The Novel Insight of SARS-CoV-2 Molecular Biology and Pathogenesis and Therapeutic Options

Abstract

On December 31, 2019, a novel coronavirus, being the third highly infective CoV and named as coronavirus disease 2019 (COVID-19) in the city of Wuhan, was announced by the World Health Organization. COVID-19 has a 2% mortality rate, is known as the third extremely infective CoV infection, and has a mortality rate less than MERS-CoV and SARS-CoV. The CoV family comprises a chief number of positive single-stranded ss (+) RNA viruses that are recognized in mammals. The 2019-nCoV patients showed that the angiotensin-converting enzyme II (ACE2) was the same for SARS-CoV. Structural proteins have an essential role in virus released and budding to various host cells. Notably, evidence indicated human-to-human transmission, along with several exported patients of virus infection worldwide. Nowadays, no licensed antivirals drugs or vaccines for being utilized against these coronavirus infections are recognized. There is an urgent requirement for an extensive research of CoV infections to disclose the route of extension, pathogenesis, and diagnosis and then to recognize the therapeutic targets to facilitate disease control and surveillance. In this article, we present an overview of the common biological criteria of CoVs and explain pathogenesis with a focus on the therapeutic approach to suggest potential goals for treating and monitoring this emerging zoonotic disease.

Full text

The Novel Insight of SARS-CoV-2 Molecular Biology
and Pathogenesis and Therapeutic Options
Arghavan Asghari,
1
Mohsen Naseri,
2
Hamidreza Safari,
3
Ehsan Saboory,
4
and Negin Parsamanesh
4
On December 31, 2019, a novel coronavirus, being the third highly infective CoV and named as coronavirus
disease 2019 (COVID-19) in the city of Wuhan, was announced by the World Health Organization. COVID-19
has a 2% mortality rate, is known as the third extremely infective CoV infection, and has a mortality rate less
than MERS-CoV and SARS-CoV. The CoV family comprises a chief number of positive single-stranded ss (+)
RNA viruses that are recognized in mammals. The 2019-nCoV patients showed that the angiotensin-converting
enzyme II (ACE2) was the same for SARS-CoV. Structural proteins have an essential role in virus released and
budding to various host cells. Notably, evidence indicated human-to-human transmission, along with several
exported patients of virus infection worldwide. Nowadays, no licensed antivirals drugs or vaccines for being
utilized against these coronavirus infections are recognized. There is an urgent requirement for an extensive
research of CoV infections to disclose the route of extension, pathogenesis, and diagnosis and then to recognize
the therapeutic targets to facilitate disease control and surveillance. In this article, we present an overview of the
common biological criteria of CoVs and explain pathogenesis with a focus on the therapeutic approach to
suggest potential goals for treating and monitoring this emerging zoonotic disease.
Keywords: coronaviruses, genome structure, pathogenesis, diagnosis, treatment
Introduction
The coronaviridae family contains a large number of
linear single-stranded positive-sense RNA viruses
(Ceraolo and Giorgi, 2020) that are found in fish, birds, and
mammals (Lea
˜oet al., 2020). Coronaviruses are genetically
categorized into four major genera: alpha coronavirus
(aCoV), beta coronavirus (bCoV), gamma coronavirus
(gCoV), and delta coronavirus (dCoV) (Li, 2016). aCoVs
and bCoVs tend to infect mammals, whereas dCoVs and
gCoVs typically infect birds. However, some dCoVs and
gCoVs can infect mammals under specific conditions. Se-
ven CoVs have been discovered in humans. Two aCoVs
(HCoV-229E and HCoV-NL63) and two bCoVs (HCoV-
OC43 and HCoV HKU1) may cause only moderate upper
respiratory disease such as common cold in immune-
competent hosts, especially a few cases of acute infection in
infants, children, and seniors (Su et al., 2016; Forni et al.,
2017) (Cui et al., 2019).
SARS-CoV-2 is a novel coronavirus with a 2% mortality
rate. It is considered the third highly infective CoV, which
has a lower mortality rate than SARS-CoV and MERS-CoV
(National Health and Health Commission and the provincial
health and health commission, including Hong Kong,
Macao, and Taiwan, 2020).
A very critical threshold associated with viral transmissi-
bility is the primary number of replication, which is usually
denoted by R0 (pronounced ‘‘R naught’’) (Organization,
2020). Transmissibility is significantly higher than SARS-
CoV-2 estimated mean R0 for SARS-CoV-2 (3.35.5) in the
early outbreak process (Zhao et al., 2020). It seems consid-
erably higher than SARS-CoV (R0: 2–5).
A recent study indicated that MERS-CoV R0 is less than
one, which means that it is impossible to trigger a pandemic
(Bauch and Oraby, 2013). The high transmission capacity of
the virus has led to 3,753,782 confirmed cases in 212 coun-
tries, areas, or territories according to the WHO statistics to
date (Thursday May 07, 2020). Proper early diagnosis and
treatment will reduce the mortality rate of this disease. Since
SARS-CoV-2 is a newly emerging disease and its diagnosis
and treatment is very controversial, a considerable number of
incorrect diagnoses caused 263,785 deaths up to now.
High transmissibility and pathogenicity of SARS-CoV-2
may be due to different genetic and protein structures such
as S protein compared with SARS-CoV and MERS-CoV
(Wang et al., 2020a). This review was conducted to study
1
Student Research Committee and
2
Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran.
3
Department of Immunology, Torbat Jam Faculty of Medical Sciences, Torbat Jam, Iran.
4
Zanjan Metabolic Diseases Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
DNA AND CELL BIOLOGY
Volume 39, Number 10, 2020
ªMary Ann Liebert, Inc.
Pp. 1–13
DOI: 10.1089/dna.2020.5703
1
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the biological structure to determine appropriate diagnosis
and therapies for SARS-CoV-2.
