Article https://doi.org/10.

1038/s41467-025-60949-1

Human protein interaction networks

of ancestral and variant SARS-CoV-2
in organ-specific cells and bodily fluids
Received: 16 May 2024 A list of authors and their affiliations appears at the end of the paper

Accepted: 9 June 2025

Understanding SARS-CoV-2 human protein-protein interactions (PPIs) and the

host response to infection is essential for developing effective COVID-19
Check for updates
antivirals. However, how the ancestral virus and its variants remodel virus-host
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protein assemblies in various organ-specific cells and bodily fluids remains

unclear. Here, we conduct 639 affinity-purifications by tagging and expressing
28 SARS-CoV-2 and spike proteins from the ancestral virus and four variants in
eight cell lines representing five mammalian organs and the immune system.
Using mass spectrometry (MS), we identify both known and previously unre-
ported SARS-CoV-2-human PPIs, highlighting similarities and differences
across organ- or immune-derived cell lines and virus strains. Besides verifying
the cell- and variant-specific PPIs, co-fractionation-MS analysis of COVID-19
patients’ saliva confirm host PPI changes between SARS-CoV-2 strains. We
discover that the NSP3 papain-like protease, a secreted protein, binds fibri-
nogen to induce abnormal blood clotting and interferon-induced proteins to
evade host innate immune responses. Leveraging deep learning, we design
peptide inhibitors that successfully blocked SARS-CoV-2 and variant replica-
tion in human liver cells, reversing virus-induced PPI alterations. Together,
these findings provide molecular insights into SARS-CoV-2 biology, uncover
reorganized viral-host protein assemblies during infection, and identify
potential host therapeutic targets and inhibitors for developing antivirals
against SARS-CoV-2 strains.

The ancestral SARS-CoV-2 virus and its variants of concern (e.g., Alpha in understanding how SARS-CoV-2 and its variants remodel host
B.1.1.7, Beta B.1.351, Gamma P.1, Delta B.1.617.2, Omicron B.1.1.529) or responses and PPIs in various organs and fluids, particularly in saliva, a
interest (e.g., Lambda C.37) causing coronavirus disease 2019 key site for SARS-CoV-2 infection and transmission2. While repurposed
(COVID-19) have been detected in various organs (e.g., liver, lung, drugs and vaccines have reduced COVID-19 deaths, the need for
kidney) and bodily fluids (e.g., saliva)1,2, with organ-specific SARS-CoV- improved therapeutics or organ-specific treatments continues. Tar-
2 evolution observed in long COVID cases3. Studies using mass spec- geting virus-host interface is a key strategy for antiviral development,
trometry (MS)-based proteomics and yeast-two hybrid (Y2H) assays with peptide-based inhibitors showing promise for their specificity and
highlight the roles for SARS-CoV-2 proteins in pathogenesis and host tolerability10. As new SARS-CoV-2 variants (e.g., KP.3, XEC, JN.1) con-
factors or processes targeted during infection4–8. Recent findings tinue to emerge, developing therapeutic peptides remains a priority.
reveal that virus-host protein-protein interactions (PPIs) differ in var- To address these gaps, we used affinity purification (AP) and MS to
iants, aiding immune evasion9. However, published studies focused on map PPIs between human proteins and ancestral SARS-CoV-2 proteins,
cell-based models or peripheral blood mononuclear cells, leaving gaps including the spike (S) protein from SARS-CoV-2 and four variants

e-mail: [email protected]

Nature Communications | (2025)16:5784 1

Article https://doi.org/10.1038/s41467-025-60949-1

(Alpha B.1.1.7, Beta B.1.351, Delta B.1.617.2, Lambda C.37) across eight liver and monkey kidney cells. These peptides restored altered host
cell lines from five mammalian organs and the immune system. PPIs in liver cells infected with ancestral or variant SARS-CoV-2.
Biochemical fractionation and MS further provided a comparative Together, this resource offers a comprehensive view of SARS-CoV-2-
analysis of human protein assemblies affected by the Alpha, Delta, and human PPIs across organ- and immune-derived cell lines and variants,
Omicron (B.1.1.529) variants compared to ancestral SARS-CoV-2 providing insights into disease mechanisms and pathobiological
infection in COVID-19 patient saliva. Analysis of 173 predicted multi- changes in ancestral SARS-CoV-2 and its variants within human hosts,
protein complexes (MPCs) from the human-SARS-CoV-2 PPI network including the physiological context of COVID-19 patients. Our findings
revealed the biological significance of the SARS-CoV-2 papain-like also establish a foundation for developing host-targeting therapies and
protease NSP3 (a non-structural protein), which binds fibrinogen broad-spectrum antivirals to prepare for future pandemics.
subunits to promote coagulation abnormalities and associates with
interferon (IFN)-induced proteins to evade immune responses. Addi- Results
tionally, our deep learning algorithm11 identified three receptor bind- AP-MS reveals organ- and immune cell line-dependent SARS-
ing domain (RBD)-targeting peptides from the S protein that inhibited CoV-2-host PPIs
entry and replication of ancestral and variant strains by blocking S-RBD To survey interactions between human and SARS-CoV-2 proteins
and ACE2 (angiotensin-converting enzyme 2) interactions in human across mammalian organ- and immune-derived cell lines, we examined

a e Overlap of SARS-CoV-2-host PPIs in BioGRID f Overlap of SARS-CoV-2-host

PPIs (x 103)
Viral-host PPIs (This study, Average RF score > 0.8)
5,000 bp 10,000 bp 15,000 bp 20,000 bp 25,000 bp 0.0 0.5 1.0 1.5 2.0 2.5

ORF Structural protein THP−1 (M1)

1a 1b
44% (1,805 THP−1 (M0)
SPIKE (S) E M N of 4,101) 56% (2,296
NSP Accessory factors of 4,101)
1 5 6 7 8 9 11 3a 6 7b 9b 10 Holdout non-human
HULEC−5a
2 3 4 10
cell lines
b 12
13
14
15
16
3b 7a 8 9c N9 microglia Low (RF ≤ 0.8)
Huh−7

N9 microglia High (RF > 0.8)

1 2 3 Human prey HEK293
Vero81 Low (RF ≤ 0.8)
SARS- proteins
CoV-2
Vero81 High (RF > 0.8)
639 affinity Viral bait VeroE6 Low (RF ≤ 0.8) Caco−2
purifications VeroE6 High (RF > 0.8)
SARS-CoV-2 ancestral Strep-tag
(29 proteins) and Lentivirus SARS-CoV-2 Strep-tag constructs
Viral bait
variants (4 ‘S’ proteins) infection in eight different cell lines
g h

THP-1 (M0)

THP-1 (M1)
protein Co-localization of False discovery

HULEC-5a
host-SARS-CoV-2 PPIs (x 102) rate (FDR)

HEK293
Human cell lines

Caco-2
Huh-7
Non-human cell lines for validation 0 2 4 6 8
Caco-2 (Colon) Huh-7 (Liver) THP-1 (M0; immune) 5e-2 2.5e-24
N9 (microglia) VeroE6 (K) Vero81 (K) Enriched bioprocesses
HEK293 (Kidney, K) HULEC-5a (Lung) THP-1 (M1; immune)
Huh−7
4 5 6 Amide synthesis
Translation
Human cell lines

HEK293 Nop56p-linked pre-rRNA complex

LC-MS 3 search ER–Golgi transport vesicles

Human interacting proteins

/MS algorithms Caco−2 Vesicle-mediated transport
Golgi vesicle transport
Organelle organization
HULEC−5a Intracellular transport
Computational Tryptic peptides MS spectra Host or viral protein identification
TRBP containing complex
scoring workflow Bait THP−1 (M0) ER stress
7 8 9 Prey TNF-α/NF-κB signaling
PPIs THP−1 (M1) Apoptosis
2 scoring Training Score Unfolded protein response
algorithms set assignment Actin cytoskeleton
RNA splicing
Spliceosome
Random Host-viral protein-protein EIF3 complex
Viral-human Endocytosis
protein associations forest (ML) (PPI) interaction network
i Interactome similarity to
SARS-CoV-2 (-log10 P-value)
Metabolism
Membrane organization

c 1.0
d Overlapping SARS-CoV2
-host PPIs
0 50 100
Caco−2
(AUC: 0.99)
HEK293
(AUC: 0.99)
Huh−7
(AUC: 0.99)
0.0 0.5 1.0
KHSV j Human prey proteins
interacting with SARS-CoV-2 baits
Zika -Log10 (P-value)
0.5 0.0 0.2 0.4 0.6
SARS-CoV-1 % coverage
Chlamydia
Multiple human P =1e-4
Sensitivity

cell lines MERS-CoV

0.0 Dengue
WNV
1.0 SARS-CoV-1
HIV
HULEC−5a THP−1 (M0) THP−1 (M1) HCV
Unthresholded Shared
(AUC: 0.99) (AUC: 0.99) (AUC: 0.98) Single human HPV
(RF < 0.8) Mtb Not shared
0.5 cell line
Thresholded Ebola
(RF > 0.8) MERS-CoV
0.0 0.5 1.0
0.0 Average RF score
Overlap of host interacting
0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 proteins (%)
1-specificity 0.0 0.2 0.4 0.6 >0.8

Fig. 1 | Organ- and immune cell-specific SARS-CoV-2-host interactions by AP- Grand, 202361), while the human protein data is sourced from the Human Protein
MS. a SARS-CoV-2 genome showing ORF1a/1b regions encoding 4 structural proteins, Atlas62. h Enrichment of shared biological processes among SARS-CoV-2-interacting
9 accessory, and 16 non-structural proteins (NSPs) used for affinity purification. b AP- human proteins across multiple human cell lines. Top enriched terms in two or more
MS workflow applied to 29 ancestral and 4 spike (S) variants (Alpha, Beta, Delta, cell lines are shown. P-values from a one-sided hypergeometric test and adjusted for
Lambda) across organ- and immune-derived human cell lines to build viral-host PPI FDR ≤ 5e−2 with the Benjamini-Hochberg (BH) method. i Coverage (%) and significance
networks; 3 non-human cell lines used for validation. MS, mass spectrometry; ML, (P = 1e−4, red dotted line) of SARS-CoV-2-interacting human proteins overlapping with
machine learning. c Performance of PPI predictions using a random forest (RF) those from other pathogens (Supplementary Data 5). P-values from one-sided
classifier, assessed by sensitivity vs. 1-specificity (area-under-the-curve, AUC) against a hypergeometric test. KHSV, Kaposi’s sarcoma herpesvirus; SCoV1, Severe acute
curated reference PPI set from BioGRID. High-confidence SARS-CoV-2-host PPI respiratory syndrome coronavirus 1; MERS, Middle East Respiratory Syndrome; WNV,
detection in one or more human cell lines (d), overlap with BioGRID (e), validation in West Nile virus; HIV, Human immunodeficiency virus; HCV, Hepatitis C virus; HPV,
non-human cell lines with RF scores below (≤0.8) or above (>0.8) the cut-off (f), and Human papillomavirus; Mtb, Mycobacterium tuberculosis. j Human proteins inter-
subcellular co-localization in the same cellular compartment (g). SARS-CoV-2 locali- acting with SARS-CoV-2 baits shared or not shared with the indicated viruses. Source
zation data is from published studies15,61 (with additional references reviewed in data are provided as a Source Data file.

Nature Communications | (2025)16:5784 2

Article https://doi.org/10.1038/s41467-025-60949-1

all 29 viral proteins (4 structural, 16 non-structural, and 9 accessory; BioGRID, while the remaining (44%, 1805 of 4101) have not been
Fig. 1a). Of these, 26 proteins, a catalytic dead mutant (NSP5 C145A), reported earlier (Fig. 1e). Among these, less than one-third (31%, 557 of
and enhanced green fluorescent protein (eGFP, as a negative control) 1805) were confirmed in multiple human cell lines in the unthre-
were expressed using a mammalian vector with a 2x Strep-epitope tag sholded data (Supplementary Data 1). Additionally, more than a dozen
at the C-terminus5 for AP-MS. Strep-tagged constructs for the SARS- were validated by co-immunoprecipitation (co-IP) in specific cell lines
CoV-2 S structural protein, two non-structural proteins (NSP3 and (Supplementary Fig. 1g) using protein-specific antibodies, emphasiz-
NSP16), and S proteins of Alpha, Beta, Delta, and Lambda variants, ing the biological relevance of our predicted PPIs. The detection of
previously unavailable in Addgene plasmid repository or analyzed5, these newly identified PPIs may have been influenced by the choice of
were cloned with a Gateway cassette carrying a versatile epitope (VA) cell lines, methodologies, and the reliance on a single cell line in earlier
tag (3× FLAG, 6× His, 2× Strep12) at the C-terminus (Supplementary SARS-CoV-2 interactome studies. Third, in the thresholded dataset,
Note 1). After confirming affinity-tagged viral protein expression by less than half (48%, 2002 of 4158) of SARS-CoV-2 interactors were
immunoblotting with an anti-Strep antibody (Supplementary Fig. 1a), identified in at least one of the three holdout non-human cell lines
ancestral SARS-CoV-2 proteins with 2x Strep or VA-tags were stably (Fig. 1f). However, this overlap increased by an average of 4.4-fold
expressed in eight mammalian cell lines representing five organs when using the unthresholded data, indicating that the chosen cut-off
(Kidney: HEK293, Vero E6/81; Liver: Huh-7; Intestinal: Caco-2; Lung: is highly stringent. Fourth, in addition to identifying SARS-CoV-2
HULEC-5a; Brain: N9 microglia) and the immune system (inactivated interactors that co-localize within the same cellular compartment
M0 and activated M1 macrophages from THP-1). Cultured cells (Fig. 1g), 166 host factors interacting with SARS-CoV-2 proteins from
expressing these viral proteins underwent Strep affinity purification in human cell lines were detected in genetic screens15–17 as enhancers or
at least two biological replicates. Purified samples were trypsinized and inhibitors of SARS-CoV-2 or common cold coronavirus infections
analyzed by MS, resulting in 639 AP-MS analyses (Fig. 1b). High (Supplementary Fig. 1h). For example, Rab GTPases (RAB2A/7 A/ 10),
reproducibility (r = 0.94) of MS2 protein spectral counts between interacting with NSP6/7 in HULEC-5a lung endothelial cells, were
replicate AP-MS experiments for each cell line (Supplementary Fig. 1b) identified as key host-dependency factors for SARS-CoV-2
validated data quality. progression18. Next, protein abundance changes during SARS-CoV-2
To define SARS-CoV-2-human PPIs, MSspectra from Strep-tagged infection19 was examined by calculating correlations between the
viral bait purifications were mapped to SARS-CoV-2 and mammalian abundance of viral proteins and their human interaction partners
host protein sequences using SEQUEST/STATQUEST, MS-GF + , and across human cell lines. Interacting partners of viral bait proteins
MaxQuant search algorithms to improve peptide identification and exhibited stronger correlations in protein level changes during SARS-
protein coverage (Supplementary Note 2). For each cell line, outputs CoV-2 infection compared to non-interacting protein pairs
from these search engines were scored independently using (1.0e−4 < P > 1.9e−2; Supplementary Fig. 1i). Additionally, these SARS-
CompPASS-Plus (Comparative Proteomic Analysis Software Suite13) CoV-2-human interacting proteins were significantly (P = 5e−2) enri-
and MiST (Mass spectrometry interaction STatistics14) scoring proce- ched in tissues such as salivary glands, pancreas, heart, and lungs
dures to assign confidence scores for viral bait-human prey associa- (Supplementary Fig. 1j; Supplementary Data 3) based on protein
tions (Fig. 1b). To enhance prediction accuracy, CompPASS-Plus and abundance across human tissues20, suggesting that PPIs relevant to
MiST scores were combined into a single probabilistic score via a target tissue and infection are context-dependent.
supervised random forest (RF) model trained on positive SARS-CoV-2- Among SARS-CoV-2 binding partners, we identified significant
human PPIs from BioGRID database and a negative dataset of non- enrichment (FDR ≤ 5e−2) of shared bioprocesses across human cell
interacting protein pairs (Supplementary Note 3). The model’s per- lines derived from various organs and immune system (Fig. 1h; Sup-
formance for each cell line, evaluated through 10-fold cross-validation plementary Data 4). These include host processes such as endoplasmic
against a BioGRID reference set of curated PPIs withheld during reticulum (ER)-Golgi vesicle transport, mRNA translation, and the
training, achieved an average false discovery rate (FDR) of 4.4e−3 with unfolded protein response, which SARS-CoV-2 exploits for
an RF score threshold > 0.8 that recovered most reference PPIs as infection21–23. Comparing SARS-CoV-2 host interaction partners with
shown by area-under-the-curve (AUC) analysis (Fig. 1c and Supple- other pathogens revealed the greatest similarity with SARS-CoV-1
mentary Fig. 1c). This yielded a high-confidence network of 4158 (SCoV1; Fig. 1i; Supplementary Data 5), reflecting their clinical and
interactions between 30 SARS-CoV-2 (including the NSP5-C145A genetic similarities. SARS-CoV-2 also shared more PPIs with SCoV1 than
mutant) and 1774 human proteins across five human cell lines with Middle East Respiratory Syndrome Coronavirus (MERS-CoV;
(Fig. 1d and Supplementary Fig. 1d; Supplementary Data 1). These Fig. 1j). Notably, human proteins interacting with SARS-CoV-2 NSP7/13
interactions were cross-validated with human orthologs of interacting and ORF7A/9B/9 C are also targeted by SCoV1, similar to the shared
proteins from non-human cell lines, such as N9 microglia (mouse interactors between MERS-CoV and SARS-CoV-2 NSP2/4/7/8 (Supple-
brain) and Vero E6/81 (African Green monkey kidney). mentary Fig. 1k), highlighting these PPIs as potential antiviral targets.
The quality of AP-MS-derived interactions was supported by sev- Together, these findings confirm that our AP-MS network, derived
eral lines of evidence. First, most (75%, 3136 of 4158) SARS-CoV-2- from diverse organ and immune cell lines, significantly expands the
human PPIs were detected across multiple human cell lines, while a number of reported PPIs, offering avenues for therapeutic
quarter (25%, 1022 of 4158) were specific to a single organ-derived cell development.
line (Fig. 1d and Supplementary Fig. 1e; Supplementary Data 2). Over
half (54%, 556 of 1022) of these were found in Huh-7 liver cells (Sup- Ancestral and variant SARS-CoV-2 S-host PPIs vary in organ- and
plementary Fig. 1f). Although detecting PPIs across multiple cell lines immune-derived cells
reinforces confidence (Fig. 1d), we were interested in the organ- Since enhanced fitness of circulating SARS-CoV-2 variants is linked to
specific interactions identified. To assess whether this trend persists in mutations in the S protein (the primary target of COVID-19 vaccines), it
the unthresholded ( <0.8) dataset, we conducted a comparative ana- is critical to comprehend how mutations in the S protein of the var-
lysis and observed a similar pattern (Fig. 1d). This confirmed that the iants, compared to ancestral SARS-CoV-2 influence virus-host protein
majority (64-75%) of PPIs occur across multiple cell lines, while a assemblies. Therefore, we examined the S regions of three variants of
smaller fraction (25-36%) remains organ-specific, regardless of whether concern (Alpha, Beta, Delta) and one variant of interest (Lambda),
the dataset was thresholded or unthresholded. which significantly affect virulence, transmission, and COVID-19
Second, over half (56%, 2296 of 4101) of SARS-CoV-2-human PPIs, severity24. Using PCR mutagenesis, we created 42 hotspot non-
excluding the NSP5-C145A mutant, were previously reported in synonymous mutations or deletions in the S protein of Alpha (9),

