The SARS-CoV-1 coronavirus is the causative agent of the outbreak of severe acute respiratory syndrome in 2003 that caused 8,098 known cases of the disease and 774 deaths. The molecular events involved in viral infection and the response of the human host to it have since been studied in detail and are annotated here (de Wit et al. 2016; Marra et al. 2003). The SARS-CoV-1 viral infection pathway here uses entries listed in the UniProt "Human SARS coronavirus (SARS-CoV) (Severe acute respiratory syndrome coronavirus)" taxonomy.
SARS-CoV-1 infection begins with the binding of viral S (spike) protein to cell surface angiotensin converting enzyme 2 (ACE2) and endocytosis of the bound virion. Within the endocytic vesicle, host proteases mediate cleavage of S protein into S1 and S2 fragments, leading to S2-mediated fusion of the viral and host endosome membranes and release of the viral capsid into the host cell cytosol. The capsid is uncoated to free the viral genomic RNA, whose cap-dependent translation produces polyprotein pp1a and, by means of a 1-base frameshift, polyprotein pp1ab. Autoproteolytic cleavage of pp1a and pp1ab generates 15 or 16 nonstructural proteins (nsps) with various functions. Importantly, the RNA dependent RNA polymerase (RdRP) activity is encoded in nsp12. Nsp3, 4, and 6 induce rearrangement of the cellular endoplasmic reticulum membrane to form cytosolic double membrane vesicles (DMVs) where the viral replication transcription complex is assembled and anchored. With viral genomic RNA as a template, viral replicase-transcriptase synthesizes a full length negative sense antigenome, which in turn serves as a template for the synthesis of new genomic RNA. The replicase-transcriptase can also switch template during discontinuous transcription of the genome at transcription regulated sequences to produce a nested set of negative-sense subgenomic (sg) RNAs, which are used as templates for the synthesis of positive-sense sgRNAs that are translated to generate viral proteins. Finally, viral particle assembly occurs in the ER Golgi intermediate compartment (ERGIC). Viral M protein provides the scaffold for virion morphogenesis (Fung & Liu 2019; Masters 2006).
View original pathway at Reactome.
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Tan YW, Hong W, Liu DX.; ''Binding of the 5'-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription.''; PubMedEurope PMCScholia
Surjit M, Kumar R, Mishra RN, Reddy MK, Chow VT, Lal SK.; ''The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation.''; PubMedEurope PMCScholia
Hatakeyama S, Matsuoka Y, Ueshiba H, Komatsu N, Itoh K, Shichijo S, Kanai T, Fukushi M, Ishida I, Kirikae T, Sasazuki T, Miyoshi-Akiyama T.; ''Dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (N) and membrane (M) proteins of SARS coronavirus.''; PubMedEurope PMCScholia
Sun H, Luo H, Yu C, Sun T, Chen J, Peng S, Qin J, Shen J, Yang Y, Xie Y, Chen K, Wang Y, Shen X, Jiang H.; ''Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus.''; PubMedEurope PMCScholia
Bhardwaj K, Sun J, Holzenburg A, Guarino LA, Kao CC.; ''RNA recognition and cleavage by the SARS coronavirus endoribonuclease.''; PubMedEurope PMCScholia
Surjit M, Lal SK.; ''The SARS-CoV nucleocapsid protein: a protein with multifarious activities.''; PubMedEurope PMCScholia
Baron SA, Devaux C, Colson P, Raoult D, Rolain JM.; ''Teicoplanin: an alternative drug for the treatment of COVID-19?''; PubMedEurope PMCScholia
Ricagno S, Egloff MP, Ulferts R, Coutard B, Nurizzo D, Campanacci V, Cambillau C, Ziebuhr J, Canard B.; ''Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family.''; PubMedEurope PMCScholia
Fung TS, Liu DX.; ''Human Coronavirus: Host-Pathogen Interaction.''; PubMedEurope PMCScholia
Toots M, Yoon JJ, Cox RM, Hart M, Sticher ZM, Makhsous N, Plesker R, Barrena AH, Reddy PG, Mitchell DG, Shean RC, Bluemling GR, Kolykhalov AA, Greninger AL, Natchus MG, Painter GR, Plemper RK.; ''Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia.''; PubMedEurope PMCScholia
Li F, Li W, Farzan M, Harrison SC.; ''Structure of SARS coronavirus spike receptor-binding domain complexed with receptor.''; PubMedEurope PMCScholia
Chen S, Jonas F, Shen C, Hilgenfeld R.; ''Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode.''; PubMedEurope PMCScholia
McBride CE, Machamer CE.; ''A single tyrosine in the severe acute respiratory syndrome coronavirus membrane protein cytoplasmic tail is important for efficient interaction with spike protein.''; PubMedEurope PMCScholia
Ujike M, Huang C, Shirato K, Matsuyama S, Makino S, Taguchi F.; ''Two palmitylated cysteine residues of the severe acute respiratory syndrome coronavirus spike (S) protein are critical for S incorporation into virus-like particles, but not for M-S co-localization.''; PubMedEurope PMCScholia
Chen JY, Chen WN, Poon KM, Zheng BJ, Lin X, Wang YX, Wen YM.; ''Interaction between SARS-CoV helicase and a multifunctional cellular protein (Ddx5) revealed by yeast and mammalian cell two-hybrid systems.''; PubMedEurope PMCScholia
Bernini A, Spiga O, Venditti V, Prischi F, Bracci L, Huang J, Tanner JA, Niccolai N.; ''Tertiary structure prediction of SARS coronavirus helicase.''; PubMedEurope PMCScholia
Huang C, Ito N, Tseng CT, Makino S.; ''Severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein.''; PubMedEurope PMCScholia
Liao Y, Lescar J, Tam JP, Liu DX.; ''Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability.''; PubMedEurope PMCScholia
Chang CK, Sue SC, Yu TH, Hsieh CM, Tsai CK, Chiang YC, Lee SJ, Hsiao HH, Wu WJ, Chang WL, Lin CH, Huang TH.; ''Modular organization of SARS coronavirus nucleocapsid protein.''; PubMedEurope PMCScholia
Song HC, Seo MY, Stadler K, Yoo BJ, Choo QL, Coates SR, Uematsu Y, Harada T, Greer CE, Polo JM, Pileri P, Eickmann M, Rappuoli R, Abrignani S, Houghton M, Han JH.; ''Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein.''; PubMedEurope PMCScholia
Yount B, Curtis KM, Fritz EA, Hensley LE, Jahrling PB, Prentice E, Denison MR, Geisbert TW, Baric RS.; ''Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus.''; PubMedEurope PMCScholia
Wu CH, Yeh SH, Tsay YG, Shieh YH, Kao CL, Chen YS, Wang SH, Kuo TJ, Chen DS, Chen PJ.; ''Glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication.''; PubMedEurope PMCScholia
Nieto-Torres JL, Dediego ML, Alvarez E, Jiménez-Guardeño JM, Regla-Nava JA, Llorente M, Kremer L, Shuo S, Enjuanes L.; ''Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein.''; PubMedEurope PMCScholia
Pan J, Peng X, Gao Y, Li Z, Lu X, Chen Y, Ishaq M, Liu D, Dediego ML, Enjuanes L, Guo D.; ''Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication.''; PubMedEurope PMCScholia
Tanner JA, Watt RM, Chai YB, Lu LY, Lin MC, Peiris JS, Poon LL, Kung HF, Huang JD.; ''The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases.''; PubMedEurope PMCScholia
Tohge T, Nishiyama Y, Hirai MY, Yano M, Nakajima J, Awazuhara M, Inoue E, Takahashi H, Goodenowe DB, Kitayama M, Noji M, Yamazaki M, Saito K.; ''Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor.''; PubMedEurope PMCScholia
Kumar S, Dare L, Vasko-Moser JA, James IE, Blake SM, Rickard DJ, Hwang SM, Tomaszek T, Yamashita DS, Marquis RW, Oh H, Jeong JU, Veber DF, Gowen M, Lark MW, Stroup G.; ''A highly potent inhibitor of cathepsin K (relacatib) reduces biomarkers of bone resorption both in vitro and in an acute model of elevated bone turnover in vivo in monkeys.''; PubMedEurope PMCScholia
Yu X, Chen S, Hou P, Wang M, Chen Y, Guo D.; ''VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability.''; PubMedEurope PMCScholia
Han DP, Lohani M, Cho MW.; ''Specific asparagine-linked glycosylation sites are critical for DC-SIGN- and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry.''; PubMedEurope PMCScholia
Vincent MJ, Bergeron E, Benjannet S, Erickson BR, Rollin PE, Ksiazek TG, Seidah NG, Nichol ST.; ''Chloroquine is a potent inhibitor of SARS coronavirus infection and spread.''; PubMedEurope PMCScholia
Ying W, Hao Y, Zhang Y, Peng W, Qin E, Cai Y, Wei K, Wang J, Chang G, Sun W, Dai S, Li X, Zhu Y, Li J, Wu S, Guo L, Dai J, Wang J, Wan P, Chen T, Du C, Li D, Wan J, Kuai X, Li W, Shi R, Wei H, Cao C, Yu M, Liu H, Dong F, Wang D, Zhang X, Qian X, Zhu Q, He F.; ''Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus.''; PubMedEurope PMCScholia
Krokhin O, Li Y, Andonov A, Feldmann H, Flick R, Jones S, Stroeher U, Bastien N, Dasuri KV, Cheng K, Simonsen JN, Perreault H, Wilkins J, Ens W, Plummer F, Standing KG.; ''Mass spectrometric characterization of proteins from the SARS virus: a preliminary report.''; PubMedEurope PMCScholia
Chen Y, Cai H, Pan J, Xiang N, Tien P, Ahola T, Guo D.; ''Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase.''; PubMedEurope PMCScholia
Eckerle LD, Becker MM, Halpin RA, Li K, Venter E, Lu X, Scherbakova S, Graham RL, Baric RS, Stockwell TB, Spiro DJ, Denison MR.; ''Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.''; PubMedEurope PMCScholia
Saikatendu KS, Joseph JS, Subramanian V, Neuman BW, Buchmeier MJ, Stevens RC, Kuhn P.; ''Ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the N-terminal domain of N protein.''; PubMedEurope PMCScholia
Agostini ML, Andres EL, Sims AC, Graham RL, Sheahan TP, Lu X, Smith EC, Case JB, Feng JY, Jordan R, Ray AS, Cihlar T, Siegel D, Mackman RL, Clarke MO, Baric RS, Denison MR.; ''Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease.''; PubMedEurope PMCScholia
Hsu MF, Kuo CJ, Chang KT, Chang HC, Chou CC, Ko TP, Shr HL, Chang GG, Wang AH, Liang PH.; ''Mechanism of the maturation process of SARS-CoV 3CL protease.''; PubMedEurope PMCScholia
Angelini MM, Akhlaghpour M, Neuman BW, Buchmeier MJ.; ''Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles.''; PubMedEurope PMCScholia
Al-Bari MA.; ''Chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases.''; PubMedEurope PMCScholia
McBride CE, Li J, Machamer CE.; ''The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein.''; PubMedEurope PMCScholia
McBride CE, Machamer CE.; ''Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein.''; PubMedEurope PMCScholia
Fehr AR, Singh SA, Kerr CM, Mukai S, Higashi H, Aikawa M.; ''The impact of PARPs and ADP-ribosylation on inflammation and host-pathogen interactions.''; PubMedEurope PMCScholia
Chakraborti S, Prabakaran P, Xiao X, Dimitrov DS.; ''The SARS coronavirus S glycoprotein receptor binding domain: fine mapping and functional characterization.''; PubMedEurope PMCScholia
Chen P, Jiang M, Hu T, Liu Q, Chen XS, Guo D.; ''Biochemical characterization of exoribonuclease encoded by SARS coronavirus.''; PubMedEurope PMCScholia
Ritchie G, Harvey DJ, Feldmann F, Stroeher U, Feldmann H, Royle L, Dwek RA, Rudd PM.; ''Identification of N-linked carbohydrates from severe acute respiratory syndrome (SARS) spike glycoprotein.''; PubMedEurope PMCScholia
The PIK3C3-containing Beclin-1 complex consists of PIK3C3 (Vps34), BECN1 (Beclin-1, Atg6), PIK3R4 (p150, Vps15) and ATG14 (Barkor) (Matsunaga et al. 2009, Zhong et al. 2009). A similar complex where ATG14 is replaced by UVRAG functions later in autophagosome maturation and endocytic traffic (Itakura et al. 2008, Liang et al. 2008). Binding of KIAA0226 to this complex negatively regulates the maturation process (Matsunaga et al. 2009).
