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Citation: Gori Savellini, G.; Anichini,
G.; Manetti, F.; Trivisani, C.I.; Cusi,
M.G. Deletion of 82–85 N-Terminal
Residues in SARS-CoV-2 Nsp1
Restricts Virus Replication. Viruses
2024,16, 689. https://doi.org/
10.3390/v16050689
Academic Editor: George Belov
Received: 3 April 2024
Accepted: 24 April 2024
Published: 26 April 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
viruses
Article
Deletion of 82–85 N-Terminal Residues in SARS-CoV-2 Nsp1
Restricts Virus Replication
Gianni Gori Savellini 1, * , Gabriele Anichini 1, Fabrizio Manetti 2, Claudia Immacolata Trivisani 2
and Maria Grazia Cusi 1, *
1Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy; gabriele.anichini2@unisi.it
2Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy;
claudia.trivisani2@unisi.it (C.I.T.)
*Correspondence: gianni.gori@unisi.it (G.G.S.); mariagrazia.cusi@unisi.it (M.G.C.);
Tel.: +39-0577-233866 (G.G.S.); +39-0577-232811 (M.G.C.)
Abstract: Non-structural protein 1 (Nsp1) represents one of the most crucial SARS-CoV-2 virulence
factors by inhibiting the translation of host mRNAs and promoting their degradation. We selected
naturally occurring virus lineages with specific Nsp1 deletions located at both the N- and C-terminus
of the protein. Our data provide new insights into how Nsp1 coordinates these functions on host and
viral mRNA recognition. Residues 82–85 in the N-terminal part of Nsp1 likely play a role in docking
the 40S mRNA entry channel, preserving the inhibition of host gene expression without affecting
cellular mRNA decay. Furthermore, this domain prevents viral mRNAs containing the 5
′
-leader
sequence to escape translational repression. These findings support the presence of distinct domains
within the Nsp1 protein that differentially modulate mRNA recognition, translation and turnover.
These insights have implications for the development of drugs targeting viral proteins and provides
new evidences of how specific mutations in SARS-CoV-2 Nsp1 could attenuate the virus.
Keywords: Nsp1; attenuated viruses; transcription and translation; host shutdown; leader sequence;
SARS-CoV-2
1. Introduction
The CoronaVIrus Disease-19 (COVID-19) pandemic, caused by Severe Acute Respi-
ratory Syndrome CoronaVirus 2 SARS-CoV-2, has resulted in over 774 million confirmed
infections and 7 million deaths [
1
]. Understanding the molecular mechanisms of viral
virulence factors and virus–host interactions is critical for the development of preventive
and therapeutic measures against COVID-19 disease. Viruses commonly employ host trans-
lation shutoff to evade innate immune responses, primarily mediated by beta Interferon
(IFN-
β
) [
2
–
5
]. Furthermore, several strategies are pursued by viruses in order to inhibit host
protein synthesis, targeting various stages of the translation process such as mRNA tran-
scription, processing, nuclear export of newly formed mRNAs, mRNA degradation, and
blocking key translation and elongation factors [
2
–
12
]. Coronaviruses (CoVs), including
SARS-CoV-2, induce this host shutoff through multiple viral proteins [
13
–
16
]. SARS-CoV-2
virion is composed of the positive-sense RNA (vRNA) genome and several structural pro-
teins (N, M, E, and Spike) [
17
–
21
]. The vRNA genome also encodes sixteen non-structural
proteins that form the replication machinery and seven accessory proteins [
17
–
21
]. Al-
though the exact functions of SARS-CoV-2 accessory proteins are not fully demonstrated,
previous studies suggest that these proteins are not essential for viral replication but they
can modulate virus pathogenesis interacting with host pathways, especially those involved
in innate immunity. Indeed, SARS-CoV-2 is also known to encode several viral proteins that
antagonize interferons (IFNs) and interferon-stimulated genes (ISGs), and hence promote
infection and early virus replication [
6
,
22
–
32
]. Coronaviruses generate a set of sub-genomic
RNAs (sgRNAs) through a discontinuous transcription mechanism, likely influenced by
Viruses 2024,16, 689. https://doi.org/10.3390/v16050689 https://www.mdpi.com/journal/viruses
Viruses 2024,16, 689 2 of 18
negative-sense RNA and a conserved 5
′
-leader sequence of about 70 nucleotides [
33
–
36
].
These sgRNAs encode structural, non-structural, and accessory viral proteins. When the
translation of cellular mRNAs is impaired, the viral mRNAs become prioritized, enhancing
viral protein synthesis, assembly, and release [
37
–
39
]. Notably, SARS-CoV-2 Nsp1 has
been extensively studied in this context. It directly binds to the 40S ribosomal subunit,
obstructing the mRNA entry channel, thus reducing cellular protein synthesis [
39
]. In our
study, we analyzed the genetic fitness of SARS-CoV-2 strains having naturally occurring
deletions within the Nsp1 protein. We assessed the impact of these Nsp1 deletion mutants
on both host cellular gene and viral mRNAs translation. Specifically, we identified deletions
at the N-terminus (aa: 82–85 and 82–86) and the C-terminus (aa: 141–143) of Nsp1 in human
samples. Our findings highlight a conserved domain in the N-terminal region (a.a. 82–85)
of Nsp1 that primarily impedes cellular mRNA translation rather than influencing mRNA
decay. Indeed, the
∆
82–85 and
∆
82–86 Nsp1 variants showed reduced degrading activity
towards GFP expression driven by the human beta-globin promoter at the transcriptional
level (mRNA). Moreover, the
∆
82–85/
∆
82–86 Nsp1 variant displayed decreased efficiency
in recognizing viral mRNAs, making these transcripts susceptible to translational inhibition
rather than decay. Therefore, this mechanism could potentially reduce SARS-CoV-2 viru-
lence by limiting viral gene expression. Indeed, the specific activities of
∆
82–85 and
∆
82–86
Nsp1 mutants were further evident as they showed a marked reduction in virus fitness and
replication, in both interferon-deficient (Vero E6) and interferon-competent (Calu-3) cells.
The specific Nsp1 domain identified in this study opens the way to drug development,
targeting the Nsp1 virulence factor as a potential therapeutic approach for infections.
2. Materials and Methods
2.1. Cell Cultures and Virus Isolates
Vero E6 (ATCC CRL-1586), human embryonic kidney HEK-293T (ATCC CRL-3216),
and Calu-3 (ATCC HTB-55) cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) (Euroclone, Milan, Italy). This medium was supplemented with 100 U/mL
penicillin/streptomycin and 10% heat-inactivated fetal bovine serum (FCS) (Euroclone)
and incubated at 37
◦
C with 5% CO
2
. Selected clinical specimens of Omicron and BQ1.1
lineages underwent Whole Genome Sequencing using the COVIDSeq test (Illumina, Milan,
Italy). The sequenced genomes have been deposited in GenBank under the accession
numbers: MT531537, ON974845, OR166017, OR166015, OR160428, and OR161044. In
accordance with the Declaration of Helsinki principles (approved by the Ethics Committee
under BIOBANK-MIU-2010, with Amendment No. 1 dated 17 February 2020), and after
obtaining written, signed, and dated informed consent from participants at the Virology
Laboratory of Santa Maria alle Scotte Hospital in Siena (Italy), the identified SARS-CoV-2
lineages were isolated using Vero E6 cells. These isolates were subsequently stored at
−80 ◦C
in individual aliquots and viral titers were determined using the Median Tissue
Culture Infectious Dose (TCID50/mL) assay.
