, in January
2020 (Bedford et al., 2020;Holshue et al., 2020). The remaining
54 genomes associate with clade B.1 and its offshoots, which
are characterized by the D614G mutation in the spike and have
prevailed globally since the spring of 2020 (Rambaut et al.,
2020a)(Figure 1B). The genetic variability in SARS-CoV-2 circu-
lating in Bozeman (April to July 2020) represents 14 independent
viral lineages, reflecting multiple introductions of the virus to the
community from many different sources (Figure S1A).
During the annotation of these genomes, we noticed a reoc-
curring (7 out of 55 genomes) 115-nt deletion in the gene-encod-
ing accessory protein ORF7a (27,549–27,644 nt). The ORF7a
D115
Figure 1. SARS-CoV-2 genomic surveillance identifies global reoccurrence of ORF7a truncations
(A) Symptom onset (purple) and PCR-based SARS-CoV-2 test results (coral) for patients in Bozeman, Montana, are shown with vertical bars. Seven-day moving
averages, shown with lines, were used to indicate epidemiological trends.
(B) Phylogenetic analysis of SARS-CoV-2 genomes sampled in Boz eman and globally. The tree was constructed from an alignment of 55 Bozeman samples and
4,871 genomes subsampled from GISAID. Subsampling was performed using Augur utility (https://nextstrain.org) by selecting 10 genomes per country per
month since the start of the pandemic. Outer ring shows SARS-CoV-2 lineages assigned to genome sequences (Rambaut et al., 2020a). Major lineages include A
(pink) that is associated with initial outbreak in China and B (blue) that emerged later in Europe. Mino r lineages (i.e., C-N) are offshoots of lineage B. Red branches
identify truncated ORF7a variants (n = 205) detected in the global data and merged into the alignment. The red dot highlights 7 of the 55 ORF7a variants that were
isolated in Bozeman between April and July (2020). White dots highlight 48 viral genomes isolated in Bozeman that have wild-type ORF7a sequences.
(C) Distribution of different mutations that occur along the ORF7a coding sequence.
Please cite this article in press as: Nemudryi et al., SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits
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mutation was found in patient swabs collected over a period of
1.5 months (Table S1). RT-PCR and Sanger sequencing veri-
fied that all these seven ORF7a
D115
variants are bona fide muta-
tions and not a sequencing artifact (Figures S1B and S1C). In
addition to the ORF7a
D115
deletion, these genomes also share
10 single-nucleotide variants (SNVs) compared with the Wu-
han-Hu-1 reference genome. Seven of the 10 SNVs are found
frequently worldwide and are a signature of the B1.1 lineage of
SARS-CoV-2 (Rambaut et al., 2020a). The remaining three muta-
tions are rare, do not co-occur in any other genomes on GISAID,
and lead to amino acid changes in ORF3a (Q38P, L95F) and N
(R195I) proteins. Interestingly, one of the genomes in the
ORF7a
D115
cluster has two additional SNVs that likely were ac-
quired during circulation in the community. This virus was
sampled from a patient 41 days after sampling the first
ORF7a
D115
variant, which agrees well with estimated SARS-
CoV-2 mutation rates (2 nt/month) (van Dorp et al., 2020).
To determine if the ORF7a
D115
genotype is unique to Boze-
man, we downloaded an alignment of 180,971 SARS-CoV-2 ge-
nomes from GISAID, extracted ORF7a sequences, and deter-
mined their mutational profiles. In total, we identified 845
unique ORF7a gene variants that are different from the Wuhan-
Hu-1 reference sequence. Next, we looked for mutations with
major effect on the ORF7a amino acid sequence. We identified
189 unique ORF7a variants (825 total sequences) with frame-
shifts, deletions, or missense mutations causing premature
stop codons (Figure 1C). To understand the evolutionary history
of these mutations, we integrated these genome sequences into
our phylogenetic analysis. Detected ORF7a variants appear to
have emerged independently on every continent and were not
confined to any single lineage (Figure 1B; Table S1). Next, we
aligned translated ORF7a sequences to look for patterns. Most
of the identified ORF7a mutations (126 out of 189) truncate the
C terminus but preserve the N-terminal half of the protein (Fig-
ure 1C; Figure S1B). A portion (36.5%) of these truncated
ORF7a variants appeared in two or more patient samples (116
maximum [max]). Although some of these ORF7a mutants
appear to have arisen on multiple independent occasions, others
come from genomes that form monophyletic clades, suggesting
that viruses with ORF7a truncations are capable of transmission
within a host population (Figure S1B).
ORF7a truncation has a loss-of-function effect
ORF7a is a type I transmembrane (TM) protein with an N-terminal
immunoglobulin-like (Ig-like) ectodomain, stalk, TM domain, and
short cytosolic tail (Figure 2A). The two bsheets of the ectodo-
main are held together by two disulfide bonds (i.e., Cys23-
Cys58 and Cys35-Cys67) (Figure 2B). The cytosolic tail of
ORF7a contains a dilysine (KRKTE) endoplasmic reticulum (ER)
retrieval signal (ERRS) that mediates protein trafficking to the
ER-Golgi intermediate compartment (ERGIC) (Nelson et al.,
2005).
The D115 nt mutation in ORF7a introduces a premature stop
codon that eliminates b5, b6, and b7, two of the cystines that
form disulfides (Cys58 and Cys67), the TM, and the cytosolic
tail (Figures 2A and 2B). This truncation is expected to destabilize
the protein structure and significantly impact protein function
and trafficking in the host cell. To determine how loss of TM
and sorting signal affects protein localization, we cloned and ex-
pressed FLAG-tagged wild-type and truncated ORF7a proteins
in HEK293T-hACE2 cells. The wild-type ORF7a accumulated in
the perinuclear region of the cell, which is consistent with previ-
ously reported ERGIC localization (Figure 2D) (Martin-Sancho
et al., 2020;Nelson et al., 2005). In contrast, the truncated
ORF7a is distributed throughout the cytoplasm and does not
associate with specific subcellular compartments, which is
consistent with the loss of the TM domain and the ERRS signals
required for protein targeting (Figure 2E).
Extent of the truncation and the defect in intracellular targeting
suggests possible loss of ORF7a function. Along with several
SARS-CoV-2 accessory proteins, ORF7a has been implicated
in suppression of host IFN response to the infection (Xia et al.,
2020). To test the effect of the truncated ORF7a protein on IFN
signaling, we generated reporter HEK293T cells with integrated
luciferase reporter gene controlled by the interferon-stimulated
response element (ISRE). In these cells, activation of interferon
response drives reporter expression, which is quantified
using a luciferase assay. To test immune suppression, we over-
expressed the wild-type and the deletion mutant of ORF7a
(ORF7a
D115
) from plasmids and the stimulated cells with IFNa2b.
