Journal of Proteins and Proteomics (2021) 12:1–13

https://doi.org/10.1007/s42485-021-00057-y

SHORT COMMUNICATION

Aggregation hot spots in the SARS‑CoV‑2 proteome may constitute

potential therapeutic targets for the suppression of the viral
replication and multiplication
Shalini Gour1 · Jay Kant Yadav1

Received: 10 October 2020 / Revised: 23 January 2021 / Accepted: 27 January 2021 / Published online: 13 February 2021
© The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. part of Springer Nature 2021

Abstract
The emergence of novel coronavirus SARS-CoV-2 is responsible for causing coronavirus disease-19 (COVID-19) impos-
ing serious threat to global public health. Infection of SARS-CoV-2 to the host cell is characterized by direct translation of
positive single stranded (+ ss) RNA to form large polyprotein polymerase 1ab (pp1ab), which acts as precursor for a number
of nonstructural and structural proteins that play vital roles in replication of viral genome and biosynthesis of new virus
particles. The maintenance of viral protein homeostasis is essential for continuation of viral life cycle in the host cell. To
test whether the protein homeostasis of SARS-CoV-2 can be disrupted by inducing specific protein aggregation, we made
an effort to examine whether the viral proteome contains any aggregation prone regions (APRs) that can be explored for
inducing toxic protein aggregation specifically in viral proteins and without affecting the host cell. This curiosity leads to
the identification of several (> 70) potential APRs in SARS-CoV-2 proteome. The length of the APRs ranges from 5 to 25
amino acid residues. Nearly 70% of total APRs investigated are relatively smaller and found to be in the range of 5–10 amino
acids. The maximum number of ARPs (> 50) was observed in pp1ab. On the other hand, the structural proteins such as,
spike (S), nucleoprotein (N), membrane (M) and envelope (E) proteins also possess APRs in their primary structures which
altogether constitute 30% of the total APRs identified. Our findings may provide new windows of opportunities to design
specific peptide-based, anti-SARS-CoV-2 therapeutic molecules against COVID-19.

Keywords SARS-CoV-2 · COVID-19 · Aggregation prone regions · Protein aggregations

Introduction 2020; Jean et al. 2020; Salvi and Patankar 2020;Srini-

vas et al. 2020;Sternberg et al. 2020]. In the meantime,
The coronavirus disease 19 (COVID-19) is caused by some novel vaccine candidates and different pharmaco-
severe acute respiratory syndrome coronavirus 2 (SARS- logical approaches are under investigation [as reviewed in
CoV-2), first emerged in Wuhan, China now imposing (Scarabelet al. 2021)]. The BNT162b2- BioNTech/Pfizer
a serious threat to human life all across the globe and and mRNA-1273-Moderna vaccines have completed their
responsible for disruption of social and economic integ- trials and been approved by FDA and EMA (Scarabelet
rity worldwide (Arabi et al. 2020; Cucinotta and Vanelli al. 2021). In India, the use of Covishield (developed by
2020; Nicola et al. 2020; Tandon 2020). So far, patients University of Oxford, AstraZeneca and produced by SII,
are mostly being managed by supportive treatment using India) has been approved and the other indigenous vac-
lopinavir/ritonavir, ribavirin, beta-interferon, glucocor- cine candidate Covaxin (ICMR-NIV-Bharat Biotech) is in
ticoid and remdesivir (Antinori et al. 2020;Chan et al. phase-III trials (ICMR report). Various other strategies
are under investigation (Sanders et al. 2020) and almost
100 different vaccine candidates have been proposed
* Jay Kant Yadav (Zhang et al. 2020) and these strategies are being validated
[email protected] through clinical studies and trials (Cao et al. 2020; Hung
1 et al. 2020; Wang et al. 2020; Chen et al. 2021). How-
Department of Biotechnology, Central University
of Rajasthan, NH‑8 Bandersindri, Kishangarh, Ajmer, ever, the post-efficacy strategies for the successful vaccine
Rajasthan 305817, India candidates are the prime requirement for the mass use of

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2 Journal of Proteins and Proteomics (2021) 12:1–13