2.2. Plasmids
Viral genomic RNA was extracted using the QIAamp Viral RNA Kit (Qiagen, Mi-
lan, Italy). HA-tagged full-length Nsp1 gene fragments were synthesized via Reverse-
Transcription Polymerase Chain Reaction (RT-PCR) using the SuperScript™ III One-Step
RT-PCR System with Platinum™ Taq High-Fidelity DNA Polymerase (Life Technologies,
Milan, Italy). Subsequently, these fragments were cloned into the EcoR1-XhoI restric-
tion sites of the pCAGGS-MCS plasmid following standard procedures. All constructed
plasmids underwent sequence verification through Sanger sequencing. The constructs
containing the human
β
-globin 5
′
-UTR, or the SARS-CoV-2 5
′
-UTR and the Leader fused
with green fluorescent protein, were generously provided by Prof. Michal Schwartz from
the Weizmann Institute of Science, Rehovot, Israel.
Viruses 2024,16, 689 3 of 18
2.3. Kinetics of Viral Replication
Monolayers of Vero E6 and Calu-3 cells were seeded at densities of
1×105
and
2×105
cells per well, respectively, in 24-well plates using complete culture medium. The following
day, the cultures were infected with a multiplicity of infection (MOI) of 0.01 of the selected
SARS-CoV-2 lineages and incubated at 37
◦
C for 1 h. After the incubation, the inoculum
was carefully removed and the cells were extensively washed. Subsequently, growth
medium containing 2% FBS was added to the wells. The cytopathic effect (CPE) was
assessed under a light microscope three days post-infection. Supernatants from the infected
cultures were harvested at 24, 48, and 72 h (h) post-infection (p.i.) and stored at
−
80
◦
C
for further analyses. Viral release from the infected cultures was quantified using a virus
microtitration assay on Vero E6 cells plated in 96-well plates. Viral titers were calculated
using the Reed–Muench formula and expressed as TCID50/mL.
2.4. Assessment of IFN-βExpression
Medium from Calu-3 cells, infected as previously described, was collected at
48, 72 h
post-infection (p.i.). IFN-
β
quantification was assessed by VeriKine-HS Human IFN Beta
TCM ELISA Kit (PBL assay science, Piscataway, NJ, USA), following manufacturer’s
instructions. The results are presented as mean fold values relative to the mock-infected
sample ±standard deviation from at least three independent experiments.
2.5. Western Blotting
HEK-293T cells co-transfected with HBB-, 5
′
-Ld-SL1- or 5
′
-UTR-GFP, and Nsp1 con-
structs were harvested, and cell pellets were lysed using RIPA buffer supplemented with
an anti-protease cocktail from Roche, Milan, Italy. Total protein content in the lysates was
quantified using the BCA assay from Pierce (Milan, Italy). A total of 25
µ
g of proteins
from each sample was prepared in Laemmli sample buffer, denatured by boiling for 5 min,
and then separated by SDS-PAGE. The resolved proteins were transferred onto nitrocellu-
lose membranes (Santa Cruz Biotechnology, Heidelberg, Germany). After blocking with
5% non-fat dry milk, membranes were probed with primary antibodies: anti-GFP (Life
Technologies, Milan, Italy), anti-HA tag (Merk-Millipore), or anti-GAPDH (Life Technolo-
gies) as a loading control. Following three washes with PBS-T (phosphate-buffered saline
containing 0.05% Tween-20), the membranes were incubated with an HRP-conjugated
anti-mouse IgG secondary antibody (Merck-Millipore) at room temperature for 1 h. The
immunoreactive bands were visualized by using the TMB-Blotting 1-Step Solution (Pierce).
2.6. Reverse-Transcription Quantitative PCR (RT-qPCR)
HEK-293T cells (1
×
10
5
/well) were plated in 24-well plates and, after O/N incubation,
cell monolayers were transfected with 200 ng of either HBB-, 5
′
-Ld-SL1-, or 5
′
-UTR-GFP
expressing plasmids in combination with 500 ng of empty vector, Wuhan-1, or mutant Nsp1
plasmids. Cells were collected at 48 h post-transfection and total RNA was isolated using
the RNAeasy Plus mini kit (Qiagen, Milan, Italy). TaqPath 1-Step Multiplex Master Mix
(Thermo Fisher Scientific, Milan, Italy) was used for RT-qPCR reactions in a QuantStudio 5
Real-Time PCR System (Thermo Fisher Scientific). TaqMan
®
Assays (Thermo Fisher Sci-
entific) for GFP and actin (ACT) were used for specific transcripts detection. Each sample
was run in duplicate and the cycle threshold (Ct) values of each gene were normalized
against the endogenous ACT gene and compared with the negative, empty vector trans-
fected, control. The results were represented as mean fold relative increments from at least
three independent experiments (2
−∆∆Ct
algorithm)
±
standard deviations (SDs). Where
indicated, cells were treated with 100
µ
g/mL of cycloheximide (CHX) (Merck-Millipore,
Milan, Italy), 5
µ
g/mL of actinomycin D (ActD) (Life Technologies, Milan, Italy) at 48 h
post-transfection or left untreated and collected at the starting point (T0), 2 h and 4 h
post-treatment. Samples were processed for RT-qPCR as described above or lysed in RIPA
buffer for subsequent immunoblotting.
Viruses 2024,16, 689 4 of 18
2.7. Cycloheximide Chase Analysis and NSs Protein Stability
Vero E6 cells were seeded in 24-well plates and infected as previously described. Forty-
eight hours post-infection, cells were collected for starting protein expression quantification
(T0), while the remaining samples were treated with 100
µ
g/mL of cycloheximide (CHX).
Samples were then collected 2 h and 4 h later for time course quantification. The amount
of Nsp1 and GAPDH loading control proteins was quantified by Western blotting and
densitometric analysis using the JmageJ 1.53t software. Relative Nsp1 intensities were
normalized with respect to the corresponding GAPDH signal value. Fold changes of each
sample were calculated relative to the corresponding T0 mock-treated sample.
2.8. Computational Details
The protein structure of the Nsp1 N-terminal domain was taken from the Protein
Data Bank using the accession code 7K7P. The
∆
82–85 protein structure was built with the
Residue and Loop mutation tool available in Maestro using the Nsp1 N-terminal domain
(7K7P) as a template. The 5
′
-Ld-SL1 mRNA belonging to the viral 5
′
-UTR was extracted
from the PDB entry 2GDT. All the structures were prepared using the Protein Preparation
Wizard, which added missing side chains and hydrogens, assigned bond orders, and
minimized hydrogen atoms. Docking was conducted using the HDOCK server. Visual
analyses were performed using PyMol Molecular Graphics System (version 2.5.0).
2.9. Statistics
Mean differences were statistically analyzed by using one-way ANOVA with Dunnett’s
multiple comparison test in GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA),
in order to compare prevalence rates among different study groups. Statistical significance
was set at p< 0.05.