SARS-CoV-1 spike (S3a) protein, as a component of the S3:M:E:encapsidated SARS coronavirus genomic RNA: 7a:O-glycosyl 3a tetramer complex, binds to glycosylated angiotensin converting enzyme 2 (ACE2) associated with the human host cell plasma membrane. Structural studies of the interaction between human ACE2 protein and the receptor-binding domain of S3a protein have identified key amino acid residues in both proteins responsible for their high-affinity interaction. These residues may be a key factor determining severity (and possibly human-to-human transmission) of SARS-CoV-1 (Li et al. 2003, 2005). The roles of S protein in viral binding to the host cell membrane and fusion of viral and host cell membranes and thus the central role of S protein in determining the host range and tissue tropisms of the virus are reviewed by Belouzard et al. (2012).
Remdesivir (GS-5734) is an investigational nucleotide analogue drug that was developed for its broad spectrum antiviral potential against Ebola and Marburg virus activity (Siegel et al. 2017). It targets and inhibitis viral RNA-dependent RNA polymerase (nsp12, RdRP), the key component of the replication transcription complex (RTC) (Agostini et al. 2018, Brown et al. 2019, Gordon et al. 2020). Remdesivir is being investigated for potential antiviral activity against SARS-CoV-2 by targeting viral replication (Agostini et al. 2018). Gordon et al. demonstrate remdesivir possesses broad antiviral activity against RNA viruses, including SARS-CoV, MERS-CoV and SARS-CoV-2 in-vitro (Gordon et al. 2020b). It could prevent asymptomatic, mild or moderate COVID-19 cases from progressing to severe disease (clinical trials NCT04252664, NCT04257656) but results so far in infected people have been mixed.
EIDD-2801, is an isopropylester prodrug of the ribonucleoside analogue N4-hydroxycytidine (NHC, EIDD-1931) that shows broad spectrum antiviral activity against various RNA viruses including Ebola, Influenza and CoV (Toots et al. 2019). NHC acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. NHC was shown to inhibit multiple genetically-distinct Bat-CoV viruses in human primary epithelial cells without affecting cell viability. Prophylactic/therapeutic oral administration of NHC reduced lung titers and prevented acute lung failure in C57B/6 mice infected with CoV. The potency of NHC against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses (Sheahan et al. 2020).
The nsp7:nsp8 heterodimer binds to the RNA-directed RNA polymerase (nsp12) of the human SARS coronavirus on the polymerase thumb domain facing the NTP entry channel. Binding in this position sandwiches the RNA polymerase index finger loop between nsp7:nsp8 and the polymerase thumbdomain. The nsp7:nsp8 heterodimer may facilitate the interaction of the viral RNA polymerase with additional components of the RNA synthesis machinery. The second subunit of nsp8 interacts with the viral RNA polymerase interface domain proximal to the finger domain and the RNA template-binding channel, and is not bound to nsp7 (Kirchdoerfer and Ward 2019). This is consistent with the stoichiometry of the complex between feline coronavirus proteins nsp7, nsp8 and nsp12 (Xiao et al. 2012).
Human SARS coronavirus nonstructural proteins nsp7 and nsp8 form a heterodimer (Kirchdoerfer and Ward 2019). The nsp7:nsp8 complex may function as a hexadecamer, composed of 8 subunits of nsp7 and 8 subunits of nsp8 (Zhai et al. 2005).
The replication transcription complex (RTC) binds to the 3' end of the viral plus strand genomic RNA to initiate synthesis of the complementary minus strand. A 36 nucleotide sequence from the 3’-UTR of the plus strand, predicted to form a stable stem-loop structure, seems to be the minimal cis-acting RNA element required for the viral RNA-directed RNA polymerase (nsp12) to initiate RNA synthesis. The polyA tail also seems to play a role in the initiation of replication of viral genomic RNA (Ahn et al. 2012).
Entry of influenza, parainfluenza and coronaviruses into airway epithelial cells requires binding of a viral spike protein to a host cell receptor, followed by cleavage and activation of the viral spike protein mediated by the host cell. Without this cleavage, fusion of the viral and host cell membranes is blocked. The primary receptor for the human SARS-CoV-1 virus is angiotensin converting enzyme 2 (ACE2) (Li et al. 2003). The resultant complex is cleaved by the protease transmembrane protease serine 2 (TMPRSS2) (Shulla et al. 2011, Heurich et al. 2014). Therefore, active site inhibitors of these airway proteases could have broad therapeutic applicability against multiple respiratory viruses (Laporte & Naesens 2017). The approved drug camostat is a protease inhibitor that may block SARS-CoV-2 entry into cells by inhibiting the actions of TMPRSS2 (Kawase et al. 2012, Hoffmann et al. 2020). Nafamostat, another serine protease inhibitor, was found to be a potent inhibitor of S-mediated membrane fusion and blocked MERS-CoV infection in vitro (Yamamoto et al. 2016).
Otamixaban (FXV673), an anticoagulant, is a potent and selective direct inhibitor of coagulation factor Xa. Virtual docking studies suggest that otamixaban may bind to the serine protease TMPRSS2 (Rensi et al. 2020, preprint). Inhibition of TMPRSS2 is being examined for antiviral activity but its inhibitory potential and/or antiviral activity have not yet been determined so it is annotated here as a candidate drug. I-432 is another inhibitor of TMPRSS2 under investigation for anti viral potential (Pászti-Gere et al. 2016).
The rep proteases that are essential for viral polyprotein processing by the coronaviruses and enteroviruses exhibit a strong preference for substrates containing Gln at P1 position, and share an active-site conformation that engages the substrate's P1 residue. Compound 11r and compound 13b are peptidomimetic α-ketoamides that function as high-affinity non-cleavable substrate analogues and thus exhibit antiviral activity against dimeric 3C-like proteinases (C3Lp dimer) of coronaviruses and enteroviruses (Chen et al. 2005, Zhang et al. 2020). Their clinical safety and efficacy in COVID-19 are under investigation.
Human glycogen synthatse kinases GSK3A and GSK3B phosphorylate SARS-CoV-1 coronavirus N (nucleocapsid) protein on multiple serine and threonine residues. GSK3-mediated phosphorylation of the N protein is needed for efficient replication of viral genomic RNA (Wu et al. 2009).
nsp8 functions as an RNA-dependent RNA polymerase (RdRp) that serves as the primase for nsp12, the main RdRp of the SARS coronavirus 1 (SARS-CoV-1) (Imbert et al. 2006), as it is capable of de novo RNA synthesis (te Velthuis et al. 2011). nsp8 synthesizes short oligonucleotides (less than 6 bases long) using genomic RNA as a template. nsp8 requires at least one cytidine residue in the template sequence for its activity. Activity is dependent on manganese ions (Imbert et al. 2006). nsp8 can also extend primers but is 20-fold less efficient than nsp12 (te Velthuis et al. 2011).
