Role of Structural and Non-Structural Proteins and Therapeutic Targets of SARS-CoV-2 for COVID-19

1In late December 2019, an acute case of respiratory diseases was reported in Wuhan, Hubei People’s Republic of China. Initially, clinical symptoms such as fever, sore throat, and respiratory distress amongst others overlapped with viral pneumonia. However, after genomic analysis of respiratory samples from patients, it was revealed as novel coronavirus (2019-nCoV) pneumonia. Later on, 2019-nCoV was finally renamed as SARS-CoV-2 by the International Committee on Taxonomy of Viruses (ICTV) owing to its genetic similarity to an earlier known coronavirus (SARS-CoV). This coronavirus-induced disease was officially announced as Coronavirus Disease-19 (COVID-19), and on 11 March 2020, World Health Organization (WHO) declared it as a pandemic owing to its vast magnitude of global spread. As of 30 March, 2021, nearly 128 million of confirmed cases and more than 2.8 million of deaths has been reported across the globe (worldometers.info/coronavirus; accessed on 30th March 2021). Coronavirus is a large family of viruses that are capable of causing moderate illness such as common cold, to more severe life-threatening diseases like acute respiratory distress syndrome (ARDS) and organ malfunctions. SARS-CoV-2 is taxonomically placed under order Nidovirales, subfamily Orthocoronavirinae, and four genera namely Alphacoronavirus (α), Betacoronavirus (β), Gammacoronavirus (γ), and Deltacoronavirus (δ). All viruses of Nidovirales order share several common features, such as they possess conserved genomic organization, replicase gene located downstream to 5′-UTR, expression of downstream genes following synthesis of multiple 3′-nested subgenomic mRNAs, expression of genes by ribosomal frameshift mechanism, and multiple unique intrinsic enzymatic activities in the large replicase-transcriptase polypeptide among others. People are susceptible to infection by seven of these viruses, including 229E (α), NL63 (α), OC43 (β), HKU1 (β), MERS-CoV (β), SARS-CoV (β), and SARS-CoV-2 (β). Among them, the four HCoVs, namely, 229E, NL63, HKU1, and OC43, cause very mild to moderate upper-respiratory tract illness, whereas SARS-CoV, MERS-CoV, and SARS-CoV-2 are quite deadly, and may cause fatal respiratory disease involving lower respiratory tract and down right to alveoli of lungs. SARS-CoV-2 shows 88% sequence identity with bat-SL-CoVZC45 and bat-SL-CoVZXC21, and 79% and around 50% sequence identity with SARS-CoV and MERS-CoV, respectively. Therefore, SARS-CoV-2 is phylogenetically closer to bat-related SARS-CoV vis-à-vis MERS-related CoV and SARS-related CoV, indicating that it might have evolved from bats. This knowledge of similarities and differences of SARS-CoV-2 with viruses, which are known to have caused earlier outbreaks, may help in developing better understanding of etiology, and consequently design curative strategy to tackle current pandemic. In contrast to SARS-CoV-2, MERS-CoV infection was first reported in Jordan, Saudi Arabia in 2012. Humans contract MERS-CoV following coming in contact with infected camel as well as infected humans. Since 2012, 27 countries have reported more than 24 thousand MERS cases, of which the majority of cases have occurred in Saudi Arabia. SARS-CoV was found in China in 2003, and has originated in bats, and was amplified in other animal reservoir before infecting humans. The outbreak was contained in mid-2003 with the implementation of infection control practices such as isolation and quarantine. Since then, a handful of cases have occurred due to laboratory accidents and related exposure which have been comprehensively reviewed. SARS-CoV-2 is a new strain of corona virus that has not been traced in humans previously. In this review, we primarily focus on multiple features of SARS-CoV-2, including genomic organization, structural and non-structural protein components, and potential prospective molecular targets, against which either various FDA approved drugs used for other diseaseare being repurposed, or novel drugs under various phases of clinical trial are being tested. In addition, we also focus on plasma therapy and antibody cocktail, as well as 13 approved/authorized vaccines being administered worldwide to tackle current pandemic.