these vaccines (Kim et al. 2021). Therefore, in addition (Pillay 2020). The M protein has three trans-membrane
to the available options, it becomes imperative to search domains and it shapes the virions, promotes membrane
novel therapeutic targets to curtail virus infection and curvature, and binds to the nucleocapsid. The E pro-
multiplication. The replication cycle of SARS-CoV-2 in tein plays a role in virus assembly and release, and it is
host cell is marked with highly synchronized processes of involved in viral pathogenesis. The N protein contains two
protein expression, protein folding, and assembly of viral domains, which bind with virus RNA genome through an
genome along with structural proteins lead to formation integrated action S, E and M proteins.
of new virus particles (Sims et al. 2008; Fehr and Perlman It has often been observed that the protein aggregation
2015; Chen et al. 2020; Lukassen et al. 2020; Lunget al. frequently disrupts the protein homeostasis leading to devel-
2020; Malik 2020). Maintenance of protein homeostasis opment of various disease conditions. Protein aggregation
in a eukaryotic cell is achieved by an integrated mecha- is generally driven by specific amino acid sequences which
nism of protein biosynthesis, folding and attainment of are interspersed within the primary structure of proteins and
native structure, and the degradation of misfolded proteins polypeptides, known as aggregation-prone regions (APRs).
(Balchin et al. 2016; Chiti and Dobson 2017; Klaips et al. The synthetic analogs of such APRs sequences contain the
2018; Zhong et al. 2019). After the entry into the host ability to self-assemble to form aggregates rich β-sheet
cell, viruses employ various strategies to hijack and regu- structures. Further, these APRs are shown to interact with
late various biochemical and molecular activities, such as similar sequences present in parent proteins and peptides
transcription and translation machineries, of the host cell through homologous interaction and induce aggregation.
to produce new viral proteins and enzymes essential for Hence, these APRs have been successfully explored for the
multiplication of the virus (Chen et al. 2020; Malik 2020; targeted disruption of protein homeostasis. Several recent
Salvi and Patankar 2020). Translation of viral genome studies have confirmed that the presence of synthetic analogs
represents a key event required for the establishment of of these sequence-stretches (i.e., APRs) effectively block the
infection and multiplication of SARS-CoV-2. We started folding of the original proteins and render them for degrada-
our prediction using the primary structures of proteins tion by the proteasomal degradation machinery of the host
emerging from all the known open reading frames (ORFs) cell (Beerten et al. 2012; De Baets et al. 2014; Gallardo
of the SARS-CoV-2. The genome structure of SARS- et al. 2016; Ganesan et al. 2016; Khodaparast et al. 2018).To
CoV-2 contains at least six ORFs. The first ORF (known explore the possibility of targeted protein aggregation to cur-
as ORF1a/b) constitutes approximately two‐thirds of the tail SARS-CoV-2 infection, we screened the viral proteome
total genome length and encodes 16 nonstructural pro- to find out presence of APRs. Our initial studies suggest that
teins (NSPS1‐16) (Gordonet al. 2020; Malik 2020). There the primary structures of many of the key proteins such as,
is a − 1 frame shift between ORF1a and ORF1b, leading polyprotein polymerase 1ab (pp1ab), envelope protein (E),
to production of two polypeptides: polypeptide 1a (pp1a) nucleoprotein (N), membrane (M) protein, etc., are marked
and polypeptide 1ab(pp1ab) having 7096 amino acid resi- by the presence of small amino acid sequence-stretches pos-
dues. These polypeptides are proteolytically cleaved to sessing high aggregation propensity. On the other hand, it
form 16 polypeptides segments that ultimately give rise has been observed that the peptide (APR)-induced protein
nonstructural proteins (NSPS). Chymotrypsin‐like pro- aggregation turns out to be a highly ordered and specific
tease (3CLpro) which is virally encoded act at specific process. Since these APRs form essential elements of the
sites and help in the formation of NSPS. Other ORFs are native proteins, in unfolded state (immediately after trans-
situated at the 3′-terminus of ORF1 constitute just 1/3rd lation), can interact with synthetic analogs of APRs and
of the viral genome and encode four major structural pro- induce aggregation of entire proteins and finally subject the
teins namely, spike (S), membrane (M), envelope (E), and protein molecules for degradation rather than their folding
nucleocapsid (N) proteins. The NSPS play specific roles into functional proteins.
during infection such as, degradation of host cell mRNA,
inhibition of interferon (IFN) signaling, blocking the host
innate immune response, promoting cytokine expression, Methods
etc. These biochemical functions of NSPS are crucial for
establishment of viral infection and multiplication. The Prediction of the potential aggregation prone
four structural proteins are vital for virion assembly and regions (APRs) in the SARS‑CoV‑2 proteome
formation of new viral particles. The S protein forms a
homotrimer and then form spikes on the viral surface that The complete genome of Wuhan-Hu-1 (NC_045512.2)
are responsible for initial attachment to the host receptors was downloaded from NCBI nucleotide database. The

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Journal of Proteins and Proteomics (2021) 12:1–13 3