Virally encoded RNA-dependent RNA polymerase (nsp12, also known as RdRP) is the key component of the replication transcription complex (RTC). As the human SARS coronavirus 1 (SARS-CoV-1) is a plus strand RNA virus, nsp12 first synthesizes the complementary minus RNA strand. The purified SARS-CoV-1 nsp12 shows both primer dependent and primer-independent RNA synthesis activities using homopolymeric RNA templates. The catalytic activity of nsp12 is strictly dependent on manganese ions (Mn2+) and primers when the template is a viral-genome-derived RNA representing part of the 3’-UTR of the plus strand with a polyA tail. A 36 nucleotide sequence from the 3’-UTR, predicted to form a stable stem-loop structure, seems to be the minimal cis-acting RNA element required for nsp12 to initiate RNA synthesis (Ahn et al. 2012). The complex of nsp7 and nsp8 confers processivity to nsp12 (Subissi et al. 2014).
After synthesizing the complementary minus RNA of the plus strand viral genomic RNA, virally encoded RNA-dependent RNA polymerase (nsp12, also known as RdRP) uses the minus strand as a template to generate viral genomic RNA that can be packaged into virions. Purified SARS-CoV-1 nsp12 shows both primer dependent and primer-independent RNA synthesis activity in vitro. nsp12 is able to initiate RNA synthesis with as little as 37 nucleotides of RNA from the 3’ end of the minus strand viral RNA (complementary to the 5’-UTR of the plus strand genomic RNA - c5’-UTR). Similar to the 3'-UTR of the plus strand, the 3' end of the minus strand (c5’-UTR) is predicted to form a stable stem-loop structure and seems to be the minimal cis-acting RNA element required for nsp12 to initiate RNA synthesis using the minus strand as a template (Ahn et al. 2012). It is unclear if replication of the minus strand is primer-dependent. The complex of nsp7 and nsp8 confers processivity to nsp12 (Subissi et al. 2014).
Human zinc finger CCHC-type and RNA-binding motif-containing protein 1 (ZCRB1, also known as MADP1) binds to the 5'UTR of the plus strand genomic RNA of the SARS-CoV-1, as well as other coronaviruses, infectious bronchitis virus (IBV) and human coronavirus OC43. ZCRB1 normally localizes to the nucleus, where it is a component of the U12-type spliceosome. Upon infection with a coronavirus, ZCRB1 appears in the cytosol. Binding of ZCRB1 to the 5'UTR stem of coronavirus genomic RNA is thought to be necessary for efficient transcription of viral genes (Tan et al. 2012).
Non-structural protein 14 (nsp14) of the human SARS coronavirus is a bifunctional enzyme bearing 3'-5 exoribonuclease activity involved in replication fidelity and RNA cap N7-guanine methyltransferase activity involved in 5'-RNA capping. nsp14 binds to the minimal replication and transcription complex (RTC), composed of nsp7, nsp8, and nsp12, by directly binding to nsp12 (the main RNA-dependent RNA polymerase). Binding of nsp14 does not affect the processivity of the RTC (Minskaia et al. 2006, Subissi et al. 2014).
The replication-transcription complex (RTC) completes synthesis of the genomic RNA complement (minus strand). The complex of nsp7 and nsp8 confers processivity to nsp12, the virally encoded RNA-dependent RNA polymerase that replicates the viral genomic RNA, enabling the RTC to complete the RNA synthesis with a very low dissociation rate. nsp7 plays a crucial role in maintaining binding of the RTC to the RNA. nsp14 subunit of the RTC does not affect the processivity (Subissi et al. 2014).
nsp10 forms a stable complex with nsp14 (Bouvet et al. 2012) and serves as a co-factor for nsp14, stimulating its 3'->5' exonuclease activity (Bouvet et al. 2012, Subissi et al. 2014, Bouvet et al. 2014).
In the presence of functional nsp14, which acts as a 3'-to-5' exonuclease, the mutation rate during human SARS coronavirus 1 (SARS-CoV-1) replication is 9 x 10^-7 (9E-7) per nucleotide per replication cycle or 2.2 x 10^-5 (2.2E-5) non-redundant substitutions per nucleotide, which translates into 2-3 nucleotide substitutions for each replicated SARS-CoV-1 genome. When nsp14 is defective, the mutation rate during SARS-CoV-1 replication increases to 1.2 x 10^-5 (1.2E-5) mutations per nucleotide per replication cycle or 3.34 x 10^-4 (3.34E-4) non-redundant substitutions per nucleotide, which translates into 12-23 nucleotide substitutions for each replicated SARS-CoV-1 genome (Eckerle et al. 2010). Here the process is annotated in two steps, nsp12- mediated misincorporation of a base (this reaction) and nsp14-mediated detection and removal of that base (next reaction).
nsp14 acts as 3'-5' exonuclease (Minskaia et al. 2006, Chen et al. 2007) that preferentially excises mismatched nucleotides from double stranded RNA (Minskaia et al. 2006, Bouvet et al. 2012). Binding to nsp10 increases the exonuclease activity of nsp14 (Bouvet et al. 2012, Subissi et al. 2014, Bouvet et al. 2014). nsp14 increases the fidelity of human SARS coronavirus 1 (SARS-CoV-1) replication by the nsp12 RNA-dependent RNA polymerase by 21-fold (Eckerle et al. 2010).
nsp13, which functions as the viral helicase, is as a part of the human SARS coronavirus 1 (SARS-CoV-1) replication-transcription complex (RTC), where it is directly bound to nsp12, the viral RNA-dependent RNA polymerase. nsp12 increases the helicase activity of nsp13 (von Brunn et al. 2007, Adedeji et al. 2012, Jia et al. 2019).
nsp13, the helicase of the human SARS coronavirus 1 (SARS-CoV-1) binds to DDX5, a host protein implicated in transcription, pre-mRNA processing, RNA degradation, RNA export, ribosome assembly and translation. DDX5 knockdown inhibits viral replication (Chen et al. 2009).
nsp13 is an ATP-dependent human SARS coronavirus 1 (SARS-CoV-1) helicase that functions in the 5'-3' direction to unwind double stranded RNAs that have a 5' single strand overhang at least 20 nucleotides long. nsp13 can also act on double strand DNA in vitro, but dsRNA is thought to be its physiological substrate. The catalytic activity of nsp13 is increased in the presence of nsp12, the viral RNA-dependent RNA polymerase. nsp13 is needed for the replication of SARS-CoV-1 and is thought to act by melting secondary structures in the genomic RNA template during replication, and also to be involved in unwinding of RNA duplexes during transcription of viral genes. nsp13 is a promising target for experimental anti-SARS-CoV-1 drugs (Tanner et al. 2003, Ivanov et al. 2004, Bernini et al. 2006, Chen et al. 2009, Lee et al. 2010, Adedeji et al. 2012).
Human SARS coronavirus 1 (SARS-CoV-1) non-structural protein 15 (nsp15) contains the LXCXE/D motif characteristic of proteins that bind to the retinoblastoma protein RB1. Binding to human RB1 increases the endonuclease activity of nsp15 but is not required for it. RB1 bound to nsp15 is retained in the cytosol. Interaction of nsp15 with RB1 likely affects the cell cycle of infected cells and probably modulates cytotoxicity of SARS-CoV-1 (Bhardwaj et al. 2012).
nsp15 forms a hexamer. Hexamer formation is necessary for the endonuclease activity of nsp15. nsp15 preferentially cleaves 3' of uridines, generating 2'-3' cyclic phosphates after cleavage. nsp15 requires Mn2+ ions for catalytic activity. C-terminal domain contains the active site, which faces away from the center of the hexamer and contains the extreme C-terminal residues. The middle and the N-terminal domains form extensive contacts with the other subunits of the hexamer. While the hexamer is likely formed by two asymmetric trimers, a trimer is not a stable intermediate. The catalytic pocket of nsp15 resembles the catalytic pocket of RNase A and their mechanism of endoribonuclease action is likely the same. Functional nsp15 is needed for production of viable virions. nsp15 is a genetic marker of the order Nidovirales, which includes the family Coronaviridae, as it is not present in other RNA viruses (Guarino et al. 2005, Ricagno et al. 2006, Bhardwaj et al. 2006, Joseph et al. 2007, Bhardwaj et al. 2008, Bhardwaj et al. 2012).
Nonstructural protein 15 (nsp15) of the SARS coronavirus (SARS-CoV-1) binds to the replication-transcription complex (RTC) through interaction with nsp8 (Imbert et al. 2008). This interaction appears to be conserved in other coronaviruses, such as mouse hepatitis virus (MHV) (Athmer et al. 2017). nsp15 is an endonuclease characteristic for the order Nidovirales that includes the family Coronaviridae. nsp15 preferentially cleaves 3' of uridines, generating 2'-3' cyclic phosphates after cleavage. nsp15 requires Mn2+ ions for catalytic activity. Functional nsp15 is needed for production of viable virions and for viral transcription (Guarino et al. 2005, Ricagno et al. 2006, Bhardwaj et al. 2006, Joseph et al. 2007, Bhardwaj et al. 2008, Bhardwaj et al. 2012). The biological role of nsp15 has not been elucidated. It may degrade host mRNAs to shut down host translation, but so far no human or viral RNA targets have been identified.
The non-structural protein 16 (nsp16) of the human SARS coronavirus is an AdoMet-dependent (nucleoside-2'O)-methyltransferase involved in capping of viral RNAs. nsp16 binds to nsp10, which serves as a cofactor for nsp16 (Bouvet et al. 2010, Lugari et al. 2010). Nsp16 alone is unstable and exhibits 2'-O-methyltransferase activity only in complex with nsp10 (Debarnot et al, 2011; Decroly et al, 2011). nsp10-mediated activation of nsp16 catalytic activity is conserved in all coronaviruses (Wang et al. 2015). The same binding surface of nsp10 interacts with nsp14 and nsp16, suggesting that binding of nsp14 and nsp16 to nsp10 is mutually exclusive. However, as nsp10 is produced in a higher number of copies than nsp14 and nsp16, and as nsp14 and nsp16 act coordinately in RNA capping, it is most likely that nsp14:nsp10 and nsp16:nsp10 complexes co-exist within the viral replication-transcription complex (RTC) (Bouvet et al. 2012, Bouvet et al. 2014). One structural study reported that nsp10 forms dodecamers (Su et al. 2006), which would potentially allow simultaneous binding of nsp14 and nsp16 to nsp10 homomeric complexes, but it is not certain if such homomeric complexes of nsp10 exist in vivo, and if the structure of the nsp10 dodecamer would be permissive for nsp16 binding (Chen et al. 2011). nsp10 contains two zinc fingers which are thought to be involved in RNA binding (Su et al. 2006, Joseph et al. 2006).
