4.

926

Article

Prion-like Domains in Spike Protein

of SARS-CoV-2 Differ across Its
Variants and Enable Changes in
Affinity to ACE2

George Tetz and Victor Tetz

Special Issue
Recent Advances in COVID-19 Receptor, Interaction, Biological Dynamic and Innovative Therapies
Edited by
Prof. Dr. Francesco Inchingolo, Prof. Dr. Gianfranco Favia, Prof. Dr. Antonio Scarano,
Prof. Dr. Gianluca M. Tartaglia, Dr. Gianna Dipalma, Dr. Felice Lorusso and Dr. Ioana-Roxana Bordea

https://doi.org/10.3390/microorganisms10020280
 microorganisms

Article
Prion-like Domains in Spike Protein of SARS-CoV-2 Differ
across Its Variants and Enable Changes in Affinity to ACE2
George Tetz 1,2, * and Victor Tetz 1

1 Department of Proteome Research, Human Microbiology Institute, New York, NY 10013, USA;
[email protected]
2 Department for Proteome Research, Tetz Laboratories, New York, NY 10013, USA
* Correspondence: [email protected]

Abstract: Currently, the world is struggling with the coronavirus disease 2019 (COVID-19) pandemic,
caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Prions are proteins
that possess a unique conformational conversion, with the ability to rapidly shift between multiple
conformations due to residue hydrophobicity and net sequence charge, and viral prion-like proteins
are known as potential regulators of viral infections. However, the prion-like domains (PrD) in the
SARS-CoV-2 proteome have not been analyzed. In this in silico study, using the PLAAC algorithm,
we identified the presence of prion-like domains in the SARS-CoV-2 spike protein. Compared with
other viruses, a striking difference was observed in the distribution of prion-like domains in the spike



protein since SARS-CoV-2 is the only coronavirus with a prion-like domain found in the receptor-

 binding domain of the S1 region of the spike protein. The presence and unique distribution of
Citation: Tetz, G.; Tetz, V. Prion-like prion-like domains in the SARS-CoV-2 receptor-binding domains of the spike protein are particularly
Domains in Spike Protein of interesting since although the SARS-CoV-2 and SARS-CoV S proteins share the same host cell receptor,
SARS-CoV-2 Differ across Its Variants
angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2 demonstrates a 10- to 20-fold higher affinity
and Enable Changes in Affinity to
for ACE2. We identified prion-like domains in the α1 helix of the ACE2 receptor that interact with
ACE2. Microorganisms 2022, 10, 280.
the viral receptor-binding domain of SARS-CoV-2. Finally, we found substantial differences in the
https://doi.org/10.3390/
prion-like domain of the S1 region of the spike protein across emerging variants including Omicron
microorganisms10020280
(B.1.1.529). Taken together, the present findings indicate that the identified PrDs in the SARS-CoV-2
Academic Editors: receptor-binding domain (RBD) and ACE2 region that interact with RBD play important functional
Francesco Inchingolo,
roles in viral adhesion and entry.
Gianfranco Favia, Antonio Scarano,
Gianluca M. Tartaglia,
Keywords: COVID-19; SARS-CoV-2; variants; prion-like domains; PrD; ACE2; Delta variant;
Gianna Dipalma, Felice Lorusso and
Omicron variant
Ioana-Roxana Bordea

Received: 28 December 2021

Accepted: 24 January 2022
Published: 25 January 2022 1. Introduction
Publisher’s Note: MDPI stays neutral The world is struggling with the pandemic caused by a novel coronavirus (now named
with regard to jurisdictional claims in severe acute respiratory syndrome-2 or SARS-CoV-2, causing coronavirus disease 2019
published maps and institutional affil- (COVID-19)) that has expanded from Wuhan throughout China and the wider world [1]. By
iations. 12 December 2021, there were over 259 million confirmed cases of the virus worldwide and it
had contributed to over 5.2 million deaths (https://www.worldometers.info/coronavirus/,
accessed on 10 January 2022).
SARS-CoV-2 is a new member of the Betacoronavirus (β-CoV) genus of large, en-
Copyright: © 2022 by the authors. veloped, single-stranded RNA viruses [2]. This genus not only includes viruses that
Licensee MDPI, Basel, Switzerland. cause deadly human infections such as severe acute respiratory syndrome (SARS) and
This article is an open access article Middle East respiratory syndrome (MERS) but also encompasses viruses that cause non-
distributed under the terms and
life-threatening common colds, including human coronavirus OC43 (HCoV-OC43) and
conditions of the Creative Commons
human coronavirus HKU1 (HCoV-HKU1) [3]. Although these viruses predominantly infect
Attribution (CC BY) license (https://
lung epithelial cells, the clinical severity and pathogenesis of the infections they cause vary
creativecommons.org/licenses/by/
between different coronaviruses [4]. While severe pneumonia and pulmonary fibrosis are
4.0/).