The ORF7a WT protein suppressed IFN response by 34%
(p = 0.0074) compared with cells transfected with control
plasmid, which is consistent with recent results by Xia et al.
(2020). In contrast, the ORF7a
D115
protein failed to suppress
IFN response and showed no significant difference to a control
(mCherry) plasmid (Figure 2F). These results indicate that the
D115 mutation in ORF7a has a loss-of-function effect that
negates antagonism of IFN signaling.
ORF7a mutation has no collateral effect on ORF7b
The ORF7a gene overlaps with the downstream ORF7b gene
(Figure S2). To determine if the D115mutationinORF7aim-
pacts ORF7b, we first examined whether it eliminates a tran-
scription-regulatory sequence (TRS) that is required for subge-
nomic RNA (sgRNA) synthesis. None of the TRSs identified via
direct RNA sequencing (RNA-seq) of SARS-CoV-2 overlap
with the D115 mutation site (Kim et al., 2020), suggesting
that ORF7b transcription is not affected (Figure S2). To
confirm this prediction, we used RT-PCR with primers that
detect ORF7a and ORF7b sgRNAs (Figure 2G). In this assay,
we infected 293T-hACE2 cells (MOI = 0.05) with ORF7a
WT
or
ORF7a
D115
viral strains and extracted total RNA from cells at
24 h postinfection (hpi). Both viruses produced specific RT-
PCR products corresponding to ORF7a and ORF7b sgRNAs.
Finally, to verify that the ORF7a
D115
variant does not impact
ORF7b translation, we probed cell lysates 24 hpi with an
anti-ORF7b antibody (Figure 2H). We detected ORF7b protein
in both viral strains with band intensities that suggest similar
expression levels. Collectively, these data indicate that the
ORF7a
D115
mutation has no collateral effect on the adjacent
gene.
ORF7a
D115
isolate displays a replication defect
To investigate the phenotype of SARS-CoV-2 D115 variant, we
infected 293T-hACE2 and Vero E6 cells with ORF7a
WT
and
ORF7a
D115
viruses at an MOI of 0.05 and measured viral RNA
Please cite this article in press as: Nemudryi et al., SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits
immune suppression, Cell Reports (2021), https://doi.org/10.1016/j.celrep.2021.109197
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replication using qRT-PCR (Figures 3A and 3B). In this assay, we
used viral strains sharing the same haplotype (C241T, C3037T,
C14408T, A23403G) that defines the SARS-CoV-2 B.1 lineage
and its derivatives (Figure S3A) (Rambaut et al., 2020a). Over
the course of 120 h, ORF7a
WT
viral RNA level increased 960-
fold in supernatants from Vero E6 cells (Figure 3A) and 270-
fold in supernatants from 293T-hACE2 cells (Figure 3B). The
ORF7a
D115
virus displayed limited replication that resulted in
only 18.6-fold and 7-fold RNA level increase at 120 hpi in VeroE6
and 293T-hACE2, respectively. This growth defect reaches
statistical significance starting at 6 hpi in Vero E6 cells (p =
0.0135) and 24 hpi in 293T-hACE2 cells (p = 6.2 310
5
).
To determine if this phenotype results from a defect in viral
egress only or if viral RNA replication in the host is also
affected, we examined early steps in the infection. We ex-
tracted total RNA from the infected 293T-hACE2 cells at 0.5,
6, and 24 hpi and measured SARS-CoV-2 RNA with qRT-
PCR. Compared with wild type, ORF7a
D115
RNA level was
reduced 2.1-fold inside the host at 6 hpi (p = 0.002) and 4.3-
fold at 24 hpi (p = 0.02) (Figure 3C). This decrease in viral
Figure 2. ORF7a truncation results in loss of function
(A) Amino acid (aa) sequence alignment of SARS-CoV-2 ORF7a
WT
, ORF7a
D115
, and SARS-CoV-1 ORF7a. Gaps show non-matching positions; red shows 17-aa
sequence resulting from a frameshift in the ORF7a mutant. Beta strands (arrows) and alpha helices (coil) are shown above the alignment.
(B) Diagram of SARS-CoV-2 ORF7a Ig-like fold. Disulfide bonds that stabilize the bsandwich structure are shown with red lines. The portion of the protein
eliminated by the deletion is shown in gray.
(C) C-terminally FLAG-tagged ORF7a
WT
and ORF7a
D115
were cloned and overexpressed in HEK293T-hACE2. Protein expression was confirmed with western
blot using anti-FLAG antibody. b-Actin (ACTB) was used as a loading control.
(D and E) FLAG-tagged ORF7a
WT
(D) or ORF7a
D115
(E) expressed in HEK293T-hACE2 cells. Immunostaining was performed using an anti-FLAG antibody (green).
Cell nuclei were stained with Hoechst 33342 (blue). White scale bar is 10 mm.
(F) HEK293T cells with integrated ISRE-luciferase reporter were transfected with pLV-mCherry (‘‘-’’), ORF7a
WT
, or ORF7a
D115
plasmids. Transfected cells were
treated with 5 ng/mL human recombinant IFN-a2b for 24 h, and induction of type I IFN signaling was measured with luciferase assay. Fold induction versus non-
treated control was determined and normalized to mCherry control. Means (n = 6) were compared with one-way ANOVA (p = 0.00162). Pairwise comparisons
were performed using post hoc Tukey’s test. Data are shown as mean ±SD. **p < 0.01;
ns
p > 0.05.
(G) ORF7a and ORF7b sgRNAs were identified by RT-PCR. Lower molecular weight band corresponding to ORF7b sgRNA is indicated with asterisk (*). Diagram
on the right shows primer (arrows) positions. Specificity of PCR products was confirmed with Sanger sequencing. GAPDH was used as a control for cDNA
synthesis.
(H) Lysates from SARS-CoV-2-infected cells were probed with antibodies raised against ORF7b protein. ACTB was used as a loading control.
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RNA in the host cell differs from the corresponding decrease in
the supernatant RNA at 24 hpi (4.3-fold versus 16.8-fold,
respectively; Figures 3B and 3C). This disparity suggests that
observed growth defect in the SARS-CoV-2 D115 variant might
result from defects in genomic RNA replication and transcrip-
tion, as well as in viral egress.