Genomic Organization
Coronaviruse genome is a 30kb single-stranded positive-
sense RNA (+ssRNA). It has a 5¢cap head and a 3¢poly(A) tail.
The 5¢end of the gene contains a leader sequence and an
untranslated region (UTR) with several stem-loop structures
that are necessary to replicate and transcribe RNA.The 3¢UTR
also includes RNA structures that are essential for viral RNA
replication and synthesis. Coronavirus genome is organized by
5¢-leader-UTR-replication-ORF1a/b-Spike-3a-3b-envelope
protein-membrane-6p-7a-7b-Nucleocapsid-3¢UTR-poly(A)
tail with additional genes interspersed in structural genes at the
3¢end of the genome (Fig. 1) (Ceraolo and Giorgi, 2020).
The SARS-CoV-2 pathogen belongs to the coronavirus
family, bCoV genus, and Sarbecovirus subgenus. Although the
origins of the virus and its intermediate host are still contro-
versial, molecular studies have demonstrated that RaTG13, the
bat coronavirus, has 96.2% nucleotide homology with the
human coronavirus (Fehr and Perlman, 2015). Mutations in
various SARS-CoV-2 primarily occur in five genes, including
S, N, ORF8, ORF3a, and ORF1ab, with around 42% of the
variants being nonsynonymous (Kahn and McIntosh, 2005).
An increased degree of viral diversity has been observed
among patients diagnosed with SARS-CoV-2, indicating that
the virus has begun adapting to the human environment and its
genomes have started developing in populations (Li, 2016).
Virion Structure
Coronaviruses are spherical with diameters of *125 nm
(Ba
´rcena et al., 2009). This virus involves four main
structural proteins, including spike (S), membrane (M),
nucleocapsid (N), and envelope (E) proteins, which are
encoded inside the 3¢end of the viral genome.
S protein
This protein is located on the virus surface and contributes
to forming a corona, and this is the reason behind the cor-
onavirus getting its name. S protein is 150 kDa with 1300
amino acids that act as homotrimers protein and N-terminal
(NT) signal sequences for 20 asparagine-linked glycan in the
endoplasmic reticulum (ER) (Fehr and Perlman, 2015; Song
et al., 2018). S protein is anchored on virus envelope functions
in attaching coronavirus receptors and internalization. This
protein in coronaviruses consists of three major domains,
including the extracellular domain, which consists of the re-
ceptor binding (S1) and membrane-fusion subunit (S2). S1 is
divided into the N-terminal domain (S1-NTD) and receptor
binding domain (RDM) (Heald-Sargent and Gallagher,
2012). Certain structures are coiled coils consisting of three a-
helices labeled heptad repeat (HR1), besides three chains
known as HR2. The complexity between the HR1 and HR2
leads to providing a structure that is resistant to cellular pro-
teases during post-fusion (Yan et al., 2020).
S proteins in all coronaviruses are cleaved by host
proteases such as TMPRSS2 and lysosomal proteases ca-
thepsins during host–virus fusion (Shang et al., 2020).
SARS-CoV-2 S protein contains 33 specific amino acids
(2.59%), with larger variations being 439–449 and 482–505,
respectively (Li et al., 2020). Moreover, various investiga-
tions found that this virus has a unique peptide (PRRA)
insertion (Wang et al., 2020a) that affects cleaving. As a
consequence, S protein indicated a specific furin cleavage
(-PRAR-) inside the S1/S2 domain that overlaps with the
mentioned insertion (Wang et al., 2020a). Due to the key
FIG. 1. Coronavirus schematic diagram. Structural proteins are encoded by the four structural genes, including spike,
envelope, membrane, and nucleocapsid genes, and also genome organization and the encoded proteins of pp1ab and pp1a
and accessory proteins (3a, 3b, 6, 7a, 7b,8a, 8b, 9b, and ORFs). ORFs, open reading frames.
2 ASGHARI ET AL.
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role of this protease in the entry of viruses into the host cell,
the inhibitory agents such as MI-1851 and camostat mesy-
late of this protease can be used to treat the SARS-CoV-2.
Another way to treat patients infected with SARS-CoV-2
is to inhibit protein S activity, which can block the virus
(Bestle et al., 2020; Hoffmann et al., 2020b).
Griffithsin is one of the drugs used to treat infected pa-
tients. It is a lectin drug that works by controlling S protein
by binding to glycoproteins (Lee, 2019). Antibody-based
therapies usually target S protein. Among them, we can
mention CR3022, which has been used to treat SARS dis-
ease. This protein acts by binding to the Receptor binding
domain site. Due to the high hematology of this site of
protein in SARS and SARS-CoV-2, it can be mentioned as a
treatment option ( Jiang et al., 2020; Tian et al., 2020).
M protein
Recent studies have shown that M protein in SARS-CoV-2
can be compared with MERS and SARS: It has a structural
identity of 39.2% and 90.1%, respectively (Ahmed et al.,
2020) M protein, a transmembrane glycoprotein type III, is an
important structural protein in coronaviruses (Arndt et al.,
2010). It is the most abundant protein on the surface of the
virus, which gives it shape. According to studies conducted
on the two viruses SARS-CoV and MERS-CoV, M protein
contains *230 amino acids in length, a 25–35 kDa, which is
the least in all structural proteins.
In silico analysis showed that M protein in the SARS-CoV-2
has a structure similar to prokaryotic sugar transport protein
and has a triple helix bundle, a single 3-transmembrane domain
(TMD).Basedonthis,itcanbeassumedthatthisproteincan
play a role in the entry of virus into the host cell and the
maturation of RNA viruses, but many studies must be done
to confirm this hypothesis (Thomas, 2020) .NT and
C-terminal domains (CTDs) are ecto domain and endo
domain, respectively. CTD has the ability to bind to RNA.