Microorganisms 2022, 10, 280. https://doi.org/10.3390/microorganisms10020280 https://www.mdpi.com/journal/microorganisms

Microorganisms 2022, 10, 280 2 of 7

fundamental to the pathogenesis of COVID-19, SARS and MERS, these symptoms are not
typical of infections caused by HCoV-OC43 and HCoV-HKU1 [5,6].
Like other β-CoVs, the genome of the novel SARS-CoV-2 virus encodes structural
proteins required for the efficient formation of infectious virions; these include the spike
(S), envelope (E), membrane (M) and nucleocapsid (N) proteins [7].
The key determinant of the host specificity of β-CoVs is the surface-located S protein,
which plays critical roles in infection by mediating viral attachment to host cell-surface
receptors and facilitating viral entry [8]. The S protein consists of two large regions: the
N-terminal S1 and C-terminal S2 [9]. S1 is responsible for recognizing host-cell receptors,
including the receptor-binding domain (RBD), and has higher sequence variability than
S2 (S1 shares around 70% identity with that of other human β-CoVs). The membrane-
embedded S2 region responsible for fusion is more highly conserved than that of S1 [8,9].
In SARS-CoV-2, the RBD in S1 allows the virus to bind directly to the peptidase domain
of the host angiotensin-converting enzyme 2 (ACE2) complex, mediating virus entry into
sensitive cells [10]. Notably, compared to SARS-CoV, SARS-CoV-2 has a higher binding
affinity to ACE2 (which is the common receptor for both SARS-CoV-2 and SARS-CoV), with
a broader interaction with ACE2 (suggested due to dynamics-based correlated motions
and the electrostatic energy perturbations) expressed not only in the lungs but also in the
kidneys, testes and heart [10–14].
Mutations in the genome sequence of SARS-CoV-2 are responsible for the emergence
of new SARS-CoV-2 variants, many of which are characterized by higher transmission
rates [15–17].
Recently, we conducted an analysis and identified for the first time viral prion-like
domains (PrDs), which we suggest are novel regulators of virion assembly with a role to
play in virus-host cell interactions [18,19]. These studies were in alignment with previous
studies, showing that in addition to the pathological role prions play in humans—being
implicated in Alzheimer’s and Parkinson’s diseases, diabetes, and many other human
pathologies—protein misfolding also plays important physiological roles in eukaryotes
and prokaryotes [20–23].
Though the detailed molecular mechanisms underlying prion formation remain elu-
sive, asparagine (Q)- and glutamine (N)-rich regions characterized by altered hydropho-
bicity and net sequence charge are known to drive prion formation. This is the basis for a
number of algorithms for identifying candidate prionogenic domains [24,25]. One such
algorithm is prion-like amino acid composition (PLAAC) analysis, which allows for the
evaluation of prion-like domains based on the hidden Markov model (HMM) that also
incorporates other prion-like domain-predictive algorithms PAPA, DIANA, and FoldIn-
dex [26–28].
Although structures for the variants of SARS-CoV-2 were extensively researched using
CryoEM, modeling, molecular dynamics and other methods to study the impact on the
binding affinity for each amino acid in contact with ACE2, the prionogenic properties of
SARS-CoV-2 have not yet been studied [29–32].
In this study, we performed the first detailed evaluation of PrDs in the spike protein of
SARS-CoV-2 and compared them to PrDs from other human-pathogenic β-CoVs. We also
analyzed PrDs in the spike protein of the variants of concern (VOC) B.1.617.2 (Delta) and
B.1.1.529 (Omicron), variants of interest (VOI) and variants being monitored (VBM), such
as B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.427 (Epsilon), B.1.617.1 (Kappa) and P.2
(Zeta), some of which are known for their ability to escape antibody neutralization [33–36].
Further analyses of these PrD-containing proteins in SARS-CoV-2 may improve our
understanding of the COVID-19 infection and provide new insights into its pathophysi-
ology and novel targets for developing therapies, including antiprion compounds with
binding properties to prion proteins.
Microorganisms 2022, 10, 280 3 of 7

2. Materials and Methods

2.1. Protein Sequences
To identify the PrDs present in viral proteomes, protein sequences were obtained from
the UniProt Knowledge Base and National Center for Biotechnology Information (NCBI)
database [37,38].