Nature Communications | (2025)16:5784 3

Article https://doi.org/10.1038/s41467-025-60949-1

a SPIKE (‘S’) protein region b Non-redundant human proteins

interacting with ‘S’ viral protein
c Redundant human proteins
interacting with ‘S’ viral protein
d Human proteins interacting
with ‘S’ viral protein
SARS-
CoV-2 0.0 0.2 0.4 0.6 0 150 300 0 150 300

PCR mutagenesis Shared across two or

Amino acid substitution SARS-CoV-2 HULEC-5a (Lung)
more CoV-2 strains
Amino acid deletion A570D P681H
SARS-CoV-2 THP-1 (M0)

Human cell lines

ΔY144 D614G Lambda
T716I

Viral ‘S’ protein

Viral ‘S’ protein

N501Y D1118H
ΔH69-V70 S982A
Delta HEK293 (Kidney)
Alpha PPI score > 0.8
Beta
Lambda PPI score ≤ 0.8 Caco-2 (Colon)
ΔA242-L243
ΔL241 E484K
K417N Delta
SARS-CoV-2 variants

D80A D215G D614G A701V Alpha Huh-7 (Liver)

N501Y
Beta Alpha THP-1 (M1)
Beta
ΔF157
G142D R158G T478K P681R

Lambda
Endoplasmic reticulum (ER) Yes ‘S’ protein abundance
D614G D950N
E156K
e

CoV-2
T19R L452R

Alpha
in HEK293 cells (%)

Delta
Beta
T95I Golgi apparatus No
Delta 0 100 200

(see Supplementary Fig. 1a)

Plasma membrane

VA -tagged ‘S’ protein

∆R246,S247,Y248, CoV-2
T76I F490S
L249,T250,P251,G252
G75V L452Q D614G T859N Alpha
Lambda
Beta

interacting proteins localized in same compartment

Human-‘S’ (SARS-CoV-2 ancestral or variants)
Delta
Lambda

f Human proteins interacting with ‘S’ viral protein

VA-tagged (Strep)-S protein
SARS-CoV-2 Alpha Beta Delta Lambda
Human cell lines
SARS-CoV-2 1

α-Strep
2
Viral ‘S’ protein

Beta ≥3

Alpha

HEK293 cells
Lamdba

ER
RF and
Delta PCA score

0.8 1.0

Merge
g -Log (FDR)
10

0 15 30

RNP
h i
Enriched complexes/processes

Alpha
Ribosome HNRNPA1
RBMXL2
HNRNPD DHX15 Unique to this study
Nop56p pre-rRNA
Pre-NOTCH transcription/translation SNRNP27 195 Overlap with Bouhaddou et al (2023)
SARS-CoV-2 and variant ‘S’ proteins 40
SNRNP200 RBMXL3 HNRNPR
COPI anterograde transport interact with RNA processing subunits SRSF5 RBM8A 43
RNA processing DHX9 Beta
mRNA metabolism RBM15B 48
TNF-α/NF-kB signaling RBM12B Delta CoV-2
Lambda 342
Spliceosome RBM44 SNRPA
SRSF1
RBM14
Myosin TUT1
SRSF2 SNRPD1
TRBP SRSF7 0.00 0.25 0.50 0.75 1.00
Spliceosomal snRNP HNRNPU
Host factors interacting with one PPIs between ancestral or variant ‘S’ protein
Mitochondrial (mt) transition pore
or more viral ‘S’ protein and host factors from various human cell lines
Mt outer membrane permeabilization Ribonucleoprotein (RNP) or DGCR8 Human proteins
Mt membrane organization spliceosomal complex 1 4
RNA metabolism Small nuclear RNP complex Viral ‘S’ protein
Drosha complex 2 5
Shared subunits 3

Fig. 2 | Comparative analysis on SARS-CoV-2 ancestral and variant S protein (normalized to GAPDH loading control), based on immunoblot (IB) quantification
interactomes. a Schematic showing substitutions/deletions in S protein variants from Supplementary Fig. 1a. Band intensity measurements (n = 10) were obtained
analyzed by AP-MS. Human proteins (n = 668) interacting with ancestral and variant from representative IB from the same experiment. Data are shown as mean ±
S proteins across viral strains (b, c) and human cell lines (d). b shows non- standard error of the mean (SEM). f Heatmap of unique/shared human prey pro-
redundant interactions independent of cell type, while panel c includes redundant teins among ancestral and variant S baits in human cell lines, filtered by random
interactions accounting for cell types. e Localization of human prey proteins (from forest (RF) and first principal component analysis (PCA) scores > 0.8. g Enrichment
the Human Protein Atlas62) with ancestral or variant S bait proteins (from this study of annotated complexes/processes (CORUM, Gene Ontology, Reactome, Wiki-
and a published report15) in the same subcellular compartment (left). Workflow (top Pathways) for S-interacting host proteins, including the components of RNP, spli-
right) for subcellular localization of VA-tagged (2x Strep) S proteins in HEK293 cells. ceosomal, Drosha-DGCR8, and small nuclear RNP complexes. P-values from one-
Representative fluorescence images (bottom right; n = 5) showing co-localization sided hypergeometric test and adjusted for FDR ≤ 5e−2 with the BH method. h Pie
of VA-tagged ancestral or variant S proteins (anti-Strep, green) with ER tracker chart of host factors interacting with ancestral and/or variant S proteins.
(blue). Additional markers is shown in Supplementary Fig. 2a. Scale bar, 20 μm. Bar i Comparison of ancestral or variant S-host PPIs unique to this study vs. a recent
plot (far right) showing consistent S protein expression in HEK293 cells report9. Source data are provided as a Source Data file.

Beta (9), Delta (11), and Lambda (13) variants (Fig. 2a; Supplementary confidence interactions. The resulting network identified 668 host
Data 6; Supplementary Note 1) as opposed to single mutations that do proteins, with a quarter (25%, 165 of 668) shared across two or more
not mimic all mutated residues. Sequence-verified plasmids were then SARS-CoV-2 S strains when thresholded, and capturing an additional
C-terminally tagged with a VA epitope containing 2x Strep using quarter (28%, 190 of 668) of human proteins interacting with the S viral
Gateway cloning and stably expressed in HEK293 cells through lenti- protein when unthresholded (Fig. 2b). The remaining (47%, 313 of 668)
viral infection (Supplementary Note 1). The specificity of expressed were distributed differently, with a fifth (21%, 142) constituting the
variant and wildtype S proteins was confirmed using an anti-Strep ancestral SARS-CoV-2 S protein, and a smaller fraction (5-8%, 33-54)
antibody (Supplementary Fig. 1a). Strep-tagged S proteins were then were exclusive to each variant (Fig. 2b; Supplementary Data 7). Further
affinity-purified from selected human cell lines for MS analysis. analysis identified 65 human proteins interacting with one or more
Using the same approach as for ancestral SARS-CoV-2 (Fig. 1b), we SARS-CoV-2 variants across 2-4 human cell lines (Supplementary
employed CompPASS-Plus and MiST algorithms to score interactions Data 7). Among these, ENO2, a glycolysis marker linked to increased
for S protein variants. Due to limited training data for the variants, SARS-CoV-2 load and inflammation25, and PARD6G, a cellular polarity
outputs from both scoring metrics were merged and analyzed using regulator exploited by SARS-CoV-2 to evade immune responses26, were
first principal component analysis (PCA14; Supplementary Note 3). PCA consistently targeted by one or more variants in four (Caco-2, HEK293,
scores for individual cell lines were averaged into a composite score HULEC-5A, THP1-M1) of six human cell lines tested. On average, the
and subjected to a stringent threshold cut-off > 0.8 to define high- ancestral SARS-CoV-2 S protein interacted with 1.4-times more host

Nature Communications | (2025)16:5784 4

Article https://doi.org/10.1038/s41467-025-60949-1

factors than variants (Fig. 2c), with interactions most frequent in lung ancestral or variant (Alpha, Delta, Omicron) SARS-CoV-2 strains
endothelial cells (HULEC-5a; Fig. 2d). Examining the localization of S (Fig. 3a). A total of 1128 biochemical fractions from 12 fractionation
protein variants in HEK293 cells revealed their presence in the ER, experiments, including replicates, were analyzed by MS, with spectra
Golgi, and plasma membrane, similar to the ancestral S protein15, but processed using multiple search algorithms, as in AP-MS, identifying
sometimes to a lesser extent (Fig. 2e and Supplementary Fig. 2a). While 9247 human proteins (Supplementary Data 13). Protein elution pat-
the ancestral S protein showed consistent localization across cell types terns and the average correlation of proteins based on MS-detected
(data shown for HEK293), variant S proteins exhibited mutation-driven spectral counts (r = 0.7) were reproducible across replicate experi-
localization changes despite unchanged S protein abundance (Fig. 2e) ments (Supplementary Fig. 3a, b). PCA analysis (Supplementary Fig. 3c)
in HEK293 cells expressing Strep-tagged S protein from each SARS- revealed clear separation of samples positive and negative for SARS-
CoV-2 strain. CoV-2 or its variants in multidimensional space.
The localization of the ancestral viral S protein aligns with that of Stably associated proteins within a MPC are expected to co-elute,
its human binding partners, but mutations in variant S proteins and their associations can be assessed by pairwise similarity of their co-
affecting localization may influence virus-host PPIs (Fig. 2e; Supple- elution profiles30. Thus, we computed nine correlation metrics (i.e.,
mentary Data 8), though do not fully account for the PPI differences Apex, Bayes correlation, cosine correlation, Jaccard index, mutual
observed across cell types. Consistently, enriched ( | Z-score | ≥ 1.96; information, Pearson correlation coefficient (PCC), PCC adjusted for
P ≤ 0.05) human interacting proteins associated with each viral S Poisson noise, P-value derived from PCC, weighted cross correlation;
protein differ significantly between compartments and across variants Supplementary Note 3) for each search engine, capturing distinct
and the ancestral strain (Supplementary Fig. 2b; Supplementary features of profile similarity in SARS-CoV-2 patients (Fig. 3a). After
Data 9). Human host factors also showed reconfiguration, with some combining the scores from all metrics, protein pairs were input into a
proteins uniquely bound and others shared among variant and RF classifier, trained using positive PPIs from BioGRID, while non-
ancestral S proteins (Fig. 2f; Supplementary Data 10). For example, interacting protein pairs from the training set were used as the nega-
host factors interacting with S proteins were enriched in cohesive tive dataset. Predicted PPIs from each search algorithm were com-
subunits linked to annotated MPCs or biological terms from CORUM, bined, and their RF scores averaged to generate a unified PPI network.
Reactome, Gene Ontology (GO), and WikiPathways (Fig. 2g; Supple- The predictive accuracy of these measurements, assessed using 10-
mentary Data 11). These included S proteins interacting with the fold cross-validation against a curated subset of BioGRID PPIs to train
components of ribonucleoprotein (RNP), spliceosomal, Drosha- the RF model in AP-MS, achieved high sensitivity and specificity with an
DGCR8, and small nuclear RNP complexes, with co-IP confirming that AUC of 0.90 (Supplementary Fig. 3d).
different S variants bind to unique host partners in specific human cell The final network comprises 2019 interactions between 17 SARS-
lines (Supplementary Fig. 2c). This suggests that, like SARS-CoV-227, CoV-2 and 880 host proteins, with each interaction having an RF score
variant S proteins can target the same cellular pathways, yet they > 0.75 in co-elution profiles (Fig. 3b and Supplementary Fig. 3d; Sup-
exploit these host factors through specific interactions, and crucially, plementary Data 14). While some viral proteins went undetected in the
distinct protein partners, facilitating replication and functioning as saliva of SARS-CoV-2-infected patients, over half (56%, 17 of 30) of the
viral RNA sensors in innate immunity27. expressed proteins showed no bias in protein abundance relative to
A small subset (7%, 48 of 668) of host proteins interacting with all their identified human interacting partners by MS (Fig. 3c). Our ana-
five SARS-CoV-2 S strains was primarily found in HULEC-5a lung lysis also revealed only 80 overlapping high-confidence PPIs between
endothelial cells (Fig. 2h and Supplementary Fig. 2d). These proteins the two methods. However, an additional 519 PPIs were found in the
were enriched (FDR ≤ 5e−2) in functions related to the mitochondrial AP-MS high-confidence dataset but fell below the CF-MS threshold, or
respiratory chain, cytokine production, and cytoskeleton organization vice versa (Fig. 3b). Regardless of the methods used, the overlap of
(Supplementary Fig. 2e), suggesting they are critical host ‘hub’ pro- high-confidence viral-host PPIs between saliva and individual cell lines
teins potentially exploited by the virus for replication, transmission, remained modest (Fig. 3d). This aligns with the limited detection of
and immune evasion. A sizable portion (40%, 267 of 668) of PPIs was host factors observed between Y2H and AP-MS methods4, suggesting
both variant- and cell type-specific, with variability observed in shared that the modest overlap may stem from underestimations in predicted
PPIs across variants (Supplementary Fig. 2f). Inactivated THP-1 M0 interaction scores due to constraints in the reference dataset used for
macrophages and HEK293 cells showed more shared interactions training. Additionally, methodological differences, such as higher viral
among variants, while HULEC-5a, Caco-2, and Huh-7 cells exhibited protein expression during natural infections compared to affinity-
fewer (Supplementary Fig. 2g). Notably, over three-fourths (79%, 526 tagged protein studies, may contribute to these discrepancies.
of 668) of interactions between ancestral or variant S proteins and host To explore the divergence in host responses to ancestral vs. var-
factors across various human cell lines (Fig. 2i; Supplementary Data 12) iant viruses, we built a differential network from the saliva of infected
were previously unreported9, suggesting cell type-specific PPIs may patients by comparing the ancestral virus to healthy subjects and the
underlie these differences. ancestral virus to each SARS-CoV-2 variant. This was achieved by cal-
culating co-elution profile similarity using PCC. For each pair of co-
Host responses remodeled by ancestral and variant infections eluting human proteins, a P-value was assigned based on differences in
in saliva correlation scores across the comparative ‘input’ samples. By applying
Prompted by notable changes in virus-host PPIs specific to organ- differential score thresholds with a FDR ≤ 5e−4 (Fig. 3e), we identified
specific cell lines or shared among variants compared to the ancestral 15,659 significant differential human PPIs when comparing the ances-
virus, we examined how these reconfigured protein assemblies man- tral virus to healthy subjects, and to variants (Fig. 3f and Supplemen-
ifest in the saliva of COVID-19 patients infected with SARS-CoV-2 and its tary Fig. 3e; Supplementary Data 15). Of these, over one-third of human
variants. This analysis helps characterize viral-host PPIs in the native protein pairs showed either positive (38%, 5968 of 15,659) or negative
physiological state during viral infection, particularly given SARS-CoV- (35%, 5535 of 15,659) differential correlations with at least one variant
2’s ease of transmission through saliva2. Since co-fractionation MS (Fig. 3g). A smaller subset were more (219 of 15,659) or less (129 of
(CF-MS) can determine native MPC membership in lysates from human 15,659) specific across all variants (Fig. 3h; Supplementary Data 16).
cells28 and model organisms29, we performed biochemical fractiona- Enrichment analysis (FDR ≤ 5e−2) revealed co-eluted human pro-
tion with size-exclusion chromatography (SEC) and MS profiling to teins from the haptoglobin-hemoglobin complex and/or the immu-
separate native macromolecules in soluble protein extracts prepared noglobulin kappa variable cluster as the most correlated across
from the saliva of healthy individuals and patients infected with variants compared to the ancestral SARS-CoV-2 strain (Fig. 3i and

Nature Communications | (2025)16:5784 5

Article https://doi.org/10.1038/s41467-025-60949-1

a 1 Saliva 2 3 4
e ≤5.0e-4
FDR
≤ 2.5e-2 Non-significant
SARS-CoV-2 vs.
variants/healthy
DF PCC
protein pairs

Healthy
Controls (3) collection ≤5.0e-3 ≤ 5.0e-2 n = 3,978
Dilution with SEC- LC-MS 1.0 1.0 Omicron

Healthy subjects (PCC)

SEC buffer HPLC /MS

Alpha patients (PCC)

n = 2,102
Alpha (2) Delta
SARS-CoV-2/Variants

(n=12; 84-96
Delta (2) fractions/sample) Cell 1,128 Mass
lysate fractions spectrometry (MS) 0.0 0.0 n = 4,156
FDR ≤ 5e-4 Healthy
Omicron (2) cut-off
Computational
CoV-2 (3) scoring workflow n = 5,423
Alpha
-1.0 -1.0
5 Fractions 6 7 -1.5 0.0 1.5
-1.0 0.0 1.0 -1.0 0.0 1.0
(viral, host)
Proteins Differential PCC score
Data 3 search 1.0
1.0