3. Results
3.1. SARS-CoV-2 Strains Bearing Nsp1 N-Terminal Deletions Are Attenuated in Replication
To evaluate the biological functions of Nsp1 deletion on virus fitness and innate
immune response to the infection, we selected SARS-CoV-2 naturally occurring virus
strains bearing 82–85, 82–86, and 141–143 amino acid deletion within the Nsp1. To this aim,
we assessed replication kinetics of selected SARS-CoV-2 strains in Vero E6 cells which are
type I IFN-deficient and represent the gold standard for SARS-CoV-2 growth. Infections
were performed at an MOI of 0.01 and supernatants were collected for virus titration by the
standard microtitration method 24, 48, and 72 h p.i. All investigated virus strains did not
show significant difference in progeny virus titer at 24 h p.i. (p> 0.05). However, replication
kinetics showed a replicative disadvantage and an evident reduction in virus progeny
release compared to the relative original strains (BA.2 and BQ1.1, respectively) for viruses
carrying the Nsp1 82–85 or 82–86 deletion at both 48 h (p= 0.0027 and p= 0.0001) and 72 h
p.i. (p= 0.003 and p= 0.001) (Figure 1A).
Viruses 2024,16, 689 5 of 18
Viruses 2024, 16, x 5 of 18
Figure 1. Kinetic growth of SARS-CoV-2 lineages bearing Nsp1 partial deletion. (A) Vero E6 cells
were infected with Omicron BA.2 and the relative ∆82–85 and ∆141–143 lineages, or with the BQ.1.1
and the ∆82–86 variants at a multiplicity of infection (MOI) of 0.01. Cell culture supernatants were
collected at 24 h, 48 h, and 72 h p.i. and viable progeny virus content was assessed by a microtitration
assay. The results are presented as Log10 of the mean viral titer expressed as plaque forming units
(PFUs) ± standard deviations (SDs) from at least three separate experiments. (B) The expression of
Nsp1 was determined by Western bloing on 50 µg of whole cell lysates (WCLs) of Vero E6 cells
infected with the indicated virus lineages and collected at 24 h and 48 h p.i. The loading control is
represented by the immuno-detection of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
protein. (C) The kinetic growth of the previously described SARS-CoV-2 lineages was also investi-
gated in type I interferon-competent Calu-3 cells. Cell culture supernatants were collected at the
indicated time points and assessed for virus replication by microtitration assay. The results are plot-
ted as Log10 of the mean PFU ± standard deviations (SDs) from at least three separate experiments.
Significance is reported as **p < 0.001, *** p < 0.0005, and **** p < 0.0001 with respect to the parental
Figure 1. Kinetic growth of SARS-CoV-2 lineages bearing Nsp1 partial deletion. (A) Vero E6 cells
were infected with Omicron BA.2 and the relative
∆
82–85 and
∆
141–143 lineages, or with the BQ.1.1
and the
∆
82–86 variants at a multiplicity of infection (MOI) of 0.01. Cell culture supernatants were
collected at 24 h, 48 h, and 72 h p.i. and viable progeny virus content was assessed by a microtitration
assay. The results are presented as Log
10
of the mean viral titer expressed as plaque forming units
(PFUs) ±standard
deviations (SDs) from at least three separate experiments. (B) The expression
of Nsp1 was determined by Western blotting on 50
µ
g of whole cell lysates (WCLs) of Vero E6
cells infected with the indicated virus lineages and collected at 24 h and 48 h p.i. The loading
control is represented by the immuno-detection of Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) protein. (C) The kinetic growth of the previously described SARS-CoV-2 lineages was also
investigated in type I interferon-competent Calu-3 cells. Cell culture supernatants were collected at
the indicated time points and assessed for virus replication by microtitration assay. The results are
Viruses 2024,16, 689 6 of 18
plotted as Log
10
of the mean PFU
±
standard deviations (SDs) from at least three separate experiments.
Significance is reported as ** p< 0.001, *** p< 0.0005, and **** p< 0.0001 with respect to the parental
strains. On the contrary, the deletion of the 141–143 domain located at the C-terminus of BA.2 Nsp1
did not affect virus replication in Vero E6 cells, leading to virus titers similar to the parental (BA.2)
strain (p> 0.05) (Figure 1A). Meanwhile, cells infected with original SARS-CoV-2 lineages (BA.2 and
BQ1.1) and with the
∆
141–143 virus variant showed similar levels of Nsp1 protein over time. Indeed,
at 24 h and 48 h p.i., Nsp1 was substantially expressed by these virus lineages, while a different
behavior was observed in CoV2-
∆
82–85- and CoV2-
∆
82–86-deleted viruses, which showed a low or
undetectable production of Nsp1, indicating a possible role of this non-structural protein in reducing
virus progeny release (Figure 1B). Moreover, replication kinetics of selected virus lineages were
investigated in Calu-3 cells, which represent a well-defined pulmonary cell system to investigate
SARS-CoV-2 fitness in a human, IFN-
β
-competent environment. The results are comparable to those
observed in Vero E6 cells, confirming that the 82–85 domain is critical for viral fitness (Figure 1C). At
late times of infections, CoV2-
∆
141–143 also showed a reduced growth in Calu-3 cells with respect to
BA.2 (p< 0.0001), albeit at a lesser extent when compared to CoV2-
∆
82–85 (p= 0.001) and CoV2-
∆
82–
86 (p= 0.0009) (Figure 1C). Additionally, it seems that the replication kinetics of these virus strains
remain unaffected in IFN-
β
-competent cells, suggesting that the reduced virus fitness in Calu-3 cells
was not a consequence of the IFN-βsusceptibility antiviral activity.
3.2. INF-βProduction by Nsp1 Deletion Strains According to Different Growth Kinetics
To explore whether the distinct replication kinetics observed in the 82–85 and
82–86 Nsp1
-
deleted strains might translate to varied innate antiviral responses, we evaluated their
capacity to stimulate IFN-
β
production in the supernatants of infected Calu-3 cells. At
48 and 72 h p.i., both the BA.2
∆
82–85- and
∆
141–143-deleted variants demonstrated the
ability to induce IFN-
β
production compared to the mock-infected control (p< 0.0001).
Notably, the
∆
141–143 variant induced higher levels of IFN-
β
compared to the other virus
variants (Figure 2).
Viruses 2024, 16, x 6 of 18
strains. On the contrary, the deletion of the 141–143 domain located at the C-terminus of BA.2 Nsp1
did not affect virus replication in Vero E6 cells, leading to virus titers similar to the parental (BA.2)
strain (p > 0.05) (Figure 1A). Meanwhile, cells infected with original SARS-CoV-2 lineages (BA.2 and
BQ1.1) and with the Δ141–143 virus variant showed similar levels of Nsp1 protein over time. Indeed,
at 24 h and 48 h p.i., Nsp1 was substantially expressed by these virus lineages, while a different
behavior was observed in CoV2-Δ82–85- and CoV2-Δ82–86-deleted viruses, which showed a low or
undetectable production of Nsp1, indicating a possible role of this non-structural protein in reduc-
ing virus progeny release (Figure 1B). Moreover, replication kinetics of selected virus lineages were
investigated in Calu-3 cells, which represent a well-defined pulmonary cell system to investigate
SARS-CoV-2 fitness in a human, IFN-β-competent environment. The results are comparable to those
observed in Vero E6 cells, confirming that the 82–85 domain is critical for viral fitness (Figure 1C).
At late times of infections, CoV2-Δ141–143 also showed a reduced growth in Calu-3 cells with re-
spect to BA.2 (p < 0.0001), albeit at a lesser extent when compared to CoV2-Δ82–85 (p = 0.001) and
CoV2-Δ82–86 (p = 0.0009) (Figure 1C). Additionally, it seems that the replication kinetics of these
virus strains remain unaffected in IFN-β-competent cells, suggesting that the reduced virus fitness
in Calu-3 cells was not a consequence of the IFN-β susceptibility antiviral activity.