This protein is translated by ribosomes, which are attached
to the rough ER (co-translational) (Arndt et al., 2010). In
almost all coronaviridae representatives, an amphipathic
region at the end of the third TMD is highly preserved
(Arndt et al., 2010).
N-linked glycosylation in a,dand O-linked glycosylation
in b(Oostra et al., 2006) are found in this glycoprotein, which
leads to an antigenic role (Braakman and Van Anken, 2000).
N protein
This protein is 43–50 kD and is presented in coronaviruses.
This helical nucleocapsid structural protein can bind to RNA
with several lysine and arginine amino acids (Chang et al.,
2006). This protein contains NTD (RNA-binding site), CTD
(dimerization domain) and is intrinsically central disordered
(serine- and arginine-rich for phosphorylation) (Kumar et al.,
2020). N protein functions in attaching and assembling the
viral RNA genome to a long helical nucleocapsid structure or
matrix of the ribonucleoprotein (Kumar et al., 2020); how-
ever, it is remarkably sensitive to proteases (Macnaughton
et al., 1978). This protein is phosphorylated in multiple spe-
cific positions in various coronaviruses, which may alter
functions such as distinguishing nonviral and viral RNA
impaired binding of the monoclonal antibody to the virus
surface as well as virus maturation and assembling (Kuo et al.,
2016; Grunewald et al., 2018; Chen et al., 2019). However,
the reason behind phosphorylation has remained uncertain.
N protein can also be involved in transcription regulation,
viral transcription and increase the performance of replicative
or genomic RNA replication in reverse genetic systems
(Hu et al., 2017; Cong et al., 2020).
Accumulating evidence has indicated that N protein that
often exists in the cytoplasm of contaminated cells leads to
an arrest in the cell cycle in the G2/M phase (Wurm et al.,
2001). Further, protein crystallography has reported that N
protein structure is extremely varied in RNA binding sites of
severe infectious coronaviruses (SARS-CoV-2, SARS-CoV,
and MERS-CoV) and mild infectious viruses (HCoV-229E,
HCoV-NL63, HCoV-HKU1, and HCoV-OC43). SARS-
CoV-2 utilized a particular pattern to bind RNA with data
on atomic resolution (Kang et al., 2020). Since N protein is
a surface protein and has less variation than S protein, it can
be suggested as a vaccine candidate for SARS-CoV-2
(Ahmed et al., 2020; Fu et al., 2020). In silico analysis
suggests that the effect of the two drugs, Glycyrrhizic acid
and the phytochemical Theaflavi, on N proteins can be ex-
amined as one of the treatment options (Ray et al., 2020).
E protein
E protein is known as a small hydrophobic protein that is
*74–109 amino acids and 8.4–109 kDa. This protein consists
of three parts: NT (negatively charged), TMD (not recharged),
and CT (negatively charged) (Nieto-Torres et al., 2011). There
are three specific sites for cysteine that undergo palmitolate
changes (Liao et al., 2006; Tseng et al., 2014). Although the
accurate function of palmitolytic protein has been still con-
troversial, various roles for this protein are considered for
coronavirus. Changes in all three amino acids in MHV-CoV
can significantly weaken this virus. Several studies have pub-
lished that this modification may contribute to SARS-CoV Ag
shedding (Lopez et al., 2008; Tseng et al.,2014).Another
conserved residue in its structure is a proline that is protected in
the CT region of the b-coil-bmotif. It plays an important role in
the maturation of the protein in Golgi (Tseng et al.,2014).
E protein contributes to ion channel, viroporin activity,
and virus assembling to release the curvature of the cell
membrane in budding virus (Schoeman and Fielding, 2019).
PDZ-binding motif (PBM) is an important protein motif in
the CT region, and it plays a critical role in pathogenicity by
interfering in cell signaling. PBM is considered as a source of
the pathology of SARS-CoV (Hung and Sheng, 2002; Javier
and Rice, 2011). Post-translational changes with critical roles,
including glycosylation, palmitoylation, myristoylation, and
ubiquitination, occur in proteins (Schoeman and Fielding,
2019). Several studies show that E protein has a role in
pathway signaling, for example, one study of E protein
showed that the E protein in SARS-CoV-2 in the CT region
had changed in several amino acids, which could affect the
PALS1, which plays a key role in tight junction, causing the
virus to become more pathogenic than other coronaviruses
(De Maio et al.,2020).
Several pangenomic studies investigating beta cor-
onavirus sequences have revealed that two functional
characteristics, including an ion channel and a PBM, are
strictly conserved in all core gene clusters in SARS and
SARS-CoV-2 variants. These characteristics stimulate a
THE NOVEL INSIGHT OF SARS-COV-2 MOLECULAR BIOLOGY 3
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cytokine storm, trigger the inflammasome, and, subse-
quently, increase edema in lungs. This process causes acute
respiratory distress syndrome and ultimately death in SARS-
CoV1 and SARS-CoV-2 infections. Most medications im-
pair this mechanism, such as Amantadine and Hexamethy-
leneamiloride (acting on ion channels) and SB203580
(effect on PBM).
E protein subcluster in SARS clade is quasi-identical for
the main functional regions of SARS-CoV1 and SARS-
CoV-2. Therefore, SARS E protein inhibitors are suggested
as an appropriate candidate for SARS-CoV-2 treatment, as
they are reported in animal models (Alam et al., 2020).
Nonstructural proteins
Sixteen nonstructural proteins (nsps) contribute to tran-
scription replication, which are originally provided by the
auto proteolytic of two primary proteins: pp1a and pp1ab.