Human von Hippel Lindau (VHL) protein, a tumor suppressor that acts as a component of an E3 ubiquitin ligase complex, interacts with the non-structural protein 16 (nsp16) of the human SARS coronavirus 1 (SARS-CoV-1) and the mouse hepatitis virus, also a coronavirus. VHL negatively regulates SARS-CoV-1 replication, but the exact mechanism is not known (Yu et al. 2015).
Chloroquine (CQ) and hydroxychloroquine (HCQ) are diprotic weak bases that can exist in both protonated and unprotonated forms. Unprotonated CQ or HCQ can diffuse freely and rapidly across the membranes of cells and organelles to acidic cytoplasmic vesicles (late endosomes and lysosomes). Agents that have this ability are known as lysosomotropic agents. Once protonated, CQ2+ or HCQ2+ are trapped in the acidic lumen of these vesicles. This leads to an irreversible accumulation of CQ or HCQ in acidic vesicles to concentrations as much as 100 fold over cytosolic ones and to an elevation of vesicle pH due to trapping of H+ ions by CQ or HCQ. Thus, CQ analogues interfere with endosomal and lysosomal acidification, which in turn inhibits proteolysis, chemotaxis, phagocytosis and antigen presentation. As a result, cells are not able to proceed with endocytosis, exosome release and phagolysosomal fusion in an orderly manner (Foley & Tilley 1998, Yang & Shen 2020). In vitro, these endosomal acidification fusion inhibitors block cellular infection by a clinical isolate of SARS-CoV-2 (Wang et al. 2020, Hu et al. 2020).
Unprotonated chloroquine (CQ) and hydroxychloroquine (HCQ) can both diffuse freely and rapidly across the membranes of cells and organelles (Chinappi et al. 2010).
There is a mixed population of mRNA3 clones having six, seven, eight and nine T stretches located 14 nt downstream of the initiation codon that occur in vivo. The transcribed Us practically act as slippery sequences in heterogenous ORFs. Translation efficiency is reduced in slipping clones, however (Thiel et al, 2003; Tan et al, 2005; Wang et al, 2006).
Nucleoprotein is capable of homodimerization in a mammalian cellular environment. It may also oligomerise transiently which is a prerequisite to forming the capsid of SARS-CoV (Surjit et al, 2004; Li et al, 2005; Chang et al, 2013).
Both a predicted beta-hairpin motif and the N-terminal part of SARS-Cov protein E are sufficient for its localization to the Golgi membrane. Although porin activity has been shown for protein E it cannot be detected in the plasma membrane of infected cells (Liao et al, 2006; Cohen et al, 2011; Nieto-Torres, 2011).
In the cis- to medial Golgis, conversion of high-mannose to complex type N-glycans side chains of Spike occurs. The N-acetylglucosaminyltransferase called GlcNAc-TI (MGAT1) adds a GlcNAc residue in the core of some high-mannose chains (Ritchie et al, 2010; Nal et al, 2005, Song et al, 2004).
Two of the four cysteine-rich clusters of the SARS-CoV-1 spike protein are modified by palmitoylation. This is required for the protein's partitioning into detergent-resistant membranes and for cell–cell fusion. In general, palmitoylation is usually non-enzymatic (Petit et al, 2007; McBride and Machamer, 2010; Veit, 2012).
N-glycan side chains on the nascent SARS-CoV-1 spike protein get their terminal glucose moieties cleaved by ER glucosidases I and II, before folding. Iminosugars inhibit this process and are good candidates for broad-spectrum anivirals (Zhao et al, 2015).
The majority of nucleoprotein is serine-phosphorylated in the cytosol and, possibly, in the nucleus where it gets immediately transported to the cytosol. Phosphorylation is catalyzed by glycogen synthase kinase 3 (GSK3) and several other host cell kinases (Surjit et al, 2005; Wu et al. 2009).
A minor proportion of the SARS-CoV E protein is modified by N-linked glycosylation at the N66 residue. This variant appears to be more likely to form multimers, and it shows a different membrane topology than the main variant (Yuan et al, 2006).
Lysine-62 is the major sumoylation site of N protein. Abolition of sumoylation of nucleoprotein significantly decreases homo-oligomerisation of the protein (Li et al, 2005)
SARS-CoV E protein is modified by palmitoylation at all three cysteine residues. In general, palmitoylation is usually non-enzymatic (Liao et al, 2006, Veit, 2012).
SARS-CoV-1 mRNA9a has a length of 1269 nt and encodes the 422 aa pre-nucleoprotein, the most abundant viral protein expressed during infection. Nucleoprotein is translated by cytosolic free ribosomes and most of it stays in the cytosol where it soon colocalizes with nsp3 and viral genomic RNA. It is involved in replication and transcription of the viral genome, but it is also a structural component of the virion (Thiel et al, 2003; Li et al, 2005; Stertz et al, 2007; Fung & Liu, 2019).
Protein 3a can form homodimers and tetramers. The homotetramer shows typical patterns of ion channels. Transport of potassium ions through this channel is effective (Lu et al, 2006). Potassium efflux by protein 3a is important for 3a-induced NLRP3 inflammasome activation (Chen et al, 2019).
Protein M is exclusively N-glycosylated at asparagine 4 by an unknown glycosyltransferase. However, further processing of N-linked glycans is prevented in SARS-CoV-infected cells. Both the glycosylated and nonglycosylated M is incorporated into the virion. In summary, glycosylation of M is neither a prerequisite for intra-cellular transport nor for recruitment into the virion (Voss et al, 2006).
Glycosyltransferases in the endoplasmatic reticulum are responsible for the attachment of numerous high-mannose N-glycans on the SARS-CoV-1 spike protein. After virion assembly and release these glycosidations are required for fusion with host cells (Krokhin et al, 2003; Nal et al, 2005; Ritchie et al, 2010).
A sialyltransferase adds a terminal sialic acid moiety to protein 3a with an O-linked glycosyl side chain. This glycosylated form later is associated with the virion (Oostra et al, 2006).
As early as 3 hours post-infection, cytoplasmic accumulations of N are formed in infected cells, they colocalize with viral RNA. From 5 hours post-infection on, N can be detected in the Golgi, the budding site (Stertz et al, 2007).
A sialyltransferase adds a terminal sialic acid moiety to protein 3a with an O-linked glycosyl side chain. This glycosylated form later is associated with the virion (Oostra et al, 2006).
Calnexin transiently binds the unfolded spike protein and prevents its aggregation and premature degradation, ensuring its correct folding (Fukushi et al, 2012)
The genomic and subgenomic (sg) mRNAs of SARS-CoV-1 coronavirus are presumed to be capped at their 5′ end, based on studies of the mouse hepatitis virus (MHV) (Lai and Stohlman 1981) and the equine torovirus (van Vliet et al. 2002). Non-structural protein 14 (nsp14) acts as an RNA guanine-N7-methyltransferase (N7-MTase) that completes the synthesis of the cap-0 on SARS-CoV-1 mRNAs. The cap-0 represents N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage, and is also known as m7G cap or m7Gppp cap. The N7-MTase domain maps to the carboxy-terminal part of nsp14 (Chen et al. 2009). Cap-0 formation requires three sequential reactions catalyzed by RNA triphosphatase (TPase), guanylyltransferase (GTase), and N7-MTase. There is no evidence that nsp14 possesses TPase and GTase activities, and no other SARS-CoV-1 proteins with these activities have been identified, so the identities of the enzymes that mediate these required steps remain unknown. Based on the study of the human coronavirus 229E, non-structural protein 13 (nsp13) may have a TPase activity in addition to its established helicase activity (Ivanov and Ziebuhr 2004).
The genomic and subgenomic mRNAs of SARS-CoV-1 coronavirus, including the plus strand genomic RNA, are presumed to be capped at their 5′ end, based on studies of the mouse hepatitis virus (MHV) (Lai and Stohlman 1981) and the equine torovirus (van Vliet et al. 2002). Non-structural protein 14 (nsp14) acts as an RNA guanine-N7-methyltransferase (N7-MTase) that completes the synthesis of the cap-0 on the SARS-CoV-1 plus strand genomic RNA. Cap-0 represents N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage, and is also known as m7G cap or m7Gppp cap. The N7-MTase domain maps to the carboxy-terminal part of nsp14 (Chen et al. 2009). Cap-0 formation requires three sequential reactions catalyzed by RNA triphosphatase (TPase), guanylyltransferase (GTase), and N7-MTase. There is no evidence that nsp14 possesses TPase and GTase activities, and no other SARS-CoV-1 proteins with these activities have been identified, so the identities of the enzymes that mediate these required steps remain unknown. Based on the study of the human coronavirus 229E, non-structural protein 13 (nsp13) may have a TPase activity in addition to its established helicase activity (Ivanov and Ziebuhr 2004).
The genomic and subgenomic mRNAs of SARS-CoV-1 coronavirus, including the minus strand genomic RNA complement, are presumed to be capped at their 5′ end, based on studies of the mouse hepatitis virus (MHV) (Lai and Stohlman 1981) and the equine torovirus (van Vliet et al. 2002). The non-structural protein 14 (nsp14) acts as an RNA guanine-N7-methyltransferase (N7-MTase) that completes the synthesis of the cap-0 on SARS-CoV-1 minus strand genomic RNA. The cap-0 represents N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage, and is also known as m7G cap or m7Gppp cap. The N7-MTase domain maps to the carboxy-terminal part of nsp14 (Chen et al. 2009). Cap-0 formation requires three sequential reactions catalyzed by RNA triphosphatase (TPase), guanylyltransferase (GTase), and N7-MTase. There is no evidence that nsp14 possesses TPase and GTase activities, and no other SARS-CoV-1 proteins with these activities have been identified, so the identities of the enzymes that mediate these required steps remain unknown. Based on the study of the human coronavirus 229E, non-structural protein 13 (nsp13) may have a TPase activity in addition to its established helicase activity (Ivanov and Ziebuhr 2004).