Figure 3. The ORF7a
D115
mutation results in replication and immune suppression defects
(A and B) VeroE6 (A) or HEK293T-hACE2 (B) cells were infected with ORF7a
WT
or ORF7a
D115
SARS-CoV-2 strain at MOI = 0.05. Viral RNA was measured in
supernatant using qRT-PCR at different time points after infection. Measured RNA levels were normalized (using DCt method) to RNA levels at 0.5 hpi.
(C) Total RNA was extracted from infected HEK293T-hACE2 cells, and viral RNA was measured. The DDCt method was used to normalize viral RNA levels to host
RNA (ACTB) and 0.5 hpi time point. Data in (A)–(C) are presented as mean ±SD of three biolo gical replicates. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001,
or
ns
p > 0.05 (no significant difference).
(D) Volcano plot showing IFN-I response in 293T-hACE2 cells infected with ORF7a
WT
SARS-CoV-2 strain at MOI = 0.05. Expression of IFN-I response genes was
studied 24 hpi using qRT-PCR array targeting 91 human transcripts (88 targets and 3 references). Experiment was performe d in three biological replicates.
Dashed lines show regulation (R2-fold) and statistical significance (p < 0.05) thresholds. Each dot represents mean (n = 3) normalized expression of a single gene
relative to non-infected host. Genes that passed the threshold are labele d.
(E) Volcano plot showing IFN-I response in ORF7a
D115
versus ORF7a
WT
infection.
(F) Data shown in (D) and Figure S3A were plotted as a heatmap. Genes were classified into three groups. Group #1 genes are oppositely regulated between
the two viral strains. Group #2 genes are upregulated by both ORF7a
WT
and ORF7a
D115
. Group #3 genes are downregulated in both. Genes that have statistically
significant difference in expression between two viral strains are marked with asterisk. Gray shows genes with no detectable expression over 40 cyclesof
qRT-PCR.
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ORF7a
D115
virus induces elevated IFN-I response in vitro
Suppressed or delayed IFN responses characterize SARS-CoV-
2 infections (Lei et al., 2020). The C-terminal truncation of ORF7a
limits suppression of the type I IFN signaling (Figure 2F). There-
fore, we hypothesized that ORF7a
D115
virus isolated from the
community is deficient in antagonizing IFN response.
To test this hypothesis, we measured expression of type I IFN
response genes upon infection with ORF7a
WT
and ORF7a
D115
strains using a qRT-PCR array targeting 91 human transcripts
(Table S2). The array included three reference genes that we
used to normalize expression of 88 target genes. In agreement
with recent transcriptomic studies, we detected a type I IFN-
mediated antiviral response after infecting HEK293T-hACE2
with SARS-CoV-2 isolates (Figure 3D; Figure S3B) (Banerjee
et al., 2020;Blanco-Melo et al., 2020). Out of 88 type I IFN
response genes included in the qRT-PCR array, 42 were differ-
entially expressed (R2-fold change, p < 0.05) in ORF7a
WT
-in-
fected cells and 55 in ORF7a
D115
infection, as compared with
the non-infected control (Figure 3D; Figure S3B). Both viruses
stimulated expression of multiple type I interferon genes, antiviral
ISGs, ILs, and proinflammatory cytokines (Table S2).
To understand the effect of ORF7a
D115
on the host type I IFN
response, we compared expression of genes targeted with
qRT-PCR array between the two viruses. In general, the ORF7a
-
D
115 variant induces elevated type I IFN response to the infec-
tion (Figures S3C and S3D). The median expression fold change
of 88 genes targeted in the qRT-PCR array was 1.72 in ORF7a
WT
and 2.51 in ORF7a
D115
virus (p = 1.4E12; Figure S3C). Analysis
of genes differentially expressed between the two viral infections
results in an asymmetric volcano plot (Figure 3E). A subset of
ISGs had expression levels that are significantly different (p <
0.05) between the two viral infections (Figures 3E and 3F; Table
S2). This subset includes sensors (TLR7), signal transducers
(MYD88, OAS2), transcriptional regulators (IRF3,IRF5), and re-
striction factors (GBP1,IFITM3,MX1) known to combat infec-
tions by RNA viruses (Schoggins, 2019). One of these differen-
tially expressed ISGs (IFITM3) restricts SARS-CoV-2 entry (Shi
et al., 2021;Zang et al., 2020). Additionally, polymorphisms in
IFITM3,MX1, and TLR7 are linked to the severity of COVID-19
(Andolfo et al., 2020;Zhang et al., 2020a;Pati et al., 2021). Upre-
gulation of ISGs indicates that the ORF7a
D115
virus is inept at
antagonizing the IFN-I response to the infection, which is consis-
tent with results from our ISRE reporter assay (Figure 2F).
DISCUSSION
In this study, we use genomic surveillance and phylogenetics to
identify ORF7a variants that have emerged globally throughout
the SARS-CoV-2 pandemic. Local occurrence of ORF7a muta-
tions has been reported by others (Addetia et al., 2020;Holland
et al., 2020;Rosenthal et al., 2020). However, the effect of these
mutations on viral fitness and host immune responses has not
been investigated. Here, we isolated a virus with a deletion mu-
tation in ORF7a (ORF7a
D115
) and show that this genotype results
in an in vitro growth defect. This growth defect is associated with
elevated IFN response to SARS-CoV-2.
Interferon systems are a frontline of host defense that signals
infection and elicits an antiviral state (McNab et al., 2015). Pre-
treatment with type I, type II, and type III IFNs restricts SARS-
CoV-2 infection and replication in cell culture (Mantlo et al.,
2020;Miorin et al., 2020). SARS-CoV-2 deploys multiple proteins
(e.g., nsp6, nsp13, ORF6, ORF7a, ORF7b, and ORF9b) to shut
down IFN signaling and dampen innate immune responses
(Jiang et al., 2020;Lei et al., 2020;Xia et al., 2020), in particular,
when overexpressed ORF7a subverts STAT2 phosphorylation,
blocking IFN-dependent transcriptional activation of antiviral
ISGs (Xia et al., 2020). Both type I and III IFNs signal through
the JAK-STAT pathway and overlap substantially in the tran-
scriptional responses they induce (Kotenko and Durbin, 2017).
Therefore, ORF7a likely suppresses both IFN types; however,
its role in suppressing the type III IFN response has yet to be veri-
fied (Xia et al., 2020). Using the ISRE reporter system, we show
that C-terminal truncation negates the ability of ORF7a to sup-
press IFN signaling (Figure 2). The ORF7a
D115
variant shows
limited replication in HEK293T-hACE2, as well as in Vero E6,
the latter of which does not express type I IFN genes (Emeny
and Morgan, 1979;Osada et al., 2014). Although type I IFN genes
are deleted in Vero E6 cells, the type III IFNs are intact and are
activated upon infection with RNA viruses, which results in ISG
upregulation (Stoltz and Klingstro
¨m, 2010;Wang et al., 2020).