Breaking down of pp1a protein leads to the production of
nsps 1–16, and the breaking down of pp1ab provides nsps
1–10 and nsps 12–16. This process is similar in all proteins,
except for those that are not produced by ribosomal fra-
meshifting. Pp1a autoproteolytic products play specific
roles, such as cell infection and assembling the RNA syn-
thesis system. Moreover, pp1ab products function in RNA
replication and transcription (Masters, 2006).
nsp1, which is located in the NT region of the pp1a
protein, contains 180 amino acids. The similarity of nsp1 in
SARS-CoV-2 is 82–84%, similar to that of SARS-CoV (Ren
et al., 2020). The difference is in the single amino acid,
which can naturally cause structural changes in the protein
(Wu et al., 2020). These changes are usually caused by
merging with the host sequence. Due to the epidemic of this
disease in human populations, point mutations increase,
which can lead to structural changes in vital proteins in the
virus.
nsp1 plays various roles, including suppression of host
gene expression through RNA degradation and 40s ribo-
somal disruption (Kamitani et al., 2006, 2009). Interest-
ingly, this protein can recognize emRNA modifications but
not a specific sequence of virus RNAs (Huang et al., 2011).
nsp1 regulates gene expression in the interferon type 1
signaling cascade by decreasing the amount of STAT1
protein phosphorylation and also through slight effects on
proteins such as STAT2, JAK1, and TYK2 (Wathelet et al.,
2007). nsp1 can be considered a virulence factor and con-
tributes to pathogenicity through favored replication of
SARS-CoV. Higher production of nsp1 influences the signal
pathway of calcineurin/NFAT, is eventually correlated with
the development of interleukin 2 (IL2), and may influence
its viral pathogenicity (Pfefferle et al., 2011). Cyclophilin
interfaces with nsp1 to alter the calcineurin signaling. The
effect of this drug on many coronaviruses has been inves-
tigated (de Wilde et al., 2013; Tanaka et al., 2013).
The degrade precursor nsp2–3 results in two nsp2 and
nsp3, which are 65 kDa and 22–240 kDa, respectively. Stu-
dies on protein nsp1 have failed to find the specific role of
this protein (Graham et al., 2005; Gadlage et al., 2008).
There are many mutations in nsp2 that have caused changes
in this protein (Menachery et al., 2017). Studies have shown
that this protein contains 61 amino acids varying from
SARS-CoV-2 and SARS-CoV (Wu et al., 2020). nsp2 was
observed as interacting with nsp8, which is involved in
replication machinery (von Brunn et al., 2007). Cor-
onaviruses create double-membrane vesicles (DMVs) in
infected cells that are filled with replication-transcription
complexes (RTCs). nsp2 is essential for the structure of
DMV-anchored RTCs (Hagemeijer et al., 2010). By inter-
acting with prohibitin 1 and prohibitin 2 cellular proteins,
nsp2 can interfere with host cell signaling, including cell
cycle death cell pathways and cell differentiation (Cornillez-
Ty et al., 2009).
One of the diagnostic methods is to suggest the identifi-
cation of coronavirus disease 2019 (COVID-19), which is
real-time reverse transcriptase PCR (RT-PCR) assay,
COVID-19-nsp2 real time, which is a more sensitive and
faster method than other conventional methods (Yip et al.,
2020).
nsp3 is a significant protein in coronaviruses and has eight
domains, including ubiquitin-like domain 1 (Ubl1), Glu-rich
acidic domain (also called ‘‘hypervariable region’’), macro-
domain (also named ‘‘X domain’’), ubiquitin-like domain 2
(Ubl2), papain-likeprotease2 (PL2
pro
), nsp3 ecto domain
(3Ecto, also called ‘‘zinc-finger domain’’), Y1 domain (with
unknown function as well as CoV-Y), and two conservation
regions, which are transmembrane (TM1 and TM2) (Lei
et al., 2018). This protein breaks down pp1a and pp1ab
through its protease activity in nsp3 (PL2
pro
) (Lei et al.,
2018).
nsp3 can disrupt the host cell cycles by affecting p53 and
calcium/calmodulin-dependent protein kinase II (Ma-Lauer
et al., 2016). This protein also plays a critical role in RTC
formation as well as in nsp2 (Angelini et al., 2013). Gen-
erally, the nsp3 multi-domain functions in infection with
coronavirus. nsp1, nsp2, and nsp3 are produced from the
polyproteins via nsp3 and cause replication/transcription
complex formation with other viral nsps as well as RNA. nsp3
performs post-translational structural changes on host protein
to suppress the host innate immunoresponse. nsp3 is affected
by 3Ecto N-glycosylation in infected cells (Lei et al., 2018).
This protein is also known as the principal target of lineage C
bCoVs development, based on a high frequency of positively
selected mutation sites (Forni et al., 2016).
Mounting evidence has shown that several structural
changes occurred in nsp2 and nsp3 during positive selection.
There are multiple varieties in the nsp2 endosome-associated
protein-like domain, which may justify the extreme conta-
gion characteristics in SARS-CoV-2. Another mutation near
the nsp3 phosphatase domain may cause the difference be-
tween SARS-CoV-2 and SARS-CoV (Angeletti et al., 2020).
SARS-CoV-2 sequencing has proposed that the papain-like
protease/deubiquitin domain, a unique protein in nsp3, can
be a promising viral inhibitor in medication (Shanker et al.,
2020). Two of the drugs that are prescribed for treatment are
lopinavir and ritonavir. These drugs inhibit the activity of the
virus by inhibiting protease activity. There is no agreement
in the articles on the use of these drugs.
Several strands of research find that these medications can
be used to cure patients (Lim et al., 2020; Xu et al., 2020).