3.2. INF-β Production by Nsp1 Deletion Strains According to Different Growth Kinetics
To explore whether the distinct replication kinetics observed in the 82–85 and 82–86
Nsp1-deleted strains might translate to varied innate antiviral responses, we evaluated
their capacity to stimulate IFN-β production in the supernatants of infected Calu-3 cells.
At 48 and 72 h p.i., both the BA.2 Δ82–85- and Δ141–143-deleted variants demonstrated
the ability to induce IFN-β production compared to the mock-infected control (p < 0.0001).
Notably, the Δ141–143 variant induced higher levels of IFN-β compared to the other virus
variants (Figure 2).
Figure 2. Induction of IFN-β release by partially deleted Nsp1 SARS-CoV-2 lineages. Secreted IFN-
β was assessed in Calu-3 cells infected (MOI = 0.01) with BA.2 and the relative ∆82–85 and ∆141–143
lineages, or with the BQ.1.1 and the ∆82–86 variants by enzyme-linked immunoassay (ELISA) at 48
h and 72 h p.i.. Negative control (Ctr-) was represented by uninfected Calu-3 cells. Quantitative
evaluation, based on relative standard curves, was performed and the results are reported as mean
concentration (pg/mL) ± standard deviations (SDs) from at least three independent experiments (n
≥ 3). Significance is reported as ns, not significant, * p < 0.05 and ** p < 0.001 with respect to the mock-
infected control (Ctr-).
Conversely, the BQ.1.1 strain, along with its Δ82–86 variant, did not trigger a signifi-
cant secretion of IFN-β expression at 48 h p.i (p > 0.05) (Figure 2). This observation was
likely aributed to their limited growth in Calu-3 cells at the specified time point, as
Figure 2. Induction of IFN-
β
release by partially deleted Nsp1 SARS-CoV-2 lineages. Secreted IFN-
β
was assessed in Calu-3 cells infected (MOI = 0.01) with BA.2 and the relative
∆
82–85 and
∆
141–143
lineages, or with the BQ.1.1 and the
∆
82–86 variants by enzyme-linked immunoassay (ELISA) at
48 h and 72 h p.i.. Negative control (Ctr-) was represented by uninfected Calu-3 cells. Quantitative
evaluation, based on relative standard curves, was performed and the results are reported as mean
concentration (pg/mL)
±
standard deviations (SDs) from at least three independent experiments
(n
≥
3). Significance is reported as ns, not significant, * p< 0.05 and ** p< 0.001 with respect to the
mock-infected control (Ctr-).
Viruses 2024,16, 689 7 of 18
Conversely, the BQ.1.1 strain, along with its
∆
82–86 variant, did not trigger a significant
secretion of IFN-
β
expression at 48 h p.i (p> 0.05) (Figure 2). This observation was likely
attributed to their limited growth in Calu-3 cells at the specified time point, as shown in
Figure 1C. However, at 72 h p.i., although no significant difference was recorded between
the two strains (p> 0.05), only the parental BQ.1.1 lineage significantly stimulated the
cytokine release compared to the mock-infected control (p= 0.0048). These results highlight
that the altered replication rates of the deleted strains do not depend on the presence of
IFN-βbut, mostly, reside in other strain-specific factors.
3.3. The N-Terminal Region of Nsp1 Emerges as Crucial for Its Interaction with the Host Cellular
Transcription/Translation Machinery
We next investigated whether the altered virus fitness was a consequence of Nsp1-
modified activity. Therefore, the naturally occurring viral protein variants were cloned,
and the activity of the viral proteins towards cellular and viral mRNA translation and
stability was investigated. It is worth mentioning that the Nsp1 protein shares the S
135
R
mutation among the Omicron sublineages with respect to the ancestral Wuhan-1; thus,
only BA.2 Nsp1 was included in subsequent experiments as a control. To this purpose,
HEK-293T cells, known as the gold standard for investigating human and viral protein
activity through transient overexpression, were selected. HEK-293T cells were transfected
to transiently express the Wuhan-1, the BA.2 or the deleted (
∆
82–85;
∆
82–86; and
∆
141–143)
Nsp1 plasmid in combination with a cellular promoter-driven (human beta globin; HBB)
Green Fluorescent Protein (GFP)-expressing plasmid. GFP-specific mRNA content was
estimated by quantitative RT-qPCR. As shown in Figure 3A, the RT-qPCR assay evidenced
that, among the SARS-CoV-2 Nsp1 variants tested, the Wuhan-1 and BA.2 SARS-CoV-2
Nsp1 markedly suppressed HBB-mediated GFP expression at the transcriptional level,
leading to a ~4-fold decrease in specific mRNA (p= 0.0003) compared to the basal, empty
plasmid transfected sample (Ctr-).
Similarly, the
∆
141–143 viral protein mutant promoted a robust HBB-driven mRNA
decay, with a mean fold reduction in GFP mRNA of 3.20
±
0.45 (p= 0.0007), (Figure 3A). A
different behavior was instead observed in the
∆
82–85 and
∆
82–86 Nsp1 mutants, which
displayed no degrading activity towards cellular mRNAs (p> 0.05) (Figure 3A). Among the
Nsp1 protein mutants tested, both the
∆
82–85 and
∆
82–86 showed a significantly reduced
degrading activity on cellular mRNAs compared to the Wuhan-1 variant, with a mean
reduction in protein activity of 4.52
±
1.43 (p= 0.012) and 2.65
±
0.59 (p= 0.037), respectively
(Figure S1A). Alteration in HBB-mediated GFP expression was also demonstrated at the
protein level on lysates of cells transfected with HBB-GFP and Nsp1 variants. The GFP
protein content was estimated by immunoblotting. As reported in Figure 3B, all protein
variants significantly reduced reporter protein translation, although the
∆
82–85 and
∆
82–86
mutants had minor effects compared to the Wuhan-1 Nsp1 (Figure S1B). These results
highlight the functional significance of 82–85 residues in modulating host cellular processes;
moreover, they indicate that the valine residue at the 86 position (V
86
) is not a determinant
for Nsp1 protein function. These insights contribute to our understanding of how SARS-
CoV-2 causes cell damage and may impact on the development of therapeutic strategies
targeting viral proteins.
Viruses 2024,16, 689 8 of 18
Viruses 2024, 16, x 8 of 18
Figure 3. Evaluation of host cell shutoff in Nsp1 variants expressing cells. The impact of Nsp1-de-
leted mutants on the human beta-globin (HBB) promoter-mediated Green Fluorescent Protein (GFP)
was assessed in HEK-293T cells. (A) The transcriptional control of GFP was examined by using total
RNA purified from HBB-GFP transfected samples, either in combination with an empty plasmid
(Ctr-) or Nsp1-expressing plasmids. Cells were collected at 48 h post-transfection and the specific
GFP mRNA content was measured by quantitative reverse-transcription polymerase chain reaction
(RT-qPCR). GAPDH gene expression served for relative quantification based on the 2−ΔΔCt method.
At least three independent experiments (n ≥ 3) were conducted, and the representative data are pre-
sented as mean values ± standard deviations. (B) The modulation of GFP expression in Nsp1 vari-
ants expressing HEK-293T cells was also evaluated as protein levels through Western bloing. Equal
amounts of total cell lysates were resolved by SDS-PAGE, and specific antibodies were used for GFP,
GAPDH, and Nsp1 proteins, with a representative image provided in the lower panel. Densitomet-
ric analysis was conducted using ImageJ software. Graph values are presented as the mean fold
change in GFP band intensity ± standard deviations (SDs). Significance is reported as ‘ns’, not sig-
nificant, ** p < 0.001 and *** p < 0.0005 between each sample and the control (Ctr-).