ISGs exert anti-viral activities that combat the infection (Schog-
gins, 2019). We hypothesize that the replication defect of the
ORF7a
D115
strain in Vero E6 and 293T-hACE2 cells results
from an IFN-dependent response that the virus fails to suppress.
Several groups have replaced ORF7a with a reporter gene
(Hou et al., 2020;Thi Nhu Thao et al., 2020;Xie et al., 2020).
Thi Nhu Thao et al. (2020) show that complete deletion of
ORF7a reduces replication of the synthetic virus, which
agrees well with growth defect in the ORF7a
D115
strain.
However, two other studies have not observed this effect.
We believe that our results with a naturally occurring
ORF7a deletion provide valuable insight to this discussion.
Finally, it should be noted that different mutations (i.e., com-
plete deletion versus partial) might have a different outcome
for the viral phenotype.
Cytokine profiling and RNA-seq of clinical samples reveal sup-
pressed or delayed IFN responses in COVID-19 patients (Aruna-
chalam et al., 2020;Hadjadj et al., 2020;Lucas et al., 2020). Dys-
regulated immune responses coupled with exacerbated
inflammatory cytokines drive pathogenesis of the disease and
correlate with its severity (Zhang et al., 2020b;Tay et al., 2020;
Zhou et al., 2020). Here, we detected enhanced IFN signaling
in cells infected with a naturally occurring ORF7a
D115
variant of
SARS-CoV-2.
Given the importance of the IFN response in COVID-19, we
anticipate that ORF7a mutations might affect SARS-CoV-2 path-
ogenicity. Clinical comparisons will be required to examine this
potential effect on COVID-19 features.
Deletions in ORF7a, ORF7b, and ORF8 have previously been
reported (Addetia et al., 2020;Holland et al., 2020;Rosenthal
et al., 2020;Su et al., 2020). Similar deletions emerged in Middle
East Respiratory Syndrome (MERS)- and SARS-CoV, which
were linked to viral attenuation (Chinese SARS Molecular Epide-
miology Consortium, 2004;Muth et al., 2018). A 382-nt deletion
in ORF8 is associated with milder SARS-CoV-2 infection in pa-
tients (Young et al., 2020). Although this mutation affects clinical
Please cite this article in press as: Nemudryi et al., SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits
immune suppression, Cell Reports (2021), https://doi.org/10.1016/j.celrep.2021.109197
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features of COVID-19, it has no evident effect on viral replication
or host transcriptional responses in human cell culture (Gamage
et al., 2020).
Several studies illustrate how persistent SARS-CoV-2 infec-
tions in immunocompromised hosts associate with accelerated
viral evolution (Avanzato et al., 2020;Choi et al., 2020). In these
hosts there is less selective pressure on viral anti-immune pro-
teins, which might permit loss-of-function mutations in accessory
genes. The ORF7a
D115
variant that we isolatedwas present in only
seven genomes over a course of 1.5 months in a local commu-
nity and then disappeared. We hypothesize that deletions in
accessory genes, such as ORF7a
D115
, might emerge in immuno-
compromised patients but do not persist in the immunocompe-
tent population. Similarly, a deletion mutation in ORF8 (i.e.,
D382), which was associated with milder COVID-19 disease, dis-
appeared soon after it was discovered (Young et al., 2020). How-
ever, founder effect or mutations that co-occur with other muta-
tions that increase transmissibility (e.g., spike mutations) can
result in fixation of an otherwise attenuating mutation. A new
more transmissible lineage of SARS-CoV-2 (i.e., B.1.1.7) has
recently emerged (Davies et al., 2021;Rambaut et al., 2020b).
Aside from changes in the Spike protein, this lineage also includes
a premature stop codon in the accessory ORF8 that truncates
most of the protein (Volz et al., 2021). This lineage might illustrate
the second scenario for fixing mutations in accessory genes.
Limitations of study
The viral replication defect and cellular immune response assay
presented here were performed using HEK293T-hACE2 and
Vero E6 cells that are not the natural targets of SARS-CoV-2.
However, both cell lines are permissive for SARS-CoV-2 infec-
tion and are used extensively to study efficacy of antiviral com-
pounds (Riva et al., 2020), virion assembly (Klein et al., 2020),
SARS-CoV-2 entry (Hoffmann et al., 2020), replication (Thi Nhu
Thao et al., 2020), and anti-immune activities of SARS-CoV-2
proteins (Xia et al., 2020). Although we acknowledge that our
conclusions are limited to Vero E6 and 293T-hACE2 cells, we
anticipate that the results presented here will provide important
context for clinical comparisons, similar to those recently pub-
lished for ORF8 (Young et al., 2020).
Studies of naturally occurring SARS-CoV-2 variants have limita-
tions, including unavailability of isogenic controls. Here, we
compared two viral isolates, ORF7a
WT
and ORF7a
D115
,thatin
addition to D115 differ at several other nucleotide positions (Fig-
ure S3A). Although ISRE reporter assay unambiguously links the
immunosuppression defect to the truncation of ORF7a, it is not
possible to rule out a role for these mutations on the viral life cycle.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
dKEY RESOURCES TABLE
dRESOURCE AVAILABILITY
BLead contact
BMaterials availability
BData and code availability
dEXPERIMENTAL MODEL AND SUBJECT DETAILS
BCell cultures
BPlasmids
BAntibodies
BHuman clinical sample collection and preparation
BProduction and titration of coronavirus stocks
BSymptom onset data and clinical test results
dMETHOD DETAILS
BQuantitative reverse transcription PCR (qRT-PCR)
BRT-PCR and SARS-CoV-2 genome sequencing
BSARS-CoV-2 genome assembly
BRT-PCR and Sanger sequencing
BPhylogenetic and ORF7a mutational analysis
BWestern blot
BImmunocytochemistry
BIFN inhibition assay
BSARS-CoV-2 replication assays
BType I interferon response assay
dQUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
celrep.2021.109197.
ACKNOWLEDGMENTS
We are grateful to members of Bozeman Health that provided de-identified pa-
tient samples, specifically C. Nero, D. Smoot, W. Wallace, C. Faurot-Daniels,
V. Lawrence, and M. Blauvelt. We are also grateful to M. Flenniken, K. Daugh-
enbaugh, and other members of the COVID task force at MSU for assistanc e
establishing the COVID testing center. We thank the Lefcort lab for generous
use of their fluorescent confocal microscope and Dr. Pincus for providing
the HEK 293T-hACE2 cells. Research in the Wiedenheft lab is supported by
the National Institutes of Health (1R35GM134867), the Montana State Univer-
sity Agricultural Experimental Station, the MJ Murdock Charitable Trust, the
Gianforte Foundation, and the MSU Office of the Vice President for Research.