Omicron patients (PCC)

compilation algorithms
f

Delta patients (PCC)

CoV-2 vs. Omicron

CoV-2 vs. healthy

CoV-2 vs. Alpha

CoV-2 vs. Delta

Co-eluted
MS spectra protein profiles
0.0 0.0
Scoring (nine PCC

Co-eluted host protein

correlation metrics)
A A B Z-score

PCC profiles
Healthy distribution
Z-score
-1.0 -1.0 DF PCC
transformation
Training CoV-2 score
-1.0 0.0 1.0 -1.0 0.0 1.0
set Variant 1.3
SARS-CoV-2 patients (PCC) SARS-CoV-2 patients (PCC)
Viral-human PPIs
B C C D

Co-eluted human protein pairs

Score
Viral FDR ≤ 5e-4 DF
DF PCC
analysis
g Human protein pairs
h 0
-1.3

Human protein pairs

assignment host PPIs -4000 -2000 0 2000 4000
Host
PPIs 5000
Random Host-viral protein-protein Differential (DF) host protein PPIs in CoV-2 vs. Alpha
forest (ML) (PPI) interaction network response to SARS-CoV-2 vs. variants 10000
Variants

b AP-MS
(HC, 4,158)
CF-MS
(HC, 2,019)
d Overlap of SARS-CoV-2-host PPIs from
saliva (CF-MS) to cell lines (AP-MS)
+ve DF correlation CoV-2 vs. Delta
15000
IGKV, HB9, H9
-ve DF correlation cluster (Supplemen
0 60 120
tary Fig. 3f)
3,851 1,647 CoV-2 vs. Omicron More specific to all variants
292 80 227 HULEC-5a Less specific to all variants
Human cell ines

HEK293
SARS-CoV-2 Variants

599
Huh-7 CF-MS data i Fold enrichment
(FDR ≤ 5e-2) +ve DF correlation +ve DF correlation
Caco-2 All 0 50 100 150 -ve DF correlation -ve DF correlation
80 HC PPIs in both AP-MS and CF-MS Average RF score > 0.75
292 HC PPIs in CF-MS but below AP-MS threshold
227 HC PPIs in AP-MS but below CF-MS threshold
THP-1 (M0)

THP-1 (M1)
Hp-Hb complex
Ig production
Adaptive immunity
j Human host
interacting proteins
Viral strains
1 2 3

c Ceruloplasmin
Enriched GO terms

Exosome 0 125 250

1000 400 Vesicle Titin
Spectral counts

(Heavy) (H2A/2B)
Histone
Human host interacting proteins
Viral-host PPIs 2+
Ca export via PM
CoV-2 vs. Alpha (1)
Protein abundance

PDE activity
PPIs (Saliva, CF-MS)
(spectral counts)

750 300
SARS-CoV-2-host

PPP activity
Ca2+ transport
Acute inflammation

IG
500 200 VGKC complex CoV-2 vs. Delta (2)
Ca2+ channel complex
Spindle microtubules
K+ channel complex
250 100

(Kappa)
More specific to variants CoV-2 vs. Omicron (3)

IG
Less specific to variants
0 0 Autocorrelation
NSP1
NSP5
NSP13

NSP12

ORF7a
NSP10

NSP14
SPIKE
NSP4

NSP16

ORF3a
NSP6

NSP8
NSP15
NSP2
NSP3
M

High autocorrelation (> 0.25)

Low autocorrelation (< 0.25) 0.7 -0.1
Ancestral SARS-CoV-2 proteins

Fig. 3 | Viral-host PPIs and host responses in saliva from SARS-CoV-2 ancestral healthy or variant-infected patients at various P-value thresholds based on PCC
vs. variant infections. a CF-MS scoring pipeline identified reconfigured host-viral score differences and two-sided Z-score transformation. Histogram (right) displays
or host-host PPIs from 1128 SEC (size-exclusion chromatography) fractions using significantly differential (DF) human protein pairs at P ≤ 1.0e−5 and BH-adjusted
soluble protein extracts from saliva of healthy individuals (n = 3) and patients FDR ≤ 5.0e−4 in the indicated comparisons. f Heatmap of positive and negative DF
infected with ancestral (n = 3), Alpha (n = 2), Delta (n = 2), and Omicron (n = 2) correlations in co-eluted human protein pairs from SARS-CoV-2- vs. healthy or
variants. b Overlap of high-confidence (HC) host-viral PPIs detected via AP-MS in variant-infected saliva. g Bar plot of positive and negative DF correlations of human
human cell lines and CF-MS in ancestral SARS-CoV-2-infected patient saliva. Host- protein pairs between SARS-CoV-2- and variant-infected individuals. h Specificity of
viral PPIs also include those detected as high-confidence in the AP-MS dataset but human protein interaction pairs across all three variants. i GO biological term
below the CF-MS threshold, and vice versa. c Distribution of protein abundance enrichment for human proteins interacting more or less specifically with variants. P-
(MS2 spectral counts) and viral-host PPIs across SARS-CoV-2 proteins detected by value from a one-sided Fisher exact test, and adjusted for FDR ≤ 5e−2 with the BH
CF-MS. d Viral-host PPIs identified in SARS-CoV-2-infected patient saliva via CF-MS, method. j Correlation profiles (autocorrelation) of co-eluted human protein inter-
and supported by AP-MS in human cell lines, analyzed with and without (i.e., All) RF actions between SARS-CoV-2 and variants, categorized as high (>0.25) or low
score thresholds. e Scatterplot (left) showing Pearson correlation coefficients (<0.25), with zoom-in examples. Source data are provided as a Source Data file.
(PCCs) for co-eluting human protein pairs in saliva from SARS-CoV-2-infected vs.

Supplementary Fig. 3f), consistent with observed changes in these findings suggest that ancestral and variant SARS-CoV-2 strains may
protein levels in COVID-19 patients31,32. In contrast, the least correlated elicit similar or distinct host responses during infection. Supporting
processes among variants involved host interacting proteins asso- this, large variations in viral protein abundance were observed in
ciated with Ca2+ signaling and other cellular functions (Fig. 3i), indi- patients’ saliva during infections with variants compared to the
cating distinct cellular responses between variants and the ancestral ancestral virus (Supplementary Fig. 3g). For instance, ORF3A levels
virus. Further analysis of differential networks revealed 224 human were higher in Delta than in Omicron, while the innate immune
proteins in a variant exhibiting varying degree of autocorrelation with antagonists ORF6 and N were elevated in Omicron compared to other
the ancestral virus (Fig. 3j). For instance, fewer than half of these variants or the ancestral virus, aligning with reported Omicron infec-
proteins (44%, 99 of 224), including those involved in epigenetics and tion patterns9. A comparison of immune, epithelial, myeloid, and
immunoglobulin heavy or kappa variable regions, showed high auto- connective tissue compartments in host salivary glands revealed that
correlation ( >0.25) with both the ancestral virus and all three variants each SARS-CoV-2 strain induced 143 unique host interacting protein
(Supplementary Data 17). Conversely, fewer than one-tenth (8%; 17 of pairs during infection (Supplementary Fig. 3h, i; Supplementary
224) of proteins, such as copper-containing ceruloplasmin and cardiac Data 18). Among these, 31 host proteins from compartments such as
titin, exhibited low autocorrelation ( < 0.25), showing marked differ- immune (Tc subtype 2), epithelial (duct, mucous acini, myoepithelia),
ences between the ancestral virus and its variants (Fig. 3j). These myeloid (M1/M2-macrophages), and connective (fibroblasts, arterial,

Nature Communications | (2025)16:5784 6

Article https://doi.org/10.1038/s41467-025-60949-1

a SARS-CoV-2 viral proteins (Merged network)

b Host factors common to SARS-CoV-2
proteins featured in ‘Panel a’ inset
c 10 d Human interacting proteins (n = 1,279) E
M

N9 Microglia
N

(AP-MS)
Fold enrichment
NSP10 N N NSP1

(FDR ≤ 5e-2)
NSP5.C145A NSP10
ORF9C NSP10 NSP10 NSP11
SPIKE NSP11 NSP11 5 NSP12
NSP7 NSP13
SARS-CoV-2 viral proteins (Merged network)

ORF6 NSP12 NSP12 NSP14

M0 inactivated M1 activated
NSP8 Nsp15
NSP15 NSP15

(AP-MS)
NSP3 NSP16
ORF8 0 NSP2
NSP4 NSP4 NSP4 NSP3
ORF7A NSP4

COPI-coated vesicle
Glycolysis
Response to IL-7
NADH metabolism
ER to Golgi vesicle
Intrinsic apoptosis
ATP biosynthesis
Respiration
Vesicle organization
Endocytosis
Cytoskeleton
Autophagy
Response to stress
NSP6 NSP5
NSP13 NSP5 NSP5 C145A
NSP14 NSP6
NSP2 NSP7

(AP-MS)
NSP1 NSP8
NSP16 NSP6 NSP6 NSP9
M ORF10
ORF3A ORF3A
ORF3B NSP7 NSP7 ORF3B
ORF10 ORF6
ORF7B
ORF7A

saliva (CF-MS)
E ORF7A ORF7A Enriched GO terms

CoV-2 patient
ORF9B ORF7B
NSP9 ORF9C ORF9C ORF8
NSP12 ORF9B
NSP11 SPIKE SPIKE ORF9C
NSP15 SPIKE
NSP5
N Average RF score

f
NSP10

ORF3B
ORF10
ORF7B
E
ORF9B
NSP9
NSP12
NSP11
NSP15
NSP5
N
NSP5.C145A
ORF9C
SPIKE
NSP7
ORF6
NSP8
NSP3
ORF8
NSP4
NSP6
NSP13
NSP14
ORF7A

NSP2
NSP1
NSP16
M
ORF3A

172 Human protein across cell lines/saliva

SARS-CoV-2 protein 0.0 1.0

Spearman correlation Drug target
Viral-host PPIs
g Human MPC subunits
targeted by drugs
e Neurological disorders
Neuroinflammation
−1 1 0 150 300

Number of drugs
CoV-2 saliva M1 activated Microglia 2-5
(CF-MS) (AP-MS) (AP-MS) 6-10
PGK1
CLIC1 11-15
EIF4G1 16-20
CCT2
ATP2B3 21-30
AAAS >30
VAPA
DHCR7
VAPB
ATP13A1
h Drugs targeting ten (or more)
human host factors
Human interacting proteins

PRKCD MPC

Hydroflumethiazide
AIFM1 subunits
SOAT1
MFN2 53 1 3

Etacrynic acid
Fostamatinib
NDUFA5 2 4

Selumetinib
Binimetinib
Trametinib

AMP-PNP
GAK

Quercetin
Artenimol
Bosutinib
NDUFA2

Miglitol
PEITC
UBQLN4

FAD
GIGYF2
LRBA
MARK1
MAP2K2

by drugs (Supplementary Data 25)

ACSL4
SNRNP70
VPS13A

MPC subunits targeted

PDIA6 Overlooked SARS-CoV-2-host multiprotein complexes (MPCs) Human protein
NEK7
SLC25A5 SARS-CoV-2 protein
TUBB 172 53 IGHV3-23 IGKV3-7
RHOT2 ATP5ME Prior knowledge
IGHG3 Src
TUBA4A SPIKE
IGKV2-30 This study BTK
PDIA3 NDUFA4 NSP10
EEF1A2 IGLV8-61
TUBB4A FGG SLC12A1
TUBA1A NSP10 FGA ORF7A NSP5 IGKV3D-7
SARS-CoV-2 viral proteins FGB IFIT5 IGLV3-19
NSP3 SLC25A42 IGHV4-59
RF score NSP3 IGLL1
0.5 1.0

Fig. 4 | Merged network links SARS-CoV-2 to neurological and druggable host absent in SARS-CoV-2 patient saliva are linked to neuroinflammation and neuro-
factors. a Spearman correlation between SARS-CoV-2 viral protein pairs based on logical disorders. f Predicted MPCs (n = 173) showing SARS-CoV-2 (yellow rhombus)
their shared human proteins in the merged (AP-MS and CF-MS) network. b Sankey and human (gray circle) proteins, with potential drug targets highlighted in red
plot showing shared host proteins among the correlated SARS-CoV-2 viral proteins circle or rhombus. Viral-host PPIs are represented by green lines. Zoomed-in views
from a. c GO biological term enrichment for host factors shared among correlated of selected MPCs illustrate both established (dashed lines) and new (thick lines)
viral proteins in b. P-values from one-sided Fisher’s exact test and adjusted for FDR interactions. g Human MPC subunits interacting with SARS-CoV-2 proteins are
≤ 5e−2 with the BH method. d Analysis of SARS-CoV-2-host PPIs (n = 1279) in M0 and targeted by at least one of 7626 drugs from DrugBank. h Heatmap of 13 drugs
M1 macrophages and N9 mouse microglia (human orthologs) vs. SARS-CoV-2 targeting 10 or more human host factors across 52 MPCs, with each MPC subunit
patient saliva. e Host-viral PPIs detected in M1 macrophages and/or microglia but targeted by at least two drugs. Source data are provided as a Source Data file.

venules, capillaries) tissues showed stronger responses during variant 30% for interactions with human proteins (Supplementary Fig. 4b),
infections compared to the ancestral SARS-CoV-2. Notably, Omicron underscoring their critical roles in the viral life cycle. Beyond co-IP
responses closely resembled Alpha strain, highlighting differences in (Supplementary Fig. 1g), we independently confirmed one-tenth (10%,
how these variants regulate host inflammatory and immune responses. 637 of 6097) of the SARS-CoV-2-human PPIs from the merged network
in blood plasma from four SARS-CoV-2-infected individuals using CF-
Merged network links SARS-CoV-2 proteins to neurological and MS (Supplementary Fig. 4c and Supplementary Data 19). Among these,
druggable host factors 139 PPIs were also detected in various human cell lines or in saliva from
To understand the shared and unique interactions of host proteins SARS-CoV-2-infected patients, strengthening the clinical relevance of
with each SARS-CoV-2 viral proteins in infected human cell lines and these interactions in a physiological context and aiding in the refine-
patient saliva, we integrated AP-MS and CF-MS datasets into a ‘merged’ ment of therapeutic target selection.
network, identifying 6097 viral-host PPIs (Supplementary Data 19). The Correlated profiles of stably associated components can predict
merged network exhibited a power-law degree distribution with fewer functional links between proteins30. We therefore calculated correla-
degrees of separation (Supplementary Fig. 4a), indicative of a scale- tions among all SARS-CoV-2 viral protein pairs based on their inter-
free network. Integration of CF-MS data with AP-MS improved overlap actions with human proteins in the merged network, identifying
with BioGRID interactors from 2296 to 2512 (Fig. 1e and Supplementary several pairs with positively correlated profiles. Notably, strong cor-
Data 20), confirming the robustness of our findings across organ cell relations (average r = 0.6) were observed, with high similarity in the
lines and SARS-CoV-2-infected patient saliva. Analysis of host interac- host proteins shared between NSP5/11/12/15 and nucleocapsid (N)
tions with each viral protein revealed that, consistent with BioGRID proteins, between NSP4/6 and ORF7A (average r = 0.7), and between
data, SARS-CoV-2 proteins NSP6, S, and ORF3A/7 A ranked in the top NSP7/10, S and ORF9C (average r = 0.7; Fig. 4a, b and Supplementary

