SARS-CoV-2 sensitive to type I interferon pretreatment.

TL;DR: Evaluating type-I Interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS -CoV indicates the novel CoV is much more sensitive to IFn-I pretreatment, while the absence of open reading frame (ORF) 3b and significant changes to ORF6 suggest the two key IFN antagonists may not maintain equivalent function in SARS.

Abstract: SARS-CoV-2, a novel coronavirus (CoV), has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While genetically distinct from the original SARS-CoV, both group 2B CoVs share similar genome organization and origins to coronaviruses harbored in bats. Importantly, initial guidance has used insights from SARS-CoV infection to inform treatment and public health strategies. In this report, we evaluate type-I Interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication kinetics to SARS-CoV in Vero cell, the novel CoV is much more sensitive to IFN-I pretreatment. Examining transcriptional factor activation and interferon stimulated gene (ISG) induction, SARS-CoV-2 in the context of type I IFN induces phosphorylation of STAT1 and increased ISG proteins. In contrast, the original SARS-CoV has no evidence for STAT1 phosphorylation or ISG protein increases even in the presence of type I IFN pretreatment. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonist. The absence of open reading frame (ORF) 3b and significant changes to ORF6 suggest the two key IFN antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to the IFN-I response between SARS-CoV and SARS-CoV-2. that could help inform disease progression, treatment options, and animal model development. Importance With the ongoing outbreak of COVID-19 disease, differences between the SARS-CoV-2 and the original SARS-CoV could be leveraged to inform disease progression and eventual treatment options. In addition, these findings could have key implications for animal model development as well as further research into how SARS-CoV-2 modulates the type I IFN response early during infection. Article Summary SARS-CoV-2 has similar replication kinetics to SARS-CoV, but demonstrates significant sensitivity to type I interferon treatment.

Summary

Summary

• SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing 2 an ongoing outbreak of viral pneumonia around the world.
• 64 In this study, the authors further characterize SARS-CoV-2 and compare it to the original SARS-65 CoV.
• In contrast, the authors find that SARS-CoV-2 is 67 significantly more sensitive to IFN-I pretreatment as compared to SARS-CoV.
• These results suggest distinct changes between SARS-74 CoV and SARS-CoV-2 in terms of IFN-I antagonism and the authors subsequently examined sequence 75 homology between the SARS-CoV and SARS-CoV-2 viral proteins that may be responsible for 76 these differences.
• In contrast, SARS-CoV-2 shows a 98 significant reduction in viral replication following IFN-I treatment.
• The authors still found a reduction in STAT1 156 phosphorylation and ISG induction in IFN-I pretreated SARS-CoV versus SARS-CoV-2 157 infection.

Figures

Figure 1. SARS-CoV-2 sensitive to type I IFN pretreatment. A) Vero E6 cells were treated with 1000 U/mL recombinant type I (hashed line) IFN or mock (solid line) for 18 hours prior to infection. Cells were subsequently infected with either SARS-CoV WT (black) or SARS-CoV-2 (blue) at an MOI of 0.01 as described above. Each point on the line graph represents the group mean, N 3. All error bars represent SD. The two tailed student’s t-test was used to determine P-values: *** P < 0.001. B) Vero cell protein lysates from IFN-I treated and untreated cells were probed 48 hours post infection by Western blotting for phosphorylated STAT1 (Y701), STAT1, IFITM1, SARS spike, and Actin.

Figure 5, Conservation of SARS-CoV IFN antagonists. Viral protein sequences of the indicated viruses were aligned according to the bounds of the SARS-CoV open reading frames for each viral protein. Sequence identities were extracted from the alignments for each viral protein, and a heat map of percent sequence identity was constructed using EvolView (www.evolgenius.info/evolview) with SARS-CoV as the reference sequence. TR = truncated

Figure 4, SARS-CoV-2 impacted by post IFN-I treatment. A-B) Vero E6 and Calu3 2B4 cells were infected with either SARS-CoV WT (black) or SARS-CoV-2 (blue) at an MOI 0.01 (A, Vero cells) or MOI 1 (B, Calu3 cells). Cells were subsequently treated with 1000 U/mL recombinant type I IFN (hashed line) or mock (solid line) for 4 hours following infection. Each point on the line

Figure 2. SARS-CoV-2 attenuated and IFN-I sensitive in Calu3 respiratory cells. A) Calu3 2B4 cells were treated with 1000 U/mL recombinant type I (hashed line) IFN or mock (solid line) for 18 hours prior to infection. Cells were subsequently infected with either SARS-CoV WT (black) or SARS-CoV-2 (blue) at an MOI of 1. Each point on the line graph represents the group mean, N 3. All error bars represent SD. The two tailed student’s t-test was used to determine P-values: *** P < 0.001. B) Calu3 cell protein lysates from IFN-I treated and untreated cells were probed 48 hours post infection by Western blotting for phosphorylated STAT1 (Y701), STAT1, IFITM1, SARS spike, and Actin.