Figure 3. Evaluation of host cell shutoff in Nsp1 variants expressing cells. The impact of Nsp1-deleted
mutants on the human beta-globin (HBB) promoter-mediated Green Fluorescent Protein (GFP) was
assessed in HEK-293T cells. (A) The transcriptional control of GFP was examined by using total
RNA purified from HBB-GFP transfected samples, either in combination with an empty plasmid
(Ctr-) or Nsp1-expressing plasmids. Cells were collected at 48 h post-transfection and the specific
GFP mRNA content was measured by quantitative reverse-transcription polymerase chain reaction
(RT-qPCR). GAPDH gene expression served for relative quantification based on the 2
−∆∆Ct
method.
At least three independent experiments (n
≥
3) were conducted, and the representative data are
presented as mean values
±
standard deviations. (B) The modulation of GFP expression in Nsp1
variants expressing HEK-293T cells was also evaluated as protein levels through Western blotting.
Equal amounts of total cell lysates were resolved by SDS-PAGE, and specific antibodies were used for
Viruses 2024,16, 689 9 of 18
GFP, GAPDH, and Nsp1 proteins, with a representative image provided in the lower panel. Den-
sitometric analysis was conducted using ImageJ software. Graph values are presented as the mean
fold change in GFP band intensity
±
standard deviations (SDs). Significance is reported as ‘ns’, not
significant, ** p< 0.001 and *** p< 0.0005 between each sample and the control (Ctr-).
3.4. The 82–85 Nsp1 N-Terminal Domain Is Involved in Viral mRNA Expression
The activity of the original and deleted versions of the Nsp1 protein towards viral
mRNA transcription, translation, and stability was investigated in HEK-293T cells trans-
fected with plasmids encoding either the Wuhan-1 and BA.2 Nsp1 or the deleted versions
(
∆
82–85;
∆
82–86, and
∆
141–143) of Nsp1, along with the viral 5
′
-leader stem loop 1 (5
′
-Ld-
SL1) or the full-length 5
′
-UTR-driven Green Fluorescent Protein (GFP). The GFP-specific
mRNA content was estimated using quantitative RT-qPCR. Among the Nsp1 proteins,
Wuhan-1 and BA.2 Nsp1 were capable of recognizing and mediating the expression of the
reporter gene downstream of either the viral 5
′
-Ld-SL1 or the 5
′
-UTR sequences, without
any evident significant difference among them (Figure S2A). An increase in GFP mRNA
under the 5
′
-Ld-SL1 or 5
′
-UTR sequence was observed both in Wuhan-1 (p= 0.0004) and
BA.2 (p= 0.0002), respectively (Figure 4A and Figure S2A).
Viruses 2024, 16, x 9 of 18
3.4. The 82–85 Nsp1 N-Terminal Domain Is Involved in Viral mRNA Expression
The activity of the original and deleted versions of the Nsp1 protein towards viral
mRNA transcription, translation, and stability was investigated in HEK-293T cells trans-
fected with plasmids encoding either the Wuhan-1 and BA.2 Nsp1 or the deleted versions
(Δ82–85; Δ82–86, and Δ141–143) of Nsp1, along with the viral 5′-leader stem loop 1 (5′-Ld-
SL1) or the full-length 5′-UTR-driven Green Fluorescent Protein (GFP). The GFP-specific
mRNA content was estimated using quantitative RT-qPCR. Among the Nsp1 proteins,
Wuhan-1 and BA.2 Nsp1 were capable of recognizing and mediating the expression of the
reporter gene downstream of either the viral 5′-Ld-SL1 or the 5′-UTR sequences, without
any evident significant difference among them (Figure S2A). An increase in GFP mRNA
under the 5′-Ld-SL1 or 5′-UTR sequence was observed both in Wuhan-1 (p = 0.0004) and
BA.2 (p = 0.0002), respectively (Figures 4A and S2A).
Figure 4. Evaluation of Nsp1 variants’ modulation of viral genes expression. The impact of Nsp1-
deleted mutants on viral 5′-Ld-SL1- or 5′-UTR-mediated GFP expression was evaluated in HEK-
293T cells. (A) Transcriptional control of GFP was examined in 5′-Ld-SL1- or 5′-UTR-GFP-
Figure 4. Evaluation of Nsp1 variants’ modulation of viral genes expression. The impact of Nsp1-
deleted mutants on viral 5
′
-Ld-SL1- or 5
′
-UTR-mediated GFP expression was evaluated in HEK-293T
Viruses 2024,16, 689 10 of 18
cells. (A) Transcriptional control of GFP was examined in 5
′
-Ld-SL1- or 5
′
-UTR-GFP-transfected
samples, either in combination with an empty plasmid (Ctr-) or Nsp1-expressing plasmids. Cells
were collected at 48 h, and total RNA was purified. GFP and GAPDH mRNAs were quantified by
RT-qPCR using the 2
−∆∆Ct
analysis. Data were presented as mean values
±
standard deviations
(SD) from different experiments. (B) GFP protein content was assessed in Nsp1 variants expressing
HEK-293T cells by Western blotting. Equal amounts of total cell lysates were resolved by SDS-PAGE,
and specific antibodies were used to probe for GFP, GAPDH, and Nsp1 proteins. A representative
image is provided in the lower panel. Densitometric analysis was performed using ImageJ software.
Graph values are presented as the mean fold change in GFP band intensity
±
standard deviations
(SDs). Significance is reported as ‘ns’, not significant, * p< 0.05, ** p< 0.001, *** p< 0.0005, and
**** p< 0.0001 between each sample and the control (Ctr-).
Among the other investigated SARS-CoV-2 mutants, the
∆
141–143 variant exhib-
ited behavior similar to that of the Wuhan-1 Nsp1, inducing GFP mRNA accumulation
using both 5
′
-Ld-SL1 and 5
′
-UTR sequences. On the contrary, the
∆
82–85 and
∆
82–86
Nsp1 proteins did not have any effect (p> 0.05) on GFP mRNA synthesis or stability
(Figures 4A and S2A).
However, the immunoblotting performed on transfected HEK-293T
cell lysates revealed an evident reduction (p< 0.0001) in GFP protein translation when
the
∆
82–85 and
∆
82–86 Nsp1 proteins were expressed
(Figures 4B and S2B).
Conversely,
neither the
∆
141–143 nor the Wuhan-1 Nsp1 displayed any impairment in GFP protein
translation (Figures 4B and S2B).
3.5. The 82–85 Nsp1 Domain Is Required for Cellular and Viral mRNAs Stability Control
The results presented above highlighted the deleted Nsp1 variants’ ability to abrogate
both cellular and viral protein translation without having any degrading activity towards
mRNA. Based on that, we further investigated the activity of these SARS-CoV-2 Nsp1
variants in HEK-293T cells, including only the Wuhan-1 control, as BA.2 Nsp1 behaved
similarly. To better understand whether the modulation of GFP protein levels previously
observed in Nsp1-expressing cells resulted from altered viral protein-degrading activity
of specific mRNAs, we treated transfected cells with actinomycin D (ActD), a well-known
transcription inhibitor that is frequently used for mRNA stability analysis. To validate that
the observed decrease in GFP protein levels in Nsp1 with HBB-GFP-expressing cells was
not attributable to enhanced mRNA decay, we assessed GFP mRNA turnover following
treatment with ActD. The impact of Wuhan-1 Nsp1 on GFP mRNA stability became notably
pronounced over time, especially 4 h post ActD treatment (fold decrease of 0.26
±
0.07;
p= 0.0004).