2.2. Identification of PrDs in Viral Proteomes

The presence of PrDs in β-CoV proteomes, found using the PLAAC algorithm, and
the output probabilities for the PrDs were constructed based on amino-acid frequencies
and similarities with PrDs in Saccharomyces cerevisiae. Using the parameter Alpha value
(which is used to allow a continuous interpolation between the tested organism-specific
background frequencies and S. cerevisiae background frequencies) of 1.0 we employed
species-independent scanning to identify the PrDs in β-CoV.
We used a 3.0 log-likelihood ratio (LLR) cutoff, which reflects the maximum sum of
per-residue log-likelihood ratios for any subsequence of length Lcore that falls partially or
entirely within the prion-like domain state in the HMM Viterbi parse within a provided
sequence [24]. Prion-like domain amino-acid positions were determined based on the
PLAAC algorithm program analysis.

2.3. Statistical Analysis

All statistical analyses were conducted using the Statistical Package for Windows
(version 5.0) (StatSoft, Inc., Tulsa, OK, USA). Data were compared between viruses using
an χ2 test or Fisher’s exact test. To detect differences in multiple comparisons, one-way
analysis of variance (ANOVA) was fitted with the standard confidence interval of 95%.
p-values < 0.05 were considered statistically significant.

3. Results
Using the prion-prediction PLAAC algorithm, we analyzed structural proteins derived
from the UniProtKB and NCBI databases and identified PrDs in the S proteins of all β-
CoVs analyzed in this study (Supplementary Figure S1). The LLR scores of PrDs of the S
proteins were practically identical within the studied β-CoVs, ranging from 2.828 to 4.856
(Supplementary Figure S2). Notably, with more precise mapping of PrDs within these
proteins, we found a striking difference in their localization, with SARS-CoV-2 being the
only virus with PrDs identified within the RBD of the S protein (Table 1).

Table 1. Comparison of the distribution of PrDs within the S protein among different β-CoV human
pathogens.

S Protein
Domain Prion-like Domain AA Position LLR Score
SARS-CoV-2 RBD 473–510 4.856
SARS-CoV HR1 900–910 4.426
MERS-CoV NA Non-detectable 4.49
HCoV-OC43 NA Non-detectable 2.828
LLR: log-likelihood ratio.

Considering that although SARS-CoV-2 and SARS-CoV (which are the closest related
human β-CoVs pathogens) share the same host-cell receptor ACE2, SARS-CoV-2 binds
tighter to it; therefore, we hypothesized that the presence of PrDs in the RBD of the SARS-
CoV-2 might explain this phenomenon [10]. Consistent with this hypothesis, we found that
SARS-CoV-2, along with other residue substitutions, has five substituted amino acids in the
RBD compared to SARS-CoV. The following are the substitutions in the RBD: S460→Q474,
T488→N481, N480→Q493, Y485→Q498 and T488→N501, which form a hydrophobic
Q/N-rich region that enables the prionogenity of the SARS-CoV-2 RBD (Figure 1).
Microorganisms 2022, 10, 280 4 of 7

Figure 1. Analysis and comparison of mutations in the RBD of SARS-CoV-2 and SARS-CoV. The
RBD of the SARS-CoV-2 (NCBIβ reference sequence: YP_009724390.1) spike protein was aligned with
the closest related human ββCoV, SARS-CoV (NCBI reference sequence: AYV99817.1). The PrDs of
SARS-CoV-2 are red. Different residues are denoted by * beneath the consensus position. The amino
acids asparagine (Q) and glutamine (N) in the PrDs of the SARS-CoV-2 RBD that differ from the amino
acids in the SARS-CoV RBD are denoted by a red ** beneath the consensus position. Amino acids of
the SARS-CoV-2 RBD that bind to ACE2 are marked with red boxes. RBD—receptor-binding domain.

We next analyzed the presence of prion-like domains in the ACE2 protein and found
α helix of ACE2 (aa 40–65 and 93–106) (Supplementary Figure S3). Using
PrDs within the α1
α
the data from previous analysis which modulated the interface between the SARS-CoV-2
RBD and ACE2, we identified a pattern in which five of the seven amino acids that interact
between the SARS-CoV-2 RBD and host cell ACE2 are localized within the PrDs of SARS-
CoV-2 RBD, ACE2 or both of them (Figure 2) [39]. Thus, Q498 and T500 from the PrD of
the SARS-CoV2 RBD interact with Q42 and Y41 within the PrD of ACE2. Meanwhile, Q474,
F486 and N501 from the PrD of the SARS-CoV2 RBD bind to Q24, M82, K343 and R357,
respectively, of a non-PrD of ACE2. Notably, K417 and Y453 were the only residues of
the SARS-CoV-2 RBD that were outside the viral PrD and bound to a non-PrD of ACE2
(Figure 2).

Figure 2. Interactions between amino acids of PrDs and non-prion-like regions of SARS-CoV-2 RBD
and ACE2. Amino acids Q498 and T500 from the PrD of the SARS-CoV2 RBD interact with Y41 and
Q42 within the PrD of ACE2, while Q474, F486 and N501 from the PrD of the SARS-CoV-2 RBD bind
to Q24, M82 and K343 from the non-PrD of ACE2. K417 and Y453 were the only amino acids of the
SARS-CoV-2 RBD that were outside the viral PrD and bound to ACE2.