We thank the GISAID’s EpiFlu Database and contributing laboratories (Table
S4). The phylogenetic analysis in this paper would not have been possible
without their willingness to share data. The graphical abstract was created
with BioRender.
AUTHOR CONTRIBUTIONS
Conceptualization, B.W., A. Nemudryi, and A. Nemudraia; methodology, B.W.,
A. Nemudraia, A. Nemudryi, D.T.S., and J.F.H.; sample acquisition, D.B. and
B.W.; investigation & data collection, A. Nemudraia, A. Nemudryi, D.T.S.,
J.F.H., J.N., H.L., and K.K.V.; genomics and bioinformatics analysis, T.W., A.
Nemudryi, and C.C.; writing – original draft, B.W., A. Nemudryi, A. Nemudraia,
and T.W.; writing – review & editing, B.W., A. Nemudryi, A. Nemudraia, T.W.,
and M.A.J.
DECLARATION OF INTERESTS
B.W. is the founder of SurGene LLC and VIRIS Detection Systems Inc. B.W., A.
Nemudryi, and A. Nemudraia are inventors on patents related to CRISPR-Cas
systems and applications thereof.
Received: February 3, 2021
Revised: April 4, 2021
Accepted: May 10, 2021
Published: June 1, 2021
Please cite this article in press as: Nemudryi et al., SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits
immune suppression, Cell Reports (2021), https://doi.org/10.1016/j.celrep.2021.109197
Cell Reports 35, 109197, June 1, 2021 7
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Biological samples
Nasopharyngeal swabs Bozeman Health Deaconess
Hospital, MT, USA
N/A
Bacterial and virus strains
SARS-CoV-2 Isolate USA-WA1/2020 BEI Resources NR-52281
Experimental models: cell lines
HEK293T-hACE2 cells BEI Resources NR-52511
Vero E6 ATCC Cat#CRL-1586
Recombinant DNA
pEGFP-N1 Clontech 6085-1
pLV-mCherry Addgene 36084
pORF7a-CoV2 This study N/A
pORF7a D115-Cov2 This study N/A
pGreenFire1-ISRE SBI TR016PA-1
Antibodies
Rabbit anti-ACTB ABclonal Cat. AC026, RRID:AB_2768234
Mouse anti-FLAG ThermoFisher Scientific Cat: MA1-91878-1MG, RRID:AB_2537619
Sheep anti-ORF7b MRC I PPU, College of Life
Sciences, University of Dundee
N/A
Goat anti-mouse IgG peroxidase-conjugated Jackson ImmunoResearch Cat: 115-035-003, RRID:AB_10015289
Goat anti-rabbit IgG peroxidase-conjugated Jackson ImmunoResearch Cat: 111-035-003, RRID:AB_2313567
Donkey Anti-Sheep IgG peroxidase-conjugated Jackson ImmunoResearch Cat: 713-035-147, RRID:AB_2340710
Mouse anti-FLAG Invitrogen Cat: MA1-91878-1MG, RRID:AB_2537619
Goat anti-mouse-AlexaFluor-488 Invitrogen Cat: A11001, RRID:AB_2534069
Chemicals, peptides, and recombinant proteins
QIAamp viral RNA mini kit QIAGEN 52904
2019-nCoV RUO kit IDT 10006713
Positive template control (PTC) plasmid IDT 10006625
TaqPath 1-Step RT-qPCR master mix Thermo Fisher Scientific A15300
SuperScript IV reverse transcriptase Thermo Fisher Scientific 18090010
R9.4.1 flow cells Nanopore Technologies FLO-MIN106
AMX, LNB, SFB, EB and SQB Nanopore Technologies SQK-LSK109
Flow cell priming kit Nanopore Technologies EXP-FLP002
NEBNext Ultra II end-prep New England Biolabs E7546S
NEBNext quick ligation module New England Biolabs E6056S
Native barcoding expansion kits Nanopore Technologies EXP-NBD104, EXP-NBD114
Q5 high-fidelity DNA polymerase New England Biolabs M0491S
DNA clean & concentrator kit Zymo Research D4005
Lipofectamine 3000 Invitrogen L3000-015
Pierce ECL western blotting substrate ThermoFisher Scientific #32106
X-Ray film Santa Cruz Biotech sc-201696
IFN-a2b InvivoGen rcyc-hifna2b
Luciferase assay system Promega E1500
(Continued on next page)
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RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Blake Wie-
denheft (bwiedenheft@gmail.com).
Materials availability
Plasmids generated in this study are available upon request from the lead contact.
Data and code availability
The complete SARS-CoV-2 genome sequences are deposited to GISAID EpiCoV and GenBank databases. GISAID accession IDs
and URLs to GenBank records are provided in Table S1. Raw data for IFN response assay presented in Figures 3 and S3 is available
in Table S2.
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Type I interferon response (SAB Target List)
H96 qPCR array
Bio-Rad N/A
SsoAdvanced universal SYBR green supermix Bio-Rad 1725270
Deposited data
URLs for SARs-CoV-2 genome sequences
are listed in the Table S1
GenBank N/A
Oligonucleotides
The oligonucleotides used in this study are
listed in the Table S3
IDT N/A
Software and algorithms
SDS software v1.4 Applied Biosystems 4379633
RStudio v1.2.1335 The R project https://www.r-project.org/
artic-ncov2019 ARTIC network https://artic.network/ncov-2019
MinKNOW software Oxford Nanopore
Technologies
https://community.nanoporetech.com/
protocols/experiment-companion-minknow/
v/mke_1013_v1_revbm_11apr2016
PrimePCR analysis software Bio-Rad https://www.bio-rad.com/en-us/category/qpcr-
analysis-software?ID=42a6560b-3ad7-43e9-
bb8d-6027371de67a
CD-HIT GitHub https://github.com/weizhongli/cdhit
BioStrings Bioconductor https://bioconductor.org/packages/release/bioc/
html/Biostrings.html
ggplot2 CRAN https://cran.r-project.org/web/packages/ggplot2/
index.html
Augur Nextstrain https://github.com/nextstrain/augur
Nextclade Nextstrain https://clades.nextstrain.org/
pangolin Github https://github.com/cov-lineages/pangolin
Trimal Github https://github.com/scapella/trimal
IQ-Tree IQ-Tree http://www.iqtree.org/
TreeTime GitHub https://github.com/jkanev/treetime
APE v5.3 CRAN https://cran.r-project.org/web/packages/ape/
index.html
tidyquant CRAN https://cran.r-project.org/web/packages/
tidyquant/index.html
stats CRAN https://stat.ethz.ch/R-manual/R-devel/library/
stats/html/00Index.html
UGENE Unipro http://ugene.net/
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EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell cultures
HEK293T-hACE2 cells (BEI, NR-52511) were generously provided by Dr. Seth Pincus (Montana State University) and maintained at
37C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO, cat. #12100-061) supplemented with 10% fetal bovine
serum (FBS, ATLAS Biologicals, Lot. #F31E18D1), sodium bicarbonate (3.7 g/L), 50 I.U./mL penicillin and 50 mg/mL streptomycin.