Nature Communications | (2025)16:5784 7

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Data 21). These correlations align with intraviral PPIs of SARS-CoV-2 human proteins (Fig. 4h and Supplementary Data 25). For example,
identified through compartmentalization-aided interaction screening fostamatinib targets Src (intracellular protein-tyrosine kinase) asso-
in cells33 and Y2H6 methods. Analysis of host proteins interacting with ciation with NSP11, and BTK (Bruton’s tyrosine kinase) interaction with
these correlated viral protein pairs revealed significant enrichment NSP9 (Supplementary Fig. 4j), consistent with prior studies high-
(FDR ≤ 5e−2) in bioprocesses like glycolysis, respiration, and ER-to-Golgi lighting these as key anti-SARS-CoV-2 and antiviral targets37,38. Among
vesicle transport, among others (Fig. 4c). These findings highlight that drugs (binimetinib, bosutinib, fostamatinib, selumetinib, trametinib)
many SARS-CoV-2 proteins target overlapping human proteins or targeting MEK2/ MAP2K2 interactions with NSP3, selumetinib, trame-
those involved in shared bioprocesses4,15. tinib, and binimetinib act as MEK inhibitors. Selumetinib reduces
In COVID-19 or long COVID patients, SARS-CoV-2 infection can SARS-CoV-2-induced lung damage39, and trametinib blocks influenza A
impair neurological and cognitive issues by targeting microglia, which virus propagation and cytokine expression40. These findings suggest
release M1-like proinflammatory cytokines, causing inflammation and host-directed drug targets from MPCs offer a viable strategy for
neurotoxicity34,35. Another critical immune component affected are prioritizing therapeutics for SARS-CoV-2 treatment.
macrophages, where functional changes, such as cytokine storms are
harmful to the host36. To understand how SARS-CoV-2 alters protein Viral-human MPCs from the merged network reveal new insights
assemblies in these immune cells, we analyzed 1279 host interacting into SARS-CoV-2 biology
proteins from 30 Strep-tagged SARS-CoV-2 viral proteins (including From the merged network, we explored two overlooked virus-host
the NSP5-C145A mutant) expressed in inactivated (M0) or activated MPCs, and discussed below in terms of the molecular mechanisms
(M1) macrophages and microglia, compared to SARS-CoV-2 patient involving previously unrecognized roles of viral proteins interacting
saliva (Supplementary Data 22). This analysis identified proteins spe- with host machinery to reveal functional insights and host-targeting
cific to macrophages (M0, M1), microglia, or saliva, as well as those mechanisms.
shared by M1 macrophages and/or microglia but not in SARS-CoV-2-
infected patient saliva (Fig. 4d and Supplementary Fig. 4d). Notably, 34 NSP3-fibrinogen complex confers coagulation defects
host factors linked to M1 macrophages and microglia, involved in We identified unexpected interactions within MPC 172 involving NSP3/
neuroinflammation (e.g., ACSL4, NEK7, ATP13A1) or neurological dis- 10, ORF7A, and S proteins associated with the fibrinogen complex,
orders (e.g., AIFM1, eEF1A2, EIF4G1, CCT2, MFN2, TUBB4A, UBQLN4, comprising fibrinogen alpha (FGA), beta (FGB), and gamma (FGG), in
VAPB) were absent in SARS-CoV-2 patient saliva (Fig. 4e). Additionally, SARS-CoV-2 patient saliva (Fig. 4f). Specifically, NSP3’s interaction with
6-28 SARS-CoV-2 viral proteins interacted with 19 host factors in M1 fibrinogen subunits, essential for blood clotting and thrombus
macrophages and/or microglia, including proteins involved in RNA formation41, led us to hypothesize that secreted NSP3 may disrupt
metabolism (DDX3Y), translation (EIF4A2, EEF1A2), cytoskeleton normal coagulation by promoting abnormal clot formation. In-silico
(actin, ß-Tubulin), cell adhesion (RAP1A), and hub functions (HSPA2, analysis revealed that NSP3 contains a secretion signal peptide
NPM1) (Supplementary Fig. 4e, f). These interaction patterns suggest (Fig. 5a), N- or O-link glycosylation sites (Fig. 5b), and four trans-
SARS-CoV-2 are likely to leverage these host factors to enhance membrane helices (Fig. 5c) necessary for secretion. Consistent with
infection, suppress IFNs to inhibit host innate immunity, and exacer- these predictions, NSP3 was secreted in HepG2 culture supernatants
bate disease severity. (Fig. 5d), similar to the ORF8 SARS-CoV-2 protein42. Since fibrinogen is
To define MPC membership, we used the ClusterONE clustering mainly produced and secreted into the bloodstream by liver hepato-
method to partition the merged network, identifying 173 distinct MPCs cytes, we investigated NSP3’s binding to fibrinogen in HepG2 human
with 29 viral and 2002 unique human proteins (Fig. 4f and Supple- hepatoma cells. Co-IP confirmed NSP3 interaction with FGA in these
mentary Data 23). ClusterONE parameters were optimized using a fibrinogen-expressing cells, but not in non-fibrinogen-expressing
composite score (maximal matching ratio, overlap, accuracy) by HEK293 cells (Fig. 5e). This interaction was further validated inde-
comparing MPCs to CORUM protein complex subunits interacting pendently by co-IP in blood plasma from SARS-CoV-2-infected patients
with SARS-CoV-2 proteins in BioGRID, selecting the highest-scoring and by immunofluorescence, which showed NSP3 co-localizing with
parameters (Supplementary Fig. 4g). Notably, two-thirds (65%, 113 of fibrinogen in SARS-CoV-2-infected Huh-7 cells (Fig. 5f, g). To examine
173) of the MPCs contain 15 or fewer subunits (Supplementary Fig. 4h). whether secreted NSP3 with protease activity might impair fibrin
Of these, 166 include 1273 BioGRID-documented host-viral PPIs, while production and contribute to coagulation defects, we performed an
seven MPCs remain unreported (Supplementary Fig. 4i), offering optical density-based coagulation kinetic assay43. Incubating varying
avenues for SARS-CoV-2 biology exploration. To identify druggable concentrations of purified NSP3 with recombinant fibrinogen showed
host factors for drug repurposing in COVID-19, we analyzed MPC that NSP3 impaired fibrin generation in a time- and concentration-
components against 7626 FDA-approved, experimental, and investi- dependent manner, albeit less effectively than thrombin, which served
gational drugs in DrugBank. Notably, one-fifth (24%, 490 of 2002) of as a positive control (Fig. 5h). Elevated fibrinogen levels were also
human MPC subunits interacting with viral proteins are targeted by at detected in the supernatants of SARS-CoV-2-infected Huh-7 cells
least one drug (Fig. 4g and Supplementary Data 24). For example, (Fig. 5i) and in the saliva of individuals infected with ancestral and
fostamatinib, a tyrosine kinase inhibitor effective in phase II variant SARS-CoV-2 strains (Fig. 5j), aligning with increased fibrinogen
(NCT04579393) and III (NCT04629703) COVID-19 trials, targets host levels reported in severe COVID-19 cases44. Notably, NSP3 when
kinase factors associated with NSPs, ORFs, M, and N proteins (Sup- transfected in HepG2 cells contributed to elevated fibrinogen levels
plementary Fig. 4j). Using co-IP, fostamatinib was shown to disrupt (Fig. 5k), reinforcing its dependence on fibrinogen. Furthermore, pro-
NSP3 interaction with MAP2K2 (mitogen-activated protein kinase inflammatory cytokines (IL-6, CCL2) were elevated in M1 macrophages
kinase 2) in Caco-2, and MAST4 (microtubule-associated serine/ (Fig. 5l), correlating with severe COVID-19 pathology45. These findings
threonine kinase family member 4) in HEK293 cells. Similarly, MARK1 support a model in which secreted NSP3 interacts with fibrinogen
(microtubule affinity regulating kinase 1), which interacts with ORF9B subunits, triggering inflammatory cytokine responses and contribut-
in M1 macrophages, was disrupted by fostamatinib but not by vehicle ing to coagulation abnormalities in severe COVID-19 patients.
(Supplementary Fig. 4k). These results suggest that fostamatinib dis-
rupts SARS-CoV-2 protein-host kinase binding to bolster host immu- NSP3 binds to and de-ISGylates IFIT5 to evade host innate
nity and support its therapeutic potential in SARS-CoV-2 infection. immunity
Conversely, we identified 55 drugs targeting four or more distinct Our network revealed interactions between SARS-CoV-2 proteins
human host factors within 98 MPCs, with 13 targeting over 10 different (NSP1/2/3/5/6/8/9/10/12/14/15, ORF7A) and IFIT (IFN-induced protein

Nature Communications | (2025)16:5784 8

Article https://doi.org/10.1038/s41467-025-60949-1

a 1.0
b AA N-link N-Glyc AA
position
O-Glyc
score
d WCE (HepG2) + –
position Glyc site score Culture supernatent – +
22 NITF 0.7148 176 0.690125
179 NQTT 0.6199 182 0.756688 kDa M NSP3-V5 NSP3-V5
SP (Sec/SPI; type of signal peptide predicted)
Probability

624 NETL 0.5803 183 0.602854

250
0.5 CS (Cleavage site) 722 NPTT 0.6518 186 0.640301
833 NHTK 0.6002 204 0.517802
IB: V5 (NSP3)
OTHER (sequence does not have any SP) 901 NKTV 0.5554 398 0.742646
1236 NPTI 0.5788 400 0.743981
1454 NSTN 0.7549 403 0.756588 100
1457 NVTI 0.5081 422 0.555038 IB: FGA (+ve)
1664 NPTD 0.5670 423 0.5
1805 NVSL 0.6367 424 0.573287 35
0.0
1335 0.529597
KSPNFSK L I N I I I WF L L L SVCLGS L I YSTAA LGV LMSNLGMPSYCTGYREGY LNSTNVT I ATYCTGS I PC 1375 0.5 IB: GAPDH (-ve)
SSSSSSSSSSSSSSSSSSSSSSCXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 1817 0.548822
e Empty NSP3
1400 1435
NSP3 protein sequence
1470 c kDa
100
M vector -V5

f 1.2
TMHMM posterior probabilities

WCE (HepG2)
Healthy plasma COVID-19 plasma IB: FGA (Input)

kDa M 1 2 1 2 3 4
h 0.4
Fibrinogen (Fgn) + Thrombin
250

Probability
(0.1 ug/ml, +ve) 0.8
250 Absorbance at 420 nm Fgn (1 mg/ml, -ve)
Transmembrane
Inside (cytoplasmic) IB: V5 (NSP3)
Fgn + NSP3 (80 ug/ml) Outside (luminal)
IP: FGA IB: NSP3 Fgn + NSP3 (20 ug/ml) 0.4 35
0.2 Fgn + NSP3 (10 ug/ml) IB: GAPDH (LC)
100 Fgn + NSP3 (3 ug/ml)
0.0 100

IP: FGA
Fgn + NSP3 (1 ug/ml)
IB: FGA (IPed, Input) Fgn + NSP3 (0.5 ug/ml) 200 600 1000 1400 1800
IB: FGA (Input)
250 0.0 Fgn + NSP3 (0.25 ug/ml) Amino acid positions of the NSP3 protein

0 60 120 250

g
IB: NSP3 Time (min) Mean FGA fluorescence
intensity in Huh-7 cells
k Mean FGA protein expression
in WCE HepG2 cells (Fig. 5e; %)
IB: V5 (NSP3)
Empty NSP3
DAPI α-NSP3 α-FGA Merge 0 20 40 kDa M vector -V5
0 5 10 15 20 25 60
Mock (uninfected)

100

WCE (HEK293)
IB: FGA

(uninfected)
250

Mock
Non transfected
cells IB: NSP3

P =2.5e-19
P =2.1e-73
35
IB: GAPDH (LC)
CoV-2 infected

SARS-CoV-2

NSP3-V5
130
infected

transfected cells

IP: FGA
IB: FGA

130

l Mean cytokine mRNA fold

IB: NSP3

i Fibrinogen (pg/ml, x103)

j WCE Mean FGA protein expression in 0
(x10³; relative to THP-1 M0)
5 10 15
COVID-1 patient saliva (%)
0 5 10 15 20
kDa M Healthy CoV-2 Alpha Delta Omicron 0 100 200 300 CCL2
THP-1 (M0)
IL-6
Mock IB: FGA

1.1e-22 ≤ P ≥ 2.9e-2
100
3.2e-9 ≤ P ≥ 9.7e-2

(uninfected) Healthy THP-1 (M1)

P = 5.4e-6
CoV-2
SARS-CoV-2 infected

1 THP-1 (M1) + NSP3

35 IB: GAPDH
Huh-7 cells (h)

(LC) Alpha
6 Saliva of healthy and COVID-19 individuals THP-1 (M0)
Delta
12

P = 7.6e-7
Omicron THP-1 (M1)

24 THP-1 (M1) + NSP3

Fig. 5 | NSP3 induces coagulation defects via fibrinogen. a NSP3 with signal fibrinogen, with thrombin-treated and untreated fibrinogen as controls.
peptide, processed by Sec translocon and cleaved by Signal Peptidase I (SPI); SPI i Fibrinogen expression in indicated Huh-7 cells was measured by ELISA over time
sequence in red, with cleavage site and non-signal regions indicated. b Glycosyla- (n = 5 technical replicates; 3.2e−9 ≤ P ≥ 9.7e−2, two-sided paired t-test). j IB of saliva
tion sites (score > 0.5) are mapped by amino acid (AA) position based on NetNGlyc WCEs using FGA antibody; mean FGA levels (n = 10) from respective IB with band
1.0 and NetOGlyc 4.0. c TMHMM predicts four transmembrane regions. d IB of intensities normalized to GAPDH (1.1e−22 ≤ P ≥ 2.9 e−2, one-tailed paired t-test). k FGA
whole cell extracts (WCEs) and supernatants from NSP3-V5 HepG2 cells using anti- levels (P = 2.5e−19, two-tailed paired t-test) in NSP3-V5 vs. empty vector HepG2 cells.
V5 antibody for NSP3, with FGA and GAPDH (1:1,000) as controls. e WCEs and Quantification (n = 10) based on WCEs from panel e using FGA antibody and GAPDH
immunoprecipitates (IPs) from NSP3-V5 HepG2 and HEK293 cells were probed with as LC. l IL-6 and CCL2 mRNA levels were measured in NSP3-transfected and
FGA, NSP3, or GAPDH antibodies, normalized to input FGA. f FGA IPs from COVID- untransfected M1 vs. M0 macrophages (n = 4 biological replicates; P = 7.6e−7 for IL-
19 (n = 4) and control (n = 2) plasma were probed with NSP3 and FGA antibodies. 6, P = 5.4e−6 for CCL2; two-tailed paired t-test). Data are mean ± SEM (g, i-l). IBs are
g Representative micrographs show (n = 7-10) NSP3 co-localization with FGA in representative (n = 2) with molecular weight markers (kDa). A clearer protein ladder
SARS-CoV-2-infected but not mock Huh-7 cells; nuclei in blue. Scale bar, 10 μm. FGA from the same blot was overlaid (dashed line). Source data are provided as a Source
fluorescence in infected vs. mock cells (n = 100, P = 2.1e−73, two-tailed paired t-test). Data file.
h Coagulation was measured at 420 nm over time using varying NSP3 with

with tetratricopeptide repeats) family members (IFIT1/2/3/5, IFIT1B) in cells at various time points (Fig. 6b, c and Supplementary Figs. 5a, b).
saliva from SARS-CoV-2-infected individuals or M0 macrophages IFIT5 mRNA levels were increased in M1 macrophages and further
(Fig. 6a). Supporting this, NSP2 has been reported to interact with IFIT5 enhanced by NSP3 compared to M0 macrophages (Fig. 6d). Given
in HEK293 cells46, and ClusterONE identified MPC 053 linking NSP3, 5 ISG15’s role in modifying IFITs via ISGylation50, a key antiviral defense
and 10 with IFIT5 (Supplementary Data 23). Evidence shows SARS-CoV- against SARS-CoV-247 and other viruses51,52, and NSP3’s de-ISGylating
2-infected macrophages secrete ISG15 (ubiquitin-like interferon- activity in suppressing antiviral responses49, we examined whether
stimulated gene 15) via NSP3, with extracellular free, non-conjugated NSP3 inhibits IFIT5 activation via de-ISGylation. While cellular ISGyla-
ISG15 acts as a cytokine, to exacerbate inflammation47. While immune tion was induced in M1 macrophages, total protein ISGylation
evasion by SARS-CoV-2 proteins is well-studied48,49, NSP3’s role in decreased following NSP3 transfection (Fig. 6e). Similarly, ISGylation
suppressing IFIT5-mediated immunity via its interaction with IFIT5 increased in A549-AT cells after IFN-γ stimulation but declined 12 h
remains unclear. post-SARS-CoV-2infection (Supplementary Fig. 5c). To determine if
We confirmed NSP3’s interaction with IFIT family proteins via co-IP IFIT5 is an ISG15 target, we immunoprecipitated IFIT5 from M1 mac-
in NSP3-transfected M1 macrophages, saliva from individuals infected rophages (Fig. 6f) and IFN-γ activated A549-AT cells (Supplementary
with ancestral or variant SARS-CoV-2 strains, plasma from COVID-19 Fig. 5d) and identified it among higher molecular weight ISGylated
patients, and SARS-CoV-2-infected A549-hACE2-TMPRSS2 (A549-AT) proteins, confirming its ISGylation. However, IFIT5 ISGylation

Nature Communications | (2025)16:5784 9

Article https://doi.org/10.1038/s41467-025-60949-1

a b c d

M1 + NSP3-V5
NSP8 1.00 ORF7A IP: NSP3 IFIT5 mRNA (fold)

SARS-CoV-2*
Sgnificant Avg. RF score
NSP5 0 40 80 120
NSP2

M1 + NSP3-V5
NSP14

SARS-CoV-2*
Omicron*
0.95

Healthy*
NSP10

Alpha*
IFIT5

Delta*
NSP12

Omicron*
NSP3 NSP8

Healthy*
0.90 NSP5 M0

Alpha*
NSP12

Delta*
NSP15 kDa M
NSP2 NSP6 250 IP: IFIT5
NSP6 NSP10

P ≤ 1.0e-4
NSP1 0.85 IB: NSP3 kDa M M1+NT siRNA
NSP14 NSP9 250 IB: NSP3
0.80 NSP1 250 IP: Beads M1+NT siRNA+NSP3
(Input)

P ≤ 7.2e-7
NSP3 IB: NSP3
IFIT1 IFIT1B IFIT2
0.75 55 IB: IFIT1 M1+IFIT5 KD
SARS-CoV-2 interactions 55 IB: IFIT5
with IFIT-family proteins (IP’ed, Input)
*Saliva from healthy or 55 IB: IFIT2 M1+IFIT5 KD+NSP3

ORF7A NSP9 NSP15 COVID-19 individuals

Viral-host PPIs (AP-MS, THP-1 M0) IFIT1 IFIT2 g IFN-ß protein release (pg/ml)
55 IB: IFIT3
M1+NSP3+IFIT5-VA OE

Viral-host PPIs (CF-MS, 0 225 450 55 IB: IFIT5 NSP3 mRNA (fold)
IFIT1B IFIT5
SARS-CoV-2-infected subjects)
Known viral-host PPIs Human proteins CoV-2 proteins
*Saliva from healthy or 0 1 2
M0 COVID-19 individuals

P ≤ 2.7e-2

P ≤ 1.0e-6
M1+NT siRNA M1+NT siRNA+NSP3

NSP3-V5 - -
WCE

+
M1+NT siRNA+NSP3 j NSP3
M1+IFIT5 KD+NSP3

P ≤ 9.5e-3
kDa M M0 M1* M1 M1+IFIT5 KD

P ≤ 4.4e-2
250 ISG15
M1+IFIT5 KD+NSP3 M1+NSP3+IFIT5-VA OE
130
*ISGylated proteins

M1+IFIT5-VA OE
100

70
M1+NSP3+IFIT5-VA OE
IFIT5
k 1 2 3 4 5 6 7 8 9

55
h mRNA fold (x102;
normalized to M0)
THP-1 (M1, -ve ctrl)
NSP3-V5
+ + + + +
- - + - +
+ + + +
- + - +
0 5 10 15 IFIT5-VA (WT)
M0 M1+NT siRNA - + + - - - - - -
35
25 M1+NT siRNA+NSP3 IFIT5 K160R-VA - - - + + - - - -
IRF3- or NF-kB mediated genes