Thus, the observed drop in reporter protein content could, at least in part, be a
consequence of diminished specific mRNA intracellular content (Figure 3B). Conversely,
both the
∆
82–85 and
∆
82–86 mutants exhibited only a minimal, non-significant effect on
GFP mRNA decay, as for the empty vector control (Ctr-) (p> 0.05) (Figure 5A). This suggests
that mechanisms other than mRNA degradation occurred to inhibit protein translation.
Furthermore, the diminished GFP protein levels observed were not due to any alterations
in protein stability or degradation. This conclusion was supported by the cycloheximide
(CHX) chase assay to measure steady-state protein stability. CHX inhibits the elongation step
in eukaryotic protein translation, thereby preventing protein synthesis upon administration.
Semiquantitative immunoblotting showed that no significant decline in GFP protein levels
over time (p> 0.05) occurred when both the Wuhan-1 and deleted Nsp1 were expressed along
the reporter gene in CHX-treated HEK-293T transfected cells (Figure 5B). Intriguingly, all the
aforementioned Nsp1 variants exhibited a marked inhibition of reporter translation from the
beginning of the CHX treatment (p< 0.0001), confirming that no post-translational events
occurred to support protein content decay (Figure 5B). Subsequently, ActD treatment of 5
′
-Ld-
SL1-mediated reporter gene expressing cells evidenced the Wuhan-1 Nsp1’s ability to maintain
GFP mRNA stability, consistent with its known protective function on viral mRNAs (Figure 5C).
Instead, Nsp1 variants carrying the
∆
82–85 and
∆
82–86 deletions did not show any particular
protective activity on the 5
′
-Ld-SL1-mediated GFP mRNA (Figure 5C). To argue the protective
effect of 5
′
-Ld-SL1-containing mRNA by the Nsp1, immunoblotting was performed on CHX-
Viruses 2024,16, 689 11 of 18
treated samples. This analysis indicated that the reporter protein translation and turnover
remained unaffected over time by CHX treatment of Wuhan-1 Nsp1-expressing cells, as well as
those concurrently expressing the
∆
82–85 and
∆
82–86 Nsp1 variants (Figure 5D). Interestingly,
the CHX chase assay revealed a significant destabilizing effect caused by the
82–85/86
deletion
on Nsp1 itself. In particular, whereas the Wuhan-1 Nsp1 demonstrated robust stability up to 4 h
from CHX treatment, the
∆
82–85 and
∆
82–86 protein variants exhibited a three-fold reduced
stability, within the same timeframe (Figure 5E). Similarly, the deleted Nsp1 proteins exhibited
significantly less stability than their corresponding original variants during viral infection
and replication in Vero E6 cells. Specifically, CHX treatment of BA.2_
∆
82–85- (fold change
0.17 ±0.04; p< 0.0001)
and BQ.1.1_
∆
82–86- (fold change 0.13
±
0.01; p< 0.0001) infected cells
resulted in drastic Nsp1 dropout at 4 h post-treatment (Figure 5F). In contrast, Vero E6 cells
infected with the corresponding original lineages showed an increased Nsp1 stability (fold
change 0.52
±
0.12; p= 0.01 and 0.57
±
0.01; p= 0.0083, respectively) at 4 h post-CHX treatment
(Figure 5F). Therefore, the 82–85/86 Nsp1 N-terminal deletion also affects its stability. Thus,
we could speculate that the reduced protein stability of the deleted Nsp1 may affect, at least in
part, its diminished protection of viral mRNAs.
Viruses 2024, 16, x 11 of 18
Figure 5. Nsp1 variants’ activity towards cellular and viral mRNAs. (A) The influence of both orig-
inal and deleted Nsp1versions on cellular mRNA decay was investigated in HEK-293T cells. A time-
course experiment was conducted using actinomycin D (ActD), a transcriptional inhibitor. Cells
were collected at indicated time points, and after total RNA isolation, GFP mRNA was quantified
Figure 5. Nsp1 variants’ activity towards cellular and viral mRNAs. (A) The influence of both
original and deleted Nsp1versions on cellular mRNA decay was investigated in HEK-293T cells. A
time-course experiment was conducted using actinomycin D (ActD), a transcriptional inhibitor. Cells
Viruses 2024,16, 689 12 of 18
were collected at indicated time points, and after total RNA isolation, GFP mRNA was quantified by
RT-qPCR using the 2
−∆∆Ct
analysis. (B) The impact of different Nsp1 variants on HBB-mediated GFP
protein expression was assessed in HEK-293T cells, either mock-treated or treated with cycloheximide
(CHX). Equal amounts of total cell lysates from samples collected at indicated time points after
CHX treatment were resolved by SDS-PAGE and specific antibodies were used to probe for GFP
and GAPDH. (C) GFP mRNA levels were quantified by RT-qPCR in HEK-293T cells expressing
Nsp1 variants along with either 5
′
-Ld-SL1- or 5
′
-UTR-GFP and treated with ActD for specified
periods. (D) HEK-293T cells expressing the GFP protein downstream of either the 5
′
-Ld-SL1- or
5
′
-UTR-GFP promoters were co-transfected in order to express different Nsp1 variants. At 48 h
post-transfection, cells were mock- or CHX-treated and collected at indicated times. Equal amounts of
total cell lysates were resolved by SDS-PAGE and GFP or GAPDH proteins were probed by Western
blotting procedure. The protein stability of Nsp1 variants was determined by Western blotting on
total cell lysates of transfected HEK-293T cells (E) or Vero E6 cells infected with indicated virus strains
(F) and subjected to a time-chase with CHX. Densitometric analysis of Western blotting images was
performed using ImageJ software. Graph bars represent mean values
±
standard deviations (SD)
from different experiments. Significance is reported as * p< 0.05, ** p< 0.001, *** p< 0.0005, and
**** p< 0.0001.
3.6. The 82–85 Nsp1 Domain Alters Viral mRNAs’ Binding Affinity
Docking calculations were performed to investigate the interactions between Nsp1 and
the viral 5
′
-Ld-SL1 mRNA and to evaluate how deletion of the 82–85 domain could affect
mRNA binding. Figure 6A shows that the backbone of Pro82 is involved in a hydrogen
bond with U101, while His83 forms a hydrogen bond network with U101, A104, and U105.
This interaction pattern confirms the direct role played by sequence 82–85 of Nsp1 in
mRNA binding.
Viruses 2024, 16, x 13 of 18
3.6. The 82–85 Nsp1 Domain Alters Viral mRNAs’ Binding Affinity
Docking calculations were performed to investigate the interactions between Nsp1
and the viral 5′-Ld-SL1 mRNA and to evaluate how deletion of the 82–85 domain could
affect mRNA binding. Figure 6A shows that the backbone of Pro82 is involved in a hydro-
gen bond with U101, while His83 forms a hydrogen bond network with U101, A104, and
U105. This interaction paern confirms the direct role played by sequence 82–85 of Nsp1
in mRNA binding.
Figure 6. Molecular docking to assess the interaction affinity between deleted Nsp1 and 5′-Ld-SL1
mRNA. (A) Graphical representation of the best-scored docked pose of the wt protein (grey, taken
from the protein data bank entry 7K7P) and 5′-SL1 mRNA (orange); (B) superimposition of the wt
(grey) and Δ82–85 variant (deep teal); (C) the best-scored docked pose of the Δ82–85 variant (deep
teal) and 5′-SL1 mRNA (orange).