Conversely, a new study consisting of a total of 199 patients
with laboratory-confirmed SARS-CoV-2 infection found
that lopinavir–ritonavir therapy outside routine care had no
positive effect in terms of clinical outcomes and deaths (Cao
et al., 2020). Disulfiram (inhibitor nsp3) and darunavir
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(conformation changes PL2pro) are one of the drugs that
cause changes in the function of this protease and that can
be considered as treatment options (Zhou et al., 2015;
Hoffmann et al., 2020a; Lin et al., 2020).
nsp4 plays a role in the development, arrangement, and
function of these complexes for viral replication (Perlman
and Netland, 2009). Amino acid sequencing in MHV nsp4
projected that this protein has four TMDs (TM1–4). MHV
nsp4 can be a critical target to overcome contagious viruses
(Sparks et al., 2007). nsp4 is necessary for the structure of
RTCs anchored with DMV as well as with nsp2 (Gadlage
et al., 2010).
nsp5, which is often known as 3C-like protease, or the
main protease, plays a significant role in the synthesis of
viral proteins and generates several nonstructural viral
proteins through its protease activity (Lai and Cavanagh,
1997; Ziebuhr et al., 2000; Masters, 2006; Perlman and
Netland, 2009). Moreover, nsp5 can inhibit interferon I
signaling processes by intervening in the NF-kB process and
breaking down STAT 1 transcription factor (Zhu et al.,
2017a, 2017b). 3C-like protease plays a crucial role in the
coronaviruses life cycle and makes it a desirable target to
produce antiviral drugs (Macchiagodena et al., 2020). Pyr-
anones 3f, 3g, and 3m can impair neuraminidases prote-
ase and 3C-like protease and can be used as proper drugs to
treat SARS-CoV-2 (Kumar et al., 2016). Other drugs that
inhibit this protease include Paritaprevir and Raltegravir
Lasinavir, Brecanavir, Telinavir, Rotigaptide, 1,3-Bis-(2-
ethoxycarbonylchromon-5-yloxy)-2-(lysyloxy) propane, and
Pimelautide, which have been suggested for treatment
(Durdagi et al., 2020).
nsp6 has a membrane proliferation potential to cause
perinuclear vesicles situated around the microtubule orga-
nizing center. This protein, besides nsp3 and nsp4, can close
the DMVs as well as infected cells (Angelini et al., 2013).
nsp6 can induce cell autophagosis by affecting two vital
proteins of the autophagy signaling pathway, including
ATG5 and PIK3C3. nsp6 restricts autophagosomal expan-
sion, either directly by nsp6 or indirectly by deprivation or
MTOR signaling chemical suppression. Inhibition happens
at the omegasome formation level and, subsequently, pro-
hibits broad autolysosome development (Benvenuto et al.,
2020; Yang and Shen, 2020). One way to treat COVID-19 is
by its effect on the cell autophagosis signaling pathway
(Yang and Shen, 2020). For example, chloroquine is con-
sidered a treatment option by affecting the pH of lysosomes
(Degtyarev et al., 2008).
nsp8 and nsp7 are 22 and 10 kDa, respectively, and they
are considered co-factor components. These proteins create
a heterodimer and function in stabilizing the RNA binding
site in nsp12. The NT residue from nsp8 is crucial in the
capacity of the protein to interact with nsp7 (Zhai et al.,
2005; Kirchdoerfer and Ward, 2019). nsp8 has RNA-
dependent RNA polymerase (RdRp) replicase subunits that
are special for CoVs and can conduct de novo RNA
synthesis only with low fidelity on ssRNA templates
(Konkolova et al., 2020). These findings characterize the
complexofSARS-CoVnsp7andnsp8asaninteresting
multimeric RNA polymerase that can extend the primers
(Snijder et al., 2016; Konkolova et al., 2020).
nsp9 is an essential protein for linking coronavirus rep-
lication to RNA. Various ways of nsp9 dimerization im-
prove their binding affinity to nucleic acid (Zeng et al.,
2018). However, nsp9 requires the presence of nsp8 for
binding to RNA (Sutton et al., 2004).
nsp10 is also recognized as a significant replication reg-
ulator. nsp10 has148 amino acids containing two zinc finger
domains for enzymatic interaction. It can interact with
nsp14 and nsp16 (Ma et al., 2015; Rosas-Lemus et al.,
2020). nsp16 requires an interaction with nsp10 to perform
powerful methyltransferase, which can transform cap-0
(7MeGpppN) specifically into the cap-1 structure (Rosas-
Lemus et al., 2020). The interaction of nsp10 with nsp14
induces exonuclease function whereas it does not affect the
function of nsp14 methyl transferase (Ma et al., 2015).
nsp11 contains two main domains, including CTD and
NTD. It exhibits endo-ribonuclease activity (Li et al., 2014)
and plays a substantial role in the viral life cycle. This
protein can break down dsRNA and ssRNA by identifying
specific sites (3¢uridylate) (Nedialkova et al., 2009).
nsp11 has an inhibitory impact on TNF-adevelopment
and IL1 signaling (He et al., 2015). RNA microarray results
have revealed that nsp11 correlates with many host cell
pathways such as cell cycle and DNA replication, histon
modification, protein kinase signaling, and proteasome
processes (Sun et al., 2014a).
nsp12 is known for its RdRp activity. This protein has an
extended NT region, which binds to two cofactors (nsp7 and
nsp8) that are necessary for nsp12polymerase function. The
NT of this protein has nucleotidyltransferase activity (Leh-
mann et al., 2015), and an RdRp domain is located at the 12
CT (te Velthuis et al., 2009). Although binding nsps can
improve the efficiency of replication and transcription, at
least the complex that can make the protein nucleation
portion is nsp12, nsp8, and nsp7 (Sevajol et al., 2014;
Lehmann et al., 2015). nsp12 has two polymerase activities:
primer-dependent and primer-independent RNA synthesis
activities using homopolymeric RNA templates (Ahn et al.,
2012).
Emerging evidence has reported that vitamin B12 (me-
thylcobalamin) may bind to the active site of nsp12 and
inhibit it. Therefore, this vitamin can be used as an antiviral
drug in SARS-CoV-2 (Narayanan and Nair, 2020). Another
study has suggested that the Pan-Janus Kinase inhibitor
might be a treatment for this disease by disabling RdRp
protein activity (Mirza and Froeyen, 2020).