We analyzed the particularities of the PrDs in the SARS-CoV-2 variants, some of which
are known to have a substantially increased binding affinity due to mutations in the S
protein, thus suggesting that they may have greater prion-forming potential. To this end,
we analyzed the correlation between the LLR score of the PrD within the RBD of the S
proteins of the VOC, VOI and VBM.
Compared with that of SARS-CoV-2 WT, we observed an elevated LLR score for the S
protein from only the Delta (B.1.617.2) variant, with the LLR of 6.025, while from the emerging
Omicron (B.1.1.529), the LLR was only 3.080 (Figure 3 and Supplementary Figure S4).
Microorganisms 2022, 10, 280 5 of 7

Figure 3. Heatmap showing PrD within the S protein in SARS-CoV-2 variants. The correlation
between the LLR scores of the identified PrDs in the S protein across different SARS-CoV-2 variants
is presented. Mean LLC scores of S protein are denoted using a color scale, ranging from white
(minimum) to saturated red (maximum). Higher LLC scores indicate a higher possibility that the
analyzed protein is a prion.

4. Discussion
This study is the most complete evaluation of PrDs in the S protein of SARS-CoV-2
variants. The results highlight some previously unknown and unique characteristics of
SARS-CoV-2 that may play a role in the pathogenesis of COVID-19.
In this study, we used a high threshold of the PLAAC score for protein identification:
only proteins with a high probability of prionogenic properties were included in the
analysis. We found that although differentβ β-CoV members contain PrDs in the S proteins,
SARS-CoV-2 is the only member that has a PrD in the RBD of the S protein that binds to the
ACE2 receptor employed for host-cell entry. Furthermore, we discovered specific amino
acids (Q474, N481, Q493, Q498 and N501) that enable the prionogenity of the SARS-CoV-2
RBD that directly interacts within ACE2.
Inspecting the atomic interaction of SARS-CoV-2 and ACE2 showed that many pair-
wise interactions occur within the intrinsic disorder, which were detected as prion-like
segments [13,14,24,33].
Notably, since five of the seven amino-acid interactions that occur between the RBD of
SARS-CoV-2 and ACE2 are within their PrDs, it is also interesting to consider whether the
prion-prion interaction between the virus and human receptor takes place in COVID-19,
and whether it adds a special value for the higher affinity to their binding. Since other
β-CoVs were shown to lack the PrDs in the RBD, this means that the presence of PrDs is
β
beneficial, but not necessary, for receptor-mediated virion attachment to the host cell. One
of the critical goals of our previous studies was to show that PrDs identified in viruses may
have important functional roles to play in virulence and are particularly associated with
viral adhesion and entry [18,19]. This study provides proof of this concept, demonstrating
that the presence of PrDs in the RBD of SARS-CoV-2 enhances viral binding to its host
receptor compared to that of SARS-CoV, which lacks PrDs in its RBD structure.
Furthermore, across all emerging SARS-CoV-2 variants, we observed the highest LLR
scores in the S protein of the SARS-CoV-2 Delta (B.1.617.2) variant. This is notable since the
Delta (B.1.617.2) variant is known for its highest transmissibility. The highest viral load is
over 1000 times higher in people infected with the Delta variant than those infected with
the original coronavirus strain, and the Delta variant is associated with higher mortality
and greater risk of hospitalization [40]. However, the emerging Omicron (B.1.1.529) variant,
although known for its high transmissibility, for now, appears to be milder than previous
strains and has the lowest LLR among all SARS-CoV-2 variants. Further analyses of
these PrD-containing proteins in SARS-CoV-2 may improve our understanding of the
COVID-19 infection and provide new insights into its pathophysiology and novel targets
for developing therapies.
Microorganisms 2022, 10, 280 6 of 7

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/microorganisms10020280/s1: Figure S1, Graphical representation
of the LLR scores in the PrDs of S proteins from different β-CoVs; Figure S2, LLR scores showing
the predicted putative PrDs in S proteins of β-CoVs; Figure S3, Graphical representation of the LLR
scores of PrDs in the ACE2 protein; Figure S4, Graphical representation of the LLR scores of PrDs of
the S proteins from different SARS-CoV-2 variants.
Author Contributions: G.T. and V.T. designed the experiments; G.T. performed the experiments and
supervised data analysis; V.T. and G.T. analyzed the data and wrote the manuscript; G.T. prepared
the images. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: This research received no specific grant from any funding agency in the public,
commercial or not-for-profit sectors. We would like to thank Gregory Andronica for his valuable input.
Conflicts of Interest: The authors declare no conflict of interest.

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