Abstract
Coronaviruses are a group of enveloped viruses with non-segmented,
single-stranded, and positive-sense RNA genomes. Human coronavirus
infection causes respiratory diseases with mild to severe outcomes. In
December 2019, a new outbreak of the novel coronavirus disease 2019
(COVID-19) emerged in Wuhan, China and spread around the world. Genomic
analysis revealed that severe acute respiratory syndrome coronavirus
(SARS-CoV-2) is phylogenetically related to SARS-like bat viruses. The
intermediate source of origin of SARS-CoV-2 and its transfer to humans
is not known; however, it acquired efficient human-to-human
transmissibility while retaining human pathogenicity. Spike protein of
SARS-CoV-2 has the potential furin-like cleavage site may play a
significant role in virus entry. Receptor binding domain (RBD) of
SARS-CoV-2 attaches with angiotensin-converting enzyme -2 (ACE2) of
epithelial cells. The SARS-CoV-2 genome encodes four major structural
proteins: the spike (S) protein, nucleocapsid (N) protein, membrane (M)
protein, and the envelope (E) protein are involved in assembly, budding,
envelope formation, and pathogenesis. Notably, E protein act as
viroporin and there is no mutation found on E protein among SARS-CoV-2
strains. At present, the case fatality rate is estimated to range from 6
to 7%. COVID-19 is now a public health emergency of international
concern. There is no clinically approved antiviral drug or vaccine
available against COVID-19. This review summarized the latest
information on the structural and molecular biology infectivity, host
immune response and molecular immunopathology of the SARS-CoV-2.
Introduction
During the 21st century, five respiratory viruses (3
coronavirus strains and 2 influenza virus strains) outbreaks have
occurred worldwide. Human respiratory tract infection by coronaviruses
first characterized in 1960 (1). A recent rapid outbreak of infection
around the globe caused by a novel coronavirus, which was first emerged
in December 2019 in Wuhan city of Hubei province, People’s Republic of
China. It was declared a pandemic on 11th March 2020
by World Health Organization (WHO) (2–4). The COVID-19 disease is
caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
(5). Within two decades COVID-19 is the 3rd outbreak
of highly contagious coronavirus in humans, followed by a severe acute
respiratory syndrome-related coronavirus (SARS-CoV) in 2002-03 and the
Middle East respiratory syndrome-related coronavirus (MERS-CoV) in 2012
(6). More than 2,600,000 people from 210 countries and territories along
with 2 international conveyance are affected by SARS-CoV-2 as of April
23, 2020 (7). The case fatality rate for COVID-19 has been estimated at
around 4-5% so far (7). Although some countries including China have
seen a decrease in new cases of COVID-19; however, the global situation
remains serious. As of now, the origin of COVID-19 has not been
determined and no specific antiviral treatment or vaccine is currently
available. During the las few months, substantial data poured in on
identifying the SARS-CoV-2, basic structure and mechanism of infection,
and immunological challenges. Based on the published data,
this review systematically discusses a general overview of COVID-19 with
latest pre-clinical issues, structural and molecular biology, host
immune response. It is believed that this review will provide the latest
information to the concerned people involved in the prevention and
control of the COVID-19 epidemic.
- SARS-CoV-2 virus
- Phylogenetics and taxonomy of SARS-CoV-2
Enveloped positive-sense single-stranded RNA virus, SARS-CoV-2 belongs
to a group of viruses known as coronaviruses that cause respiratory,
enteric, hepatic and neurological diseases in mammals and birds (8). So
far, 7 coronavirus species are identified those can infect humans(9).
Four of them (HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E) are known to
infect immunocompromised individuals (8). Other two zoonotic origin
species (SARS-CoV and MERS-CoV) from Betacoronavirus genus are
lethal to humans (10,11). SARS-CoV-2 is the new strain of SARS-CoV
species (12). In December 2019, SARS-CoV-2 caused an outbreak
of coronavirus disease 2019 (COVID-19). Phylogenetic analysis has shown
that like other SARS-related coronavirus strains (including the strain
of 2003 SARS outbreak), SARS-CoV-2 is also the member of subgenusSarbecovirus and species SARS-CoV (13,14). SARS-CoV-2 genome has
the following similarities: 96% identity to the sequence of
Yunnan-RaTG13 (nearest bat precursor), 90% identity to the sequences of
Guangdong-1/PS2 (nearest pangolin precursors), 88% identity to the
sequences of SL-CoVZC45/ZXC21 (bat precursor), 80% SARS-CoV and only
50% identity to the sequence of MERS-CoV (14,15). Even despite
mutations at the genome or amino acid (AA) level, SARS-CoV-2 strains are
highly homologous (~99.99%) (16).