A significant group of drugs used for viral diseases are
nucleotide analogues (Remdesivir), which function by af-
fecting nsp12 (Wang et al., 2020b). Hence, this protein can
be regarded as one of the antiviral medications ( Ju et al.,
2020). Nucleoside analogues are usually derivatives of
adenosine and guanosine, which inhibit RdRp. This group of
drugs is commonly used in viral infections (De Clercq,
2019). Favipiravir, from the family of Nucleoside ana-
logues, inhibits the virus by targeting the catalytic domain of
nsp12 (Furuta et al., 2017). A study of patients with SARS-
CoV-2 showed that reducing the time to clear the virus and a
CT of the lungs of patients taking the drug were better than
those of those who did not take the drug (Cai et al., 2020).
Remedisir is another member of the nucleoside analog
family that is currently being recommended for the treat-
ment of infected individuals. It has previously been used to
treat MERS and SARS (Al-Tawfiq et al., 2020; Singh and
Sharma, 2020). It works by its effects on NSP12 and
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Replicase polyprotein 1ab (Agostini et al., 2018; Elfiky,
2020). This drug reduces the symptoms of the disease in
people infected with the SARS-CoV-2, such as: temperature
normalization, respiratory rate, oxygen saturation, and
cough alleviation (Al-Tawfiq et al., 2020).
nsp13 is characterized by helicase activity and belongs
tosuperfamily1(SF1)(Begumet al., 2020). This protein
can unwind both double-stranded DNA and RNA by hy-
drolyzing deoxyribonucleotide triphosphates (dNTPs) and
ribonucleotide triphosphates. nsp12 can enhance the un-
winding activity of nsp13 (Jia et al., 2019). nsp13 has
several domains, including NT Cys/His-rich domain (CH)
with three zinc atoms, b-barrel domain and CT SF1 heli-
case center with two RecA-like subdomains. This protein is
one of the most conserved ancestral proteins in nido-
viruses, rendering it an essential drug discovery (Hao et al.,
2017).
Mounting evidence has revealed that nsp13 can hydrolyze
all forms of NTPs and unwind NTP-dependent RNA helices
by its NTPase and RNA helicase activity. Some divalent
actions, such as Ca2
+
, Zn2
+
, and Mg2
+
, impair nsp13 ac-
tivity. A research report has shown that bismuth salts such
as potassium citrate (BPC), ranitidine bismuth citrate
(RBC), and bismuth citrate would inhibit NTPase and RNA
helix-unwinding behaviors of SARS-CoV-2 nsp13. Among
them, BPC or RBC can significantly impair SARS-CoV-2
nsp13-dependent NTPase and RNA helicase activities (Shu
et al., 2020).
Mounting evidence has revealed that nsp13 can hydrolyze
all forms of NTPs and unwind NTP-dependent RNA helices
by its NTPase and RNA helicase activity. Certain bismuth
salts can effectively inhibit nsp13 NTPase and RNA heli-
case activity (Shu et al., 2020).
nsp14 is known as an exonuclease; it functions in a 3¢-5¢
direction on ssRNAs and dsRNAs that depend on conserved
residues in the DEDD exonuclease super family ( Minskaia
et al., 2006). Further, it plays a role in RNA viral modifi-
cation (N7-methylguanosine [m7G]) (Chen et al., 2009).
The N7-MTase activity of nsp14 is related to protein
carboxy-terminal (Chen et al., 2009). Three natural and
microbial extracts of PF35468, PA48202, and PA48523
products may inhibit SARS-CoVnsp14 (Sun et al., 2014b).
One of the opposite approaches to COVID-19 can be to
inhibit this protein, which is one of the treatment options
based on the adenine dinucleoside SAM analogues (Ahmed-
Belkacem et al., 2020).
nsp15 is made of 345 amino acids and has three domains:
NT, central, and CT (catalytic NendoU) domain. This is a
hexameric endoribonucleases protein, and it is called En-
doU. Endoribonuclease breaks down the double-stranded
and single-stranded RNA by identifying uridine sites and
cleaves uridines 3¢(Bhardwaj et al., 2008).
nsp15 can detect multiple sensors such as MDA5, PKR,
and OAS/RNaseL and it can delay interferon signaling
(Deng et al., 2017; Kindler et al., 2017). nsp15 in COVID-
19 is a conserved protein that has 88% and 95% sequence
homology with other coronaviruses and SARS-CoV, re-
spectively. It means that certain variations can be related to
SARS-CoV-2 virulence (Kim et al., 2020). Recent research
on inhaled corticosteroid ciclesonide revealed that nsp15
inhibition might be an appropriate candidate for treating
SARS-CoV-2 (Matsuyama et al., 2020).
nsp16 is an S-adenosylmethionine-dependent operation
(nucleoside-2¢-O)-methyltransferase and generates nsp16/
nsp10 complex (Lugari et al., 2010). This protein, besides
the interfering interferon band ISRE signaling pathway, can
downregulate RIG1 and MDA5 proteins. nsp10 can also
accelerate this process (Shi et al., 2019); nsp10-derived
peptides K12 and K29 block nsp16 dose-dependent action in
SARS-CoV and suppress full MTase action of nsp10/nsp16
(Lugari et al., 2010). Ribavirin, known as an antiviral drug,
inhibits the nsp16. Ribavirincan can be blocked by 2¢-O-
methyltransferase activity of nsp16, which effectively in-
hibits the 5¢messenger RNA (mRNA) cappig (Te et al.,
2007; Elfiky, 2020) and contributes to a decrease in viral
gene expression by targeting the nucleotide binding site
(Tam et al., 2001). It also affects the immune system and
activates the antiviral response in the body (Tam, 2002)
Dolutegravir and Bictegravir are two drugs that are re-
commended to inhibit this enzyme in patients with SARS-
CoV-2 (Beck et al., 2020).