Furthermore, superimposing the structures of Wuhan-1 and Δ82–85 proteins reveals
differences in spatial and conformational arrangements, suggesting that the shorter loop
formed by Thr80 and the hydrophobic amino acids Ala81 and Val86 may engage in differ-
ent interactions with mRNA (Figure 6B). Although Δ82–85 Nsp1 retains the ability to bind
viral mRNA, the deletion of the four amino acids results in the loss of loop interactions
with nucleic acid bases (Figure 6C). Consequently, the modified Nsp1 protein exhibits a
decreased affinity for mRNA binding compared to Wuhan-1 Nsp1, as supported by the
docking scores (Wuhan-1: −299 kcal/mol; Δ82–85: −248 kcal/mol). In summary, molecular
docking simulations and superposition of three-dimensional structures of Wuhan-1 and
Δ82–85 Nsp1 reveal significantly different abilities to bind viral mRNA, consistent with
the reduced capacity of the protein to recognize and translate viral mRNA.
4. Discussion
Viruses have developed various mechanisms in order to overcome host defenses and
exploit cellular metabolic pathways for their own genome replication and protein synthe-
sis. Among these strategies, virus-induced host protein shut-down is a crucial step for
Figure 6. Molecular docking to assess the interaction affinity between deleted Nsp1 and 5
′
-Ld-SL1
mRNA. (A) Graphical representation of the best-scored docked pose of the wt protein (grey, taken
from the protein data bank entry 7K7P) and 5
′
-SL1 mRNA (orange); (B) superimposition of the wt
(grey) and
∆
82–85 variant (deep teal); (C) the best-scored docked pose of the
∆
82–85 variant (deep
teal) and 5′-SL1 mRNA (orange).
Viruses 2024,16, 689 13 of 18
Furthermore, superimposing the structures of Wuhan-1 and
∆
82–85 proteins reveals
differences in spatial and conformational arrangements, suggesting that the shorter loop
formed by Thr80 and the hydrophobic amino acids Ala81 and Val86 may engage in different
interactions with mRNA (Figure 6B). Although
∆
82–85 Nsp1 retains the ability to bind
viral mRNA, the deletion of the four amino acids results in the loss of loop interactions
with nucleic acid bases (Figure 6C). Consequently, the modified Nsp1 protein exhibits a
decreased affinity for mRNA binding compared to Wuhan-1 Nsp1, as supported by the
docking scores (Wuhan-1:
−
299 kcal/mol;
∆
82–85:
−
248 kcal/mol). In summary, molecular
docking simulations and superposition of three-dimensional structures of Wuhan-1 and
∆
82–85 Nsp1 reveal significantly different abilities to bind viral mRNA, consistent with the
reduced capacity of the protein to recognize and translate viral mRNA.
4. Discussion
Viruses have developed various mechanisms in order to overcome host defenses and
exploit cellular metabolic pathways for their own genome replication and protein synthesis.
Among these strategies, virus-induced host protein shut-down is a crucial step for enhanc-
ing viral pathogenesis. This phenomenon has been demonstrated for several pathogens,
including the Influenza virus and Coronaviruses (CoVs) [
40
–
44
]. Simultaneously, viral
transcripts must maintain robust expression during host shutoff, which can be achieved
through the employment of alternative RNA processing and ribosome recruitment mech-
anisms [
45
,
46
]. In this context, previous studies on SARS-CoV-1 indicated that the Nsp1
viral protein is involved in both viral replication and the innate immune system hindrance.
Numerous studies have also investigated the translational repression and the degrading
functions of SARS-CoV-2 Nsp1 on cellular mRNAs. However, the mechanisms by which
the viral Nsp1 protein coordinates both activities against cellular mRNA while sparing
viral transcripts remain largely elusive. Virus variants containing various deletions in the
Nsp1 N-terminus have been previously described by Lin et al. [
42
]. The deletion identified
in that study (a.a. 79–89) was likely not important for Nsp1–ribosome binding ability but
significantly suppressed the type I IFN response and attenuated virus replication [
47
]. In
this study, we analyzed mutational variations in the SARS-CoV-2 Nsp1 protein through
the use of naturally occurring Nsp1 partially deleted virus lineages. Virus strains bear-
ing N- (a.a. 82–85/82–86) or C-terminal (a.a. 141–143) Nsp1 deletions were isolated and
tested
in vitro
by using cell-based assays to evaluate their respective fitness. Despite the
considerable number of sequences deposited so far, the global prevalence of the
∆
82–85
and
∆
82–86 Nsp1-deleted strains is 0.82% and 0.78%, respectively, and they are distributed
worldwide. Within the indicated domain, the methionine at position 85 was found to
be the most frequently mutated or deleted among all SARS-CoV-2 strains, with almost
2.9% prevalence of changes. Conversely, other residues in the selected domain were less
frequently targeted by changes (1.5%), suggesting their reduced impact on protein function
(Table S1). Unfortunately, experimental investigation of such mutants was not feasible,
as clinical specimens carrying these mutations were identified and isolated for further
in vitro
analysis. Thus, we could only speculate about their implications in virus fitness. In
contrast, a higher prevalence (2.02%) was observed for virus lineages carrying the
∆
141–143
deletion. Nonetheless, these deletions were equally reported by Outbreak.info [
48
] as
circulating SARS-CoV-2 lineages or variants. This study may significantly contribute to
the knowledge of Nsp1 function in terms of virus gene translation, virus replication, and
innate immunity modulation. In comparison to previous studies, we identified a more
specific amino-acid sequence (82–85) as critical for Nsp1 function during viral infection
and replication. We observed that virus lineages with the N-terminal deletion (
∆
82–85
and
∆
82–86) exhibited a more pronounced attenuation in virus replication compared to
their corresponding original lineage counterparts. This was demonstrated in both type
I interferon (IFN-
β
)-deficient (Vero E6 cells) and competent (Calu-3 cells) cell systems,
supporting the evidence that the reduced replication proficiency was not influenced by
the antiviral activity of secreted IFN-
β
. Indeed, despite the markedly reduced progeny
Viruses 2024,16, 689 14 of 18
virus release in
∆
82–85 and
∆
82–86 Calu-3-infected cells, a sustained release of IFN-
β
was
observed in original and deleted virus strains, regardless of the strain used. However, the
augment secretion of IFN-
β
by both the BA.2 deleted variants infected Calu-3 cells evi-
denced the Nsp1 negligible activity towards the cytokine control. Thus, factors other than
the Nsp1 protein were involved in the induction of IFN-
β
in a strain-specific manner, as
clearly evidenced by previous
reports [2–6,27,28].
The replication efficiency and production
of intracellular dsRNAs, for instance, play a central role in pattern-recognition receptors’
(PRRs) activation and downstream IFN-
β
induction [
28
]. We then investigated the host
shutoff activities of selected deleted Nsp1 proteins. N-terminal domain (NTD)-deleted
Nsp1 proteins (amino acid residues 82–85) retained the ability to block the translation
of proteins under a human promoter (HBB-GFP) without affecting mRNA abundance.