aggregation propensity of all the SARS-CoV-2 proteins helpful in enhancing host mediated destruction of the virus.
primary structure was assessed by using in silico predic- Similarly, the polypeptide regions corresponding to NSP-3
tions. These primary structures of the proteins were sequen- and NSP-4 consist of large number of APRs (Fig. 1).
tially submitted to different computation algorithms namely Apart from pp1ab, the structural proteins also contain
FoldAmyloid (http://bioin ​ fo.protr ​ e s.ru/FoldA ​ myloi ​ d /) sequence stretches of high significant aggregation score.
(Garbuzynskiy et al. 2010), TANGO (http://tango​.crg.es/) There were six potential APRs identified in S protein, how-
(Fernandez-Escamilla et al. 2004), AGGRESCAN (http:// ever, the length of APRs use to be relatively shorter except
bioin​f.uab.es/aggre​scan/) (Conchillo-Soleet al. 2007), and the APR present at N-terminus of the protein. The E-protein
AMYLPRED (http://aias.biol.uoa.gr/AMYLP​RED/input​ is the smallest structural protein (75 residues) and known
.php) (Frousios et al. 2009) with the default setting. The to play essential role in the virus morphogenesis (Liu et al.
scores were compared with classical aggregating peptide 2007), consists of a single potential APR. The M-protein
i.e., Amyloid beta (Aβ) peptide. constitutes an essential component of virus along with other
structural proteins and plays a central role in virus morpho-
genesis and assembly via its interactions with other viral
Result and discussion proteins (Neuman et al. 2011). It consists of five APRs rang-
ing from 7 to 18 amino acid residues. The N-protein consists
Prediction of APRs in different proteins emerging of relatively less number of shorter APRs compared to other
from different ORFs of SARS‑CoV‑2 structural proteins. Among all the four structural proteins
the S-protein and M-proteins are comparatively richer in the
Table 1 summarizes the locations of the predicted APRs APRs compared to N and E-proteins. The score of individual
in different structural and nonstructural proteins of SARS- APRs range from 20 to 100. However, most of the APRs
CoV-2. The APRs are found to be asymmetrically distrib- have aggregation score above 50, indicative of less chance
uted in the different regions of all the proteins investigated. to give false positive values.
As mentioned earlier, pp1a and pp1ab are the two large The lengths of most of the APRs identified in the SARS-
polypeptides that formed from direct translation of virus CoV-2 proteome are in the range of 5–8 residues (Table 1).
genome after its entry into host cells. Given the fact that Most of the APRs in pp1ab possess are found to be relatively
2/3rd proportion of the total virus genome is utilized for the shorter in length compared to the one observed in structural
synthesis of NSPS, they are very crucial for the continuation proteins. It is observed that the shorter APR peptides (of ≈
of virus replication cycle (Masters 2006; Chen et al. 2020). 6 residues) found to be giving better prediction reliability
NSP-1 is the first non-structural protein formed from pp1ab, compared to the larger one. On the other hand, it has also
obstructs translation of host mRNA by interfering with the been established that the longer APRs possesses greater
40S ribosomal subunit (Raj 2021). The primary structure tendency to display false positives compared to the shorter
of NSP-1 contains 180 residues and it was found to be free ones. It has been observed that APRs of shorter length pos-
from any APRs in it. Similarly, the region corresponding to sess high aggregation propensity and interact more effi-
NSP-13 spanning from 5325 to 5925 residues does not con- ciently with the identical sequences in the large peptides or
tain any aggregation prone regions in it. In the polypeptide proteins compared to longer APRs.
segments corresponding to NSP-2 to NSP-12 and NSP-14 The legitimacy of the predicted APRs is based on the
to NSP-16 contain several APRs. NSP-2, 637 residues in its reliability of mathematical and statistical lucidities. The
primary structure, is the second nonstructural protein and computational algorithm TANGO uses a statistical mechan-
found to have 6 potential APRs ranging from 6 to 13 resi- ics approach to make predictions of different secondary
dues in length. The maximum numbers of APRs are found structures present in different regions for a given proteins
in the segment spanning from 3570 to 3859 residues, which (Pande 2004). The algorithm assumes a particular amino
corresponds to NSP-6. The total APRs in this region consti- acid sequence (of at least five consecutive residues) is aggre-
tute > 35% residues of the total protein. Along with NSP-3 gation-prone if it has high propensity to form β-sheet struc-
and NSP-4, NSP-6 plays vital role in creation of cytoplasmic ture and when this sequence form aggregate all the residues
double-membrane vesicles essential for viral replication. On of the β-region are buried in the hydrophobic interior. It
the other hand, NSP-6 also plays important role in prevent- predicts the aggregation propensity in a sequence specific
ing delivery of the viral components to lysosomes of the host manner and presents the data in the form of beta-aggrega-
cell and hence protects the virus from lysosomal inactivation tion score and its value range from 1 to 100. It is reported
(Gordon et al. 2020). Hence, truncating NSP-6 would be that the TANGO score of 5 per residue gives a Matthews

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4 Journal of Proteins and Proteomics (2021) 12:1–13

correlation coefficient between prediction and experiment of different viral proteins by all the four algorithms are found
0.92 (Fernandez-Escamillaet al. 2004). Further, it has been to be unanimous.
shown that the false-positive rate of TANGO is below 5%
for a TANGO score of more than 15 (Bednarskaet al. 2016). Mechanistic outlook of APRs‑induced disruption
Most of the classical amyloidogenic peptides possess the SARS‑CoV‑2 protein homeostasis
aggregation score above 50 and hence we gathered all the
sequence stretches displaying the score above it. The overall For the first time the mechanism of APR-induced disrup-
score above 90 suggest high aggregation propensity with tion of protein homeostasis action was proposed by Balch
less probability of getting false positive. The data obtained et al. (2008). They showed that the disruption of bacterial
from Tango were further analyzed by using other analogous protein homeostasis can be induced by small aggregating
algorithms such as Aggrescan, AmylPred and FoldAmyoid. peptides resulting into formation of toxic protein aggregates
In all the predictions we used amyloid beta (Aβ1-42) peptide in the bacterial cell. Generally, the ordered protein aggrega-
as a reference due to its ability to form classical aggregates tion is facilitated through the formation of intermolecular
rich in β-structures. The AGGRESCAN program predicts β-structures by short polypeptide sequence with high aggre-
the aggregation prone regions in a protein as “hot spot” gation propensity. Presence of such sequences define the
sequences of 5 to 11 residues that can nucleate aggregation basis of amyloid formation in various disease conditions,
in peptides and proteins. The aggregation propensity of the particularly the most debilitating Alzheimer’s and Parkin-
hot spots is determined largely by amino acid composition, son’s diseases. Similar sequences are commonly present in
which is based on the experimentally determined aggrega- various globular proteins that constitute their hydrophobic
tion propensity scale for individual amino acids. The Fol- core and confer structural stability. They also assist oligo-
dAmyloid program predicts short amino acid sequences (≥ 5 meric proteins by forming protein–protein interfaces.
residues) based on the contacts, packing density, backbone Despite the fact that these sequences participate in pro-
H-bonds of acceptors or donors for prediction of aggregation viding stability to the native proteins, they can self-assemble
prone regions. AmylPred combines the data from SecStr, with identical sequences to form β-structured aggregates in
a secondary structure prediction tool, to predict the amino unfolded state. While forming the β-structured aggregates,
acid sequence in protein that can act as potential conforma- it is often observed that their interactions with identical
tional switch. As shown in Fig. 2, the APRs identified in sequences in denatured proteins use to be more efficient than