Replication and Pathogenesis
Attachment and entry
At this stage, S protein, which contains S1 and S2, plays a
critical role. S1 includes CTD (receptor binding and pro-
moting membrane fusion and has RBD) and NTD (sugar
receptors recognized).
S protein has two forms: the pre-fusion, which is observed
in mature virus and has a hemotrymeric arrangement; the
post-fusion, which is completed after membrane fusion
(Li et al., 2006). Coiled-coil structure is observed only in S2
and not S1 (Li et al., 2006; Walls et al., 2017). SARS-CoV-2
recognizes angiotensin-converting enzyme II (ACE2) recep-
tor protein by using the RBD motif of the S protein and binds
to the host cell (Minskaia et al., 2006; Chen et al., 2009; Sun
et al., 2014a). The binding of ACE2 to SARS-CoV-2 has
15 nM affinity, which is *10–20-fold higher than SARS-
CoV unexpectedly. This can be an important reason that
SARS-CoV-2 is exceedingly contagious (Wrapp et al., 2020).
ACE2 plays a significant role in controlling blood pressure
and it is found in tissues such as heart, liver, kidneys, etc.
The specialty of the host receptors triggers host tropism
and host selection (Bhardwaj et al., 2008). This is cleaved in
two places after attaching Sprotein to ACE2 via host pro-
teases, including the S1/S2 boundary by furin and the S2¢
site by extracellular proteases such as trypsin or TMPRSS2.
Furin is expressed in brain, lung, gastrointestinal tract, liver,
pancreas, and reproductive tissues. It can cut a particular
pattern (-PRRA-) at the border of S1 and S2 proteins that
activate the S protein for membrane fusion. It can be another
reason for high virulence and organ infections of COVID-19
(Wang et al., 2020a).
The unstable pre-fusion form is altered to the post-fusion
form via proteases and binds to viral receptors. Then, S2
helps the virus entering the host cell by creating a six-helix
bundle (Lee et al., 2016).
After binding the S protein to the ACE2 receptor, the
signaling pathway occurs through the phosphorylation of the
receptor by CK2. It can activate AP1 and ERK1/2 and
eventually leads to CCL2 expression, which refers to pul-
monary fibrosis (Fig. 2) (Heurich et al., 2014).
6 ASGHARI ET AL.
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Uncoating
Virus membrane fused with host membrane resulted
in SARS-CoV-2 entering the host cell as enodocytosis
through clathrin-dependent or clathrin-independent ways
(We˛ drowska et al., 2020; Algarroba et al., 2020). Then, the
viral genome is released into the cytosolic space via lyso-
somal enzymes that are referred to as catapsin L and trypsin
(Hoffmann et al., 2020a).
Translation and the unfolded protein response
in coronavirus-infected cells
After releasing the viral genome into the host cell cyto-
solic space, the virus alters the transcription process in its
favor by disrupting the transcription process of the host cell.
Synthesized viral proteins are utilized instead of host cell
proteins, which contribute toward cellular pathogenesis
(Fung et al., 2016). Transmission of the virus genome oc-
curs after entering the 5¢region of the oRf1a and oRF1b and
products pp1a and pp1ab (Perlman and Netland, 2009).
Polyproteins generate nsps by affecting nsp3 (papain pro-
tease), nsp5 (3C protease), and cellular protease (Lai and
Cavanagh, 1997; Ziebuhr et al., 2000; Masters, 2006;
Perlman and Netland, 2009; Lei et al., 2018). The replica-
tion and transcription complex, which is produced in DMVs,
involves various proteins such as RdRp (nsp12), helicase
(nsp13), RNA cap-modifying methyltransferases (nsp14 and
nsp16), and an exoribonuclease (nsp14) (Perlman and Net-
land, 2009; Hao et al., 2017).
The RTC synthesizes a cluster, including subgenomic
RNAs in a discontinuous transcription manner. These sub-
genomic mRNAs have specific sequences of 5¢-leaders and
3¢-terminals. Several RNA processing enzymes, such as the
nsp14 exoribonuclease 3¢-5¢, are special among all RNA
viruses to CoVs and are likely to provide the RTC proof-
reading feature. The RTC instead uses the genome to syn-
thesize progeny genomes and a series of subgenomic
mRNAs, using negative-stranded intermediates. Structural
proteins such as M, S and accessory protein are translated by
ribosomes attached to the ER membrane and then, they
transit to the ER-Golgi intermediate compartment (ERGIC).
The N protein covers the progeny genomes by encapsidation
of these components along with the membrane-bound
components, creating virions by budding in the ERGIC.
Eventually, the vesicles, which include the viral particles,
combine with the plasma membrane to release the virus.
Diagnostic approach. Current screening and character-
istic ways help in epidemiologic monitoring, along with
successful control (Ozma et al., 2020). We have evaluated
the various procedures that are currently being prepared for
coronavirus detection.
CT imaging examination. The chest computerized to-
mography (CT) scan is one of the useful radiology tech-
niques or ways to observe pulmonary imaging variations in
suspected cases (Pan et al., 2020; Shi et al., 2020). The CT
scan is a complementary method to molecular-based meth-
ods and is a less costly and more successful treatment or
follow-up for a patient (Shi et al., 2020). Radiologists of the
patients with confirmed SARS-CoV-2 can obtain general
information regarding the infectious steps (Shi et al., 2020).