***

15 ISG15 M1+IFIT5 KD M953 Y916

IL-5 IFIT5 K197R-VA - - - - - + + - -
**

M1+IFIT5 KD+NSP3
** *

10 IB: ISG15 IFIT5 K206R-VA - - - - - - - + +

N88 K206
M1+IFIT5-VA OE
250 M1+NSP3+IFIT5-VA OE F149 1
IB: V5 (NSP3) L145
**
**

IFNG
K197
*P ≤ 5e-2 **P ≤ 1e-2 ***P ≤ 1e-3 kDa 1 2 3 4 5 6 7 8 9
35 IB: GAPDH (LC) E948
IB: ISG15
*

70
**

IFNB1
**

THP-1 K160
IP: FLAG (IFIT5)
f IP: IFIT5
*

IFNA17 2
**
**

2 3 1 2 3 4 5 6 7 8 9
NSP3-V5 - - + P ≤ 8.0e-21
Band intensties (normalized

(normalized to IFIT5 WT; %)

IB: V5
to IFIT5 WT+ NSP3; %)

kDa M M0 M1 M1 150 130

P ≤ 1.4e-13 150 (NSP3)
250

i NF-kB activity IP: FLAG (IFIT5)

Band intensties
130
100 (%; relative to M1)
70 Modified 75
0 75 150 75 3
IFIT5 1 2 3 4 5 6 7 8 9
55
IB: FLAG
M0 55 (IFIT5, Input)
35 0 0 IP: FLAG (IFIT5)
IFIT5-VA (WT)+NSP3-V5

IFIT5 K160R-VA+NSP3-V5

IFIT5 K197R-VA+NSP3-V5

IFIT5 K206R-VA+NSP3-V5
P ≤ 6.5e-5

IFIT5-VA (WT)

IFIT5 K160R-VA

IFIT5 K197R-VA

IFIT5 K206R-VA
M1+NT siRNA
25 M1+NT siRNA+NSP3
P ≤ 2.0e-2

M1+IFIT5 KD
P ≤ 4.1e-2

IB: ISG15
15
M1+IFIT5 KD+NSP3
10
IB: IFIT5 M1+IFIT5-VA OE
55
(Input) M1+NSP3+IFIT5-VA OE
THP-1

Fig. 6 | NSP3 antagonizes IFIT5 to evade innate immunity. a IFITs targeted by PCR (h), and NF-κB activity by ELISA (i) were measured in samples from panel
SARS-CoV-2 proteins via known and newly identified interactions by AP-MS (M0 d (data are mean ± SEM; n = 3-6 biological replicates; 6.5e−5 ≤ P ≥ 4.4e−2 or sig-
macrophages) and CF-MS (SARS-CoV-2-infected patient saliva). Scatter plot shows nificance with asterisks by one or two-sided paired t-tests). j Structural docking of
SARS-CoV-2 interactions with IFITs (RF > 0.75). IFIT5 (b) or NSP3 (c) IPs from indi- the ISG15-IFIT5 interface with NSP3 (PDB ID: 6YVA); full data available in Source file.
cated samples were probed with NSP3 or IFIT antibodies. d qRT-PCR of IFIT5 and k IPs of wild-type (WT) and mutant IFIT5-VA with or without NSP3-V5 in M1 mac-
NSP3 transcripts in IFIT5 knockdown (KD) and non-targeting (NT) siRNA M1 mac- rophages were probed using ISG15 (k-1), V5 (NSP3, k-2), and FLAG (IFIT5-VA, k-3)
rophages, with or without NSP3, and in NSP3 M1 cells overexpressing (OE) IFIT5-VA; antibodies. Plot k-2 shows NSP3-V5 band intensities (Lanes 3, 5, 7, 9) matched to
M0 served as negative control (data are mean ± SEM; n = 3-4 biological replicates; corresponding IFIT5-FLAG samples, normalized to WT IFIT5-VA transfected with
7.2e−7 ≤ P ≥ 1.0e−4; two-sided paired t-test). e ISG15 IB of WCEs from M1 and NSP3-V5 NSP3-V5. Plot k-3 shows IFIT5-FLAG intensities (Lanes 2, 4, 6, 8) for WT and mutants,
M1 macrophages shows ISGylation/de-ISGylation; NSP3 and GAPDH (LC) were normalized to WT IFIT5-VA (Lane 2). Data are mean ± SEM (n = 10; 4.6e−24 ≤ P ≥
probed with V5 and LC antibodies. A clearer protein ladder from the same blot is 8.0e−21; 1.5e−19 ≤ P ≥ 1.4 e−13 by one or two-sided paired t-tests). Representative IBs
overlaid on the membrane (dashed line). f IFIT5 IPs probed with ISG15 show IFIT5 (n = 2) include molecular weight markers (kDa). Source data are provided as a
ISGylation in M1 macrophages, and reduced in NSP3-V5 cells. IFIT5 levels verified by Source Data file.
IFIT5 antibody. IFN-β by ELISA (g), IRF3 and NF-κB target gene expression by qRT-

decreased in SARS-CoV-2-infected A549-AT cells at 12 h post-infection responses. This finding is supported by the observation that IFIT5
(Supplementary Fig. 5d), which is due to NSP3, as a similar reduction knockdown diminished IFN-β production and IRF3/NF-κB-driven gene
(Fig. 6f) was observed in NSP3-transfected M1 macrophages. transcription in NSP3-transfected cells (Fig. 6g, h). NF-κB activity was
To assess whether NSP3 mediates the decrease in IFIT5 ISGylation, also declined in NSP3-transfected M1 macrophages regardless of IFIT5,
we depleted IFIT5 in NSP3-challenged M1 macrophages and observed a but the ectopic expression of IFIT5 restored NF-κB levels (Fig. 6i).
greater reduction in IFIT5 mRNA levels compared to NSP3 transfection These results establish NSP3 as a negative regulator and IFIT5 as a
alone (Fig. 6d and Supplementary Fig. 5e). Silencing IFIT5 in M1 cells positive regulator of IRF3 and NF-κB activation in innate immune
with NSP3 significantly reduced NSP3 transcript levels, indicating that response.
NSP3 depends on IFIT5 (Fig. 6d). Given NSP3’s role in regulating IFN Since IFIT5 potentiates IFN and IRF3 signaling in antiviral defense,
and NF-κB pathways49, we evaluated its effect on IFN-β production, we probed whether IFIT5 serves as a key link between MAVS and TBK1
IRF3 (IFN regulatory factor-3)-activated INF-I (IFNA17, B1) and INF-II in the signalosome. Immunoprecipitation of IFIT5 from M1 macro-
(IFNG), and NF-κB-mediated pro-inflammatory cytokine (IL-5) gene phage lysates, with and without NSP3, revealed enhanced binding of
expression in M1 macrophages with or without IFIT5. NSP3- IFIT5 to key IFN signaling effectors, including IκB kinase (IKK α/β/γ/ε),
transfection reduced IFN-β release and mRNA levels of IRF3- or NF- MAVS, TBK1, and IRF3, in the presence of NSP3 (Supplementary
κB-responsive genes, while IFIT5 overexpression enhanced these Fig. 5f, g). These factors are crucial for IRF3 and NF-κB pathway

Nature Communications | (2025)16:5784 10

Article https://doi.org/10.1038/s41467-025-60949-1

activation, suggesting IFIT5 bridges MAVS and TBK1. In contrast, IFIT5 Peptide treatment also reduced mRNA levels of proinflammatory
depletion reduced NSP3’s binding to these effectors, but over- cytokines (IL-1β, IL-6, TNF-α, PGE2) and the inflammatory marker
expression of IFIT5 in NSP3-transfected M1 macrophages restored (eNOS) in SARS-CoV-2-infected Huh-7-ACE2 cells compared to
effector interactions (Supplementary Fig. 5h, i), suggesting that IFIT5 untreated controls (Fig. 7c). Peptides decreased infectivity of ancestral
binding is critical for NSP3 to suppress IRF3 and NF-κB activation. and variant SARS-CoV-2 strains in Vero E6-TMPRSS2 cells dose-
Although human IFIT proteins share 40-56% sequence identity dependently by plaque assays (Fig. 7d), and inhibited S protein activ-
(Supplementary Fig. 5j), IFIT5 and its orthologs exhibit high similarity ity in HEK293T cells over time using a pseudovirus entry assay (Sup-
(72-99%; Supplementary Fig. 5k), indicating a shared ancestral gene. plementary Fig. 6c). Similarly, peptide-treated SARS-CoV-2-infected
Molecular docking using AlphaFold structures of ISG15 and IFIT5 with Huh-7 cells showed marked inhibition of cytoplasmic S protein loca-
the NSP3 crystal structure identified three conserved lysine residues lization (Supplementary Fig. 6d), confirming their ability to block S
(K160, K197, K206) on IFIT5 at the interaction interface with ISG15 and protein entry. The peptides showed specificity, with no toxicity (in
NSP3 (Fig. 6j). This supports the IFIT5-ISG15-NSP3 association and Vero E6-TMPRSS2 or A549-AT cells (Supplementary Fig. 6e), and no
ISG15’s ability to conjugate lysines through ISGylation53. To confirm RNA-level changes in Huh-7 cells transfected with other human cor-
these lysines at IFIT5-ISG15 interface as ISGylation sites, we generated onaviruses (HCoV-229E, OC43; Supplementary Fig. 6f). ProtParam
three single-site IFIT5 mutants by substituting lysine residues with analysis revealed favorable physicochemical properties of these AVPs,
arginine (K160R, K197R, K206R). Mutants VA-IFIT5 K197R and K206R including an aliphatic index of 66.4-87.4, a half-life of 1.4-100 hrs, and
showed reduced ISGylation with or without NSP3, while K160R negative GRAVY scores (Supplementary Fig. 6g), suggesting they are
exhibited wild-type IFIT5 ISGylation levels, but only in response to thermally stable and hydrophilic.
NSP3 (Fig. 6k-1), indicating K197 and K206 as key ISGylation sites on To confirm that peptides block SARS-CoV-2 or variant S protein
IFIT5. Notably, these IFIT5 mutants impacted SARS-CoV-2 infection, RBD binding to ACE2, surface plasmon resonance was performed
affecting viral replication and titers in supernatants and lysates in using C-terminal histidine-tagged recombinant wild-type and mutated
CRISPR IFIT5 knockout and CRISPR-resistant IFIT5 single-site mutants (Delta variant with T478K L452R mutations, Omicron variant with 16
(K197R, K206R) in IFIT5 knockouts at 4, 8, and 12 h post-infection in mutations) S RBD proteins, expressed in Expi293 cells. The peptides
A549-AT cells. However, these effects were not seen in IFIT5 knockout exhibited high-affinity binding (KD = ~ 30.5 ± 3.5 nM; Fig. 7e) to all S
cells complemented with wild-type CRISPR-resistant IFIT5 (Supple- RBDs, and effectively inhibited S-RBD:ACE2 interactions. In silico
mentary Fig. 5l, m). modeling of SARS-CoV-2 RBD-ACE2 crystal structures57 showed pep-
We then assessed how these mutations affect IFIT5-NSP3 binding tides bind to conserved S RBD residues (N487, T500, Y449/453/489/
by immunoprecipitating wild-type and mutant VA-IFIT5 from M1 505) and SARS-CoV-2 or variant-specific residues (E484, F456, L455,
macrophages, while probing for NSP3. The IFIT5 mutants disrupted N501, Q493, Y473), forming interfaces with ACE2 (RBD:E484-ACE2:K31-
IFIT5-NSP3 binding at the interface, albeit with lower affinity than wild- AVP2:S4/T3; RBD:Y453-ACE2:H34-AVP3:Y18; Supplementary Fig. 7a).
type IFIT5, as shown by quantified band intensities (Fig. 6k-2). This These findings underscore peptide residues’ role in disrupting S-
disruption was specific, as IFIT5 mutants showed slightly higher RBD:ACE2 binding.
expression levels than the wild-type IFIT5 (Fig. 6k-3), suggesting Using a CF-MS framework similar to that applied to SARS-CoV-2-
ISGylation affects IFIT5 stability. These findings support a model where infected patients’ saliva (Fig. 3a), we evaluated whether altered co-
NSP3 interacts with ISG15 and IFIT proteins to suppress ISG15- eluting human proteins in SARS-CoV-2-infected Huh-7 cells could be
dependent ISGylation of IFIT5 at K197 and K206. This suppression restored by treating them with peptides AVP1 and AVP2. SEC-MS
frees ISG15 to enhance pro-inflammatory cytokine secretion linked to analysis of 384 fractions from soluble protein extracts showed that
COVID-19 cytokine storms. Furthermore, while IFIT5 acts as an adaptor the hierarchical clustering of 1189 human proteins profiles (Fig. 7f;
linking IFN effectors to activate IRF3 and NF-κB, NSP3 inhibits these Supplementary Data 26) from peptide-treated cells had elution pat-
pathways in the presence of IFIT5. terns more similar to mock (uninfected) than virus-infected cells.
PCC analysis of co-eluted human protein profiles identified 4539
Deep learning-based peptides impede ancestral SARS-CoV-2 and protein pairs grouped into three clusters, with positively (cluster 1)
variant replication and negatively (cluster 3) correlated PPI pairs in peptide-treated
While vaccines boost immunity and protect against COVID-19, their cells, resembling those in mock cells more closely than in untreated
efficacy may be reduced by emerging SARS-CoV-2 variants. Antiviral SARS-CoV-2-infected cells (Fig. 7g; Supplementary Data 27). GO cel-
peptides (AVPs), on the other hand, offer prophylactic and therapeutic lular component analysis of clusters 1 and 3 showed enrichment for
benefits due to their specificity and low toxicity. AVPs target viral proteins in endosomal sorting, pre-mRNA splicing, and mitochon-
proteins like the S protein to block host receptor binding (e.g., ACE2, drial biogenesis (Supplementary Fig. 7b). These findings suggest
TMPRSS2), inhibit viral infection or replication, disrupt PPIs, and peptides restore host protein assemblies hijacked by SARS-CoV-2,
compete for binding by mimicking protein surfaces54. Currently, reflecting antiviral efficacy by targeting virus-dependent host
21 synthetic peptides are under implementation for COVID-19 pathways.
treatment55, indicating the potential for discovering effective pep- To examine changes in viral-host PPIs, we compared correlation
tides as promising therapeutics for this virus and future outbreaks. scores of protein pairs in SCoV-2-infected Huh-7 cells treated with or
Since the S glycoprotein RBD binds the human ACE2 receptor for viral without peptides. Applying a 5% FDR, we generated a differential viral-
entry56, we postulated that AVPs blocking this interaction could counter host PPI network (Fig. 7h; Supplementary Data 28), identifying 48
emerging variants. Using the In-Silico Protein Synthesizer (InSiPS11), we protein pairs highly correlated in untreated SCoV-2-infected Huh-7
designed synthetic peptides targeting the RBD or S1/S2 cleavage site of cells but weakly correlated in peptide-treated cells (Fig. 7h). For
the S protein, critical for viral entry. InSiPS generates random sequen- example, the strong correlation (r = 0.6) between SARS-CoV-2 ORF9B
ces and predicts high-affinity peptides without requiring 3D structural and the mitochondrial antiviral signaling protein MAVS in untreated
input (Supplementary Fig. 6a). Among 27 peptides designed (15 tar- SARS-CoV-2-infected cells reflected their physical coupling and role in
geting RBD, 12 at the S1/S2; Fig. 7a), three RBD-binding peptides suppressing IFN production57. However, in peptide-treated cells, this
reduced SARS-CoV-2 RNA by 54-95% and S protein expression in Huh-7 association was poorly correlated (r = -0.08 to 0.06), suggesting that
cells at 0.27-2.7 µM, as confirmed by real-time PCR and immuno- peptides disrupt ORF9B-MAVS binding, thereby preserving host innate
fluorescence (Fig. 7b and Supplementary Fig. 6b). immune signaling cascades.