Table 1  Location of newly identified aggregation prone regions in different proteins of SARS-CoV-2
Amino acid sequences Positions Residues Amino acid Sequence of APRs Length of
APRs

Polyprotein polymerase 1ab

Nsp1 MESLVPGFNEKTHVQLSLPVLQVRDVLVRGFGDS- 1–180 180 Nil
VEEVLSEARQHLKDGTCGLVEVEKGVLPQLEQPY-
VFIKRSDARTAPHGHVMVELVAELEGIQYGRSGETL-
GVLVPHVGEIPVAYRKVLLRKNGNKGAGGH-
SYGADLKSFDLGDELGTDPYEDFQENWNTKHSSG-
VTRELMRELNGG
Nsp2 AVTRYVDNNFCGPDGYPLDCIKDFLARAGKSMCTLSE- 181–818 637 409CVFAYV415 6
QLDYIESKRGVYCCRDHEHEIAWFTERSDKSYEHQTP- 473VAIILASF480 8
FEIKSAKKFDTFKGECPKFVFPLNSKVKVIQPRVEKK- 565AAVTIL570 6
KTEGFMGRIRSVYPVASPQECNNMHLSTLMKCNHCDE- 595VIIMAYVTG603 9
VSWQTCDFLKATCEHCGTENLVIEGPTTCGYLPTNAV- 645AWEILKFLITGVF657 13
VKMPCPACQDPEIGPEHSVADYHNHSNIETRLRKGGR​ 675VKCFIDVV682 8
TRC​FGGCVFAYVGCYNKRAYWVPRASADIGSGHT-
GITGDNVETLNEDLLEILSRERVNINIVGDFHLNEEVAI-
ILASFSASTSAFIDTIKSLDYKSFKTIVESCGNYKVTK-
GKPVKGAWNIGQQRSVLTPLCGFPSQAAGVIRSIFAR-
TLDAANHSIPDLQRAAVTILDGISEQSLRLVDAMVYTS-
DLLTNSVIIMAYVTGGLVQQTSQWLSNLLGTTVEKL-
RPIFEWIEAKLSAGVEFLKDAWEILKFLITGVFDIVKG-
QIQVASDNIKDCVKCFIDVVNKALEMCIDQVTIAGAK-
LRSLNLGEVFIAQSKGLYRQCIRGKEQLQLLMPLKAP-
KEVTFLEGDSHDTVLTSEEVVLKNGELEALETPVDS-
FTNGAIVGTPVCVNGLMLLEIKDKEQYCALSPGL-
LATNNVFRLKGG

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Journal of Proteins and Proteomics (2021) 12:1–13 5

Table 1  (continued)
Amino acid sequences Positions Residues Amino acid Sequence of APRs Length of
APRs

Nsp3 APTKVTFGDDTVIEVQGYKSVNITFELDERIDKVL- 819–2763 1945 1173VYLAVF1178 6

NEKCSAYTVELGTEVNEFACVVADAVIKTLQPV- 1295VLTAVV1300 6
SELLTPLGIDLDEWSMATYYLFDESGEFKLASH- 1570VFTTV1574 5
MYCSFYPPDEDEEEGDCEEEEFEPSTQYEYGTED- 1676LATALLT1682 7
DYQGKPLEFGATSAALQPEEEQEEDWLDDDSQQT- 1710FCALILAY1717 8
VGQQDGSEDNQTTTIQTIVEVQPQLEMELTPVVQ- 2171YFFTLLL2177 7
TIEVNSFSGYLKLTDNVYIKNADIVEEAKKVKPTV- 2229IIIWFLLLSVCLGSLI2244 16
VVNAANVYLKHGGGVAGALNKATNNAMQVESD- 2324VAEWFLAYILFTRFFYV2340 17
DYIATNGPLKVGGSCVLSGHNLAKHCLHVVGP- 2363WLMWLIINLV2372 10
NVNKGEDIQLLKSAYENFNQHEVLLAPLLSAGIF- 2384YIFFASFYYVW2394 11
GADPIHSLRVCVDTVRTNVYLAVFDKNLYDKLVS- 2538INVIVF2543 6
SFLEMKSEKQVEQKIAEIPKEEVKPFITESKPS- 2709IALIWNV2715 7
VEQRKQDDKKIKACVEEVTTTLEETKFLTENLL-
LYIDINGNLHPDSATLVSDIDITFLKKDAPYIVGDV-
VQEGVLTAVVIPTKKAGGTTEMLAKALRKVPTDNY-
ITTYPGQGLNGYTVEEAKTVLKKCKSAFYILPSIIS-
NEKQEILGTVSWNLREMLAHAEETRKLMPVCVET-
KAIVSTIQRKYKGIKIQEGVVDYGARFYFYT-
SKTTVASLINTLNDLNETLVTMPLGYVTHGLN-
LEEAARYMRSLKVPATVSVSSPDAVTAYNGYLTSSSK-
TPEEHFIETISLAGSYKDWSYSGQSTQLGIEFLKRG-
DKSVYYTSNPTTFHLDGEVITFDNLKTLLSLREVR-
TIKVFTTVDNINLHTQVVDMSMTYGQQFGPTYLD-
GADVTKIKPHNSHEGKTFYVLPNDDTLRVEAFEYY-
HTTDPSFLGRYMSALNHTKKWKYPQVNGLTSIK-
WADNNCYLATALLTLQQIELKFNPPALQDAYY​
RAR​AGEAANFCALILAYCNKTVGELGDVRETM-
SYLFQHANLDSCKRVLNVVCKTCGQQQTTLKGVEAV-
MYMGTLSYEQFKKGVQIPCTCGKQATKYLVQQESP-
FVMMSAPPAQYELKHGTFTCASEYTGNYQCGHYKH-
ITSKETLYCIDGALLTKSSEYKGPITDVFYKENSYTT-
TIKPVTYKLDGVVCTEIDPKLDNYYKKDNSYFTE-
QPIDLVPNQPYPNASFDNFKFVCDNIKFADDLNQLT-
GYKKPASRELKVTFFPDLNGDVVAIDYKHYTPSFK-
KGAKLLHKPIVWHVNNATNKATYKPNTWCIR-
CLWSTKPVETSNSFDVLKSEDAQGMDNLACEDLK-
PVSEEVVENPTIQKDVLECNVKTTEVVGDIILK-
PANNSLKITEEVGHTDLMAAYVDNSSLTIKKPNELSRV-
LGLKTLATHGLAAVNSVPWDTIANYAKPFLNKV-
VSTTTNIVTRCLNRVCTNYMPYFFTLLLQLCT-
FTRSTNSRIKASMPTTIAKNTVKSVGKFCLEASF-
NYLKSPNFSKLINIIIWFLLLSVCLGSLIYSTAAL-
GVLMSNLGMPSYCT​GYR​EGYLNSTNVTIATY​CTG​
SIPCSVCLSGLDSLDTYPSLETIQITISSFKWDLTAFGL-
VAEWFLAYILFTRFFYVLGLAAIMQLFFSYFAVHFIS-
NSWLMWLIINLVQMAPISAMVRMYIFFASFYYVWK-
SYVHVVDGCNSSTCMMCYKRNRATRVECTTIVNG-
VRRSFYVYANGGKGFCKLHNWNCVNCDTFCAGST-
FISDEVARDLSLQFKRPINPTDQSSYIVDSVTVKNG-
SIHLYFDKAGQKTYERHSLSHFVNLDNLRANNT-
KGSLPINVIVFDGKSKCEESSAKSASVYYSQLMC-
QPILLLDQALVSDVGDSAEVAVKMFDAYVNTFSSTFN-
VPMEKLKTLVATAEAELAKNVSLDNVLSTFISAARQG-
FVDSDVETKDVVECLKLSHQSDIEVTGDSCNNYML-
TYNKVENMTPRDLGACIDCSARHINAQVAKSHNIALI-
WNVKDFMSLSEQLRKQIRSAAKKNNLPFKLTCA​TTR​
QVVNVVTTKIALKGG