Full text

Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV.
Kumari G. Lokugamage
1*
, Adam Hage
1*
, Maren de Vries
3
, Ana M. Valero-Jimenez
3
, Craig
Schindewolf
1
, Meike Dittmann
3
, Ricardo Rajsbaum
1,2
, Vineet D. Menachery
1,2
1
Department of Microbiology and Immunology,
2
Institute for Human Infection and Immunity,
University of Texas Medical Branch, Galveston TX, USA
3
Department of Microbiology, New York University School of Medicine, New York, NY 10016,
USA
*Equal contributions
Corresponding Author: Vineet D. Menachery
Address: University of Texas Medical Branch, 301 University Blvd, Route #0610 Galveston, TX
77555
Email: Vimenach@utmb.edu
Article Summary: SARS-CoV-2 has similar replication kinetics to SARS-CoV, but
demonstrates significant sensitivity to type I interferon treatment.
Running title: SARS-CoV-2 sensitive to type I IFN pretreatment
Keywords: Coronavirus, 2019-nCoV, SARS-CoV-2, COVID-19, SARS-CoV, type I interferon,
IFN
.CC-BY-NC 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 13, 2020. ; https://doi.org/10.1101/2020.03.07.982264doi: bioRxiv preprint

Abstract
1
SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing
2
an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both
3
group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of
4
immune antagonists. In this report, we evaluate type-I interferon (IFN-I) sensitivity of SARS-
5
CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2
6
maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I.
7
In Vero and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I
8
pretreatment, while SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to
9
counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able
10
to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial
11
cultures (HAEC), we observe the absence of IFN-I stimulation by SARS-CoV-2 alone, but detect
12
failure to counteract STAT1 phosphorylation upon IFN-I pretreatment resulting in near ablation
13
of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment post infection and found SARS-
14
CoV-2 was sensitive even after establishing infection. Finally, we examined homology between
15
SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence
16
of an equivalent open reading frame (ORF) 3b and changes to ORF6 suggest the two key IFN-I
17
antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify
18
key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that
19
may help inform disease progression, treatment options, and animal model development.
20
Importance
21
With the ongoing outbreak of COVID-19, differences between SARS-CoV-2 and the original
22
SARS-CoV could be leveraged to inform disease progression and eventual treatment options.
23
In addition, these findings could have key implications for animal model development as well as
24
further research into how SARS-CoV-2 modulates the type I IFN response early during
25
infection.
26
.CC-BY-NC 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 13, 2020. ; https://doi.org/10.1101/2020.03.07.982264doi: bioRxiv preprint

Introduction
27
At the end of 2019, a cluster of patients in Hubei Province, China was diagnosed with a
28
viral pneumonia of unknown origins. With community links to the Huanan seafood market in
29
Wuhan, the disease cluster had echoes of the severe acute respiratory syndrome coronavirus
30
(SARS-CoV) outbreak that emerged at the beginning of the century (1). The 2019 etiologic
31
agent was identified as a novel coronavirus, 2019-nCoV, and subsequently renamed SARS-
32
CoV-2 (2). The new virus has nearly 80% nucleotide identity to the original SARS-CoV and the
33
corresponding CoV disease, COVID-19, has many of the hallmarks of SARS-CoV disease
34
including fever, breathing difficulty, bilateral lung infiltration, and death in the most extreme
35
cases (3, 4). In addition, the most severe SARS-CoV-2 disease corresponded to old age (>50
36
years old), health status, and healthcare workers, similar to both SARS- and MERS-CoV (5).
37
Together, the results indicate SARS-CoV-2 infection and disease have strong similarity to the
38
original SARS-CoV epidemic occurring nearly two decades earlier.
39
In the wake of the outbreak, major research efforts have sought to rapidly characterize
40
the novel CoV to aid in treatment and control. Initial modeling studies predicted (6) and
41
subsequent cell culture studies confirmed that spike protein of SARS-CoV-2 utilizes human
42
angiotensin converting enzyme 2 (ACE2) for entry, the same receptor as SARS-CoV (7, 8).
43
Extensive case studies indicated a similar range of disease onset and severe symptoms seen
44
with SARS-CoV (5). Notably, less severe SARS-CoV-2 cases have also been observed and
45
were not captured in the original SARS-CoV outbreak. Importantly, screening and treatment
46
guidance has relied on previous CoV data generated with SARS-CoV and MERS-CoV.
47
Treatments with both protease inhibitors and type-I interferon (IFN-I) have been employed (4);
48
similarly, remdesivir, a drug targeting viral polymerases, has been reported to have efficacy
49
against SARS-CoV-2 similar to findings with both SARS- and MERS-CoV (9-12). Importantly,
50
several vaccine efforts have been initiated with a focus on the SARS-CoV-2 spike protein as the
51
.CC-BY-NC 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 13, 2020. ; https://doi.org/10.1101/2020.03.07.982264doi: bioRxiv preprint