Nature Communications | (2025)16:5784 11

Article https://doi.org/10.1038/s41467-025-60949-1

a b Intracellular SARS-CoV-2 RNA

levels in Huh-7 cells (Fold)
d 80
SARS-CoV-2 AVP1 (IC50 : 1.27 μM)
AVP2 (IC50 : 1.42 μM) 100
Alpha AVP1 (IC50 : 6.24 μM)
AVP2 (IC50 : 2.46 μM)

Avg. plaque reduction

S2’ 0 2 4 AVP3 (IC50 : 9.62 μM) 80 AVP3 (IC50 : 1.99 μM)
SS RBD SD2 HR1 CD TM 60
60

P ≤1.1e-4
Vehicle (DMSO) 40
NTD SD1 FP CH HR2 CT
40
S1/S2 SARS-CoV-2
20
20

P ≤ 2.7e-2
15 antiviral peptides (AVP 1-3) CoV-2 + AVP1 (0.13 μM)
12 AVPs 0 0
CoV-2+ AVP2 (0.27 μM) -10 -8 -6 -4 -2 -10 -8 -6 -4 -2

c PGE2
e CoV-2 + AVP3 (2.27 μM)
Beta AVP1 (IC50 : 5.55 μM) Delta AVP1 (IC50 : 6.60 μM)
eNOS mRNA levels of cytokines or inflammatory 80 AVP2 (IC50 : 3.29 μM) 100 AVP2 (IC50 : 1.67 μM)

AVPs inhibit S-RBD

:ACE2 binding (RU)

CoV-2 S-RBD:ACE2
100 1800

or AVP binding (RU)

CoV-2 RBD + AVP1

Avg. plaque reduction

IL-6 marker in Huh-7-ACE2 cells (Fold) AVP3 (IC50 : 8.66 μM) AVP3 (IC50 : 2.49 μM)
CoV-2 RBD + AVP2 80
IL-1β 60
0.0 0.5 1.0 1.5 CoV-2 RBD + AVP3
TNF-α 60
50 900
KD= 44.3 ± 7.66 nm 40

P ≤ 1.6e-5
AVP3 + CoV-2 21.7 ± 2.31 nm 40
AVP2 + CoV-2 24.5 ± 2.30 nm
P ≤ 7.5e-6

AVP1 + CoV-2 23.3 ± 1.55 nm 20 20

SARS-CoV-2 0 0
Vehicle 0 200 400 600 0 200 400 600 0 0
AVP3 + CoV-2
P ≤ 6.7e-8

AVP2 + CoV-2 -10 -8 -6 -4 -10 -8 -6 -4 -2

P ≤ 1.6e-6 P ≤ 6.0e-6

AVP1 + CoV-2
SARS-CoV-2
AVPs inhibit S-RBD
:ACE2 binding (RU) 100 Delta RBD + AVP1 1800 Gamma AVP1 (IC50 : 7.21μM) Omicron AVP1 (IC50 : 7.43 μM)

or AVP binding (RU)

Vehicle

Delta S-RBD:ACE2
120 80
AVP3 + CoV-2 Delta RBD + AVP2 AVP2 (IC50 : 3.65μM) AVP2 (IC50 : 3.04 μM)

Avg. plaque reduction

P ≤ 7.9e-6 P ≤ 7.9e-5 P ≤ 1.8e-4

AVP2 + CoV-2 Delta RBD + AVP3 AVP3 (IC50 : 8.74 μM) AVP3 (IC50 : 8.27 μM)
AVP1 + CoV-2 50 900 KD= 34.4 ± 2.12 nm 60
28.1 ± 9.45 nm 80
SARS-CoV-2
Vehicle 14.1 ± 1.37 nm 40
AVP3 + CoV-2 44.0 ± 5.26 nm
AVP2 + CoV-2 0 0 40
20
P ≤ 2.0e-4 P ≤ 7.8e-5

AVP1 + CoV-2 0 200 400 600 0 200 400 600

SARS-CoV-2
Vehicle 0 0
AVP3 + CoV-2 -10 -8 -6 -4 -2 -10 -8 -6 -4
AVPs inhibit S-RBD
:ACE2 binding (RU)

AVP2 + CoV-2 100 Omicron RBD + AVP1 1800

Omicron S-RBD:ACE2
Log10 [Peptide, M] Log10 [Peptide, M]

or AVP binding (RU)

AVP1 + CoV-2 Omicron RBD + AVP2
SARS-CoV-2
Vehicle Omicron RBD + AVP3
900 KD= 48.1 ± 3.48 nm ACE2 (20 μg/ml; +ve ctrl) SARS-CoV2 S-RBD (50 μM)
f CoV-2 + - + +
50
11.2 ± 1.94 nm
15.6 ± 1.73 nm
56.8 ± 2.68 nm
AVP1 (50 μM)
AVP2 (50 μM)
Delta S-RBD (50 μM)
Omicron S-RBD (50 μM)
AVP1 - - + - 0
AVP3 (50 μM)
0
AVP2 - - - + 0 200 400 600 0 200 400 600
Huh-7 + + + + SRP9 SLC25A13 P4HA1
SLC36A1
1 2 3 4 g Time (sec) Time (sec)
h EIF5A
N
EPS15L1
SSR1

SARS-CoV-2 HNRNPAB TFG ITSN1 SF3B3 SCAMP1

Huh-7 cells

Uninfected/untreated CMPK1 CTSC USP5 ORF9B

CARHSP1 FAU BSG RALY
ATP1B1 GSTK1 ERO1A
1,189 human proteins

SARS-CoV-2 + AVP1 M
RBM14 HUWE1 ROGDI HSPA4L CALD1 CFAP47
SARS-CoV-2 + AVP2 SRRM2 GRIPAP1
POLR1G
?

3 2 1 UBXN4 CGN PCMT1 MAVS

SRRT ITPA EPS15
Clusters based on pairwise FAM3C
SPIKE
co-elution profile correlations HEBP2
Pearson correlation coefficient APIP CEMIP2 PRKDC PPM1G
-0.32

1.00

SARS-CoV-2 proteins ORF8

NSP4 NSP16
Spectral counts 384 SEC-HPLC fractions Human proteins
SMIM20
Viral-host PPIs correlated in SARS-CoV-2-infected
Huh-7 cells but not with AVP1 and AVP2 treatment
0

50
100

200

300

Fig. 7 | Inhibitory peptides reduce ancestral and variant SARS-CoV-2 infectivity. dependent AVP inhibition of ancestral and variant SARS-CoV-2 (n = 2 biological
a SARS-CoV-2 S protein structure shows domains and InSiPS-derived peptides replicates per dose). e Surface plasmon resonance (SPR, right) shows ACE2 (posi-
(arrows) targeting the receptor-binding domain (RBD) and S1/S2 protease cleavage tive control) or AVP binding to His-tagged S-RBD (wild-type, Delta with T478K
site. SS, single sequence; NTD, N-terminal domain; SD1, subdomain 1; SD2, sub- L452R mutations, and Omicron with 16 mutations). SPR (left) reveals AVPs inhibit
domain 2; S2’ (black arrow), protease cleavage site; FP, fusion peptide; HR1, heptad ACE2 binding to wild-type or mutated S-RBD. f SEC-HPLC/MS-based hierarchical
repeat 1; CH, central helix; CD, connector domain; HR2, heptad repeat 2; TM, clustering of co-fractionated protein profiles in SARS-CoV-2-infected Huh-7 cells
transmembrane domain; CT, cytoplasmic tail. b qRT-PCR of viral RNA in Huh-7 cells treated with AVPs vs. mock (uninfected/untreated) control. g Heatmap shows AVP-
treated with SARS-CoV-2 and RBD-binding antiviral peptides (AVPs) at specified treated SARS-CoV-2-infected Huh-7 cells exhibit protein coelution profiles more
concentrations were compared to virus-only or vehicle (DMSO) controls (data are similar to mock (uninfected/untreated) control than to untreated virus-infected
mean ± SEM (n = 3–13 biological samples; P = 8.3e−5 ≤ P ≥ 2.7e−2 or P ≤ 1.1e−4 by one cells. h Viral-host protein pairs (n = 48) show altered correlations in AVP-treated
or two-sided, paired or unpaired t-test). c mRNA expression of markers in SARS- SARS-CoV-2-infected Huh-7 cells, but not in those treated with AVP 1 or AVP 2
CoV-2-infected Huh-7-ACE2 cells treated with AVPs measured by qRT-PCR and (PCC < 0.25). PCC differences for each co-eluted human protein pair were calcu-
compared to untreated controls (n = 3 biological replicates; 6.7e−8 ≤ P ≥ 2.0e−4 by lated across samples, with two-sided Z-transformed P-values adjusted for FDR ≤ 5e−2
two-sided, paired t-test). d Plaque assays in Vero E6-TMPRSS2 cells show dose- using the BH method. Source data are provided as a Source Data file.

Discussion Differential network analysis of saliva from individuals infected

Ancestral SARS-CoV-2 and its variants impact various organs, tissues, with SARS-CoV-2 and its variants revealed notable variations in viral
and bodily fluids, enabling viral transmission and immune evasion, protein abundance and host responses, particularly in inflammatory or
potentially leading to multiorgan failure and death. Despite extensive immune pathways like the immunoglobulin kappa variable cluster.
studies on SARS-CoV-2-human PPIs using experimental methods4–7, Certain host interacting proteins from immune cells, epithelial, myeloid,
our understanding of context-dependent host interactions with SARS- and connective tissues in the salivary glands were markedly induced
CoV-2 and its variants at physiological infection sites remains incom- during variant infections. The merged network showed NSP6, S, and
plete. To address this, we employed AP-MS across eight cell lines from ORF3A/7A relying heavily on host factors, while other SARS-CoV-2
five mammalian organs susceptible to SARS-CoV-2 infection. This proteins (E, M, N, NSPs, S, ORFs) linked to M1 macrophage- or microglia-
analysis revealed hundreds of PPIs, shared or unique to different organ specific human proteins were associated with neuroinflammation or
cell lines, including those with the S protein of SARS-CoV-2 and its neurological disorders. Overall, identifying viral proteins dependent on
variants. Rigorous validation using complementary analyses, such as host factors altered during SARS-CoV-2 or variant infections is essential
CF-MS profiling of patient saliva carrying SARS-CoV-2 or its variants, for understanding COVID-19 pathophysiology, developing host-
mapped reconfigured protein assemblies during infection. directed therapies, and uncovering mechanisms of action. Mapping

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SARS-CoV-2 ancestral and variant-host PPIs revealed 173 human protein board, adhered to University of Regina ethical standards, with written
subunits within predicted MPCs targeted by at least one of the 7626 informed consent obtained from all participants. For blood collection,
cataloged drugs. This includes fostamatinib, a phase 3 oral spleen tyr- BD Vacutainer tubes (Thermo Fisher Scientific) treated with lithium
osine kinase inhibitor for COVID-19, which targets 25 kinases associated heparin were used for plasma separation. Stimulated whole saliva were
with NSPs, ORFs, M and N SARS-CoV-2 proteins in MPCs. Additionally, 13 collected into sterile polystyrene tubes from both healthy and COVID-
drugs target 10 or more subunits within 52 MPCs connected to various 19 patients after chewing paraffin, sugar-free, or sugared chewing gum
SARS-CoV-2 proteins. While further investigation is needed, targeting for five min, then cooled on ice, and processed for cellular assays.
these host factors may disrupt their interactions with viral proteins,
potentially aiding in SARS-CoV-2 or variant infection treatment. Cell lines, viruses, plasmids, and reagents
Among the context-specific host-viral PPIs identified, our prob- Mammalian cell lines and coronaviruses (HCoV-OC43, 229E) were
abilistic network revealed previously unnoticed associations in SARS- obtained from the American Type Culture Collection, USA. Vero E6
CoV-2 and variant biology, supported by biochemical, genetic, and cells expressing the transmembrane serine protease TMPRSS2 (Vero
cell-based assays. Key findings include the interaction of the secreted E6-TMPRSS2) were sourced from the Japanese Collection of Research
NSP3 viral protein with human fibrinogen complex subunits (FGA, FGB, Bioresources Cell Bank (JCRB1819), and the human alveolar epithelial
FGG), crucial for initiating inflammatory cytokine responses and cell line A549, expressing angiotensin-converting enzyme 2 (ACE2) and
causing coagulation abnormalities in COVID-19 patients. Our inquiry TMPRSS2 (A549-AT) was from InvivoGen. Ancestral SARS-CoV-2
into NSP3 binding with IFN-induced proteins, particularly IFIT5, in LPS- (VIDO/01/2020) was provided by Darryl Falzarano from VIDO-INTER-
IFN-γ-activated or NSP3-transfected M1 macrophages and saliva from VAC, and variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta
ancestral or variant SARS-CoV-2-infected individuals, revealed sig- (B.1.617.2), and Omicron (B.1.1.529) were isolated from COVID-19
nificant findings. Specifically: (1) NSP3 reduces ISGylation of IFIT5; (2) patients. Viruses were propagated at a low multiplicity of infection
IFIT5 acts as an adaptor linking MAVS, TBK1, and IFN effectors to (MOI = 0.0001) in HCoV-OC43 and 229E-infected Huh-7 cells, and
trigger IRF3 and NF-κB pathways, suppressed by NSP3-IFIT5 interac- ancestral or variant SARS-CoV-2-infected Huh-7-ACE2, Vero E6-
tion; (3) IFIT5 lysine residues (K160, K197, K206) interface with ISG15 TMPRSS2, and A549-AT cells with media containing 2% heat-
and NSP3; and (4) NSP3 inhibits IFIT5 ISGylation at K197 and K206, inactivated fetal bovine serum (FBS). Viral stocks were stored at
releasing free ISG15 to promote pro-inflammatory cytokine secretion, −80 °C and titrated via TCID50 (50% tissue culture infective dose) or
driving cytokine storms linked to COVID-19 severity. These insights plaque-forming assays. Expression plasmids for SARS-CoV-2 open
into NSP3 dependency on IFIT5 reveal a key immune evasion reading frames and an eGFP control in the pLVX-EF1alpha-2xStrep-
mechanism and identify a promising molecular target for antiviral IRES-Puro vector5, were gifts from Nevan Krogan (Addgene 141367-
development. 141381; 141383-141395). Gateway-compatible entry plasmids
Our deep learning-driven InSiPS analysis designed synthetic (pDONR207, 223) for S (Addgene 152988), NSP3 (Addgene 141257), and
inhibitory peptides targeting the RBD or S1/S2 cleavage region of the S NSP16 (Addgene 141269) were from Frederick Roth (Supplementary
protein, identifying three potent RBD-binding peptides (AVP1-3) that Note 1). Detailed information on antibodies, plasmids, oligonucleo-
strongly inhibit infectivity of both ancestral and variant SARS-CoV-2 tides, and reagents is provided in Supplementary Data 29. All work
strains by blocking RBD-ACE2 binding, critical for viral entry. The with ancestral or variant SARS-CoV-2 strains was conducted in bio-
identification of 48 highly correlated viral-host interacting protein safety level 3 facilities at the University of Alberta, University of Tor-
pairs in SARS-CoV-2-infected Huh-7 cells, absent with AVP treatment, onto, University of Pennsylvania, University of Saskatchewan/VIDO-
further supports the antiviral activity of these peptides in disrupting INTERVAC in Saskatoon, and Canada’s National Microbiology Labora-
viral-host PPIs, interrupting the SARS-CoV-2 lifecycle, and preserving tory in Winnipeg.
host innate immunity. Together, this study offers a comprehensive
biochemical map of the SARS-CoV-2 and variant interactome across Cell culture
organ or immune-derived cell lines and biofluids. The host-directed HEK293T, Huh-7, HepG2, N9 microglia, Huh-7 expressing ACE2
interventions and antiviral candidates presented here advance receptor (Huh-7-ACE2), Vero (Vero E6, 81, E6-TMPRSS2), and A549-AT
understanding of COVID pathophysiology and inform pan-viral cells were cultured in high-glucose Dulbecco’s modified Eagle medium
therapeutic development. By providing host-viral PPI data via a (DMEM) with 2–4 mM L-glutamine. Caco-2 cells were grown in Eagle’s
public repository (MassIVE: MSV000092774, MSV000096435, minimum essential medium (MEM) with 2 mM L-glutamine, while
MSV000097310) and a web portal (https://babulab-uofr.shinyapps.io/ HULEC-5a cells were maintained in MCDB131 medium with 10 ng/mL
scov2db/), we offer this resource to support further exploration of epidermal growth factor, 1 µg/mL hydrocortisone, and 10 mM gluta-
context-specific interactions critical to the distinct biology of SARS- mine. Human monocytic THP-1 cells were cultured in Roswell Park
CoV-2 and other viruses. Memorial Institute (RPMI) 1640 medium with 2 mM L-glutamine and
differentiated into M0 macrophages by treating them with RPMI
Methods medium supplemented with 150 nM phorbol 12-myristate 13-acetate
Saliva and blood plasma collection for 24 h, followed by incubation in RPMI medium without supplements
Saliva specimens were collected from COVID-19 patients infected with for an additional 24 h. To induce M1 polarization, M0 macrophages
the ancestral SARS-CoV-2 strain (n = 16), or the Alpha (n = 4), Delta were stimulated with 20 ng/mL IFN-γ and 100 ng/mL LPS for 48 h in
(n = 2), and Omicron (n = 10) variants during three consecutive waves. RPMI medium. Expi293F cells (Thermo Fisher Scientific) were cultured
Additionally, blood samples (n = 6) were taken from SARS-CoV-2 in Expi293 expression medium. All cell lines were maintained at 37 °C
patients in the first wave. All COVID-19 cases were verified via naso- and 5% CO2 in their respective media with antibiotics (100 U/mL
pharyngeal or throat swabs, with SARS-CoV-2 RNA detected by real- penicillin, 100 µg/mL streptomycin) and 10% heat-inactivated FBS,
time quantitative polymerase chain reaction (qRT-PCR) and variants except for Caco-2 cells, which were cultured with 20% FBS.
identified via whole-genome sequencing. COVID-19 patients were
admitted to Regina General Hospital, Canada, from June 2020 to Lentivector production and transduction into mammalian cells
December 2022. Control blood and saliva samples were obtained from HEK293T cells were seeded at 4 ×106 cells per 10 cm dish. At 60%
6-10 healthy volunteers without clinical symptoms for four weeks, confluence, 7.5 µg psPAX2 packaging plasmid (Addgene 12260), 750 ng
confirmed negative for SARS-CoV-2 RNA by qRT-PCR. The study, pMD2.G envelope plasmid (Addgene 12259), 7.5 µg lentiviral expres-
approved by the Saskatchewan Health Authority research ethics sion plasmid (Supplementary Note 1), and 15 µL PLUS reagent