Vero E6 cells (ATCCcat. #CRL-1586) were maintained at 37C and 5% CO2 in Eagle’s Modified Eagle Medium (EMEM,
ATCC, cat. #30-2003) supplemented with 10% fetal bovine serum (FBS, ATLAS Biologicals, Lot. #F31E18D1) and 50 I.U./mL
penicillin and 50 mg/mL streptomycin. All cell lines tested negative for mycoplasma.
Plasmids
Human-codon optimized dsDNA gene fragments (gBlocks) encoding for SARS-CoV-2 C-terminally FLAG-tagged ORF7aWT and
ORF7aD115 proteins were synthesized by Integrated DNA Technologies (IDT). Sequence of ORF7a from Wuhan-Hu-1 reference
(GenBank MN908947.3) was used for ORF7aWT gBlock. The gene fragments were cloned into the pEGFP-N1 (Clontech cat#
6085-1) backbone to replace the eGFP gene. pLV-mCherry was a gift from Pantelis Tsoulfas (Addgene plasmid # 36084; http://
www.addgene.org/36084/; RRID:Addgene_36084).
Antibodies
Western blotting: Rabbit anti-ACTB (Cat: AC026, 1:20,000) antibodies were from ABclonal, Mouse anti-FLAG antibodies (Cat:
MA1-91878-1MG, 1:1000) were from ThermoFisher Scientific, sheep anti-ORF7b antibodies (1:120) were from MRC I PPU, Col-
lege of Life Sciences, University of Dundee, Scotland; Goat anti-mouse IgG peroxidase-conjugated (Cat: 115-035-003,
1:10,000), Goat anti-rabbit IgG peroxidase-conjugated (Cat: 111-035-003, 1:10,000) and Donkey Anti-Sheep IgG peroxidase-
conjugated (Cat: 713-035-147, 1:10,000) antibodies were from Jackson ImmunoResearch. Immunocytochemistry: Mouse
anti-FLAG antibodies (Cat: MA1-91878-1MG, 1:200), Goat anti-mouse-AlexaFluor-488 (Cat: A11001, 1:2,000) antibodies
were from Invitrogen.
Human clinical sample collection and preparation
Clinical samples were obtained with local IRB approval (protocol #DB033020) and informed consent from patients undergoing testing
for SARS-CoV-2 at Bozeman Health Deaconess Hospital. Patients tested for SARS-CoV-2 included both in-patients and out-pa-
tients. The latter included individuals who developed symptoms and sought medical care and asymptomatic (at the time of testing)
individuals who were exposed to known COVID-19 case and therefore were tested. Nasopharyngeal swabs from patients that tested
positive for SARS-CoV-2 were collected in viral transport media. RNA was extracted from all patient samples using QIAamp Viral RNA
Mini Kit (QIAGEN) a biosafety level 3 (BSL3) laboratory. All samples were heat-inactivated before removing from BSL3. Information
about age and sex of the patients is provided in Table S1.
Production and titration of coronavirus stocks
The nasopharyngeal swabs in the viral transport media were used to generate viral stocks as previously described (Harcourt et al.,
2020). Briefly, 100 ul of the viral transport media was two-fold serially diluted and applied to Vero E6 cells When infected cells
showed extensive cytopathic effect, the media was collected and utilized to generate a greater volume second passage in
Vero E6 cells. When CPE was apparent, supernatants were centrifuged 1,000 RCF for 5 minutes to remove cellular debris. Virus
titer in clarified supernatants was determined with plaque assay in Vero E6 cells as described (Loveday et al., 2021). For plaque
assay, Vero E6 cells were incubated with viral inoculum at limiting dilutions. Inoculated cells were overlayed with either 0.75%
methylcellulose, DMEM supplemented with 2% FBS and 1% pen-strep and incubated for 4 days. Cells were fixed and stained
with 0.5% methylene blue in 70% ethanol. Plaques were counted and the overall titer was calculated. Viral stock’s identity
was confirmed via whole genome sequencing on Oxford Nanopore. All SARS-CoV-2 experiments were performed in a BSL3
laboratory.
Symptom onset data and clinical test results
Suspect cases of COVID-19 were tested in a CLIA lab and instructed to self-quarantine until notified of the RT-qPCR test results. All
laboratory confirmed positive cases of COVID-19 were contacted via telephone by local public health nurses to complete contact
tracing. During this interview, the nurses collected recorded symptoms, symptom onset date, travel history, contact with other known
laboratory confirmed cases, close contacts and activities on the two days before symptom onset up until notification of a positive
test. Data collection was conducted as part of a public health response. The study was reviewed by the Montana State University
Institutional Review Board (IRB) For the Protection of Human Subjects (FWA 00000165) and was exempt from IRB oversight in accor-
dance with Code of Federal regulations, Part 46, section 101. All necessary patient/participant consent has been obtained and the
appropriate institutional forms have been archived.
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METHOD DETAILS
Quantitative reverse transcription PCR (qRT-PCR)
qRT-PCR was performed using CDC primers (N1 and N2) and probes from the 2019-nCoV RUO Kit (IDT# 10006713). SARS-CoV-2
RNA was quantified using one-step qRT-PCR in ABI 7500 Fast Real-Time PCR System according to CDC protocol (https://www.fda.
gov/media/134922/download). In brief, 20 mL reactions included 8.5 mL of Nuclease-free Water, 1.5 mL of Primer and Probe mix (IDT,
10006713), 5 mL of TaqPath 1-Step RT-qPCR Master Mix (ThermoFisher, A15299) and 5 mL of the template. Nuclease-free water was
used as negative template control (NTC). Amplification was performed using following program: 25C for 2 min, 50C for 15 min, 95C
for 2 min followed by 45 cycles of 95C for 3 s and 55C for 30 s. Run data was analyzed in SDS software v1.4 (Applied Biosystems).