Structural biology of SARS-CoV-2
SARS-CoV-2 virions are around 50-200 nm in diameter, contains four main
structural proteins: spike protein (S protein), envelope protein (E
protein), nucleocapsid protein (N protein) and membrane protein (M
protein) (17–19). The single-stranded RNA genomes of SARS-CoV and
MERS-CoV encode two large genes, the ORF1a and ORF1b. Figure-1shows the structural proteins of SARS-CoV and SARS-CoV-2. S, E
and M proteins form viral envelope/surface together, while N protein
holds RNA genome (18). SARS-CoV-2 virus nucleotide is around 30k long
(14). Like SARS-CoV, SAR-CoV-2 genome contains around ten open reading
frames (ORFs), and 2/3 of the viral genome is first ORFs (ORF1a/b),
those are translated into polyproteins required for viral replication
and transcription (20). One-third of SARS-CoV-2 genome contains other
ORFs, which encode both structural, and accessory proteins. These
accessory proteins interrupt the innate immune response of the host(20).
13 variation sites in ORFs (1a, 1b, S, 3a, M, 8 and N regions) have so
far been identified in SARS-CoV-2 (16). The functions of all accessory
proteins encoded by ORFs of SARS-CoV-2 are still not well elucidated.
Non-structural proteins (NSPs) of SARS-CoV-2
Table-1 summarises the non-structural proteins of SARS-CoV-2
virus. ORF1 encodes pp1a and pp1ab polyproteins those are responsible
for the formation of replicase-transcriptase complex (20,21). Following
the virus entry, 16 non-structural proteins (NSPs) are produced from
these polyproteins by essential viral proteases, e.g. main protease and
papain-like protease (20,22). Main protease and papain-like protease act
like scissors, snipping the links between the different NSPs and freeing
them to do their jobs.
Unique class I spike glycoprotein of SARS-CoV-2
Primary coronavirus tropism, the crown (Latin ”corona”) shaped S protein
(~1200 AA) long) resides on the surface of SARS-CoV-2,
which allows the virus to attach and fuse with the host cell membrane
(18,23,24). Structural proteins also help assemble and release of new
copies of the virus. Like other human infecting coronaviruses, following
cleavage class I , S protein of SARS-CoV-2 generates 2 subunits: S1
(attachment domain) subunit and S2 (fusion & transmembrane domain)
subunit (24–26). Figure-2 describes the S protein of
SARS-CoV-2 with a focus on putative favourable sites . SARS-CoV-2
virus is highly pathogenic and transmissible comparing with otherbetacoronaviruses of linage b, due to its polybasic furin-like
cleavage site in S protein (24,26,27). Also, the receptor-binding domain
(RBD) of SARS-CoV-2 S protein has a high affinity for human angiotensin
converting enzyme 2 (ACE2) receptor, which increases the virulence (26).
Also, S protein of SARS-CoV-2 can interact with CD147 for cellular
invasion (28). So, targeting spike protein of SARS-CoV-2 could be
potential for drug or vaccine designing.
Attachment capability of S1 subunit of SARS-CoV-2
The S protein mediates viral entry into host cells by first binding to a
host receptor through the RBD in the S1 subunit. N terminal S1 subunit
comprises 2 distinct domains: N-terminal domain(NTD) and RBD (24,29).
Details of various domains of S1 subunit are described in
Figure-2. Both domains are critical as receptor recognition
(29). Although the RBD is known to bind the cell surface receptor ACE2,
the function of NTD of SARS-CoV-2 is not well investigated. NTD has been
suggested to facilitate the initial attachment by recognizing sialic
acids (as receptor determinants) in the host cell membrane (25,30,31).
Further studies however are required for definitive conclusions.
Fusion capability and viral infectivity of S2 subunit of
SARS-CoV-2
Several viral proteins such as fusion peptide (FP), second proteolytic
site (S2’), internal fusion peptide (IFP), heptad repeat 1& 2 (HR1 &
2) and transmembrane domain (TM) form S2 subunit complex (24). FP and
IFP are thought to be responsible for virus entry (32). Like another
type I fusion proteins such as Ebola GP and HIV gp41, SARS-CoV-2 HR1 and
HR2 may form canonical six-helix bundle (32). The detailed functions of
FP, IFP, HR1 & 2 and TM are still not known to conclude whether they
work individually or synergistically in the fusion with the host.