This was observed through molecular investigations using RT-qPCR and immunoblotting
for the GFP reporter protein. While the
∆
82–85 and
∆
82–86 Nsp1 proteins showed an
evident reduction in the degrading activity of cellular mRNAs, ribosome recruitment and
translational machinery hindrance still occurred, leading to the partial suppression of host
protein translation (Figure 4). In addition, molecular docking analysis of deleted Nsp1 and
5
′
-Ld-SL1 viral mRNAs revealed a reduced capability of the Nsp1 variants to recognize
and efficiently bind viral mRNAs (Figure 6). However, the cycloheximide (CHX) chase
supported the evidence that the decreased viral replication could be partly attributable
to decreased protein stability of deleted Nsp1 rather than enhanced RNA degradation
(Figure 5). Indeed, the stability of deleted Nsp1 was found to be altered compared to
the parental protein
in vitro
in both transfected and infected cells, potentially affecting
protein function in terms of viral mRNA protection and ribosome binding for translation.
Although 5
′
-Ld-SL1 mRNAs were not degraded, they were neither stabilized nor engaged
by ribosomes for efficient translation into viral components. This aspect warrants further
investigation to understand the impact of mutated viral proteins on virus fitness. These
observations align with the findings of other studies. It appears that the Nsp1 NTD pro-
motes RNA decay, and R
99
and R
124
/K
125
residues are believed to play a fundamental role
in this mechanism, presumably by recruiting or stabilizing host nucleases at the Nsp1–
ribosome–mRNA complex site [
11
,
26
,
43
]. However, it is necessary to further investigate
the role of Nsp1. Moreover, the aforementioned mutations rescued cellular mRNA content,
confirming that Nsp1 domains responsible for mRNA stability and translation are located
in distinct parts of the protein. Unlike cellular mRNAs, viral mRNAs are highly translated
in the presence of Nsp1 [
46
]. For Coronaviruses, including SARS-CoV-2, a conserved stem
loop (SL1) within the 5
′
-UnTranslated Region (5
′
-UTR) present on viral mRNAs is required
for Nsp1-mediated viral gene translation [
34
,
45
,
46
]. Several models have been proposed
to explain the escape of viral mRNA from degradation. The most reliable one suggests
that viral mRNAs containing the SL1 interact with Nsp1 and, in association with cellular
factor(s), induce a conformational change in Nsp1 that unplugs its C-terminal domain from
the 40S entry channel, thereby allowing mRNA translation [
36
,
47
–
49
]. Specific mutations
within NTD of Nsp1 (R
99
A and R
124
A/K
125
A) have negative effects on the translation
of SARS-CoV-2 leader mRNA, instead. In particular, these residues are crucial for the
SL1-triggered conformational changes to Nsp1 or may prevent proper assembly of this
multicomplex that enables viral mRNA translation [
11
]. In this study, our findings highlight
that the
∆
82–85 Nsp1 variant can diminish the translation of viral mRNA containing SL1
without impacting its decay but significantly affecting its recognition by ribosomes. This
discovery holds significant implications to understand the role of Nsp1 in virus pathogene-
sis. It suggests that specific Nsp1 mutations or deletions, such as the 82–85 domain, may
impair viral pathogenesis due to translational suppression of viral transcripts, although
the shutoff of host genes still occurs. Notably, among the eleven Nsp1 N-terminal domain
(NTD) amino-acid deletions (a.a. 79–89) previously identified as critical for virus fitness
by Lin et al., only four (a.a. 82–85) are associated with the lower viral load. This insight
has implications for potential antiviral drugs targeting the Nsp1 protein, particularly the
82–85 region.
Despite Nsp1 being a validated target for therapeutic action, there have been
Viruses 2024,16, 689 15 of 18
few studies conducted on Nsp1 for structure-based drug discovery through in silico screen-
ing and identification of potential inhibitors. Only a few studies have been conducted,
resulting in the identification of potential antiviral molecules. Among them, Montelukast
and Mitoxantrone dihydrochloride have been proven to efficiently bind to the C-terminus
of SARS-CoV-2 Nsp1 [50,51].
The Nsp1 N-terminal domain can also be a valid target for antiviral drug design.
Specifically, since the N-terminal domain of Nsp1 interacts with the stem-loop 1 (SL1) on
the 5
′
-UTR of viral mRNA, targeting the SL1 could prevent its binding to Nsp1 and, conse-
quently, decrease the viral translation of its mRNAs. Several drugs have been shown to bind
the SL1 region, including glycyrrhizic acid, lobaric acid, garcinolic acid, and tirilazad [
52
,
53
].
Therefore, our results pave the way for an alternative approach to directly target the N-
terminal domain of the viral Nsp1 protein rather than the SL1 genomic elements.
Furthermore, the reported data provide valuable information about Nsp1 activity and
functional domains which could be examined to reduce the virus virulence and enhance
our knowledge of the proteins that could attenuate the virus.
5. Conclusions
In this study, our findings highlight that the 82–85 domain within the N-terminus
of the SARS-CoV-2 Nsp1 protein is crucial in inhibiting the translation of viral mRNAs
containing the 5
′
-SL1 sequence, without affecting their decay. This evidence provides new
insights into virus biology and carries significant implications for understanding the role
of Nsp1 in virus pathogenesis. Our data suggest that specific mutations or deletions of
the 82–85 domain of Nsp1 may impair viral pathogenesis by suppressing the translation
of viral proteins, in agreement with the observed lower viral load in naturally occurring
virus lineages having the indicated Nsp1 deletion. In detail, the deletion of this domain
negatively impacts on viral mRNAs recognition by the viral protein and its interaction with
ribosomes. This insight paves the way for the development of antiviral drugs targeting the
Nsp1 protein. Furthermore, the reported data provide valuable information about Nsp1
activity and functional domains that could be explored to mitigate virus virulence and
enhance our knowledge of proteins capable of attenuating the virus.
6. Limitation of the Study
The analysis of the correlation of virus variants with clinical profiles of the disease
was hindered by the lack of clinical data concerning patients infected with the specific
SARS-CoV-2 lineages investigated. This study relied on experiments that serve as a proxy
for cellular or viral transcripts.
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/v16050689/s1,
Figure S1: Evaluation of host cell shutoff
in Nsp1 variants expressing cells; Figure S2: Evaluation of Nsp1 variants modulation of viral genes
expression; Table S1: Prevalence of mutational changes in the N- and C-terminal domains of SARS-
CoV-2 Nsp1 protein.
Author Contributions: Conceptualization, G.G.S. and M.G.C.; Data curation, G.G.S. and G.A.; Formal
analysis, G.G.S., F.M., C.I.T. and G.A.; Investigation, G.G.S., F.M., C.I.T. and G.A.; Methodology, G.G.S.
and G.A.; Resources, M.G.C.; Funding acquisition, M.G.C.; Writing—original draft, G.G.S., G.A. and
M.G.C.; Writing—review and editing, G.G.S. and M.G.C. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by Piano di Sostegno alla Ricerca (PSR) F-CUR 2022, University
of Siena, Italy (G.G.S.); Italian Ministry of University and Research (PNRR-National Center for
Gene Therapy and Drugs based on RNA Technology) CN00000041 (M.G.C.); EU funding within the
NextGenerationEU with Italian Ministry of University and Research (MUR) PRIN 2022 2022F37JRF
(M.G.C.) and Italian MIUR PNRR “One Health Basic and Translational Research Actions addressing
Unmet Needs on Emerging Infectious Diseases” CUP B63C22001400007 (F.M. and C.I.T.).
Institutional Review Board Statement: Not applicable.
Viruses 2024,16, 689 16 of 18
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article or Supplementary Material. The
data presented in this study are available upon request.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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