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6 Journal of Proteins and Proteomics (2021) 12:1–13

Table 1  (continued)
Amino acid sequences Positions Residues Amino acid Sequence of APRs Length of
APRs

Nsp4 KIVNNWLKQLIKVTLVFLFVAAIFYLITPVH- 2764–3263 500 2776VTLVFLFVAAIFYLI2790 15

VMSKHTDFSSEIIGYKAIDGGVTRDIASTDTC- 2853LIAAVIT2859 7
FANKHADFDTWFSQRGGSYTNDKACPLIAAVIT- 2975VVTTF2979 5
REVGFVVPGLPGTILRTTNGDFLHFLPRVF- 3052IVAIVVTCLAYYF3064 13
SAVGNICYTPSKLIEYTDFATSACVLAAECTIFK- 3077VAFNTLLFLMSFTVLCL3094 17
DASGKPVPYCYDTNVLEGSVAYESLRPDTRY- 3104VYSVIYLYLTFYL3116 13
VLMDGSIIQFPNTYLEGSVRVVTTFDSEYCRH- 3138FWITIAYIICI3148 11
GTCERSEAGVCVSTSGRWVLNNDYYRSLPG- 3153FYWFF3157 5
VFCGVDAVNLLTNMFTPLIQPIGALDISASIVAGG​ 3180LCTFLL3185 6
IVAIVVTCLAYYFMRFRRAFGEYSHVVAFNTLL-
FLMSFTVLCLTPVYSFLPGVYSVIYLYLTFYLTNDVS-
FLAHIQWMVMFTPLVPFWITIAYIICISTKHFYWFF-
SNYLKRRVVFNGVSFSTFEEAALCTFLLNKEMYLK-
LRSDVLLPLTQYNRYLALYNKYKYFSGAMDTTSY-
REAACCHLAKALNDFSNSGSDVLYQPPQTSITSAVLQ
Nsp5 SGFRKMAFPSGKVEGCMVQVTCG​TTT​ 3264–3569 306 3463ITVNVLAWLYAAVI3476 14
LNGLWLDDVVYCPRHVICTSEDML-
NPNYEDLLIRKSNHNFLVQAGNVQL-
RVIGHSMQNCVLKLKVDTANPKTPKYKFVRIQPGQTF-
SVLACYNGSPSGVYQCAMRPNFTIKGSFLNGSCGSVG-
FNIDYDCVSFCYMHHMELPTGVHAGTDLEGNFYGP-
FVDRQTAQAAGTDTTITVNVLAWLYAAVINGDRWFL-
NRFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQT-
GIAVLDMCASLKELLQNGMNGRTILGSALLEDEFTP-
FDVVRQCSGVTFQ
Nsp6 SAVKRTIKGTHHWLLLTILTSLLVLVQSTQWSLFFF- 3570–3859 290 3582WLLLTILTSLLVLV3595 14
LYENAFLPFAMGIIAMSAFAMMFVKHKHAFL- 3616MGIIAMSAFAMMFV3629 14
CLFLLPSLATVAYFNMVYMPASWVMRIMTWLDM- 3635FLCLFL3640 6
VDTSLSGFKLKDCVMYASAVVLLILMTARTVYDD- 3644LATVAYFNMVY3654 11
GARRVWTLMNVLTLVYKVYYGNALDQAISMWALI- 3683VMYASAVVLLILMT3696 14
ISVTSNYSGVVTTVMFLARGIVFMCVEYCPIFFIT- 3709WTLMNVLTLVY3719 11
GNTLQCIMLVYCFLGYFCTCYFGLFCLLNRYFRLTLG- 3733MWALIISV3740 8
VYDYLVSTQEFRYMNSQGLLPPKNSIDAFKLNIKLLGV- 3747VVTTVMFLA3755 9
GGKPCIKVATVQ 3758IVFMCV3763 6
3779IMLVYCFLGYFCTCYF3794 16
Nsp7 SKMSDVKCTSVVLLSVLQQLRVESSSKLWAQCVQL- 3860–3942 83 3870VVLLSVL3876 7
HNDILLAKDTTEAFEKMVSLLSVLLSMQGAVDINKL- 3911MVSLLSVLL3919 9
CEEMLDNRATLQ
Nsp8 AIASEFSSLPSYAAFATAQEAYEQAVANGDSEVVLK- 3943–4140 198 128LMVVI132 5
KLKKSLNVAKSEFDRDAAMQRKLEKMADQAMTQ- 184LIVTAL189 6
MYKQARSEDKRAKVTSAMQTMLFTMLRKLDNDALN-
NIINNARDGCVPLNIIPLTTAAKLMVVIPDYNTYK-
NTCDGTTFTYASALWEIQQVVDADSKIVQLSEISM-
DNSPNLAWPLIVTALRANSAVKLQ
Nsp9 NNELSPVALRQMSCAA​GTT​QTACTDDNALAYYNTTK- 4141–4253 113 4180FVLALL4185 6
GGR​FVLALLSDLQDLKWARFPKSDGTGTIYTELEP-
PCRFVTDTPKGPKVKYLYFIKGLNNLNRGMVLGS-
LAATVRLQ
Nsp10 AGNATEVPANSTVLSFCAFAVDAAKAYKDY- 4254–4392 139 4266VLSFCAFA4273 8
LASGGQPITNCVKMLCTHTGTGQAITVTPEANM-
DQESFGGASCCLYCRCHIDHPNPKGFCDLKGKYV-
QIPTTCANDPVGFTLKNTVCTVCGMWKGYGCSCDQL-
REPMLQ