Further investigation is required for better understanding of
functionality of S2 subunit.
- Multiple cleavage sites of SARS-CoV-2 glycoprotein
- Identical polybasic furin-like cleavage sites of
SARS-CoV-2
Proteolytic cleavage regulates processes in viral-host interaction. One
key player is the ubiquitously expressed serine protease furin, which
cleaves a plethora of proteins at polybasic recognition motifs. The
enzymatic activity of furin is exploited by numerous viral pathogens to
enhance their virulence and spreading capacity. Seven mammalian
secretory preprotein convertases (PC1, PC2, furin, PC4, PC5, PACE4 and
PC7) cleave precursor proteins at specific single or paired basic amino
acids (AA) within the canonical motif
(R/K )-(2X )n -(R/K )↓, wheren = 0, 1, 2, or 3 and X = AA.(33). The preprotein
convertases, especially furin (highly expressed in the lung) is known to
cleave viral envelope glycoproteins to enhance cellular tropism and
pathogenesis (24). And polybasic motif in viral envelope glycoproteins
is furin-like cleavage specific and associated with the hyper-virulence
of the SARS-CoV-2 (24). Different pathogenic RNA viruses, e.g. HIV,
Ebola virus with polybasic envelop protein cleavage site(s) are on focus
due to their higher pathogenicity, as shown in Table-2 . Similar
to MERS-CoV, 2 polybasic furin-like cleavage sites (S1/S2 and S2’) are
discovered in SARS-CoV-2 (24). But the role of furin-like cleavage sites
in S protein of SARS-CoV-2 on viral pathogenesis or replication is still
not well investigated.
Other protease-mediated S1/S2 cleavage site of
SARS-CoV-2
Like SARS-CoV, SARS-CoV-2 contains the conserved site sequenceAYT↓M between RBD and FP, which can be cleaved by other
proteases (e.g. cathepsin L, TMPRSS2) rather than proprotein convertases
(24,25). The conserved sequence AYT↓M may be cleaved following
furin cleavage at S1/S2 site. This possibly demonstrates if S protein of
SARS-CoV-2 would not cleave at furin-like cleavage sites during viral
endocytosis, S protein would certainly cleave at conserved sequenceAYT↓M.
Glycobiology of SARS-CoV-2
Insertion of proline before furin cleavage site (-RRAR-) results in the
addition of O-linked glycans which flank at the cleavage site ( S1/S2)
(25,26). Figure-3 shows the cleavage site of closely relatedbetacoronaviruses. The function of O-linked glycan of SARS-CoV-2
has not been studied much. It is predicted that O-linked glycan could
create mucin-like domain to protect epitopes or key residues on S
protein. Different viruses use the mucin-like domain to conserve immune
evasion (34). Therefore, O-linked glycan could be a crucial determinant
for vaccine discovery of SARS-CoV-2.
Mutations in the receptor-binding domain
Surface proteins of SARS-CoV-2 have around 98% and 76% identical AA
sequence of Yunnan-RaTG13 (bat precursor) and SARS-CoV, respectively
(14). The most variable part of the coronavirus genome seems to be RBD
in the S protein (26). Figure-4 describes the RBD of closely
related betacoronaviruses. With 30 mutations (13
interface mutations) in RBD indicates how changes could allow switching
the host from bat to human (14). As positive-sense RNA virus, COVID-19
virus can mutate in human for its adaptation. Although second closest
relative, Guangdong-1/PS2 (pangolin precursors) has approximately 91%
similarity with S protein of SARS-CoV-2, RBD of Guangdong-1/PS2 is much
closer to SARS-CoV-2 (99% identical) than Yunnan-RaTG13 (14,35,36). So,
the probable pangolin origin of COVID-19 outbreak may not be ruled out.
Seemingly natural selection in animal host before zoonotic transfer or
natural selection in humans following zoonotic transfer can be the
reason of origin of human SARS-CoV-2 infection (26). Additionally, these
genomic comparisons suggest that the SARS-CoV-2 could be the result of a
recombination between Yunnan-RaTG13 and Guangdong-1/PS2 (pangolin
precursors) viruses. For recombination, two divergent positive-sense RNA
viruses must have infected the same organism simultaneously (37,38). The
recombination mechanism was described earlier section to explain the
origin of SARS-CoV (39). Enough data are still not available to conclude
whether the recombination occurred in bat, pangolin, or any another
species. Further research is needed for definitive conclusions. However,
the two features of the SARS-CoV-2 virus such as its distinct backbone
and the mutations in the RBD of the S protein, probably rule out
laboratory manipulation as a potential origin for SARS-CoV-2.