The NTC showed no amplification throughout the 40 cycles of qPCR.
RT-PCR and SARS-CoV-2 genome sequencing
For sequencing 10 mL of RNA from SARS-CoV-2 positive patient sample was reverse transcribed using SuperScript IV (Thermo Fisher
Scientific) according to the supplier’s protocol. The protocol developed by ARTIC Network was used to generate and sequence am-
plicon library that covers whole SARS-CoV-2 genome on Oxford Nanopore using ligation sequencing kit (SQK-LSK109) (https://artic.
network/ncov-2019)(Grubaugh et al., 2019;Tyson et al., 2020). Briefly, two multiplex PCR reactions were performed with primer
pools from ARTIC nCoV-2019 V3 Panel (IDT; Table S3) using Q5 High-Fidelity DNA Polymerase (New England Biolabs). Reactions
were performed with the following thermocycling conditions: 98C for 2min, 30 cycles of 98C for 15 s and 65C for 5 min. Two re-
sulting amplicon pools for each patient sample were combined and used for library preparation. After end repair (NEB E7546) sam-
ples were barcoded using Native Barcoding Expansion Kits EXP-NBD104 and EXP-NBD114 from Oxford Nanopore. A total of 24
barcoded samples were pooled together and Nanopore adaptors were ligated. After clean-up 20 ng of multiplexed library DNA
was loaded onto the MinION flowcell for sequencing. A total of 0.3 Gb of raw sequencing data was collected per patient sample.
Nuclease-free water was used as a negative control for library preparation and sequencing. Non-specific amplification in negative
control was additionally checked using an agarose gel. SARS-CoV-2 Isolate USA-WA1/2020 (BEI, NR-52281) was used as a positive
control for genome sequencing.
SARS-CoV-2 genome assembly
MinKNOW software was used to basecall raw Nanopore reads in high-accuracy mode. ARTIC bioinformatic pipeline for COVID-19
was used to analyze reads (https://artic.network/ncov-2019). Pipeline included demultiplexing with guppy barcoder and generating
consensus sequences with minimap2 and calling single nucleotide variants with nanopolish relative to Wuhan-Hu-1/2019 reference
genome (Li, 2018;Quick et al., 2017;Wu et al., 2020). 20X coverage was used as a threshold both for consensus and variant calling.
Nucleotide positions with less than 20X coverage were masked with Ns in the final consensuses. Consensus sequences were up-
loaded to GISAID (https://www.gisaid.org/), accession IDs are provided in Table S1.
RT-PCR and Sanger sequencing
To verify the ORF7aD115 mutation we used RT-PCR with primers that flank the deletion region (Table S3). Reactions were performed
with Q5 High-Fidelity DNA Polymerase in 25 mL volume (New England Biolabs) using following program: 98C for 2min, 35 cycles of
98C for 15 s and 65C for 5 min, 35 cycles. PCR products were analyzed on 1% agarose gels stained with SYBR Safe (Thermo Fisher
Scientific). The remaining volume of the reaction was purified using DNA Clean & Concentrator kit (Zymo Research) and sent to Pso-
magen for Sanger sequencing. Each PCR product was sequenced with both forward and reverse primers used for PCR. To detect
sgRNAs of ORF7a and ORF7b primers annealing to Leader region of SARS-CoV-2 and inside the ORF were used (Table S3). PCR
products were cut out from 1% agarose gel and purified with Zymoclean Gel DNA Recovery Kit. Purified products were sequenced
with forward and reverse primers. Sequence was verified by aligning to SARS-CoV-2 genome in UGENE software (Unipro).
Phylogenetic and ORF7a mutational analysis
An alignment of 181,003 SARS-CoV-2 genomes was downloaded from the GISAID database at 9:11 AM on 2020-11-11, and
sequences without corresponding metadata entries were removed. The nucleotide positions encoding ORF7a in the reference
sequence (Wuhan-Hu-1; EP_ISL_402125) were extracted for the remaining 180,971 sequences, and ORF7a sequences with
100% sequence identity were clustered using CD-HIT (with settings: -c 1 -aL 1). One representative was randomly selected from
each of the 846 ORF7a sequence clusters and mutations in each nucleotide and translated sequence were determined using the
Biostrings package in R. Graphs of these mutations were rendered with the ggplot2 package. To construct a phylogenetic tree of
global and Bozeman SARS-CoV-2 sequences, up to ten sequences from each country in the world were selected for each month
of the pandemic using the Filter utility in the Augur pipeline (–group-by country year month–sequences-per-group 10) (Bedford
et al., 2020;Lemieux et al., 2021). This resulted in an alignment of 4,235 GISAID sequences. Then, the Nextclade online tool was
used to determine the quality of 73 SARS-CoV-2 genomes (% genome covered) sequenced from Bozeman patients. 55 Bozeman
sequences that were determined to be of ‘‘Good’’ quality were merged with the alignment of GISAID sequences, as well as an align-
ment of 669 representative SARS-CoV-2 genomes that had non-synonymous mutations in ORF7a. The bat coronavirus RaTG13
(MN996532.2) and the Wuhan- Hu-1 SARS-CoV-2 sequences were also merged, resulting in an alignment of 4,959 full genome
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sequences, and positions that are hypervariable or prone to sequencing artifacts were masked with a VCF file downloaded from
EMBL at 12:45PM on 2020-05- 29. The alignment was trimmed of columns composed of 90% or more gaps using Trimal (-gt
0.9), and IqTree was used to construct a maximum likelihood phylogeny (-m GTR -ninit 2 -n 2 -me 0.05). Branch lengths and internal
nodes were rescaled on the resulting tree using TreeTime, and tree was re-rooted to the RaTG13 sequence using the APE package
before being visualized with the ggTree package in R. Clades were assigned to SARS-CoV-2 genomes using Nextclade and pangolin
(https://cov-lineages.org/) utilities.
Western blot
293T-hACE2 cells were transfected using Lipofectamine 3000 (Invitrogen, L3000-015) with plasmids encoding for ORF7aWT,
ORF7aD115 or control pEGFP-N1 plasmid. To detect ORF7b expression cells were infected with ORF7aWT or ORF7aD115
SARS-CoV-2 strains at MOI = 0.05. After 24 hours, cells were washed two times with PBS and lysed in RIPA buffer (150 mM sodium
chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.2% SDS, 50 mM Tris, pH 8.0) at 4C for 30 min. The lysates were clarified by
centrifugation (10’000 g, 20 min) and stored at 80C. For western blot lysates were mixed with 6xLaemmli SDS-PAGE buffer
and heated at 98C for 5 min, resolved in 12% SDS-PAGE gel and transferred onto a PVDF (ORF7b) or nitrocellulose (ORF7a) mem-
brane using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, #1703930). Membranes were blocked and probed with indicated
antibodies. The proteins were visualized using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, #32106) and
exposed to X-Ray film (sc-201696, Santa Cruz Biotech).