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Journal of Proteins and Proteomics (2021) 12:1–13 7

Table 1  (continued)
Amino acid sequences Positions Residues Amino acid Sequence of APRs Length of
APRs

Nsp12 SADAQSFLNRVCGVSAARLTPCGTGTSTDVVYRAFDI- 4393–5324 932 4593IVGVL4597 5

YNDKVAGFAKFLKTNCCRFQEKDEDDNLIDSYFV- 4763LLVYA4767 5
VKRHTFSNYQHEETIYNLLKDCPAVAKHDFFK- 4861LLFVV4865 5
FRIDGDMVPHISRQRLTKYTMADLVYALRHFDE-
GNCDTLKEILVTYNCCDDDYFNKKDWYDFVENP-
DILRVYANLGERVRQALLKTVQFCDAMRNAGIVG-
VLTLDNQDLNGNWYDFGDFIQTTPGSGVPV-
VDSYYSLLMPILTLTRALTAESHVDTDLTKPYIK-
WDLLKYDFTEERLKLFDRYFKYWDQTYHPNCVN-
CLDDRCILHCANFNVLFSTVFPPTSFGPLVRKIFVDG-
VPFVVSTGYHFRELGVVHNQDVNLHSSRLSFKELLVY-
AADPAMHAASGNLLLDKRTTCFSVAALTNNVAFQT-
VKPGNFNKDFYDFAVSKGFFKEGSSVELKHFF-
FAQDGNAAISDYDYYRYNLPTMCDIRQLLFVVEVVD-
KYFDCYDGGCINANQVIVNNLDKSAGFPFNKWGKAR-
LYYDSMSYEDQDALFAYTKRNVIPTITQMNLKYAI-
SAKNRARTVAGVSICSTMTNRQFHQKLLKSIAAT​RGA​
TVVIGTSKFYGGWHNMLKTVYSDVENPHLMGWDYP-
KCDRAMPNMLRIMASLVLARKHTTCCSLSHRFYR-
LANECAQVLSEMVMCGGSLYVKPGGTSSGDATT​AYA​
NSVFNICQAVTANVNALLSTDGNKIADKYVRNLQHR-
LYECLYRNRDVDTDFVNEFYAYLRKHFSMMILSD-
DAVVCFNSTYASQGLVASIKNFKSVLYYQNNVFM-
SEAKCWTETDLTKGPHEFCSQHTMLVKQGDDYVY-
LPYPDPSRILGAGCFVDDIVKTDGTLMIERFVSLAID-
AYPLTKHPNQEYADVFHLYLQYIRKLHDELTGHMLD-
MYSVMLTNDNTSRYWEPEFYEAMYTPHTVLQ
Nsp13 AVGACVLCNSQTSLRCGACIRRPFLCCKCCYDHVIST- 5325–5925 601
SHKLVLSVNPYVCNAPGCDVTDVTQLYLGGMSYY-
CKSHKPPISFPLCANGQVFGLYKNTCVGSDNVTD-
FNAIATCDWTNAGDYILANTCTERLKLFAAETLKA-
TEETFKLSYGIATVREVLSDRELHLSWEVGKPRPPLN-
RNYVFTGYRVTKNSKVQIGEYTFEKGDYGDAVVYRG​
TTT​YKLNVGDYFVLTSHTVMPLSAPTLVPQEHY-
VRITGLYPTLNISDEFSSNVANYQKVGMQKYSTLQGP-
PGTGKSHFAIGLALYYPSARIVYTACSHAAVDAL-
CEKALKYLPIDKCSRIIPARARVECFDKFKVN-
STLEQYVFCTVNALPETTADIVVFDEISMATNY-
DLSVVNARLRAKHYVYIGDPAQLPAPRTLLT-
KGTLEPEYFNSVCRLMKTIGPDMFLGTC​RRC​
PAEIVDTVSALVYDNKLKAHKDKSAQCFKMFYKG-
VITHDVSSAINRPQIGVVREFLTRNPAWRKAVFISPYN-
SQNAVASKILGLPTQTVDSSQGSEYDYVIFTQTTE-
TAHSCNVNRFNVAITRAKVGILCIMSDRDLYDKLQFTS-
LEIPRRNVATLQ
Nsp14 AENVTGLFKDCSKVITGLHPTQAPTHLSVDTKFKTEGL- 5926–6452 527 6106VVFVLW6111 6
CVDIPGIPKDMTYRRLISMMGFKMNYQVNGYPN- 6306VCLFW6310 5
MFITREEAIRHVRAWIGFDVEGCHATREAVGTNL- 6431FSLWVY6436 6
PLQLGFSTGVNLVAVPTGYVDTPNNTDFSRVSAKPP-
PGDQFKHLIPLMYKGLPWNVVRIKIVQMLSDTLKN-
LSDRVVFVLWAHGFELTSMKYFVKIGPERTCCLCDR-
RATCFSTASDTYACWHHSIGFDYVYNPFMIDVQQW-
GFTGNLQSNHDLYCQVHGNAHVASCDAIMTR-
CLAVHECFVKRVDWTIEYPIIGDELKINAACRKVQHM-
VVKAALLADKFPVLHDIGNPKAIKCVPQADVEWKFY-
DAQPCSDKAYKIEELFYSYATHSDKFTDGVCLFWNC-
NVDRYPANSIVCRFDTRVLSNLNLPGCDGGSLY-
VNKHAFHTPAFDKSAFVNLKQLPFFYYSDSPCESHG-
KQVVSDIDYVPLKSATCITRCNLGGAVCRHHANEYR-
LYLDAYNMMISAGFSLWVYKQFDTYNLWNTFTRLQ
Nsp15 SLENVAFNVVNKGHFDGQQGEVPVSIINNTVYT- 6453–6798 346 6457VAFNVV6462 6
KVDGVDVELFENKTTLPVNVAFELWAKRNIKPVPE- 6571LTVFF6575 5
VKILNNLGVDIAANTVIWDYKRDAPAHISTIGVCSMT- 6779ISFMLW6784 6
DIAKKPTETICAPLTVFFDGRVDGQVDLFRNARNG-
VLITEGSVKGLQPSVGPKQASLNGVTLIGEAVK-
TQFNYYKKVDGVVQQLPETYFTQSRNLQEFKPR-
SQMEIDFLELAMDEFIERYKLEGYAFEHIVYGDFSH-
SQLGGLHLLIGLAKRFKESPFELEDFIPMDSTVKNY-
FITDAQTGSSKCVCSVIDLLLDDFVEIIKSQDLSVVSKV-
VKVTIDYTEISFMLWCKDGHVETFYPKLQ

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8 Journal of Proteins and Proteomics (2021) 12:1–13

Table 1  (continued)
Amino acid sequences Positions Residues Amino acid Sequence of APRs Length of
APRs

Nsp16 SSQAWQPGVAMPNLYKMQRMLLEKCDLQNYGDS- 6799–7096 298 6947FFTYICGFI6955 9

ATLPKGIMMNVAKYTQLCQYLNTLTLAVPYNMRVI- 6985FAWWTAFV6992 8
HFGAGSDKGVAPGTAVLRQWLPTGTLLVDSDLND- 7069ILSLL7073 5
FVSDADSTLIGDCATVHTANKWDLIISDMYDPKT-
KNVTKENDSKEGFFTYICGFIQQKLALGGSVAI-
KITEHSWNADLYKLMGHFAWWTAFVTNVNASS-
SEAFLIGCNYLGKPREQIDGYVMHANYIFWRN-