2The nucleotide length of genome of SARS-CoV-2 is ranging from 26.0 kb to 32.0 kb, having variable number of open reading frame (ORFs), and its genomic organization is considerably similar to other known HCoVs. For instance, coronaviruses characterized till date are enveloped viruses with numerous surface-projected club-like spikes, and they possess an unsegmented, single-stranded (ss), +ve sense RNA genome with 5′-cap and 3′-poly(A) tail, allowing it to act as functional mRNA for translational synthesis of the replicase polyproteins. Two-thirds of the viral genomic region i.e., initial 20 kb lying downstream to 5′-end, occupies replicase gene referred to as Open Reading Frame 1a and ab (ORF1ab), which potentially encode the nonstructural proteins (nsps) referred to as pp1a and pp1ab polyproteins, respectively. The pp1a non-structural protein corresponds to NSP1 to NSP11 and pp1ab non-structural protein comprises of NSP12 to NSP16. The remaining 10 kb region preceding 3′-end encodes various structural proteins involving surface (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Additionally, the structural genes encode nine accessory proteins, encoded by ORF3a, ORF3d, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF14, and ORF10 genes. Furthermore, the genomic region lying immediate to 5′-end possesses two distinct domains, leader sequence and untranslated region (UTR), capable of forming multitude of stem loop structures prerequisite for replication and transcription of viral genome. The transcriptional regulatory sequences (TRSs) precede each structural and accessory gene which are prerequisite for their functional expression. The structural component of 3′-UTR is required for replication of viral RNA (Figure 1).

3The SARS-CoV-2 are spherical, enveloped, around 80–120 nm in diameter, with multiple outwardly projected club-like homotrimeric, glycosylated S proteins imparting them incredible appearance of a solar corona, prompting their popular name, CoVs. Enclosed within the lipid bilayer envelope of the virion is helically symmetrical nucleocapsids comprising complex of +ssRNA and capsid proteins. There are four important structural proteins—spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins—that are encoded by structural genes located within the region preceeding 3′ end of genome. Apart from above-mentioned structural proteins, there are several non-structural and/or accessory proteins, which together are responsible for the structural and functional aspects of virus. The various structural and functional aspects of aforementioned proteins are summarized below.

4The S protein of virion is a type I transmembrane N-linked glycosylated protein (150–200 kDa) consisting of 1273 amino acids, and displays varying degree of conservation across the Coronaviridae family. Following synthesis, three polypeptide chains of S protein associate with each other, forming homotrimeric assemblage. Each monomeric S protein consists of S1 and S2 subunits, which together possess multiple functional domains, from N to C-termini as follow: N-terminal domain (NTD), receptor binding motif (RBM) containing receptor binding domain (RBD), furin cleavage site (S1/S2, which is likely to be cleaved by the TMPRSS2 protease), fusion peptide (FP), central helix (CH), connecting domain (CD), heptad repeat (HR1/2) domain, transmembrane domain (TM), and cytoplasmic tail (CT). Furthermore, each monomeric S protein folds into a three-dimensional structure with three distict topological domains—i.e., head, stalk, and cytoplasmic tail. The RBD/S1 (~200 amino acids) of S protein undergoes down-to-up conformational transition during interaction with membrane bound ACE2 recepetor, facilitating cell recognition and binding. In addition, RBD substantially contributes towards the overall mechanical stability of homotrimeric spike. The S2 subunit with its fusion peptide (FP), central helix (CH), connecting domain (CD), heptad repeat (HR1/2) domain mediates integration between viral and host cell membrane. Evetually, the interaction between receptor binding motif of RBD of S1 subunit and ACE2 receptor leads to entry into host cells. Another remarkable feature of the S protein is its substantial N-linked glycosylation covering large proportion of protein’s surface area, and shows conformation-dependent dyanamic changes. Apart from shielding, N-glycans especially at N165 and N234 positions are involved in modulation of RBD conformation, which may be used as potential therapeutic target. Besides, the S protein consists of disulfide bonded extracellular N-terminus, a transmembrane domain, and a short intracellular C-terminal part with palmitoylation. The S protein plays major determinant of host immune response, and is involved in viral pathogenesis through activation of endoplasmic reticulum (ER) stress response, and therefore any mutational change may lead to altered pathogenesis.