Promising envelop protein of SARS-CoV-2
Like in SARS-CoV, E protein may be involved in SARS-CoV-2 assembly,
budding and envelope formation (40). Interestingly no mutation has been
observed so far among E protein of SARS-CoV-2 strains (16). Similar to
SARS-CoV, E protein of SARS-CoV-2 may form channels in ERGIC/Golgi
membranes to permeabilize calcium ions to trigger NOD-like receptor 3
(NLRP3) inflammasome, leading to IL-1β overproduction (41). E protein
latches onto proteins that help turn host genes on and off. Ultimately,
E protein has a significant role in the viral life cycle which could be
a promising target for vaccine development (42).
Nucleocapsid protein of SARS-CoV-2
Nucleocapsid protein (N protein) protects the viral RNA by stabilising
it inside the virus (43). N protein induces apoptosis and inhibits type
I interferon production, cell cytokinesis and proliferation (44). N
protein also regulates several pathways, e.g. AP-1 signal transduction
pathway, nuclear factor kappa B (NFκB) pathway and transforming growth
factor-beta signalling (44). These cytokines associated with
inflammation in COVID-19 may be useful in developing the therapeutic
strategy.
Accessory proteins of SARS-CoV-2
Accessory proteins modulate the environment in infected cells to make
easier for viral replication. Therefore, their roles in pathogenesis
are important. The roles of the accessory proteins such as ORF3a, ORF6,
ORF7a, ORF8, ORF9b and ORF10 of SARS-CoV-2 in viral replication are yet
to be elucidated fully. KAUST metagenomic 154 analysis platform (KMAP,
www.cbrc.kaust.edu.sa/kmap)
has shown that accessory proteins of SARS-CoV-2 encoded by 5 gene
clusters (ORF3, ORF6, ORF7, ORF8, ORF9) are similar to those of otherbetacoronaviruses (40). The endoplasmic reticulum-induced ORF3a
protein is thought to be responsible for apoptosis and suppression of
type I interferon (IFN) signalling through the PERK pathway (45).
Whereas ORF3a upregulates fibrinogen expression and activates NFκB and
NLRP3 inflammasome to trigger inflammation (45,46). ORF3a makes holes in
the infected cell membrane for new viruses to escape out. Vesicle-bound
ORF6 may play a vital role in the viral replication (47). It also blocks
some of host cells’s virus-fighting proteins (48). ORF7a binds
lymphocyte function-associated antigen 1 (LFA-1), induces apoptosis and
activates NFκB and IL-8 promoters (47). ORF7a cuts down infected cell’s
supply of tetherin to allow more of the viruses to escape. ORF8 of
SARS-CoV-2 was distinct from other betacoronaviruses (49).
Intracellular stress pathways and NLRP3 inflammasomes triggering
aggregation motif VLVVL is absent in the SARS-CoV-2 ORF8 (49).
More research is needed to define the function of ORF8. Cellular protein
Crm1 interacts with ORF9b encoded protein to induce apoptosis (47).
ORF9b can block interferon. Unlike SARS-CoV, host immune response
modulating ORF3b seems to be absent in SARS-CoV-2 due to overlap the
same sequence of ORF3a. Multi-localized accessory protein encoded by
ORF3b upregulates cytokines (except type I IFNs) and chemokines by
modulating transcription factor RUNX1b, induces AP1 transcriptional
activity through activation of JNK and ERK pathways, arrests cell growth
in G0/G1 phase and induces apoptosis and necrosis (47). Surprisingly
lacking of ORF3b doesn’t affect much in the infective ability of the
virus (47). ORF7b overlaps the same sequence of ORF7a in RNA genome, but
it is not clear what, if anything, the gene does. Although ORF7b encoded
non-structural (type III integral transmembrane) protein is responsible
for apoptosis, IFN dependent reporter gene expression and type I IFN
response, but deletion of ORF7b enhances viral growth may explain the
reason of more tissue damage by SARS-CoV-2 (51). The non-conserved ORF7b
in SARS-CoV-2 may affect its infection property. However, unique ORF10
may be an artefact annotation for SARS-CoV-2 (40).