TNPIQLSSYSLFDMSKFPLKLRGTAVMSLKEGQINDM-
ILSLLSKGRLIIRENNRVVISSDVLVNN
Spike MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYP- 1–1273 1273 2FVFLVL7 6
Protein DKVFRSSVHSTQDLFLPFFSNVTWFHAIHVSGTNGT- 140FLGVYY145 6
KRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSK- 510VVVLSF515 6
TQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK- 1060VVFL1063 4
SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN- 1128VVIGIV1133 6
FKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSA- 1215YIWLGFIAGLIAIVMVTI1232 18
LEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWT​
AGAA​ AYYVGYLQPRTFLLKYNENGTITDAVDCALDPL-
SETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITN-
LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYN-
SASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEV-
RQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLD-
SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAG-
STPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV-
VVLSFELLHAPATVCGPKKSTNLVKNKCVNFN-
FNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRD-
PQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQD-
VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGC-
LIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARS-
VASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT-
TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSF-
CTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIK-
DFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAG-
FIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMI-
AQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYR-
FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTA-
SALGKLQDVVNQNAQALNTLVKQLSSNFGAISS-
VLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQ-
LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGY-
HLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH-
DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNT-
FVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKY-
FKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNL-
NESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVM-
VTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLK-
GVKLHYT
E-protein MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRL- 1–75 75 17VLLFLAFVVFLLVTLAIL34 18
CAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV
M-protein MADSNGTITVEELKKLLEQWNLVIGFL- 1–222 222 22LVIGFLFLTWICLLQFA38 17
FLTWICLLQFAYANRNRFLYIIKLIFL- 51LIFLWLL57 7
WLLWPVTLACFVLAAVYRINWITGG​ 60VTLACFVLAAVY71 12
IAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPET- 80IAIAMACLVGLMWLSYFI97 18
NILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGH- 138LVIGAVIL145 8
HLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAG-
DSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ
N-protein MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGAR- 1–419 419 108WYFYYL113 6
SKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQG- 129GIIWV133 5
VPINTNSSPDDQIGYY​RRA​TRRIRGGDGKMKDLSPRW- 219LALLLL224 6
YFYYLGTGPEAGLPYGANKDGIIWVATEGALNT-
PKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEG-
SRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMA-
GNGGDAALALLLLDRLNQLESKMSGKGQQQQGQT-
VTKKSAAEASKKPRQKRTATKAYNVTQAFGRRG-
PEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASA-
FFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFK-
DQVILLNKHIDAYKTFPPTEPKKDKKKKADETQAL-
PQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA
Aβ (1–42) peptide DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG- 1–42 42 17LVFFA21 5
GVVIA 30AIIGLMVGGVVI41 12