5The membrane (M) protein is O-linked glycoprotein of around 25–30 kDa, and is most abundant amongst various structural proteins, and possesses three distinct transmembrane domains. The homodimeric M protein associates with other viral structural proteins, including nucleocapsid, facilitating the molecular assembly of virus particles as well as may be involved during pathogenesis. Although M protein possesses diverse amino acid composition, but it is structurally preserved across the various genera. Except β-CoVs and δ-CoVs which shows O-linked glycosylation, other coronavirus M protein undergoes N-linked glycosylation. The glycosylation plays vital role in organ tropism and IFN signalling.

6Envelope (E) protein is smallest amongst all the structural proteins, around 8–12 kDa, and plays major role in pathogenesis, virus assembly, and release. Using solid-state NMR spectroscopy, one study demonstrated the structure and drug binding of SARS-CoV-2 E protein. The E protein topology of SARS-CoV-2 represented a five-helix bundle surrounding adehydrated narrow pore with bipartite channel. Although, E proteins are highly divergent in terms of amino acid composition, but structurally preserved across various genera of β-coronaviruses with a short hydrophilic N-terminus, a large hydrophobic region, followed by hydrophilic C-terminal tail.

7The N (nucleocapsid) protein is solely complexed in structural organization of the nucleocapsid. It distinctly possesses three highly conserved domains; an N-terminal domain, an RNA-binding domain or a linker region, and a C-terminal domain. It has been observed that these three domains may together orchestrate RNA binding, and its phosphorylated status is prerequisite for triggering a structural dynamism facilitating the affinity for viral versus non-viral RNA. N protein participates in RNA packaging in a beads-on-a-string type conformation. In addition to be involved in organization of viral genome, N protein also facilitates virion assembly and enhances virus transcription efficiency amongst others. Owing to considerably high immunogenic nature, N protein may be useful as potential vaccine target. Most importantly, the M, E, and S proteins possess trafficking signal sequence, which enable their translocation to the endoplasmic reticulum (ER).

8Apart from aforementioned structural proteins there are several non-structural proteins, namely NSP1 to NSP 10 and NSP12 to NSP16, encoded by genes located within the 5′-region of viral RNA genome. These non-structural proteins with their corresponding functions along with other associated molecular features are tabulated below (Table 1).

9There are nine accessory proteins—ORF3a, 3d, 6, 7a, 7b, 8, 9b, 14, and 10—produced from at least five ORFs encoding accessory genes (ORF3a, ORF6, ORF7a, ORF7b, and ORF8), novel overlapping ORF3d (earlier known as 3b), leaky scanning of sgRNA of N gene (ORF9b and 14), and ORF10 from downstream of N gene (Figure 1). Aforementioned accessory genes show considerable variability amongst coronavirus group. However, number of exact ORFs is still debated and awaits further experimental verification. These proteins along with abovementioned NSPs play very crucial role in viral replication.

10The accessory factor 3a is encoded by ORF3a located in between the S and E genes, and is the largest accessory proteins of SARS-CoV-2, consisting of 274 amino acid residues. Hydrophobicity analysis and topology studies have revealed that 3a protein is an O-linked glycosylated, possessing three transmembrane domains. ORF3a forms dimer and its six transmembrane helices together create ion channelin the host cell membrane, which is highly conducive for Ca2+/K+ cations compared to Na+ ion. It is also involved in virus release, apoptosis and pathogenesis. Similarly, ORF3d (one amongst youngest genes) encodes 3d protein which consists of 154-aa long polypeptide chain, and is found to be located in the nucleolus and mitochondria. However, the presence of ORF3d in SARS-CoV-2 has also been confirmed.