major antigenic determinant (13). Together, the similarities with SARS-CoV have been useful in
52
responding to the newest CoV outbreak.
53
The host innate immune response is initiated when viral products are recognized by host
54
cell pattern recognition receptors, including Toll-like receptors (TLRs) and RIG-I-like receptors
55
(RLRs) (14, 15). This response ultimately results in production of IFN-I and other cytokines,
56
which together are essential for an effective antiviral response (16). IFN-I then triggers its own
57
signaling cascade via its receptor, in an autocrine or paracrine manner, which induces
58
phosphorylation of signal transducers and activators of transcription 1 (STAT1) and STAT2.
59
Together, STAT1, STAT2, and a third transcription factor, IRF9, form the Interferon Stimulated
60
Gene Factor 3 (ISGF3) complex, which is essential for induction of many IFN-stimulated genes
61
(ISGs), and ultimately elicit an effective antiviral response (17, 18). To establish productive
62
replication, viruses have developed different mechanisms to escape this antiviral response
63
targeting different parts of the IFN-I response machinery (19).
64
In this study, we further characterize SARS-CoV-2 and compare it to the original SARS-
65
CoV. Using Vero E6 cells, we demonstrate that SARS-CoV-2 maintains similar viral replication
66
kinetics as SARS-CoV following a low dose infection. In contrast, we find that SARS-CoV-2 is
67
significantly more sensitive to IFN-I pretreatment as compared to SARS-CoV. Infection of IFN-I
68
competent Calu3 2B4 cells resulted in reduced SARS-CoV-2 replication compared to SARS-
69
CoV. Similar to Vero cells, Calu3 cells pretreated with IFN-I had a greater reduction of
70
replication of SARS-CoV-2 compared to SARS-CoV. In human airway epithelial cultures, SARS-
71
CoV-2 showed robust replication and an absence of IFN-I stimulation contrasting influenza A
72
virus. However, pretreatment with IFN-I confirmed SARS-CoV-2 sensitivity and inability to
73
control IFN-I responses once initiated. These results suggest distinct changes between SARS-
74
CoV and SARS-CoV-2 in terms of IFN-I antagonism and we subsequently examined sequence
75
homology between the SARS-CoV and SARS-CoV-2 viral proteins that may be responsible for
76
.CC-BY-NC 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 13, 2020. ; https://doi.org/10.1101/2020.03.07.982264doi: bioRxiv preprint

these differences. Together, the results suggest SARS-CoV-2 lacks the same capacity to
77
control the IFN-I response as SARS-CoV.
78
Results
79
SARS-CoV-2 is sensitive to IFN-I pre-treatment
80
Our initial studies infected Vero E6 cells using a low multiplicity of infection (MOI) to
81
explore the viral replication kinetics of SARS-CoV-2 relative to SARS-CoV. Following infection,
82
we found that both SARS-CoV and SARS-CoV-2 replicate with similar kinetics, peaking 48
83
hours post infection (Fig. 1A). While SARS-CoV-2 titer was slightly lower than that of SARS-
84
CoV at 24 hours post infection, the results were not statistically different. By 48 hours,
85
replication of both viruses had plateaued and significant cytopathic effect (CPE) was observed
86
for both SARS-CoV and SARS-CoV-2 infections. Together, the results indicated that SARS-CoV
87
and SARS-CoV-2 replicate with similar replication kinetics in Vero E6 cells.
88
We next evaluated the susceptibility of SARS-CoV-2 to IFN-I pretreatment. Treatment
89
with IFN-I (recombinant IFN-
α
) has been attempted as an antiviral approach for a wide variety of
90
pathogens including hepatitis B and C viruses as well as HIV (20). During both the SARS and
91
MERS-CoV outbreaks, IFN-I has been employed with limited effect (21, 22). In this study, we
92
pretreated Vero E6 cells with 1000 U/mL of recombinant IFN-I (IFN-
α
) 18 hours prior to
93
infection. Vero E6 lack the capacity to produce IFN-I, but are able to respond to exogenous
94
treatment (23). Following pretreatment with IFN-I, SARS-CoV infection has a modest reduction
95
in viral titer of 1.5 log
10
plaque forming units (PFU) as compared to untreated control 24 hours
96
post infection (Fig. 1A). However, by 48 hours, SARS-CoV has nearly equivalent viral yields as
97
the untreated conditions (7.2 log
10
PFU versus 7.5 log
10
PFU). In contrast, SARS-CoV-2 shows a
98
significant reduction in viral replication following IFN-I treatment. At both 24 and 48 hours post
99
infection, SARS-CoV-2 had massive 3-log
10
(24 HPI) and 4-log
10
(48 HPI) drops in viral titer as
100
.CC-BY-NC 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 13, 2020. ; https://doi.org/10.1101/2020.03.07.982264doi: bioRxiv preprint

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Type i interferon susceptibility distinguishes sars-cov-2 from sars-cov" ?

In this report, the authors evaluate type-I interferon ( IFN-I ) sensitivity of SARS5 CoV-2 relative to the original SARS-CoV. Finally, the authors examined homology between 15 SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. 26. CC-BY-NC 4. 0 International license available under a was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. The absence 16 of an equivalent open reading frame ( ORF ) 3b and changes to ORF6 suggest the two key IFN-I 17 antagonists may not maintain equivalent function in SARS-CoV-2. In addition, these findings could have key implications for animal model development as well as 24 further research into how SARS-CoV-2 modulates the type I IFN response early during 25 infection.