Immunocytochemistry
HEK293T-hACE2 were transfected with plasmid DNA using Lipofectamine 3000 (Invitrogen REF: L3000-015) according to manufac-
turer’s instructions. Cells were either transfected with expression vectors for SARS-CoV-2 ORF7aWT, SARS-CoV-2 ORF7aD115, or
mock transfected. 24 hours after transfection, cells were detached and seeded at 50% confluency on glass coverslips (Fisherbrand,
#12-545-80) pre-coated with poly-D-lysine (Cultrex, #3439-100-01) in a 24 well plate. The next day, media was removed, and cells
were washed with PBS and fixed with ice-cold 100% methanol on ice for 10 minutes. Half of the methanol was replaced with PBS
three times before the methanol solution was discarded and cells were washed three times in PBS (5 minutes each wash). Cells were
blocked with 1% BSA (Fisher Scientific BP9703-100) in PBS + 0.1% Tween-20 (PBST) for 30 minutes at room temperature. After
blocking, cells were stained with mouse monoclonal anti-Flag antibody (Invitrogen, MA1-91878) (1:200 in 1% BSA in PBST) overnight
at 4C. The next day, cells were washed 3X in PBS (5 min each) and stained with secondary antibody diluted 1:2000 in 1% BSA in
PBST for 1 h at RT in the dark (Goat anti-mouse AlexaFluor488; Invitrogen, A11001). After 3x PBS washes nuclei were stained with
Hoechst 33342 in PBS for 5 minutes at RT. Coverslips were then mounted with ProLong Gold antifade mounting media (Invitrogen,
P36934) on Superfrost Plus microscope slides (#22-037-246). Immunostained cells were imaged using a Leica SP8 confocal
microscope.
IFN inhibition assay
To generate ISRE reporter system, HEK293T cells were transduced with lentiviruses carrying pGreenFire1-ISRE (Cat. TR016PA-1,
SBI) construct and selected with puromycin (1 ug/ml) for 2 days. After selection cells (1x105 cells/well, 48-well plate) were transfected
with expression plasmids (250 ng) for SARS-CoV-2 ORF7aWT, SARS-CoV-2 ORF7aD115, or control plasmid pLV-mCherry. 16 hours
post transfection, cells were treated for 24 h with 5 ng/ml of human IFN-a2b (Cat. rcyc-hifna2b, InvivoGen) according to manufac-
turer’s recommendations. After treatment activation of IFN signaling was measured using Luciferase Assay System (Cat. E1500,
Promega) on Cytation 5 (BioTek) plate reader. Assay was performed in six replicates for each condition.
SARS-CoV-2 replication assays
HEK293T-hACE2 and Vero E6 cells were seeded in the 48-well plate and infected with ORF7aWT or ORF7aD115 SARS-CoV-2
strains at MOI = 0.05 in 50 mL of the cell culture media. Infections were performed in triplicates. Next, 0.5 h post infection (hpi)
500 mL of the fresh cell culture media was added to each well. The supernatants (140 ul) from the infected cells were harvested at
the following time points 0.5, 6, 24, 48, 72 and 120 hpi. The RNAs from the supernatants were extracted using QIAamp Viral RNA
Mini Kit (QIAGEN) and used as an input in qRT-PCR with CDC N1 and N2 primers and probes as described above. Relative quan-
tification was used (DCt method) to calculate viral replication versus 0.5 hpi time point as 2-DCt.
The infected cells were harvested 0.5, 6 and 24 hpi and washed with PBS. Total RNA from cells was extracted using RNeasy kit
(QIAGEN, Cat No./ID: 74104) with on-column DNase digestion step (QIAGEN, 79254) according to the manufacturers protocol. Viral
RNA was quantified using qRT-PCR CDC N1 primers and probe. Human ACTB endogenous control was quantified with TaqMan
assay (ThermoFisher, 4333762T). DDCt method was used to normalize viral RNA level to host RNA and 0.5 hpi. Viral replication
was calculated for each replicate as 2-DDCt.
Type I interferon response assay
HEK293T-hACE2 were seeded in the 48-well plate and infected with ORF7aWT or ORF7aD115 SARS-CoV-2 strains at MOI = 0.05 in
50 ul of the media. After 30 min on infection the 500 mL of the cell culture media was added to each well. At 24 hpi the cells were
washed once with PBS, detached using Trypsin and the total RNA was extracted using RNeasy kit (QIAGEN, Cat No./ID: 74104)
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with on-column DNase digestion (QIAGEN, Cat No./ID: 79254) according to the manufacturer’s protocol. Non-infected cells were
processed using the same protocol with mock infection. After extraction, 0,5-1 mg of total RNA was reverse transcribed Reverse us-
ing SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) according to the supplier’s protocol. The cDNA was diluted 10-
fold in water and analyzed with Type I interferon response (SAB Target List) H96 qPCR array. Each reaction contained 2 mL of cDNA,
10 mL of 2X SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 8 mL of water. Reactions were performed on QuantStudio 3
Real-Time PCR System instrument with following thermocycling conditions: 95C for 2min, 40 cycles of 95C for 5 s and 60C for 30
s. After amplification, melt curve analysis was performed to examine product specificity. Assay was performed in triplicates for non-
infected cells and both viruses. Run data was analyzed in PrimePCR Analysis software (Bio-Rad). Expression of IFN-I response genes
was normalized to geometric mean of 3 reference transcripts (TBP, GAPDH, HPRT1) and non-infected control (DDCt method).
QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical analyses were performed in RStudio v1.2.1335. Moving averages (n = 7) for symptom onset and positive COVID-19 tests
data were calculated using geom_ma function from tidyquant package and plotted with ggplot2 in RStudio. qRT-PCR data is shown
as mean of three biological replicates (each with three technical replicates) ±standard deviation (SD). Data in ISRE reporter assay
was analyzed with one-way ANOVA with Tukey’s post hoc pairwise comparisons. Replication of viruses and expression of IFN-I
response genes was compared using Student’s t test. Medians of IFN-I response between two viruses were compared with Wilcoxon
signed-rank test. Significance levels in figures: * p < 0.05, ** p < 0.01, *** p < 0.001 or ns (no significant difference, p > 0.05).
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