11SARS-CoV ORF6 protein is a 61-amino acid long membrane-associated protein. The expression of this protein was confirmed in virus-infected Vero E6 cells, and also in the lung and intestine tissues of patients. It is actually placed in the ER and Golgi compartments in expressing cells and virus infected cells.

12ORF7a and ORF7b accessory proteins are synthesized from the bicistronic subgenomic RNA 7 of SARS-CoV-2. The 122-aa ORF7aprotein is a type-I transmembrane protein containing a 15 aa signal peptide sequence, an 81aa luminal domain, 21aa transmembrane domain and a short C-terminal tail.

13The ORF7b protein consists of 44-amino acids, and is an integral membrane protein, expressed in SARS-CoV-infected cells wherein it remains localized in the Golgi compartment. Furthermore, there is production of anti-7b antibody in SARS patient serum, indicative of its expression in infected patients. In addition, 7b protein was found to be closely associated with intracellular virus particles, strengthening the findings related to its expression and importance.

14ORF8, one amongst youngest genes, shows low homology to SARS-CoV due to deletion. This protein consists of 121 amino acid residues, and its shape resemble immunoglobulin (Ig)-like fold owing to β-strand core (18–121 residues). The 1–17 residues comprise N-terminal siganal sequence, prerequisite for transport to ER. ORF8 has been found to interact with major histocompatibility complex-I (MHC-I), thereby mediating their degradation in cell culture, and therefore may help in immune evasion.

15It consists of 97 amino acid residues, and is probably expressed by leaky scanning of sgRNA of N gene. It tends to associate with adaptor protein, TOM70, and therby suppress IFN-I mediated antiviral response. Therefore, developing greater insight into 9b-TOM70 interaction may help in desiging therapeutic molecule.

16Gene encoding ORF10 protein is predicted to be located downstream of the N gene. Although its corresponding sgRNA is rarely detected, however ORF 10 protein has been found in infected cells.

17It is made up of 73 amino acid residues, and is also likely to be synthesized by leaky scanning of sgRNA of N gene. However, its function is not clearly understood.

18Apart from crucial role in virus replication, accessory proteins may also be involved in host immune escape. For instance, during infection of MERS-CoV, accessory ORFs 3–5 inhibit the host’s innate immune response, including perturbation of type I interferon, blockade of NF-κB and RNaseL activation, among others. Study from Li J.Y. et al. has demonstrated the role of ORF6, ORF8, and nucleocapsid protein in inhibiting type I interferon (IFN-β) and NF-κB-responsive promoter, and impede interferon signaling. Following study from Miorin et al. demonstrates the impairment in nuclear translocation of STAT1 and STAT2 leading to inhibition of transcriptional induction of IFN-stimulated genes. Another report has shown the association of SARS-CoV-2 ORF9b to host mitochondrial import receptor subunit (TOM70), and thereby suppresses type I interferon signaling.

19Life cycle of SARS-CoV-2 consists of cellular invasion of virus, expression of viral genes, and formation of progeny and eventual exit. It can roughly be divided into following 5 steps (Figure 2).

20The S protein system is homotrimeric, consisting of three monomeric S polypeptides. Each monomeric polypeptide contains S1 and S2 subunits with multiple functional domains and motifs, which undergo several conformational changes. SARS-CoV-2 via its receptor binding domain (RBD)of S1 subunit (RBD/S1 with down-to-up conformation) of homotrimeric spike glycoprotein binds host cell receptor ACE2 (angiotensin-converting enzyme 2) zinc-binding carboxypeptidase, which is normally involved in cardiac function and blood pressure regulation. ACE2 is primarily expressed by epithelial cells of the lungs and small intestine as well as kidney, heart, and other tissues. In addition, a recent molecular simulation-based work has proposed that SARS-CoV-2′s S protein can also bind to nicotinic acetylcholine receptors (nAChRs), indicative of its diverse binding potential, and may be one of the underlying reasons for multi-organ pathogenesis. Whereas the S2 domain of S protein possesses heptad repeat region and fusion peptide, mediating fusion following conformational rearrangement. Following the binding of S protein of SARS-CoV-2 to the host protein ACE2, the spike protein undergoes protease-mediated cleavage at the S1/S2 cleavage site for priming and a cleavage for activation at the S′2 site, a position adjacent to a fusion peptide within the S2 subunit. The S1/S2 site are also subjected to cleavage by other proteases such as transmembrane protease serine, 2 (TMPRSS2), cathepsin L and/or other proteases. Subsequent cleavage at the S′2 site presumably triggers membrane fusion via irreversible, conformational changes and thereby facilitates access to host cell cytosol. In addition to the involvement in infection and cross-species transmission, the S protein is crucial target for anti-virus neutralizing antibodies, and crucial mutation in it may lead to considerable alteration in pathogenesis.