The genomic and subgenomic mRNAs of SARS-CoV-1 coronavirus, including the minus strand genomic RNA, are presumed to be capped at their 5′ end, based on studies of the mouse hepatitis virus (MHV) (Lai and Stohlman 1981) and the equine torovirus (van Vliet et al. 2002). Non-structural protein 16 (nsp16) acts as a 2'O-methyltransferase that converts coronavirus cap-0 to cap-1, which was first demonstrated with nsp16 cloned from the feline coronavirus (FCV) (Decroly et al. 2008). Cap-0 represents N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage (also known as m7G cap or m7Gppp cap). Cap-1 is generated by an additional methylation on the 2′O position of the initiating nucleotide, and is also known as m7GpppNm. Non-structural protein 10 (nsp10) acts as an activator of nsp16 and is necessary for cap-1 synthesis (Bouvet et al. 2010, Decroly et al. 2011). Coronavirus RNAs with cap-1 are protected from IFIT-mediated interferon response. IFITs are interferon-induced proteins with tetratricopeptide repeats that recognize unmethylated 2'-O RNAs and act to inhibit expression of virally encoded mRNAs (Menachery et al. 2014).
The genomic and subgenomic mRNAs of SARS-CoV-1 coronavirus, including the plus strand genomic RNA, are presumed to be capped at their 5′ end, based on studies of the mouse hepatitis virus (MHV) (Lai and Stohlman 1981) and the equine torovirus (van Vliet et al. 2002). The non-structural protein 16 (nsp16) acts as a 2'O-methyltransferase that converts coronavirus cap-0 to cap-1, which was first demonstrated with nsp16 cloned from the feline coronavirus (FCV) (Decroly et al. 2008). Cap-0 represents N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage (also known as m7G cap or m7Gppp cap). Cap-1 is generated by an additional methylation on the 2′O position of the initiating nucleotide, and is also known as m7GpppNm. Non-structural protein 10 (nsp10) acts as an activator of nsp16 and is necessary for cap-1 synthesis (Bouvet et al. 2010, Decroly et al. 2011). Coronavirus RNAs with cap-1 are protected from IFIT-mediated interferon response, as IFITs recognize unmethylated 2'-O RNAs. IFITs are interferon-induced proteins with tetratricopeptide repeats that recognize unmethylated 2'-O RNAs and act to inhibit expression of virally encoded mRNAs (Menachery et al. 2014).
The subgenomic mRNAs of SARS-CoV-1 coronavirus are presumed to be capped at their 5′ ends, based on studies of the mouse hepatitis virus (MHV) (Lai and Stohlman 1981) and the equine torovirus (van Vliet et al. 2002). The non-structural protein 16 (nsp16) acts as a 2'O-methyltransferase that converts coronavirus cap-0 to cap-1, which was first demonstrated with nsp16 cloned from the feline coronavirus (FCV) (Decroly et al. 2008). Cap-0 represents N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage (also known as m7G cap or m7Gppp cap). Cap-1 is generated by an additional methylation on the 2′O position of the initiating nucleotide, and is also known as m7GpppNm. The non-structural protein 10 (nsp10) acts as an activator of nsp16 and is necessary for cap-1 synthesis (Bouvet et al. 2010, Decroly et al. 2011). Coronavirus RNAs with cap-1 are protected from IFIT-mediated interferon response, as IFITs recognize unmethylated 2'-O RNAs. IFITs are interferon-induced proteins with tetratricopeptide repeats that recognize unmethylated 2'-O RNAs and act to inhibit expression of virally encoded mRNAs (Menachery et al. 2014).
M protein is the most abundant component of the mature virionm and contributes to the shape of the virus. It consists of three transmembrane domains with an N-terminus outside the virus and an internal C-terminus (N-exo, C-endo conformation). Homotypic interactions between M proteins contribute to the initial formation of a nascent virus by forming a lattice, consistent with what is seen in other coronavirus systems (Tseng et al, 2010; de Hann et al, 1998; de Haan et al, 2000; Locker et al, 1995). Multiple segments of M are required for oligomerization (Tseng et al, 2010; de Hann et al, 1998; de Haan et al, 2000; reviewed in Masters, 2006). Both glycosylated and non-glycosylated forms of M are incorporated into the virion, and the significance of the N-linked glycosylation is not clear (Voss et al, 2006; Voss et al, 2009). Despite its importance, expression of M alone is not sufficient to drive formation of a mature virus (reviewed in Masters, 2006). Protein-protein interactions between M and S, N and E, among other components, are required for assembly of a mature virus and for membrane curvature. Many studies have examined the minimal system required for release of viral like particles (VLPs) with sometimes contradictory results, but interactions between M, N and E are sufficient to promote release of significant numbers of VLPs (Ho et al, 2004; Huang et al, 2004; Mortola and Roy, 2004; Hsieh et al, 2005; Siu et al, 2008; Hatakeyama et al, 2008; Tseng et al, 2013; reviewed in Masters, 2006)
N protein is synthesized in the cytosol of the host cell and then moves adjacent to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane, where mature virions are assembled. The primary function of the SARS-CoV nucleocapsid (N) protein is to encapsulate the positive-strand 5'-capped genomic RNA into a nucleocapsid for export. Nucleocapsid formation is depends on multiple weak protein-protein and protein-RNA interactions (reviewed in Chang et al, 2014). The SARS-CoV N protein has globular N-terminal and C-terminal domains separated by three intrinsically disordered regions (IDRs) (Chang et al, 2006; Chang et al, 2009). N protein forms a weak dimer in the absence of RNA mediated by residues in the middle and C-terminal IDR (He et al, 2004a; Luo et al, 2006; Chang et al, 2005; Surjit et al, 2004; Yu et al, 2005; Chang et al, 2006; Chang et al, 2013; Surjit and Lal, 2008). Positive residues in the middle IDR are subject to phosphorylation, which may affect the function of N (Surjit et al, 2005; Peng et al, 2008). Binding of the genomic RNA to one or a small number of N-N dimers may be the initiating event in nucleocapsid formation. Both the NTD and the CTD of N have been shown to have RNA-binding activity (Huang et al, 2004a; Huang et al, 2004b; Chen et al, 2007; Chang et al, 2009; Takeda et al, 2008), and the IDRs seem likely to also contribute (Chang et al, 2009). These initial binding events may nucleate nucleocapsid formation through further recruitment of N protein dimers (reviewed in Chang et al, 2014).
The final ribonucleoprotein complex is a hollow helical structure with an approximate diameter of 9-16nm, with the C-terminal domain of N protein forming the inner core and the N-terminal domain forming the outer surface (Neuman et al, 2006; Chen et al, 2007). Oliomerization of the N protein capsid coat is likely nucleated through both protein-RNA and protein-protein interactions by the first few N-protein dimers on the genomic RNA (Saikatendu et al, 2007; Chang et al, 2013; reviewed in Chang et al, 2014). Each N dimer may make contact with up to 7 bases of the RNA (reviewed in Chang et al, 2014).
Formation of an M lattice nucleates recruitment of other structural proteins including N, E and S (reviewed in Masters, 2006; Fung and Liu, 2019; Ujike and Taguchi, 2015). Expression of M and N or M and E have been shown to be sufficient to support release of viral like particles (VLPs), with co-expression of all three promoting release of significant numbers of VLPs (Huang et al, 2004; Ho et al, 2004; Hsieh et al, 2005; Mortola and Roy, 2004; Siu et al, 2008). Neither the S protein nor the genomic RNA are required for release of these otherwise morphologically normal particles, but both are incorporated if co-expressed (Siu et al, 2008). E protein is a small, integral membrane protein that is present in low amounts in the mature virion. It forms homo-oligomers and may exist as a homopentamer (Torres et al, 2005). E is palmitoylated and N-glycosylated, although the significance of these modifications is not clear (Liao et al, 2006; Yuan et al, 2006). E is recruited to the M lattice through interactions between the transmembrane domains of both proteins, and plays multiple roles in virion assembly and host interactions, including membrane budding, induction of apoptosis and membrane permeability (Hsieh et al, 2008; Chen et al, 2009; reviewed in Schoeman and Fielding, 2019; Liu et al, 2016). The ribonucleoprotein complex is recruited to the assembling virion through interactions with the M C-terminal tail, and appears to be independent of viral RNA (Hsieh et al, 2008; Hatakeyama et al, 2008; He et al, 2004; Luo et al, 2006; reviewed in Ujike and Taguchi, 2015).
S trimers are recruited to the assembling virion through interaction with M protein (reviewed in Ujike and Taguchi, 2015). Multiple regions of M contribute to the recruitment of S, with a single tyrosine residue in the C-terminal domain of M playing a critical role (McBride et al, 2010a; Hsieh et al, 2008). Interaction with M is aided by a dibasic motif in the C-terminus of S, which promotes retrieval of the spike protein from the cell surface by binding the COPI coat (McBride et al, 2007; Ujike et al, 2016). Palmitoylation of the C-terminus of S appears dispensible for the interaction with M in SARS-COV-1, unlike the case in other coronaviruses (Ujike et al, 2012; McBride 2010b; reviewed in Ujike and Taguchi, 2015). Size estimates and modelling suggest the mature virion has approximately 300 S trimers (Neuman et al, 2006; reviewed in Chang et al, 2014).
Main protease cleaves all cleavage sites of pp1a and ppa1b starting with nsp4/5, thus cleaving itself, and all the cytosolic RTC proteins (Fan et al, 2004)
In about 15% of translation attempts of the genomic viral mRNA1 in the host cell cytosol, a -1 frameshift happens after the nsp10 gene that leads to translation of the full 7,033 aa pp1ab polyprotein (Baranov et al, 2005).