Polarity of the RNA of SARS-CoV-2
SARS-CoV-2 belongs to the group IV according to the Baltimore
classification (52). Positive-sense (5’-3’) viral RNA genome directly
serves as messenger RNA, which is capable of translating into viral
proteins (skipping transcription step) in the host cell during viral
replication. Vulnerable SARS-CoV-2 RNA genome protects it’s genome
integrity from environmental factors and host factors (attack by
nucleases and other RNA-modifying enzymes that comprise the cellular
intrinsic or innate immune response) through different mechanisms (37).
- Transmission immunopathology of SARS-CoV-2 virus
- Reservoir of SARS-CoV-2
Bat is thought to be the primary reservoir for all coronavirus related
human outbreaks (10), but the original source of SARS-CoV-2 is
yet to be established (53,54). SARS-CoV-2 shares 96% of its genome with
RaTG13 from Rhinolophus affinis in Yunnan province, China (55).
Coronavirus recovered from malayan pangolin also have higher similarity
with SARS-CoV-2, which indicates significant recombination event and
more complex origin of SARS-CoV-2 (56).
Routes of transmission of SARS-CoV-2
Transmission of COVID-19 occurs via respiratory droplets (within the
range of approximately 27 feet) and fomites during unprotected contact
between the infector and infectee (54,57,58). Although the possibility
of airborne transmission cannot be ruled out (59,60). Still, it is not
known how much concentration of viable SARS-CoV-2 is needed to infect a
human being. van Doremalen et al. showed that viability of
SARS-CoV-2 in the air (up to 3 hours), copper (4 hours), cupboard (24
hours), plastic and stainless steel (up to 3 days) (61). Chin et
al. could not detect viable infectious SARS-CoV-2 from printing and
tissue paper after 3 hours, wood or cloths on day 2 and glass or
banknote on day 4, but infectious SARS-CoV-2 was detectable on the outer
surface of the surgical mask on day 7 (62). COVID-19 transmission was
suggested to be reduced at high temperature and high humidity (63).
SARS-CoV-2 is extremely stable at 4°C, but at 70°C SARS-CoV-2 virus
inactivates within 5 min (62). As viable SARS-CoV-2 is present on stool
sample and ultimately sewage water, so the faecal-oral route may be
considered for COVID-19 transmission (54,64). COVID-19 transmission may
occur at subclinical infection or asymptomatic conditions (65–67).
- Molecular biology of infection
- Entry of SARS-CoV-2 virus
In order to reproduce and establish infection the virus must enter the
host organism. S protein of SARS-CoV-2 is the vital determinant of viral
entry, which binds to ACE2 expressing host epithelial cell through RBD
(Figure- 5) (20). S protein of SARS-CoV-2 has 10-20 times
stronger affinity for human ACE2 receptor than that of SARS-CoV (68).
ACE2 regulates renin-angiotensin system homeostasis (69). Along with
angiotensin II receptor type 2, ACE2 protects from severe acute lung
injury (69). The potential target ACE2 is expressed in upper and lower
respiratory tract (polarized epithelial cells and type II pneumocytes),
oral cavity (epithelial cells), oesophagus (upper and stratified
epithelial cells), intestine (enterocytes), gall bladder
(cholangiocytes), heart (myocardial cells), breast, adrenal gland,
kidney (proximal tubular cells), urinary bladder (urothelial cells), eye
(conjunctival fibroblast & epithelial cells and corneal epithelial
cells), brain (neuronal cells) and male organ (testis and seminal
vesicle) (70–76). Direct or indirect contact with mucus membrane in the
nose, mouth and eyes can transmit COVID-19 virus (77). Droplets and
fomites containing SARS-CoV-2 find their prospective target ACE2
receptors at respiratory tract primarily as a portal of entry. But due
to the highest expression of ACE2 receptor into enterocytes (and
cholangiocytes), the intestinal wall cannot be ignored as the portal of
entry of COVID-19 virus (70). Studies found that stools were positive
for SARS-CoV-2 for 1 to 12 days in patients following negative results
in respiratory samples (78). Gastric pH may reduce the infective
capacity of SARS-CoV-2, as previous studies showed how pH influenced the
viability of SARS-CoV in the stool (79). Although Chin et al. claimed of
extreme stability of SARS-CoV-2 in a wide range of pH values (3 -10) at
room temperature (22°C) (62).