21After fusion of viral spike glycoprotein with ACE2 there is subtle conformational changes, releasing the viral nucleocapsid into the cell cytosol. This process is aided by several host factors, including type II transmembrane protease serine 2 (TMPRSS2) protease and cathepsin L.

22Immediately after release of viral nucleocapsid, +ssRNA serves as functional mRNA with respect to ORF1a and ORF1b encoding polyprotein pp1a (440–500 kDa) and pp1ab (740–810 kDa), respectively. However, pp1a is 1.2–2.2 folds more expressed compared to pp1ab owing to differential efficiency of frameshift between ORF1a and ORF1b genes. These two polyproteins undergo autoproteolytic processing yielding 16 nsps, which together form the RTC for viral RNA synthesis. This functional RTC results into formation of a nested set of sgRNAs via discontinuous transcription.

23The formation of RTC sets molecular process in motion leading to synthesis of multiple copies of viral RNA. These -ssRNA (negative ssRNA) serves as intermediate template. Meanwhile, polymerase switches template at short motifs, transcription regulated sequences (TRS) during -ssRNA synthesis, thereby producing a multiple 5′-nested set of negative sense sgRNAs which, in turn, used as templates to form a 3′-nested set of positive sense sgRNAs. Thereafter, they associate with host ribosome, synthesizing various structural and accessory proteins building multiple virus structure.

24Most of the structural and accessory proteins associated with membrane such as S, M, and E are synthesized by endoplasmic reticulum-bound ribosomes, whereas other viral proteins, including N protein, are translated by free cytosolic ribosomes of host cells. In addition, these structural proteins also undergo posttranslational modification that modulate their functions. The assembly of virion converges at site of endoplasmic reticulum–Golgi intermediate compartment (ERGIC), wherein M protein provides scaffold and orchestrate virion morphogenesis by heterotypic interaction with other structural proteins, such as M-S and M-E, thereby facilitating molecular incorporation. Furthermore, M-N interactions mediates condensation of the nucleocapsid with the envelope along with E protein. Post molecular assembly, progeny virions are transported in smooth-wall vesicle and using secretory pathway they are trafficked to plasma membrane and eventually exit though exocytosis and spread to other parts of body.

25Currently, SARS-CoV-2 has rapidly spread across world beginning from Wuhan, capital of Central China’s Hubei province, causing a fatal outbreak of acute infectious pneumonia. Till date, considerable success has not been achieved regarding development of specific anti-virus drugs or vaccines for the treatment of this disease despite consistent efforts made by researchers around the world. Nonethless, lots of efforts are being made to drug repurposing of FDA-approved/clinical-trial drugs in order to use them against COVID-19. In this direction, various pharmaceutical companies and government agencies have reportedly succeeded to a certain extent, necessitating further research work to find the cure. There are various potential therapeutic drug molecules, compounds and antibodies under clinical trial, offering considerable benefits are listed in Table 2 below.