Formation of the nsp9 dimer is necessary for viral viabilit. While the dimer retains only a slight advantage over the monomer in RNA binding the nsp9 monomer does not function in vivo, probably because of the correct positioning of RNA in the replication complex requiring a properly dimerized nsp9 (Miknis et al, 2009)0
In most translation attempts the genomic viral mRNA1 in the cytosol is translated to a shortened polyprotein, pp1a (4,382 aa), that does not contain genome replication enzymes (Baranov et al, 2005).
Main protease cleaves all cleavage sites of pp1a and ppa1b starting with nsp4/5, thus cleaving itself, and all the cytosolic RTC proteins (Fan et al, 2004)
The main protease has no post-translational modifications. Most of it is in monomeric form, but only the dimer shows cysteine endopeptidase activity (Sun et al, 2003; Fan et al, 2004)
Main protease cleaves all cleavage sites of pp1a and ppa1b starting with nsp4/5, thus cleaving itself, and all the cytosolic RTC proteins (Fan et al, 2004)
The crucial step of autocleavage of pp1a involves the formation of an "intermediate" pp1a dimer which has weak protease activity. This "embedded" 3CLp liberates itself by cleaving the ends off its monomer in trans. Only after that the cleaved 3CLp forms a dimer, the most efficient form of the enzyme (Hsu et al, 2005; Chen et al, 2010; Muramatsu et al, 2016).
SARS-CoV-1 plus strand genomic RNA, like genomic RNAs of other coronaviruses, possesses a polyadenylation signal in its 3'UTR and is polyadenylated by an undetermined viral RNA polymerase, possibly nsp8 or nsp12 (Spagnolo and Hogue 2000, Peng et al. 2016, Tvarogova et al. 2019).
Interaction of the ribonucleocapsid and the structural proteins in the ERGIC membrane promotes the formation of virions by budding into to the ERGIC lumen (reviewed in Masters, 2006). Coronavirus membrane curvature is driven by M lattice formation, interaction with the nucleocapsid and by E protein (de Haan et al, 1998; de Haan et al, 2000; Hsieh et al, 2005; Hsieh et al, 2008; reviewed in Masters, 2006; Ujike and Taguchi, 2015; Schoeman and Fielding 2019).
Similar to other coronaviruses, SARS-CoV-1 virions are released from the host cell by exocytosis in smooth-walled vesicles (reviewed in Ujike and Taguchi, 2015; Masters, 2006; Fung and Liu, 2019). The nature and details of this export remain to be elucidated.
After synthesizing the complementary minus RNA of the plus strand viral genomic RNA, SARS-CoV-1 replication-transcription complex (RTC) associates with the minus strand to initiate plus strand synthesis and to initiate transcription of subgenomic (sg) mRNAs (Ahn et al. 2012).
SARS-CoV-1 encodes eight subgenomic RNAs, mRNA2 to mRNA9. mRNA1 corresponds to the genomic RNA. The 5' and 3' ends of subgenomic RNAs are identical, in accordance with the template switch model of coronavirus RNA transcription (Snijder et al. 2003, Thiel et al. 2003, Yount et al. 2003). Therefore, consistent with this and the studies of the murine hepatitis virus (MHV), which is closely related to SARS-CoV-1, genomic positive strand RNA is first transcribed into negative sense (minus strand) subgenomic mRNAs, that subsequently serve as templates for the synthesis of positive strand subgenomic mRNAs. Negative-sense virus RNAs are present in much smaller amounts than positive-sense RNAs (Irigoyen et al. 2016). Each subgenomic RNA contains a leader transcription regulatory sequence (leader TRS) that is identical to the leader of the genome, appended via polymerase “jumping� during negative strand synthesis to the body transcription regulatory sequence (body TRS), a short, AU-rich motif of about 10 nucleotides found upstream of each ORF that is destined to become 5' proximal in one of the subgenome-length mRNAs. The 3' and 5'UTRs may interact through RNA–RNA and/or RNA–protein plus protein–protein interactions to promote circularization of the coronavirus genome, placing the elongating minus strand in a favorable topology for leader-body joining. The host protein PABP was found to bind to the coronavirus 3' poly(A) tail and to interact with the host protein eIF-4G, a component of the three-subunit complex that binds to mRNA cap structures, which may promote the circularization of the coronavirus genome. Two viral proteins that bind to the coronavirus 5'UTR, the N protein and nsp1, may play a role in template switching. The poly(A) tail is necessary for the initiation of minus-strand RNA synthesis at the 3' end of genomic RNA. For review, please refer to Sawicki et al. 2007 and Yang and Leibowitz 2015.
Lysosomes play critical roles in human biology receiving, trafficking, processing, and degrading biological molecules from cellular processes such as endocytosis, phagocytosis, autophagy and secretion. Lysosomes house around sixty proteolytic enzymes, among them cathepsins. Cathepsins are involved in many processes involving cell death, protein degradation, post-translational modifications of proteins, extracellular matrix (ECM) remodeling, autophagy, and immune signaling. The early stages of the viral life cycle involve the cleavage of the viral spike protein by cathepsin L (CTSL) in late endosomes, facilitating viral RNA release to continue viral replication. Teicoplanin, a glycopeptide antibiotic used to treat Gram-positive bacterial infection, especially in Staphylococcal infections, was shown to have efficacy in vitro against Ebola Virus, MERS and SARS-CoV-1 (Zhou et al. 2016).
Teicoplanin is thought to inhibit the low pH cleavage of the viral spike protein by CTSL in late endosomes thereby preventing the release of genomic viral RNA and the continuation of virus replication cycle (Baron et al. 2020). The target sequence that serve as the cleavage site for CTSL is conserved in the SARS-CoV-2 spike protein (Zhou et al. 2020 [preprint]). Further investigation is required to determine the therapeutic potential of teicoplanin in COVID-19 patients.
Relacatib is an investigational drug trialed for the treatment of osteoporosis (Duong et al. 2016). It is a potent CTSK inhibitor but also shows activity against CTSL (Kumar et al. 2007) so could potentially be investigated for Covid-19 patients. The antileprotic drug clofazimine and the antituberculous drugs rifampicin and isoniazid have been shown to inhibit cathepsins B, H and L from purified goat and bovine brains (Kamboj et al. 2003).
SARS-CoV-1 encodes eight subgenomic RNAs, mRNA2 to mRNA9. mRNA1 corresponds to the genomic RNA. mRNA2 encodes the S protein. mRNA3 is bicistronic and encodes proteins 3a and 3b. mRNA4 encodes the E protein. mRNA5 encodes the M protein. mRNA6 encodes the protein 6. mRNA7, mRNA8 and mRNA9 are bicistronic, with mRNA7 encoding proteins 7a and 7b, mRNA8 encoding proteins 8a and 8b, and mRNA 9 encoding proteins 9a and N. The 5' and 3' ends of subgenomic RNAs are identical, in accordance with the template switch model of coronavirus RNA transcription (Snijder et al. 2003, Thiel et al. 2003, Yount et al. 2003). Based on studies of the murine hepatitis virus (MHV), which is closely related to SARS-CoV-1, positive-sense virus mRNAs are present at much higher amounts than negative-sense mRNAs (Irigoyen et al. 2016).
SARS-CoV-1 plus strand subgenomic mRNAs share a 3'UTR with the plus strand genomic RNA, and as this 3'UTR possesses a polyadenylation signal, they undergo polyadenylation by an undetermined viral RNA polymerase, possibly nsp8 or nsp12 (Spagnolo and Hogue 2000, Peng et al. 2016, Tvarogova et al. 2019).
As it contains cargo sorting motifs in its cytoplasmic domain, protein 3a gets localized by the cell's protein transport system to the cell membrane where it functions as an ion channel (Tan et al, 2004). This ion channel function is necessary for the protein's pro-apoptotic function (Chan et al, 2009).
Protein 3a is rapidly internalized into cells by endocytosis. It contains a Yxxφ motif and also diacidic motifs which are typically found in internalized membrane proteins (Tan et al, 2004). The ability to be internalized is necessary for the protein's pro-apoptotic function (Wong et al, 2006; Chan et al, 2009).
On the final assembly, proteomics data suggest that the NAB−βSM−TM1 domains of nsp3 can interact with nsp7 − 8, as well as nsps 12–16, and the domain Y1 plus CoV-Y interacts with nsp9 and nsp12 (Imbert et al., 2008). Also a PL2pro−NAB−βSM−TM1 construct of Nsp3 can bind Nsp4 and Nsp12, while the region from TM1 to the end of Nsp3 only binds Nsp8 (Pan et al., 2008).
nsp3 and nsp4 alone caused considerable ER membrane deformation, producing a perinuclear double-walled maze-like body (MLB), and the nsp3–nsp4 interaction was shown to be absolutely necessary for such membrane rearrangement. Further necessary factors are nsp6 and unidentified host factors (Angelini et al 2013, Sakai et al 2017). nsp6 by itself can form Atg5 and LC3II-positive vesicles classically observed in autophagy (Cottam et al, 2014). However, in mouse hepatitis virus (MHV) infections, EDEM1 and OS9 of the ER-associated degradation system have been shown to be necessary co-factors (Reggiori et al, 2010).
Nucleoprotein (N) is ADP-ribosylated. The modification is maintained both in the cell and in virions (Grunewald et al, 2018). Members of the protein mono-ADP-ribosyltransferase (PARP) enzyme family are thought to catalyze this reaction (Fehr et al. 2020).