- Replication of SARS-CoV-2
- Two-step cleavage of S Protein of SARS-CoV-2
Following binding to ACE2 receptor, 2 steps sequential protease cleavage
occurs in S protein of SARS-CoV-2 by host proteases: priming cleavage on
S1/S2 and activating cleavage on S2’ sites (20,31) Priming cleavage on
S1/S2 site is regulated by host proteases, e.g. cathepsin L and TMPRSS2
(20,25,80). Also, ACE2 receptor is proteolytically cleaved by TMPRSS2
and/or ADAM17 to propagate SARS-CoV-2 coronavirus entry (81).
Syncytium formation and virus entry
Figure-5 shows the schematic diagram of SARS-CoV-2 entry to the
host cells. Phosphatidylinositol-3,5-bisphosphate
(PI(3,5)P2) is important for early to late lysosomal
maturation. As the critical SARS-CoV-2 viral entry regulators,
phosphatidylinositol 3-phosphate 5-kinase primarily synthesizes
PI(3,5)P2 in the early endosome, while two-pore channel
subtype 2 (TPC2) in endosome is one of the fundamental downstream
effector of PI(3,5)P2 (31). Cathepsin L (highly
expressed in lung and bronchus) is essential for endosomal cell entry
and might be responsible for activation of membrane fusion through
proteolysis on S1/S2 site (31,71,82). The FP can expose
and insert into the host cell membrane due to the acidic environment of
the endosome (80). HR1 and HR2 domains of S2 subunit form canonical
six-helical fusion core between the viral and host cell endosomal
membrane for syncytium formation (68). Trypsin priming or activation of
S protein may trigger directly by endocytosis for rapid disease
progression (31). Membrane-bound TMPRSS2 may play the role for viral
entry in the gastrointestinal tract (25,71). At neutral pH,
transmembrane TMPRSS2 activates S protein that results in syncytium
formation and the release of the viral RNA genome into the host cell
cytoplasm (25,68,80). The involvement of furin/furin-like protease in
SARS-CoV-2 entry is obvious. Similar to MERS-CoV, the SARS-CoV-2 has
polybasic furin-like cleavage sites and expected to have a similar
mechanism of action (24). Endogenous furin expression in various tissues
(e.g. upper respiratory tract, salivary gland) may influence viral entry
through cleavage of S2’ site during fusion mediated viral entry
(71,83,84). However, one cannot rule out the possibility of cross-talk
between heparan sulphate proteoglycans in the host cell surface, and
furin-like cleavage sites in SARS-CoV-2 (84,85), as high level of
heparan sulphate proteoglycans may cause coagulopathy during COVID-19
(86). More studies on furin-like cleavage sites are urgently needed.
Virus replication
Virus replication process starts after hijacking the host’s
translational machinery (22). The released SARS-CoV-2 RNA genome in host
cell cytoplasm is translated into 2 polyproteins, structural and
accessory proteins (20,22). This translational microenvironment protects
viral RNA from host nucleases and innate immune response (22). The
cleavage of the polyproteins forms 16 NSPs (20,49). The NSPs assembled
replicase-transcriptase complex in double-membraned vesicles is
responsible for synthesizing a full-length negative RNA strand template
(20,21). The RNA strand replicates to genomic RNA and individual
sub-genomic RNA continuously (21,87). Sub-genomic RNA serves as mRNA,
which directly translated to viral structural proteins and trafficked
into the endoplasmic reticulum-Golgi intermediate compartment (21). It
is noteworthy to mention that furin is thought to cleave S1/S2 site
during S protein biosynthesis (83).
Packaging and release of SARS-CoV-2
S protein, E protein, N protein and relatively abundant membrane protein
(M protein) along with newly generated complete RNA genome form virion
in budding Golgi vesicles (21). Virion containing vesicles fuse with the
host cell membrane, and mature SARS-CoV-2 is released by exocytosis
(20,87).
Local spread and dissemination at the portal of entry
The newly formed SARS-CoV-2 virus attacks another neighbouring cell, and
this goes on. Immune and lung systems, pulmonary epithelium and immune
cells are the primary targets for SARS-CoV-2 (20,88). Similar to
SARS-CoV and MERS-CoV, the studies showed widespread immunopathology
and/or extrapulmonary dissemination and replication of SARS-CoV-2 among
infected patients (88,89). Post mortem study analysis of deceased
SARS-CoV-2 and SARS-CoV infected patients found other potential target
sites such as neurons, epithelium of renal tubules, intestinal mucosa,
macrophages in various organs (88–91). As part of the upper respiratory
tract, the olfactory epithelium has also come into focus due to COVID-19
associated anosmia (92).