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Journal of Proteins and Proteomics (2021) 12:1–13 9

Fig. 1  Identification of aggregation prone regions (APRs) in the major proteins of SARS-CoV-2. The aggregation score and propensity in the
predicted APRs found to be equivalent to the Abeta peptide, which serves as a classical β-structured aggregates

the partially similar sequences. Therefore, we hypothesize that Conclusion

the amino acid sequence stretches with high aggregation pro-
pensity derived from SARS-CoV-2 proteins could be able to The maintenance of viral protein homeostasis remains as
induce specific protein aggregation leading to virucidal activ- one of the most crucial steps for continuation of viral life
ity against the virus. Although, these APRs self-assemble to cycle. The presence of APRs in the SARS-CoV-2 proteins
form β-structured aggregates and initiate seeding of identical constitutes susceptible proteomic segments that might act
peptides or the denatured proteins who’s APRs are exposed. as hot spots for the commencement of the viral protein
Therefore, to target a specific protein using APRs, it is a pre- homeostasis failure. Taking the advantage of distinctive
requisite that the protein must remain in unfolded state so that viral protein expression, folding and assembly of viral
the interaction between homologous APRs becomes feasible. proteins, we propose a hypothesis that the disruption
Following the viral entry, the direct translation of the pp1ab is of protein homeostasis during viral replication will be
one of the most essential steps for initiation of virus replication able to prevent formation of new viral particles. Mainte-
cycle. The APRs present in the polypeptide remain transiently nance of integrated protein homeostasis is essential and
exposed during translation. It is that time point when the syn- remains at the highest risk during translation. Targeted
thetic analogs of APRs can be effectively be used to target aggregation of viral proteins, specifically during transla-
specific proteins by interfering the protein folding reactions tion, would be able deplete the functional proteins and
of polypeptide chains into functional proteins. As shown in imposes an explicit inhibitory effect on viral replication
Fig. 3, the repeated interruption of protein folding, aggregation and multiplication. The primary structures of SARS-
and degradation will lead to deprivation of key proteins lead- CoV-2 proteins are marked by the presence of small
ing to suppression of the viral replication and multiplication. unique sequences that would play vital role in inhibiting
On the other hand the proteome of SARS-CoV-2 is highly the formation of functional proteins and hence prevent
specific and hence these APRs are not likely to interfere with the viral replication and multiplication. A recent study
the protein homeostasis of the host cells. has shown that viral translation, splicing and nucleic

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10 Journal of Proteins and Proteomics (2021) 12:1–13

Fig. 2  Consensus among differ-

ent methods for the prediction
of aggregation prone regions in
the different proteins of SARS-
CoV-2

acid metabolism constitute viable therapeutic targets for actively engaged in synthesizing all peptides analogous to
COVID-19 (Bojkova et al. 2020). Hence, the develop- the identified APRs and characterizing their biophysical
ment of an exclusive and multi-target strategy to disrupt characteristics and we hope that the APR-induced proteo-
the protein homeostasis will represent an attractive and static disruptions will provide an innovative approach to
potential anti-SARS-CoV-2 strategy. At present, we are fight with COVID-19.

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Journal of Proteins and Proteomics (2021) 12:1–13 11

Fig. 2  (continued)

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12 Journal of Proteins and Proteomics (2021) 12:1–13

Fig. 3  Schematic representation of APR peptide-based inhibition to form new viral particles. The events depicted in the region 2 (left
of viral replication. The events in the region one represents usual side) depict the events leading to APR peptide-based targeting of pro-
cycle of infection, release of viral + ssRNA AND its direct trans- teins formed from ORF1a/ORF1ab (pp1ab). Addition of APR pep-
lation to form pp1ab which subsequently forms all the nonstruc- tides will interfere the protein folding reaction of viral proteins and
tural proteins (NSPS). The NSPS, are used in amplification of viral subject them for proteasomal degradation in the host cell. Depletion
genomic + ssRNA, formation of structural and other accessory pro- of essential viral proteins will lead to complete halt of the viral repli-
teins. At the end genomic + ssRNA assemble with structural proteins cation and formation of new viral particles

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