In addition to the main structural proteins and the nucleocapsid, the mature virion may also contain low proportions of accessory proteins, including protein 3a and 7a (reviewed in McBride and Fielding, 2012). Protein 3a has been shown to interact with E, M, S and protein 7a and is estimated to be present in the virion at 2/3 the molar ratio of E protein (Ito et al, 2005; Shen et al, 2005; Tan et al, 2004). Although 3a tetramers are predicted to act as ion channels in the host plasma membrane, increasing cell permeability, the role of 3a in the mature virion is not clear (Lu et al, 2006; reviewed in McBride and Fielding, 2012). Protein 7a is type 1 transmembrane protein that interacts with M, E, S and protein 3a and may be incorporated into the mature virion. What functional role protein 7a may play in the assembled virion is unclear (Huang et al, 2006; Fielding et al, 2004; Tan et al, 2004).
The SARS-CoV-1 nucleocapsid is released from the host cell endosome into the cytosol. Molecular details of this step are not well worked out. Studies of the infection of the human cultured cells with HCoV-229E coronavirus established a requirement for VCP (transitional endoplasmic reticulum ATPase) protein function for release to occur (Wong et al. 2015). A similar requirement for VCP involvement in SARS-CoV-1 nucleocapsid release is inferred here.
Spike protein S1: attaches the virion to the cell membrane by interacting with host receptor, initiating the infection.
Spike protein S2: Acts as a viral fusion peptide which is unmasked following S2 cleavage occurring upon virus endocytosis.
Spike protein S2: mediates fusion of the virion and cellular membranes by acting as a class I viral fusion protein. Under the current model, the protein has at least three conformational states: pre-fusion native state, pre-hairpin intermediate state, and post-fusion hairpin state. During viral and target cell membrane fusion, the coiled coil regions (heptad repeats) assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes.
Within the host cell endocytic vesicle, SARS-CoV-1 Spike (S) protein is cleaved between residues 797 and 798 by cathepsin L1 (CTSL) (Huang et al. 2006). The roles of S protein in viral binding to the host cell membrane and fusion of viral and host cell membranes and thus the central role of S protein in determining the host range and tissue tropisms of the virus are reviewed by Belouzard et al. (2012).
SARS-CoV-1 virions attached to the host cell surface via a complex involving viral spike (S) protein and host angiotensin-converting enzyme 2 (ACE2) undergo endocytosis. Studies with pseudoviruses have established that S protein is necessary and sufficient for mediating viral attachment and entry. Inhibition of this SARS-CoV-1 S protein-mediated transduction by two different classes of lysosomotropic agents in multiple cell lines strongly suggests that acidification of endosomes is needed for viral entry (Hofmann et al. 2004; Simmons et al. 2004; Yang et al. 2004). The roles of S protein in viral binding to the host cell membrane and fusion of viral and host cell membranes and thus the central role of S protein in determining the host range and tissue tropisms of the virus are reviewed by Belouzard et al. (2012).
Transmembrane protease serine 2 (TMPRSS2), associated with the plasma membrane of the host cell, mediates the hydrolytic cleavage of SARS-CoV-1 Spike (S) protein component of the viral membrane-associate S3:M:E:encapsidated SARS coronavirus genomic RNA: 7a:O-glycosyl 3a tetramer complex associated with ACE2 (Matsuyama et al. 2010; Glowacka et al. 2011; Shulla et al. 2011).
Inhibition of host cellular functions required for viral replication is considered another host-targeting antiviral strategy. Extensive pharmacological studies have validated ER glucosidases as valuable host antiviral targets against many enveloped viruses (Chang et al. 2013). Most known ER glucosidase inhibitors are imino sugars like 1-deoxynojirimycin (DNJ) and castanopermine (CAST) derivatives (Taylor et al. 1994).
It is generally believed that inhibition of ER glucosidase I and/or II prevents the removal of the terminal glucose moieties on N-linked glycans and results in misfolding and retention of glycoproteins in the ER and ultimate degradation via the ER-associated degradation (ERAD) pathway (Simsek et al. 2005, Alonzi et al. 2013). As a consequence of the abnormal trafficking and degradation of viral glycoproteins, virion assembly and secretion are inhibited (Chang et al. 2009, Taylor et al. 1998).
Long-term suppression of ER glucosidases I and/or II with more potent inhibitors may cause significant side effects, particularly in nerve and immune systems (Sadat et al. 2014).
It is known that non-structural proteins (nsp) in SARS-CoV viruses induce the formation of ER-bound double membrane vesicles (DMV) in host cells post infection. These DMVs are decorated with microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) proteins that are involved in autophagosome formation. However, there is no evidence that DMVs are recruited to the autophagy machinery. Studies show that in some cells sars8b (8b) colocalizes with MAP1LC3B. However, little is known about underlying molecular mechanisms (Shi C S. et al 2019).
The replicase polyprotein 1a of the human severe acute respiratory syndrome coronavirus (SARS-CoV) is post-translationally cleaved by virally encoded proteases to generate non-structural proteins (nsps). Viral nsps induce the formation of ER-bound double membrane vesicles (DMV) in host cells post infection. These DMVs are decorated with microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) proteins that are involved in autophagosome formation. However, there is no evidence that DMVs are recruited to the autophagy machinery. Immunofluorescence studies show that nsp8 colocalizes with MAP1LC3B suggesting a binding event (Prentice E. et al 2004).
Remdesivir (GS-5734) is an investigational nucleotide analogue drug that was developed for its broad spectrum antiviral potential against Ebola and Marburg virus activity (Siegel et al. 2017). It targets and inhibitis viral RNA-dependent RNA polymerase (nsp12, RdRP), the key component of the replication transcription complex (RTC) (Agostini et al. 2018, Brown et al. 2019, Gordon et al. 2020). Remdesivir is being investigated for potential antiviral activity against SARS-CoV-2 by targeting viral replication (Agostini et al. 2018). Gordon et al. demonstrate remdesivir possesses broad antiviral activity against RNA viruses, including SARS-CoV, MERS-CoV and SARS-CoV-2 in-vitro (Gordon et al. 2020b). It could prevent asymptomatic, mild or moderate COVID-19 cases from progressing to severe disease (clinical trials NCT04252664, NCT04257656) but results so far in infected people have been mixed.
EIDD-2801, is an isopropylester prodrug of the ribonucleoside analogue N4-hydroxycytidine (NHC, EIDD-1931) that shows broad spectrum antiviral activity against various RNA viruses including Ebola, Influenza and CoV (Toots et al. 2019). NHC acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. NHC was shown to inhibit multiple genetically-distinct Bat-CoV viruses in human primary epithelial cells without affecting cell viability. Prophylactic/therapeutic oral administration of NHC reduced lung titers and prevented acute lung failure in C57B/6 mice infected with CoV. The potency of NHC against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses (Sheahan et al. 2020).
Remdesivir (GS-5734) is an investigational nucleotide analogue drug that was developed for its broad spectrum antiviral potential against Ebola and Marburg virus activity (Siegel et al. 2017). It targets and inhibitis viral RNA-dependent RNA polymerase (nsp12, RdRP), the key component of the replication transcription complex (RTC) (Agostini et al. 2018, Brown et al. 2019, Gordon et al. 2020). Remdesivir is being investigated for potential antiviral activity against SARS-CoV-2 by targeting viral replication (Agostini et al. 2018). Gordon et al. demonstrate remdesivir possesses broad antiviral activity against RNA viruses, including SARS-CoV, MERS-CoV and SARS-CoV-2 in-vitro (Gordon et al. 2020b). It could prevent asymptomatic, mild or moderate COVID-19 cases from progressing to severe disease (clinical trials NCT04252664, NCT04257656) but results so far in infected people have been mixed.
EIDD-2801, is an isopropylester prodrug of the ribonucleoside analogue N4-hydroxycytidine (NHC, EIDD-1931) that shows broad spectrum antiviral activity against various RNA viruses including Ebola, Influenza and CoV (Toots et al. 2019). NHC acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. NHC was shown to inhibit multiple genetically-distinct Bat-CoV viruses in human primary epithelial cells without affecting cell viability. Prophylactic/therapeutic oral administration of NHC reduced lung titers and prevented acute lung failure in C57B/6 mice infected with CoV. The potency of NHC against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses (Sheahan et al. 2020).
The replicase polyprotein 1a of the human severe acute respiratory syndrome coronavirus (SARS-CoV) is post-translationally cleaved by virally encoded proteases to generate non-structural proteins (nsps). Viral nsps induce the formation of ER-bound double membrane vesicles (DMV) in host cells post infection. These DMVs are decorated with microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) proteins that are involved in autophagosome formation. However, there is no evidence that DMVs are recruited to the autophagy machinery. Immunofluorescence studies show that nsp6 (Cottam E M. et al 2011) and nsp8 (Prentice E. et al 2004) colocalizes with MAP1LC3B suggesting a binding event. In some cell types, expression of sars9b (9b) triggers the formation of autophagosomes and underlying molecular mechanisms are unclear (Shi C S. et al 2014). Studies also show that sars8b (8b) can trigger cellular stress, which results in a calcineurin dependent Transcription Factor EB (TFEB) activation and its target genes. This leads to an increase in autophagic flux (Shi C S. et al 2019).
Many GSK-3β inhibitors (GSKi) have been identified. They are known to induce apoptosis in leukemia and pancreatic cancer cells, and can destabilize p53, which may promote cellular death in response to DNA damaging agents (Wang et al, 2008; Beurel et al, 2009). Administration of GSKi inhibited cochlear destruction in cisplatin-injected mice (Park et al, 2009).
Li is a selective ATP competitive inhibitor of GSK-3 (Ryves and Harwood, 2001). Lithium carbonate has been and continously is in clinical trials with bipolar disorder patients (Moore et al, 2009). LY2090314 has been in clinical trials for metastatic pancreatic cancer and acute leukemia ([NCT01632306], [NCT01287520], [NCT01214603]). Clinical trials of GSKi for Alzheimer's disease were unsuccessful.