The CT finding taken from the SARS-CoV-2 patients con-
firmed various abnormalities in the lungs, such as consoli-
dation, centrilobular nodules, bronchial wall thickening,
vascular enlargement, crazy paving pattern, architectural
distortion, traction bronchiectasis, subpleural bands, and
reticulation, that cause pulmonary disease and need fast
FIG. 2. Schematic representation of SARS-CoV-2 and ACE2 receptor interaction. Interaction of spike protein on the
surface of the coronavirus and the cellular ACE2 receptors is needed for entrance into the target cell. ACE2, angiotensin-
converting enzyme II.
THE NOVEL INSIGHT OF SARS-COV-2 MOLECULAR BIOLOGY 7
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diagnosis and viral therapy (Chung et al., 2020). However,
chest CT is a fast and easy way of diagnosis, beginning with
SARS-CoV-2 infection with high sensitivity for suitable
detection. Specific attention should be paid to the role of
radiologists in detecting this recent infectious disease (Shi
et al., 2020).
Molecular technique
PCR-based technique. PCR is the most common and
accurate technique that is used to detect pathogenic viruses
in blood and respiratory secretions (Uhlenhaut et al., 2012;
Setianingsih et al., 2019). The PCR-based technique has a
wide range of applications with high specificity and sensi-
tivity (Wan et al., 2016). This approach has become a
standard and effective method for the detection of cor-
onavirus. Coronavirus RNA is usually extracted, and then
PCR is done by a particular detection technique or instru-
ment (Lu et al., 2014).
RT-PCR is usually recommended for coronavirus detec-
tion because of its advantages as a precise and easy quan-
titative test (Wan et al., 2016). Also, real-time RT-PCR is
more specific and sensitive than traditional RT-PCR assays,
which aid in primary infection diagnosis (Corman et al.,
2012). For the SARS-CoV-2 molecular analysis, various
specimens such as oropharyngeal swabs, throat swabs, and
rectal swabs are examined by using RT-PCR as golden
clinical diagnosis method for virus detection according to
classical Koch‘s postulates (Corman et al., 2020).
Loop-mediated isothermal amplification-based tech-
nique. The Loop-mediated isothermal amplification
(LAMP) is a new isothermal nucleic acid amplification
technique with high efficiency. This is used to amplify
RNAs and DNAs with high specificity and sensitivity due to
its exponential amplification feature and six particular target
sequences diagnosed by four separate primers (Notomi
et al., 2000). The LAMP assay is rapid and does not need
high-priced reagents or equipment. In addition, the LAMP
test will help to decrease the cost of coronavirus detection.
Several strategies for the detection of coronavirus based on
LAMP will be defined here, which have been developed and
performed in clinical diagnosis. The gel electrophoresis
method is widely utilized for its study of the amplified items
to detect endpoints (Enosawa et al., 2003). Poon and co-
workers indicated a simple LAMP test for the SARS study
and showed the feasibility of the usage of these methods for
the SARS-CoV finding. The aim of the study was for the
amplified items to detect endpoints (Enosawa et al., 2003).
Microarray-based technique. The microarray-based
method is a quick and high-throughput detection device. For
this method, the SARS-CoV-2 RNA will first produce
cDNA by reverse transcription with different probes and
these are then hybridized with solid-phase oligonucleotides
into each well (Chen et al., 2010). Afterward, free DNAs are
separated by washing the solution. Because of this advan-
tage, the microarray test was usually used for coronavirus
detection (Li et al., 2014). According to the sequence of
TOR2, Shi and coworkers used a 60mer oligonucleotide
microarray and effectively utilized it for the SARS detection
of coronavirus in patient samples (Chen et al., 2010).
CRISPR technique. The evidence showed that RNA-
targeting detection with CRISPR-Cas13 for fast detection
and portable sensing of nucleic acids can be effective in
virus epidemiology, diagnosis, and prevention (Wright
et al., 2016; Gootenberg et al., 2017). Zhang et al. reported
a protocol for SARS-CoV-2 detection by CRISPR, that
Cas13/C2c2 can be programmed to target and destroy the
genomes of live cells and different diseases (Freije et al.,
2019). The test is carried out by isothermal amplification of
the nucleic acid extraction of patient clinical samples and
then viral RNA sequence amplified through Cas13/C2c2
and, consequently, readout in less than an hour by paper
dipstick (Freije et al., 2019). This method provides quick,
manageable, multiplexable, and quantitative detection plat-
forms of viral nucleic acids (Gootenberg et al., 2018).
In conclusion, this novel virus outbreak emerged and
spread immediately and has posed a challenge to medicine,
economy, and public health worldwide. Several strands of
evidence of this virus are proposed from the unknown in-
termediate host to cross-species, and human-to-human
transmission is established and is an alarm. Even so, assessing
the viral nature of COVID-19 and considering a significant
impairment of the human immune response in serious forms,
it is essential to balance the risk and benefit ratio before
beginning anti-inflammatory treatment. We also speculate on
a number of diagnostic approaches that contribute to the new
COVID-19 pneumonia, such as radiographic or laboratory
result, PCR, Microarray, LAMP, and CRISPR strategies.
To date, the number of infected cases is rapidly elevated.
This necessitates uncovering the viral mystery of the mo-
lecular pathways to provide a future investigation for
emerging targeted vaccines and antiviral treatment. We
expect that these strategies, in combination with the follow-
up laboratory studies aimed at assessing the computationally
anticipated antiviral agents, would allow us to have a
broader range of potential repurposed drugs in the event of
any possible pandemic virus.
Disclosure Statement
No competing financial interests exist.
Funding Information
No funding was received for this article.
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Address correspondence to:
Negin Parsamanesh, PhD
Zanjan Metabolic Diseases Research Center
Zanjan University of Medical Science
Zanjan 4213956184
Iran
E-mail: neginparsa.684@gmail.com;
parsamanesh@zums.ac.ir
Received for publication May 13, 2020; received in revised
form June 24, 2020; accepted July 7, 2020.
THE NOVEL INSIGHT OF SARS-COV-2 MOLECULAR BIOLOGY 13
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