26Plasma therapy is being tested and adopted as one of the crucial therapeutic regimes to treat COVID-19 patients. This is based on fact that polyclonal antibodies produced in convalescent persons following infection may help neutralize viruses, and substantially reduces the duration of viremia and hospitalization. During this therapy, isolated anti-SARS-CoV-2 antibodies from recovered patients are administered in patient in order to effectively neutralize the virus through multiple immunological mechanisms. Besides, it could also provide prophylactic immunity prior to the occurrence of viral infection. Furthermore, various epitopes on the spike protein can be targeted using antibody cocktails. This line of supportive treatment has shown quite promising results in preliminary investigations carried out across the world, and therefore, is being scaled up to an unprecedented level. However, it requires further detailed investigation and optimization at multiple levels, including antibody concentration, dose and frequency of administration. Besides, genetic engineering has enabled development of single-domain antibodies (sdAbs), or nanobodies against S and N proteins in order to prevent viral attachment to host cells by blocking ACE2 binding. One of the central hypotheses regrading coronavirus disease is based on the empirical observation that complications and death are the consequences of viral load whose reversal may bring clinical benefits to the patient concerned. Following this hypothesis, a recent study involving RGEN-COV2 (antibody cocktail consisting of two fully human antibody, noncompeting IgG1 against RBD of SARS-CoV-2 S protein) has shown substantial benefit in term of enhanced viral clearance and hence, may be adopted as antiviral therapy, particularly in patient with weakened immune response. Cytokine storm following SARS-CoV-2 infection leads to substantial complications and tissue damage leading to ARDS (Acute Respiratory Distress Syndrome). Therefore, controlling cytokine storm by using therapeutic antibodies, which can target TLR4 (EB05), CXCL10 (EB06), and IL6 (Levilimab) amongst others, may suppress proinflammatory response and hence consequent histological damage in multiple tissues following infection.

27The worldwide endeavor for manufacturing vaccines to tackle SARS-CoV-2 infection has been growing since the beginning of the current pandemic. Vaccine is administered to all age groups of people to help them develop and strengthen both humoral and cell-mediated immunity so that they can fight infection. Until now, there have been the development and authorization/approval of 13 vaccines worldwide, and around 58 vaccines are under various phases of trial (https://www.raps.org/news-and-articles/news-articles/2020/3/covid-19-vaccine-tracker; accessed on 30 March 2021). Inactivated vaccine accounts for 38% of total approved vaccines worldwide (Figure 3). The various aspects of 13 approved vaccines are mentioned in Table 3.

28The SARS-CoV-2 is the causative pathogen for the current pandemic, and is evolving through recombination and mutation into several strains over a period of time. Owing to free geopolitical borders, lack of knowledge regarding its spread, and initial negligence by various stakeholders have led to quick spread of COVID-19, causing millions of deaths and debilitation, as well as huge burden on socio-economic and health system of nations, and territories worldwide. Nevertheless, technological and methodological advances in the field of virology, molecular biology, and pharmacology haves helped us understand the structural and genomic organization as well as mechanism underlying cellular entry, lifecycle, and pathophysiological characteristics of SARS-CoV-2 to some extent. However, there is still need for establishing cellular and animal models for SARS-CoV-2 to develop even greater insights into mechanisms underlying viral replication, pathogenesis, and transmission dynamics. Globally, such studies are being carried out, aiming at the development of therapeutic strategies against the zoonotic coronavirus epidemic. The development of therapeutic strategies could rely upon studying the molecular mechanisms underlying host–pathogen interaction, ever-evolving virus genome through recombination and mutation, host immune response involving innate and adaptive immunity, and so forth. Amongst such therapeutic studies are the development and/or repurposing of various drugs (Table 2), targeting cellular entry and replication to reduce the severity of the disease. In addition, plasm therapy is also being used as a supportive treatment to neutralize the virus and as a prophylactic measure as it involves mixture of antibodies against multiple epitopes on various structural moleculessuch as S and N proteins. Genetically engineered nanobodies and antibodies suppressing cytokine storms have also shown promising results. The worldwide effort has also resulted in the development of various types of vaccines, which are being administered, and promising results have been obtained. Therefore, considering above advancement in the field of etiology, drug, and vaccine development, we may hope for promising outcome in near future.