Nature Communications | (2025)16:5784 13

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(Lipofectamine LTX kit) were mixed in 750 µL Opti-MEM I reduced MPC prediction
serum medium (Thermo Fisher Scientific). In another sterile tube, High-confidence viral-host PPIs from the merged network were ana-
52.5 μL Lipofectamine LTX was mixed with 697.5 μL Opti-MEM I. The lyzed using the ClusterONE algorithm60 to delineate complex mem-
contents of both tubes were combined, incubated at room tempera- bership. The algorithm’s settings were optimized using various
ture for 15 min, added to HEK293T cells, and incubated at 37 °C in 5% parameter combinations (density, d; overlap, o) and complementary
CO2 in DMEM without antibiotics. After 16 h, the medium was replaced evaluation metrics (accuracy, maximum matching ratio, overlap) as
with DMEM containing 100 U/mL penicillin, 100 µg/mL streptomycin, previously described30. Predicted complex sets were benchmarked
and 30% FBS. Lentiviral particles were collected at 24 and 48 h post- against CORUM human protein assemblies interacting with SARS-CoV-
transfection and filtered through a 0.4 µm low protein-binding poly- 2 proteins in the BioGRID, at various density (0.20-0.70) and overlap
ethersulfone membrane syringe filter. (0.5–0.8) settings. The parameter combination yielding the highest
Lentiviral supernatants were transduced into mammalian cells at composite score (i.e., sum of overlap, accuracy, and maximal matching
10% confluence with an MOI of 0.5 for 48 h using filter-sterilized (0.22 µm ratio) was deemed as the most effective for predicting MPCs. Only
syringe filter) polybrene (10 µg/mL) to enhance infection. After 48 h, cells clusters involving four or more human proteins interacting with SARS-
were transferred to 10 cm dishes and selected with puromycin (2 µg/ mL) CoV-2 proteins were considered (Supplementary Data 23). All viral-
for three days. For pLX304-hACE2 in Huh-7 cells, blasticidin (5 µg/mL) host or host-host PPIs, and MPCs were visualized using Cytoscape
was used for two weeks to ensure stable expression of VA-tagged (ver.3.9.1) software and are accessible through our web portal (https://
ancestral SARS-CoV-2 or variants, pLEX-307-LgBiT, or pcDNA3.1-SARS2- babulab-uofr.shinyapps.io/scov2db/).
Spike-HiBiT. VA-tag expression was verified by immunoblotting using an
anti-Strep antibody. Stable transformants were expanded to five 10 cm Whole-cell salivary proteomics
dishes, reaching 80% confluency ( ~5 × 107 cells). Cells were harvested in Stimulated saliva (1 mL) from healthy individuals and patients infected
batches, yielding up to six biological replicates, and pellets were either with SARS-CoV-2 or its variants was mixed with 200 µL of lysis buffer
processed immediately or stored at -80 °C for future use. (6 M urea, 2 M thiourea, protease inhibitor cocktail (PIC), 0.05% Pro-
tease MaxTM surfactant) and incubated on ice for 30 min. The mixture
Affinity purification and SEC fractionation of saliva or plasma was centrifuged at 3000 x g for 10 min at 4 °C, and the supernatant was
protein extracts filtered through a 0.22-μm filter. About 50 µg of protein per sample was
Fresh or frozen cell pellets were crosslinked using dithiobis succini- trypsin digested for LC-MS/MS analysis.
midyl propionate as described in studies on human-SARS-CoV-2
PPIs58,59. Crosslinked cells expressing Strep or VA-tagged SARS-CoV-2 Authenticating viral-host or host-host PPIs
proteins were lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), We evaluated our viral-host and host-host PPIs from AP-MS and CF-MS
150 mM NaCl, 1 mM EDTA, 0.5% Nonidet-P40 (NP-40), and protease/ methods against known interactions in BioGRID. SARS-CoV-2-human
phosphatase inhibitors. For VA-tagged SARS-CoV-2 ‘S’ bait proteins, 1% PPIs were also assessed against non-human cell lines using InParanoid
n-dodecyl-β-D-maltoside (DDM) was replaced with NP-40. Lysates orthology predictions with default BLASTP and BLOSUM62 settings.
were chilled on ice for 30 min, centrifuged at 13,000 x g for 15 min at We examined the overlap of SARS-CoV-2-human PPIs with those
4 °C, and the supernatant was incubated with 30 µL MagStrep ‘type3’ identified in a previous interactome study of human proteins inter-
Strep-Tactin beads for purification as detailed previously5. Proteins acting between pathogen pairs (references in Supplementary Data 5).
were eluted with 50 mM D-biotin (Sigma), dried with a Savant Speed- The co-localization of viral-host PPIs was assessed using localization
vac, and then denatured, reduced, and alkylated following established data from prior studies15,61 (with additional references reviewed in
protocol12. Strep-tagged protein expression was confirmed via immu- Grand, 202361) and the Human Protein Atlas62, considering only main
noblotting with an anti-Strep antibody. Proteins were digested over- subcellular locations where proteins are localized in the cell. Further-
night with sequencing-grade trypsin (Promega) at room temperature more, host interacting protein pairs within the salivary glands during
on a rocking shaker, and digestion was quenched with 0.1% (v/v) formic infection were investigated using data from oral atlases that include
acid. Peptides were purified using Zip-Tip C18 tips (Millipore)12, air- human minor salivary glands and gingival/palatal mucosae2.
dried, resuspended in 0.1% formic acid, and analyzed by liquid
chromatography-tandem MS/MS (LC-MS/MS), as detailed in Supple- Enrichment analysis
mentary Note 2. We identified the most enriched terms in the viral-host or host-host
Saliva or plasma samples from healthy individuals, patients PPIs using protein subunits from MPCs from databases like CORUM,
infected with SARS-CoV-2 or its variants, or antiviral peptide-treated Reactome, WikiPathways, and GO categories using the g:Profiler R
Huh-7 cells (with and without SARS-CoV-2 infection) were mixed with package. Enrichment analysis utilized a hypergeometric test
an equal volume of SEC buffer (25% v/v acetonitrile, 50 mM triethyla- (P ≤ 5.0e−2) with Benjamini-Hochberg FDR correction. For Gene ontol-
mine phosphate, 50 mM sodium perchlorate)30, lysed by syringe with a ogy analysis, we prioritized terms with fewer than 500 proteins,
23-gauge needle, and centrifuged at 3000 x g for 10 min at 4 °C to focusing on those with the lowest adjusted P-values to highlight hits.
remove debris. The supernatant was filtered through a 0.22-μm poly-
ethersulfone filter. About 500 μg of protein per sample was separated Site-directed mutagenesis
using an Agilent 1100 high performance liquid chromatography sys- The pDONR223 Gateway-compatible entry plasmid encoding IFIT5
tem with a binary pump and SEC buffer on a Yarra SEC-4000 column cDNA (HsCD00512366) from the human ORFeome collection was
(300 × 7.8 mm i.d., 3 μm). Proteins were eluted at 0.5 mL/min for obtained and sequence-verified with M13 forward and reverse primers
40 min, monitored at 280 nm, and collected in 84-96 fractions. HPLC at the DNASU Plasmid Repository. Catalytically inactive IFIT5 single
fractions were acid-precipitated and digested with sequencing-grade mutants (Lys160Arg, Lys197Arg, Lys206Arg) were generated in the
trypsin, and resuspended in 0.1% formic acid, following the above pDONR223 plasmid using the Phusion site-directed mutagenesis kit
described protocol. Scoring of PPIs from AP-MS and CF-MS datasets, (Thermo Fisher Scientific) by a PCR-based method with primers listed
along with the construction of differential networks comparing saliva in Supplementary Data 29. Modified pDONR223 plasmids, each con-
from SARS-CoV-2-infected patients to healthy subjects, and between taining one of the IFIT5 single mutants, were recombined with the pLD-
the ancestral virus and its variants, are detailed in Supplemen- puro-CcVA destination vector using the Gateway cloning LR II enzyme
tary Note 3. mix. IFIT5 mutant construct accuracy was verified by Sanger DNA
sequencing with CMV forward and cPPT reverse primers at Toronto’s

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TCAG DNA sequencing facility. To produce mutated SARS-CoV-2 Emulsiflex (Avestin) homogenizer at 30,000 psi, and the lysates were
S-RBD proteins, Delta variant double mutations (T478K, L452R) were centrifuged at 20,000 x g for 30 min at 4 °C. The supernatant was
engineered into the pcDNA3.1-SARS-CoV-2-S-RBD-8xHis-tag mamma- incubated with HisPur Ni-NTA resin at 4 °C for 2 h. After washing with
lian expression plasmid (Addgene 145145) using the QuikChange increasing concentrations of imidazole, His-tagged proteins were
Lightning mutagenesis kit (Agilent) with primers from Supplementary eluted in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl,
Data 29. Omicron mutations (G339D, S371L, S373P, S375F, K417N, 250 mM imidazole, and 2 mM DTT. Purity of the eluted proteins was
N440K, G446S, S477N, T478K, E484A, Q493R, Q498R, G496S, N501Y, confirmed by Coomassie-stained 10% SDS-PAGE, and pooled fractions
Y505H, T547K) in the SARS-CoV-2 S-RBD protein (amino acids 319–591) were dialyzed at 4 °C for 6 h in dialysis buffer (50 mM Tris, pH 7.8,
were synthesized by Genscript and cloned into the pcDNA 3.1 plasmid 150 mM NaCl, 2 mM DTT). Dialyzed proteins were concentrated, flash
with a C-terminal mouse Fc and 6xHis-tag. frozen, and stored at −80 °C.

IFIT5 knockdown and knockout Expression and purification of human ACE2

siRNA targeting human IFIT5 sourced from Horizon Discovery was Full-length human ACE2 with C-terminal Myc and FLAG tags in pCMV6-
used for gene knockdown in cells cultured from 48 h to 7 days, entry vector (Origene RC208442) was expressed in Expi293 cells and
depending on the experiment. To transiently knockdown IFIT5 in M1 purified as described previously63. Four days post-transfection, cell pel-
macrophages, 2 ×106 cells were seeded in 10 mL of RPMI medium in a lets were centrifuged at 500 x g for 20 min at 4 °C, resuspended in 30 mL
10 cm dish and transfected with IFIT5 siRNA using Lipofectamine ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM PMSF),
RNAiMAX (Thermo Fisher Scientific), following the manufacturer’s lysed with an Emulsiflex homogenizer at 30,000 psi, and solubilized with
instructions. Non-targeting siRNA served as a control. For IFIT5 0.1% Triton X-100. Lysates were clarified by centrifugation at 20,000 x g
knockdown in NSP3-expressing cells, 2 ×106 M1 macrophages in a for 30 min at 4 °C. The supernatant was treated with 0.05% DDM, and
10 cm dish were transduced with V5- or VA-tagged NSP3 at an MOI of incubated with anti-FLAG resin in Tris-buffer saline (TBS) containing
0.4 for 5 days with blasticidin selection. The next day, IFIT5 was 0.05% DDM at 4 °C for 1 h. The resin was transferred to a gravity flow
knockdown or overexpressed for 48 h in the same dish using Lipo- column, washed with TBS containing 0.05% DDM, and eluted with 0.1 M
fectamine RNAiMAX or LTX as per the manufacturer’s protocol. bicine (pH 3.5). Protein purity was confirmed by Coomassie-stained 10%
Knockdown efficacy was verified by immunoblotting with tag- or SDS-PAGE. Pooled eluted fractions were dialyzed for 6 h at 4 °C in buffer
protein-specific antibodies prior to qRT-PCR or immunoprecipitation. containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 5% Glycerol. The
IFIT5 knockout was generated using oligonucleotide pairs concentrated proteins were aliquoted, flash frozen in liquid nitrogen,
encoding 20-nt guide RNAs targeting IFIT5. sgRNAs were designed and stored at −80 °C for later use.
with Synthego and CHOPCHOP software and synthesized by Inte-
grated DNA Technologies (USA). The sgRNA was annealed with its RNA isolation and qRT-PCR
complementary ssDNA at 95 °C for 5 min, then cooled to 20 °C to form Total RNA was extracted from SARS-CoV-2 protein-treated cell lysates
dsDNA. This dsDNA was ligated into a BamH1-digested lentiCRISPR v2- (with or without antiviral peptides), and siRNA knockdown or over-
Blast (Addgene 83480) plasmid and transformed into SURE 2 super- expressed genes in M1 macrophages or Huh-7 cells transduced with
competent cells (Agilent). Positive transformants were selected with viral proteins using the Qiagen RNeasy kit with DNase treatment.
ampicillin and sequence verified at Toronto’s TCAG facility. The Reverse transcription was performed using the oligo (dT) primers and
sequence-confirmed IFIT5-KO lentiCRISPR v2-Blast plasmid was co- RNase H + MMLV (Moloney Murine Leukemia Virus) reverse tran-
transfected into A549-TMPRSS2-ACE2 cells with packaging and scriptase. Target mRNA transcripts were quantified in triplicate using
envelope plasmids, followed by seven days of blasticidin selection to FastStart SYBR Green I dye (Roche) on a LightCycler 96 SW qRT-PCR
establish stable IFIT5 knockout. To create CRISPR-resistant (CR) IFIT5 instrument. Transcript abundance was calculated by the 2−ΔΔCT
mutants (K206R, K197R), the IFIT5-VA overexpression plasmid with method, normalized to housekeeping genes (β-Tubulin, GAPDH), and
puromycin and the QuikChange II mutagenesis kit (Agilent) were used expressed as fold change in target gene expression relative to the
to engineer single-site mutations conferring resistance to CRISPR control. Primers were synthesized as single-stranded oligomers and
cleavage. Briefly, the IFIT5-VA CR wildtype and mutants were amplified purified by standard desalting (IDT, USA).
with CR-IFIT5 primers using QuikSolution reagent and QuikChange
lightning enzyme, following manufacturer’s instructions. The ampli- Enzyme-Linked Immunosorbent Assay (ELISA)
con, containing non-mutated plasmids, was digested with DpnI Total IFN-β in M1 macrophage supernatants was measured with the
restriction enzyme, and the mutated plasmids were transformed into VeriKine Human Interferon Beta ELISA kit (PBL Assay Science), and
SURE 2 competent cells, with positive transformants selected over- fibrinogen levels in uninfected and SARS-CoV-2-infected Huh7 cell
night using ampicillin. Sequence-verified IFIT5-VA CR wild-type and supernatants were assessed at various time points using the Human
mutant plasmids were transfected into A549-TMPRSS2-ACE2 IFIT5 Fibrinogen ELISA Kit (Abcam), following manufacturer’s protocol.
knockout cells with 72 h of blasticidin and puromycin selection. Diluted supernatants were incubated in assay diluent at room tem-
perature for 1 h, followed by the addition of antibody and horseradish
Expression and purification of recombinant SARS-CoV-2 S peroxidase. Samples were incubated with 3,3′,5,5′-tetra-
protein RBD methylbenzidine (TMB) substrate for 60 min (IFN-β) or 15 min (fibri-
Plasmid pcDNA3.1 (Addgene 145145) encoding SARS-CoV-2 S RBDs nogen) in the dark at room temperature. After adding the stop
(amino acids 333-529) with C-terminal tags (8xHis for ancestral and solution, absorbance at 450 nm was measured with a Multiskan FC
Delta variant, and 6xHis for Omicron), was expressed in Expi293 cells. microplate reader (Thermo Fisher Scientific). Triplicate samples were
For transfection, 30 mL of Expi293 cells at 3 × 106 cells/mL in expres- blank-corrected, and protein concentrations were determined using
sion medium were prepared following the Expi293 system user guide IFN-β and fibrinogen standards, with linear regression used to fit the
(Thermo Fisher Scientific). Three days post-transfection, cells were standard curve. NF-κB p65 activity in M1 macrophages with silenced or
harvested by centrifugation at 500 x g for 20 min at 4 °C, and the overexpressed IFIT5, challenged with or without NSP3, was assessed
supernatant was discarded. Cell pellets were resuspended in 30 mL using an ELISA-based NF-κB p65 kit (Abcam) as instructed. M0 mac-
lysis buffer (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 5% glycerol for rophages served as controls. Cells (1 × 104/well) seeded in a 24-well
ancestral/Delta; 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM PMSF for plate were lysed in Abcam’s cell extraction buffer and centrifuged at
Omicron) and incubated at 4 °C for 1 h. Cells were lysed using an 10,000 x g for 15 min at 4 °C. Supernatants were transferred to a 96-