- Host immune response
- Antigen presentation in SARS-CoV-2 infection
Currently, there is very limited knowledge of the host immune response
to SARS-CoV-2. However, based on the accumulated clinical and
experimental data on these previous viruses some suggestions can be
made. Following virus entry into the host cell, the antigen presentation
is the key determinant of T-cell immune response. T cells identify major
histocompatibility complex (MHC) / human leukocyte antigen (HLA) bound
processed cell surface antigens only. Mainly with the help of MHC class
I (e.g. HLA-A, HLA-B, HLA-C) molecules virus-infected host cells are
presented to CD8+ / cytotoxic T cells, but the MHC class II (HLA-DM,
HLA-DR, HLA-DP, HLA-DQ, HLA-DOA and HLA-DOB) also contributes in antigen
presentation to CD4+ / helper T cells (93,94). Not only macrophages,
dendritic cells or B cells, but also aerodigestive epithelial cells
(enterocytes, columnar ciliated epithelial cells and type II
pneumocytes) present MHC class II (93). Variability of HLA genes makes
the differences in immune response and disease severity among
individuals (95). Same as SARS-CoV, studies across 145 HLA alleles
showed that individuals with HLA-B*46:01 allele is vulnerable to
COVID-19 disease (20,95). HLA-A*25:01 and HLA-C*01:02 alleles are also
shown to be related to the susceptibility of SARS-CoV-2, while
HLA-A*02:02, HLA-B*15:03, and HLA-C*12:03 might be related to the
protection from SARS-CoV-2 infection (95). Due to limited comprehensive
COVID-19 pathogenesis study. HLA-DR B1*1202 and HLA-DR0301 are least and
top, binding MHC class II molecules for SAR-CoV (20). Even
HLA-DRB1*11:01 and HLA-DQB1*02:0 are associated with MERS-CoV infection
(20).
Lymphocytopenia with hyperactivity
Following antigen presentation, B and T cell-mediated humoral and
cellular immune response occurs respectively to eliminate the virus and
disease progression. The humoral immune response plays a vital role to
limit infection at later phase and prevent reinfection in the future.
Like other acute viral infections, a typical pattern of IgM and IgG
antibodies against SARS-CoV-2 are reported. SARS-CoV-2 induced
seroconversion starts gradually from approximately day 5 and switches to
IgG by around day 14 (96). At the end of week 12, the SARS-specific IgM
antibodies disappears, while the IgG antibody against SARS-specific S,
M, E and N proteins can last long (20,97). In vitrocross-reactivity of SARS-CoV-2 sera with SARS-CoV suggests possible
mounting of the humoral immune response (97). As COVID-19 disease is
associated with lymphocytopenia, the number of blood CD4+ and CD8+ T
cells of COVID-19 patients are significantly reduced (20,98). But CD4+
and CD8+ T cells are overactivated (88). Similar results were observed
in acute phase response in SARS-CoV virus infection where CD4+ and CD8+
memory T cells sustained for 4 years in recovered patients to perform T
cell proliferation, DTH response and production of IFN-γ (20). CD8+ T
cell responses are higher in SARS-CoV infection (which is crucial for
lung pathology) than CD4+ T cell responses (97). In severe COVID-19
cases, higher levels of multifunctional CD4+ T cells (IFNγ, TNFα, and
IL-2), CD8+ T cells (IFNγ, TNFα and degranulated state) and Th2
cytokines (IL-4, IL-5, IL-10) were detected (97).
The first line of defence against SARS-CoV-2
Only a few studies are available on host innate immune response to
SARS-CoV-2 infection. Gradual increment of total neutrophil counts,
C-reactive protein and many innate cytokines suggesting highly
pro-inflammatory condition in COVID-19 disease progression and severity
(97). First-line defence against RNA viruses is comprised of toll-like
receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors
(RLRs) and NLRPs to detect viral genome and it’s replication
intermediates (99). Innate immune cells recognise viral invasion by
either the endosomal TLRs (TLR3, TLR7 and TLR8) and the cytosolic
double-stranded RNA sensor RIG-I/MDA-5 (97,100,101). Adaptor proteins
(MyD88 and MAVS) induced downstream signalling are recruited upon
recognition by TLRs and RLRs (101). As the first line of defence, the
recognition events lead to interferon regulatory transcription factor 3
or 7 (IRF3/7), and NFκB induced type I IFN (IFN-α and IFN-β) and other
pro-inflammatory cytokines (e.g. TNF-α, IL-6) expression (97,101). Type
I induction is essential in the early phase of the disease to halt virus
propagation within the host and modulate innate and adaptive immune
responses. STAT1/2-IRF9 complex-mediated IFN-stimulated genes (e.g.
anti-viral enzyme RNAse L, pro-inflammatory chemokine CXCL10)
transcription follows activation of the JAK-STAT pathway via type I IFN
(97). During the early phase of infection, SARS-CoV-2 viral (structural
and non-structural) proteins could use multiple strategies to dampen
type I IFN mediated immune response (97). But the influx of
hyperinflammatory neutrophils, monocytes, and macrophages due to
delayed-type I IFN immune response compensates early viral control could
result in deteriorating consequences to the infected host. Additionally,
SARS-CoV-2 E and ORF3a proteins may trigger inflammasome sensor NLRP3 to
secrete IL-1β and induce pyroptosis (an inflammatory form of cell death)
(41,102). Simply saying, the innate immune response could be protective
or destructive for SARS-CoV-2 infected patients and may open the window
for immune intervention. Due to highly effective innate immunity, very
few severe cases were reported in young patients. So, the innate immune
response may be the key determinant for disease outcome
Immune evasion by SARS-CoV-2
Clinical and experimental data showed that SARS-CoV-2 deals with host
immune response (97). This virus can evade immune detection and dampen
human immune responses, which explain why the incubation period of
SARS-CoV-2 is higher than the influenza virus. Innate immune responses
are inhibited at the level of type I interferon recognition and
signalling, while adaptive immune evasion sis processed by MHC class I
and class II downregulation markedly (97). Like other
betacoronaviruses, intermediate products of SARS-CoV-2 may avoid host
recognition within double-membraned vesicles during replication process
(103,104). SARS-CoV-2 M protein may suppress RIG‐I‐induced IRF3
triggering, while deubiquitinase (DUB) activity in the infected cell and
IRF3 inhibitory activity of SARS-CoV-2 NSP3 may play a vital role in
immune evasion (105–108).
NSP1 and NSP6 of SARS-CoV-2 may block the phosphorylation of STAT1 and
the translocation of the STAT1/2/IRF9 complex to block antiviral
activity and IFN response (109,110). Therefore, downregulating IFN may
cause an imbalanced production of pro-inflammatory cytokines and
infiltration of inflammatory cells that observed in severe COVID-19.
Cytokine release syndrome on COVID-19
Acute respiratory distress syndrome (ARDS) and multiple organ
dysfunction are the fundamental causes of COVID-19 deaths (20). As the
initiator of ARDS, deadly uncontrolled cytokine storm results from the
release of large amounts of pro-inflammatory cytokines (IFN-α, IFN-γ,
IL-1β, IL-6, IL-12, IL-18, IL-33, TNF-α, TGFβ etc.) and chemokines
(CCL2, CCL3, CCL5, CXCL8, CXCL9, CXCL10 etc.) by the host immune
effector dendritic cells, macrophages, lymphocytes, and natural killer
cells (20).
Conclusions
A better understanding of structural, molecular biology of SARS-CoV-2 is
critically important to develop effective prevention and therapeutic
strategies such as vaccine and antiviral drugs against COVID-19 disease.
Rapid response to the current COVID-19 outbreaks with diagnostic and
therapeutic measures are urgently required to save thousands of lives.
However, there are still several unanswered questions remain regarding
the COVID-19 despite massive pouring of research and clinical data on a
daily basis. The presence of most significant percentages of COVID-19
are asymptomatic or mildly symptomatic infections underscore the
critical importance of elucidating the structure, function of the
SARS-CoV-2 and the host-immune response. This sort of information can be
exploited in the development of vaccine against COVID-19. For example, E
protein can be exploited for vaccine preparation as it is being involved
in multiple aspects of the viral replication cycle: from assembly and
induction of membrane curvature to scission or budding and release to
apoptosis, inflammation and even autophagy.
In this review, we summarized the latest information in these aspects,
although information regarding the virology, epidemiology, and
transmission of SARS-CoV-2 continues to evolve.