Nature Communications | (2025)16:5784 15

Article https://doi.org/10.1038/s41467-025-60949-1

well plate and incubated with an NF-κB antibody for 1 h at room tem- Zen Blue software (Zeiss), and FGA fluorescent intensity was quantified
perature. After three 5-min phosphate-buffered saline with Tween 20 in ImageJ. Brightness and contrast were uniformly adjusted across
(PBS-T) washes, TMB substrate was added, and absorbance at 600 nm entire images in Adobe Photoshop, and cropped for presentation. All
was recorded every 40 s for 20 min using a Multiskan FC plate reader. original fluorescence microscopy images have been deposited on
NF-κB activity was quantified with a four-parameter logistic Figshare.
regression model.
Detection of SARS-CoV-2 NSP3 protein secretion
Co-IP and immunoblotting HepG2 cells expressing NSP3-V5 tag were seeded at 60% confluency in
Saliva and blood plasma from healthy and SARS-CoV-2-infected indi- DMEM with 10% FBS. After 48 h, cell pellets and supernatants were
viduals, SARS-CoV-2-infected A549-TMPRSS2-ACE2 cells, along with collected and clarified by centrifugation at 700 x g for 5 min to remove
various cell lines (HepG2, THP-1 M0 or M1 macrophages, HEK293, debris. Both supernatants and pellets were analyzed by immunoblot-
Caco-2, and HULEC-5a) transfected with wild-type or mutant VA-IFIT5 ting using an anti-V5 antibody. FGA was used as a positive control, and
(with or without V5/VA-NSP3 or Strep-S/ORF3A/7 A/NSP5) were used Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a negative
for immunoblotting and co-IP experiments. For co-IP, Tag-or protein- control for secreted protein. Blots were imaged using the Odyssey Fc
specific polyclonal antibodies raised in rabbit for protein A magnetic imager (LI-COR) and quantified with ImageJ plugin.
microbeads, or in mouse for protein G microbeads (Miltenyi) were
used if validated by Antibodypedia or commercial vendors. After 48 h Coagulation kinetic assay
of transfection, samples were lysed in RIPA buffer (150 mM NaCl, Thrombin (positive control) was obtained from Sigma-Aldrich, human
50 mM Tris-HCl pH 7.5, 0.1% sodium dodecyl sulfate (SDS), 1% Na plasma fibrinogen from Millipore Sigma, and partial NSP3 recombinant
deoxycholate, 1% NP-40, 1 mM EDTA) with 1X protease inhibitor protein (papain-like protease domain, amino acids 746-1060) with an
cocktail, centrifuged at 13,000 x g for 20 min at 4 °C, and supernatants N-terminal 10xHis-tag from CUSABIO. Briefly, fibrinogen diluted in PBS
were incubated with antibodies for 2 h at 4 °C and μMACS protein A or to 1 mg/mL was treated with 0.25 U thrombin or varying NSP3 con-
G magnetic microbeads overnight at 4 °C. Bead suspensions were centrations, while fibrinogen in PBS without thrombin or NSP3 served
passed through μMACS columns pre-equilibrated with RIPA and PIC, as the negative control. Enzyme kinetics were monitored at 37 °C in a
washed twice with RIPA containing 0.1% detergents and PIC, and finally 96-well plate using a Multiskan FC plate reader, with absorbance at
with detergent-free buffer. Proteins bound to the beads were eluted 425 nm recorded every minute for 2 h or until thrombin reached a
with pre-heated 2x Laemmli buffer at 95 °C, separated by Bis-Tris SDS- plateau, indicating complete coagulation.
PAGE, and transferred onto nitrocellulose membranes using the iBlot
system (Thermo Fisher Scientific). Immunoreactive proteins were Peptide design and synthesis
detected using IRDye 680RD goat anti-rabbit IgG or IRDye 800 CW All 15 RBD and 12 S1/S2-targeting-synthetic peptides of the SARS-CoV-2
conjugated goat anti-mouse IgG secondary antibodies (LI-COR), and S protein were computationally designed using InSiPS, as described
the protein bands were visualized with an Odyssey Fc imaging system previously11. Peptides were custom-synthesized by Shanghai Royo-
(LI-COR). Bands were digitized and quantified using ImageJ, with pixel biotech at >95% purity and characterized using reverse-phase HPLC
intensities measured and normalized to loading or positive controls. and MS analysis as per the company’s quality control standards. Lyo-
Immunoblots were converted to grayscale using Adobe Photoshop philized peptides were dissolved in dimethylsulfoxide (DMSO) to a
(ver. 26.5.0), with linear exposure or contrast adjustments applied as final concentration of 0.5% (v/v) before use.
necessary. Blot images were cropped for visual clarity. In instances
where the original protein ladder was of low quality, a clearer ladder SARS-CoV-2 ‘S’pseudotyped lentivirus production
from the same blot was overlaid for presentation, and is indicated by a For transfection, 7.5 µg psPAX2 packaging plasmid, 750 ng pcDNA3.1-
dashed line. All original, unprocessed Western blot images are pro- SARS2-Spike or E484K Spike-HiBiT envelope plasmid, and 7.5 µg pLV-
vided in the Source Data corresponding to each figure. eGFP (Addgene 36083) were used. Lentiviral supernatant was con-
centrated via sucrose cushion centrifugation to produce high-titer
Immunofluorescence and confocal imaging lentivirus particles. Briefly, 30 mL of supernatant was layered onto a
Approximately 30,000 HEK293 cells expressing plasmids for SARS- 20% sucrose buffer (50 mM Tris-HCl, pH 7.4; 100 mM NaCl; 0.5 mM
CoV-2 ancestral or variant S proteins-Strep, and Huh-7 cells infected EDTA) at a 4:1 v/v ratio and centrifuged at 8000 x g for 3 h at 4 °C. The
with and without SARS-CoV-2, were seeded onto 18 mm glass cover- pellet was resuspended in 150 µL PBS, and pseudotype virus infectivity
slips in 6-well plates. After 24 h at 37 °C, cells were washed with PBS, was assessed in HEK293T cells 24 h post-infection by counting GFP-
fixed with 4% paraformaldehyde for 20 min at room temperature, positive cells under a microscope.
permeabilized with 0.2% Triton X-100 in PBS for 5 min, and blocked
with PBS-T containing 5% FBS for 2 h at room temperature. Cells were SARS-CoV-2 S-mediated pseudovirus entry assay
incubated overnight at 4 °C with primary antibodies against Strep-tag We developed a nanoluciferase complementation bioreporter-based
(LSBio, C387305), S (Sino Biological, 40150-R007), NSP3 (GeneTex, cell assay to evaluate peptides targeting the RBD and S1/S2
GTX135589; Cell signaling, 88086S), FGA (Abcam, ab34269), regions of the S protein, aimed at blocking ACE2-RBD binding.
E-cadherin (Abcam, ab40772), or Golgin-97 (Thermo Fisher Scientific, HEK293T cells expressing pLEX-307-LgBiT were seeded in a black
PA5-30048) in PBS supplemented with 5% FBS. After three 10 min PBS 96-well plate at 5 × 104 cells per well. After 24 h, these cells were treated
washes, cells were incubated with Goat anti-mouse Alexa Fluor 488 with pseudotyped lentivirus (either pcDNA3.1-SARS2-Spike-HiBiT or
(Invitrogen, A32723) and/or Donkey anti-rabbit Alexa Fluor 647 (Invi- pcDNA3.1-SARS2-E484K Spike-HiBiT), with or without 15 RBD or
trogen, A32795) secondary antibodies for 2 h at room temperature in 12 S1/S2 peptides. Subsequently, 100 µL of furimazine (Chemshuttle),
PBS-T with 5% FBS. For ER staining, cells were treated with 100 nM ER- diluted in DMEM to 10 µM, was added to each well. Luminescence was
Tracker blue-white DPX (Thermo Fisher Scientific, E12353) in DMEM recorded every 30 s for 1 h using an EnSpire multimode plate reader
with 10% FBS for 30 min at 37 °C in the dark, followed by fixation, (Perkin Elmer), and results were presented in relative units over time.
permeabilization, and antibody labeling. Coverslips were mounted in
100% glycerol, or mounting medium with DAPI, and images were Infection with authentic SARS-CoV-2
captured using a Zeiss LSM 900 confocal microscope with a 63X, 1.4 Huh-7 or Huh-7-ACE2 cells were seeded at 5 × 104 cells/mL in 96-well
NA oil objective, and Airyscan detector. Images were processed using plates using serum-free DMEM. These cells were infected with an SARS-

Nature Communications | (2025)16:5784 16

Article https://doi.org/10.1038/s41467-025-60949-1

CoV-2 isolate at an MOI of 0.0001 pfu/cell, with or without specified Omicron variant with multiple RBD mutations, each tagged with either
doses of RBD-targeting peptides for 1 h at 37 °C. Post-infection, cells 6x or 8x-His tags. Recombinant wild-type or mutant S RBD-His proteins
were washed three times with PBS and cultured in DMEM with 2% FBS. were immobilized on an NTA sensor chip (Nicoya Lifesciences), and
At 48 h post-infection, viral RNA was extracted from the supernatant antiviral peptides were injected at specified concentrations into the
for qRT-PCR, using primers to quantify intracellular SARS-CoV-2 RNA. SPR system. To evaluate the inhibition of SARS-CoV-2 S protein’s
mRNA levels of proinflammatory cytokines and inflammatory marker binding to ACE2, recombinant ACE2-FLAG protein was immobilized on
were also measured by qRT-PCR using KiCqStart SYBR Green Primers a carboxyl sensor chip, and antiviral peptides along with SARS-CoV-2
(Sigma-Aldrich, KSPQ12012). The efficacy of the peptides in inhibiting wild-type or mutated S RBD-His proteins were injected into the SPR
SARS-CoV-2 infectivity was evaluated using a plaque-forming assay. To system. Measurements were taken at a flow rate of 40 μL/min with an
assess the effect of RBD-targeting peptides on SARS-CoV-2 S protein immobilization time of 600 s at 20 °C using a running buffer of 10 mM
expression, immunofluorescence was performed on SARS-CoV-2- HEPES (pH 7.5), 150 mM NaCl, 3 mM EDTA, and 0.005% (v/v) surfactant
infected Huh-7 cells treated with or without peptides. Cells were P20 (pH 7.4). Steady-state responses were plotted against time on a
stained with an S protein antibody and Hoechst 33342 for nuclear sensorgram to generate binding curves, and kinetic parameters (ka
staining. Fluorescence was measured using an EnSpire plate reader and kd) were analyzed by TraceDrawer ver. 1.9.2 software (Ridgeview
(Perkin Elmer) at an emission of 461 nm and excitation at 358 nm. S instruments) to estimate the dissociation constant (KD).
protein fluorescence in peptide-treated cells, normalized to untreated
cells, were reported relative to DMSO control. The peptides’ inhibition Structural modeling
of S protein entry was also assessed using immunofluorescence with To assess whether candidate peptides bind the same SARS-CoV-2 S
infectious SARS-CoV-2 particles, with a primary antibody against the S RBD residues and interface with the human ACE2 receptor, we
protein (Sino Biological, 40150-R007) at a 1:400 dilution and a Goat employed a molecular docking strategy with trRosetta for predicting
anti-rabbit Alexa Fluor 488 (Abcam Ab150077) secondary antibody. the 3D structures of SARS-CoV-2 peptide inhibitors. These structures
To assess viral replication and titers, IFIT5 knockout, along with were docked onto the trimeric S protein RBD (PDB ID: 7CWL) using
IFIT5-VA CR wild-type and mutants in IFIT5 knockout A549-TMPRSS2- ZDOCK server (ver. 3.0.2) to generate 10 scoring models and identify
ACE2 cells infected with ancestral SARS-CoV-2 at MOI of 0.1 pfu/cell, interface residues between the SARS-CoV-2 peptide inhibitors and the
were collected at various time points. Total RNA was extracted using the S protein trimeric RBD. The best model was chosen based on the
TRIzol method. Supernatants were used to determine viral titers, while lowest binding free energy and interaction specificity (i.e., salt bridges,
cell pellets were resuspended in 30 µL of RNAase free water for viral van der Waals forces, and hydrogen bonds). Interface residues of the
replication analysis. qRT-PCR was conducted using the Luna Probe One- trimeric SARS-CoV-2 S protein-peptide inhibitor were mapped onto
Step RT-qPCR Mix (New England Biolabs) with N1 and N2 primer-probe the crystal structure of the S RBD-ACE2 complex64. Similarly, we
sets, following the manufacturer’s instructions. All reactions were per- identified interface residues in the NSP3-IFIT5-ISG15 complex using the
formed in triplicate on an Applied Biosystems 7500 Fast Real-Time PCR resolved NSP3 structure (PDB ID: 6YVA) for docking AlphaFold struc-
detection system with automatic threshold and baseline settings. A tures of ISG15 and IFIT5 via Z-Dock. Interface residues across all studied
standard curve was generated using a series of 5-fold dilutions starting protein-peptide inhibitors and complexes were visually examined
from a 106 copies/uL standard. Using the standard curves for the N1 and using PyMOL (ver. 3.4).
N2 genes, mean cycle threshold (Ct) values from the samples were
converted into absolute viral copy numbers. Web portal overview
The interactive web portal (https://babulab-uofr.shinyapps.io/scov2db/)
IC50 determination of SARS-CoV-2 peptides by plaque assay provides access to SARS-CoV-2-human PPI data from AP-MS and CF-MS
Vero E6-TMPRSS2 cells were seeded in 12-well plates at 5 ×105 cells/well experiments. The portal is built with Shiny for Python and hosted on
and infected with ancestral or variant SARS-CoV-2 strains at MOI Shiny-Apps server. Multiprotein complexes are visualized using Pyvis
0.0001 pfu/cell. The infection medium included DMEM with 1x non- and includes five sections: Project description, AP-MS, CF-MS, Merged
essential amino acids, 10 mM HEPES, 2% FBS, and 50 U/mL penicillin network, Multiprotein complex, and Contact. The AP-MS tab allows
and streptomycin, and varying doses of RBD or S1/S2-targeting pep- users to explore high-confidence SARS-CoV-2–human PPIs, including
tides. After 1 h, the medium was replaced with MEM containing 10 mM those identified in wild-type (WT) and variant (Var) strains. Each SARS-
HEPES and 1.2% Avicel RC-591 (DuPont), with suitable peptide dose. CoV-2 bait protein is linked to its human prey protein, accompanied by a
After 48 h, cells were fixed in 10% formaldehyde and stained with 0.5% brief description, a direct link to the UniProt database, and information
(w/v) crystal violet. Plaques were counted and data plotted as percent on the cell line source of the interaction. Additionally, the ‘Cell line score’
inhibition vs. Log10 [peptide] in GraphPad Prism (ver. 10.1.1.323). IC50 button allows users to retrieve the predicted interaction score for each
values were calculated using a non-linear regression model. cell line. Users can also extract high-confidence interactions from AP-MS
studies for specific or all human cell lines. In the Spike WT/Var-human
Cytotoxicity of SARS-CoV-2 peptide inhibitors PPI tab, they can filter spike-human PPIs across all variants and wild-type
Cell viability was evaluated using the CellTiter-Glo ATP content assay strains or focus on specific viral strains. The CF-MS tab provides high-
kit (Promega) or the Cell Counting Kit-8 (CCK-8, Sigma-Aldrich) fol- confidence SARS-CoV-2-human PPIs obtained from the saliva of COVID-
lowing manufacturer’s guidelines. A549-AT and Vero E6-TMPRSS2 cells 19 patients infected with wild-type SARS-CoV-2, along with their pre-
were seeded at 5 × 103 cells/well in 96-well plates and incubated over- dicted interaction scores. The Merged network tab presents all high-
night. RBD-targeting peptides of S protein, dissolved in DMSO, were confidence SARS-CoV-2-human PPIs from both AP-MS and CF-MS data-
applied at 5 µM and 50 µM. After a 24 h incubation at 37 °C with 5% CO2, sets, with a “More info” button providing details on the source of each
the substrate was added, and cell viability was assessed by measuring interaction. The Multiprotein complex tab displays predicted protein
ATP activity or dye uptake relative to DMSO control. complexes containing both SARS-CoV-2 and human proteins, listing the
complex ID, total number of subunits, and viral and host proteins
Surface plasmon resonance (SPR) forming the complex. A “Show network” button allows users to visualize
SPR kinetic experiments were conducted on an OpenSPR Rev4 system and zoom in on each complex, highlighting SARS-CoV-2 and human
(Nicoya Lifesciences) to measure the binding affinities of InSiPS- proteins as well as drug targets as nodes, while the edges represent viral-
generated peptides to wild-type and mutant SARS-CoV-2 S RBD pro- host PPIs. Lastly, the Contact tab provides details on whom to reach out
teins, including the Delta variant (T478K, L452R mutations), and the to for inquiries, additional data requests, or technical support.

Nature Communications | (2025)16:5784 17

Article https://doi.org/10.1038/s41467-025-60949-1

Reporting summary 12. Malty, R. H. et al. A map of human mitochondrial protein interac-
Further information on research design is available in the Nature tions linked to neurodegeneration reveals new mechanisms of
Portfolio Reporting Summary linked to this article. redox homeostasis and NF-kappaB signaling. Cell Syst. 5,
1–14 (2017).
Data availability 13. Huttlin, E. L. et al. The BioPlex Network: A Systematic Exploration of
AP-MS and CF-MS raw data and search results have been deposited in the Human Interactome. Cell 162, 425–440 (2015).
the MassIVE repository under accession codes MSV000092774, 14. Verschueren, E. et al. Scoring large-scale affinity purification mass
MSV000096435, and MSV000097310. High-confidence SARS-CoV-2- spectrometry datasets with MiST. Curr. Protoc. Bioinforma. 49, 8 19
human PPIs from AP-MS, CF-MS, and the merged network are acces- 11–18 19 16 (2015).
sible through our interactive web portal (https://babulab-uofr. 15. Gordon, D. E. et al. Comparative host-coronavirus protein interac-
shinyapps.io/scov2db/), which includes predicted scores, cell line/ tion networks reveal pan-viral disease mechanisms. Science 370,
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format. These PPIs are also archived in the open-access BioGRID 16. Wang, R. et al. Genetic Screens Identify Host Factors for SARS-CoV-
database. Source data are provided with this paper. Analyzed data and 2 and Common Cold Coronaviruses. Cell 184, 106–119 e114 (2021).
uncropped representative immunoblots are provided in the Supple- 17. Daniloski, Z. et al. Identification of required host factors for SARS-
mentary Data and Source Data files. Original microscopy images are CoV-2 Infection in human cells. Cell 184, 92–105 e116 (2021).
publicly available on Figshare (https://doi.org/10.6084/m9.figshare. 18. Baggen, J., Vanstreels, E., Jansen, S. & Daelemans, D. Cellular host
28929278). Expression plasmids used in this study are available factors for SARS-CoV-2 infection. Nat. Microbiol 6, 1219–1232 (2021).
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USA 118, e2024202118 (2021). The authors declare no competing interests.

Nature Communications | (2025)16:5784 19

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Kirsten Broderick1,14, Mohamed Taha Moutaoufik1,2,14, Tatiana Saccon1,14, Ramy Malty1,14, Shahreen Amin1,14,
Sadhna Phanse 1, Thomson Patrick Joseph1, Mara Zilocchi 1, Ali Hosseinnia1, Zoe Istace 1, Maryam Hajikarimlou 3
,
Sakib Abrar 1, Jade Fisher 1, Raelynn Brassard4, Ranawaka Perera5, Anil Kumar6, Hiroyuki Aoki 1,
Matineh Rahmatbakhsh1, Matthew Jessulat1, Darwyn Kobasa 7,8, Frank Dehne9, Bhanu Prasad10, Alla Gagarinova1,11,
M. Joanne Lemieux4, Alan Cochrane12, Walid A. Houry 13, Khaled A. Aly 1, Ashkan Golshani3 & Mohan Babu 1

1
Department of Biochemistry, University of Regina, Regina, SK, Canada. 2Faculty of Medical Sciences, University Mohammed VI Polytechnic,
Benguerir, Morocco. 3Department of Biology, Carleton University, Ontario, ON, Canada. 4Department of Biochemistry, University of Alberta, Edmonton,
AL, Canada. 5Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 6Department of Biochemistry,
Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada. 7National Microbiology Laboratory, Public Health Agency of Canada,
Winnipeg, MB, Canada. 8Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, MB, Canada. 9School of Computer
Science, Carleton University, Ottawa, ON, Canada. 10Department of Medicine, Regina Qu’Appelle Health Region, Regina, SK, Canada. 11Department of
Biology, University of New Brunswick, New Brunswick, N.B., Canada. 12Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
13
Department of Biochemistry and Department of Chemistry, University of Toronto, Toronto, ON, Canada. 14These authors contributed equally: Kirsten
Broderick, Mohamed Taha Moutaoufik, Tatiana Saccon, Ramy Malty, Shahreen Amin. e-mail: [email protected]

Nature Communications | (2025)16:5784 20