The use of GSKi remains controversial because of their possibly oncogenic properties. Evaluation of GSKi in clinical trials has been hampered by the fear that inhibition of GSK-3 may stimulate or aid in malignant transformation as GSK-3 can phosphorylate pro-oncogenic factors such as beta-catenin, c-Jun and c-Myc which targets them for degradation (Patel & Woodgett, 2008). However, no studies have been reported suggesting that treatment of mice with GSKi resulted in an increase in cancer incidence. In fact, many patients with bi-polar disorder have been treated with lithium for prolonged periods of time. There does not appear to be any evidence that these patients have increased incidences of cancer (McCubrey et al, 2014).
The GSKi kenpaullone and lithium chloride were found to reduce viral Nucleoprotein phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic symptoms. Effect of GSK-3 inhibition were reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus (Wu et al, 2009).
The CORDITE database contains aggregated information from published and preprint articles about potential drugs, their targets and their interactions (Martin et al. 2020). Different computational approaches reveal drug candidates that may be repurposed for the Covid-19 pandemic. The data provide by this database should be treated as interesting starting points for approved drug candidates that would require clinical testing to determine their efficacy specifically in Covid-19 patients. Here, potential drug candidates for the human ACE2 receptor are described.
Fan et al. constructed a pangolin coronavirus model to screen 2406 approved drugs for their ability to inhibit cytopathic effects and thereby identify candidates for treating Covid-19 infection (Fan et al. 2020). Three drugs, cepharanthine, selamectin and mefloquine hydrochloride, exhibited complete inhibition of cytopathic effects in cell culture. Selamectin is excluded from inclusion here as it is a vetinary drug not approved for human use.
Using Human Pluripotent Stem Cell-derived Colonic Organoids (hPSC-COs) and humanized mouse models, Duan et al. 2020 screened 1280 FDA-approved drugs, which uncovered mycophenolic acid and quinacrine dihydrochloride as promising candidates for SARS-CoV-2 entry inhibition, with greater efficacy than drugs currently being investigated for therapeutic use in COVID-19 (preprint https://www.biorxiv.org/content/10.1101/2020.05.02.073320v1.full).
Molecular dynamic simulations of SARS-CoV-2 spike protein and human ACE2 receptor complexes with stilbenoid analogues potentially having activities against these targets revealed resveratrol to have good affinity for the spike:ACE2 complex. Resveratrol could be a promising anti-COVID-19 drug candidate acting through disruption of the spike protein (Wahedi et al. 2020).
Using a virtual screen of the main targets involved in Covid-19 infection with 7922 FDA-approved drugs, Durdagi et al. 2020 ranked compounds based on their docking scores. Promising ACE2 receptor-binding domain inhibitors included denopamine and rotigaptide amongst the top 5 hits. These compounds could be clinically tested to check whether they may be considered to be use for the treatment of COVID-19 patients (preprint https://chemrxiv.org/articles/preprint/Screening_of_Clinically_Approved_and_Investigation_Drugs_as_Potential_Inhibitors_of_COVID-19_Main_Protease_A_Virtual_Drug_Repurposing_Study/12032712).
Using an in-silico structure-based virtual screening approach, Choudhary et al. 2020 found the FDA-approved drug eptifibatide acetate bound to the virus binding motifs of the ACE2 receptor (preprint https://chemrxiv.org/articles/preprint/Identification_of_SARS-CoV-2_Cell_Entry_Inhibitors_by_Drug_Repurposing_Using_in_Silico_Structure-Based_Virtual_Screening_Approach/12005988).
Redka et al. 2020 utilised a deep learning drug design platform to interrogate the polypharmacological profiles of FDA-approved small molecule drugs or going through clinical trials, with the goal of identifying molecules predicted to modulate targets relevant for COVID-19 treatment. Top drug hits predicted to bind to the ACE2 receptor included a number of broad-spectrum antibiotics such as latamoxef, cefazolin, cefoxitin, enoxacin and pheneticillin, amongst others. This study may identify and prioritise candidates for testing in Covid-19 patients (preprint https://chemrxiv.org/articles/preprint/PolypharmDB_a_Deep_Learning-Based_Resource_Quickly_Identifies_Repurposed_Drug_Candidates_for_COVID-19/12071271).
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lattice:E
protein:encapsidated SARS coronavirus genomic RNA7a:O-glycosyl 3a
tetramerSARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramer:glycosylated-ACE2SARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramer:glycosylated-ACE2SARS coronavirus genomic RNA:7a:O-glycosyl
3a tetramerSARS coronavirus genomic RNA:7a:O-glycosyl
3a tetramerSARS coronavirus genomic RNA:O-glycosyl 3a
tetramerSARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramercoronavirus gRNA with secondary
structure:RTC:nascent RNA minus strandgRNA with secondary
structure:RTCgRNA:RTC:RNA primer:RTC
inhibitorsgRNA:RTC:nascent RNA minus strand with mismatched
nucleotidegRNA:RTC:nascent RNA minus strand:RTC
inhibitorsgRNA:RTC:nascent
RNA minus strandcomplement (minus strand):RTC:RTC
inhibitorscomplement (minus
strand):RTCRNA complement
(minus strand)strand subgenomic
mRNAsstrand subgenomic
mRNAsdimer:SARS coronavirus genomic
RNAgenomic RNA (plus
strand)N-glycan-PALM-Spike
trimercoronavirus genomic
RNA (plus strand)coronavirus genomic
RNASARS-CoV-1 genomic
RNA (plus strand)SARS-CoV-1 genomic RNA complement
(minus strand)polyadenylated SARS-CoV-1 subgenomic mRNAs
(plus strand)polyadenylated SARS-CoV-1 genomic
RNA (plus strand)plus strand
subgenomic mRNAsSARS-CoV-1 genomic
RNA (plus strand)SARS-CoV-1 genomic RNA complement
(minus strand)N-glycan-PALM-Spike
trimerN-glycan-PALM-Spike
trimerAnnotated Interactions
lattice:E
protein:encapsidated SARS coronavirus genomic RNAlattice:E
protein:encapsidated SARS coronavirus genomic RNAEIDD-2801, is an isopropylester prodrug of the ribonucleoside analogue N4-hydroxycytidine (NHC, EIDD-1931) that shows broad spectrum antiviral activity against various RNA viruses including Ebola, Influenza and CoV (Toots et al. 2019). NHC acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. NHC was shown to inhibit multiple genetically-distinct Bat-CoV viruses in human primary epithelial cells without affecting cell viability. Prophylactic/therapeutic oral administration of NHC reduced lung titers and prevented acute lung failure in C57B/6 mice infected with CoV. The potency of NHC against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses (Sheahan et al. 2020).
Otamixaban (FXV673), an anticoagulant, is a potent and selective direct inhibitor of coagulation factor Xa. Virtual docking studies suggest that otamixaban may bind to the serine protease TMPRSS2 (Rensi et al. 2020, preprint). Inhibition of TMPRSS2 is being examined for antiviral activity but its inhibitory potential and/or antiviral activity have not yet been determined so it is annotated here as a candidate drug. I-432 is another inhibitor of TMPRSS2 under investigation for anti viral potential (Pászti-Gere et al. 2016).
Despite its importance, expression of M alone is not sufficient to drive formation of a mature virus (reviewed in Masters, 2006). Protein-protein interactions between M and S, N and E, among other components, are required for assembly of a mature virus and for membrane curvature. Many studies have examined the minimal system required for release of viral like particles (VLPs) with sometimes contradictory results, but interactions between M, N and E are sufficient to promote release of significant numbers of VLPs (Ho et al, 2004; Huang et al, 2004; Mortola and Roy, 2004; Hsieh et al, 2005; Siu et al, 2008; Hatakeyama et al, 2008; Tseng et al, 2013; reviewed in Masters, 2006)
The SARS-CoV N protein has globular N-terminal and C-terminal domains separated by three intrinsically disordered regions (IDRs) (Chang et al, 2006; Chang et al, 2009). N protein forms a weak dimer in the absence of RNA mediated by residues in the middle and C-terminal IDR (He et al, 2004a; Luo et al, 2006; Chang et al, 2005; Surjit et al, 2004; Yu et al, 2005; Chang et al, 2006; Chang et al, 2013; Surjit and Lal, 2008). Positive residues in the middle IDR are subject to phosphorylation, which may affect the function of N (Surjit et al, 2005; Peng et al, 2008).
Binding of the genomic RNA to one or a small number of N-N dimers may be the initiating event in nucleocapsid formation. Both the NTD and the CTD of N have been shown to have RNA-binding activity (Huang et al, 2004a; Huang et al, 2004b; Chen et al, 2007; Chang et al, 2009; Takeda et al, 2008), and the IDRs seem likely to also contribute (Chang et al, 2009). These initial binding events may nucleate nucleocapsid formation through further recruitment of N protein dimers (reviewed in Chang et al, 2014).
E protein is a small, integral membrane protein that is present in low amounts in the mature virion. It forms homo-oligomers and may exist as a homopentamer (Torres et al, 2005). E is palmitoylated and N-glycosylated, although the significance of these modifications is not clear (Liao et al, 2006; Yuan et al, 2006). E is recruited to the M lattice through interactions between the transmembrane domains of both proteins, and plays multiple roles in virion assembly and host interactions, including membrane budding, induction of apoptosis and membrane permeability (Hsieh et al, 2008; Chen et al, 2009; reviewed in Schoeman and Fielding, 2019; Liu et al, 2016).
The ribonucleoprotein complex is recruited to the assembling virion through interactions with the M C-terminal tail, and appears to be independent of viral RNA (Hsieh et al, 2008; Hatakeyama et al, 2008; He et al, 2004; Luo et al, 2006; reviewed in Ujike and Taguchi, 2015).
Teicoplanin is thought to inhibit the low pH cleavage of the viral spike protein by CTSL in late endosomes thereby preventing the release of genomic viral RNA and the continuation of virus replication cycle (Baron et al. 2020). The target sequence that serve as the cleavage site for CTSL is conserved in the SARS-CoV-2 spike protein (Zhou et al. 2020 [preprint]). Further investigation is required to determine the therapeutic potential of teicoplanin in COVID-19 patients.
Relacatib is an investigational drug trialed for the treatment of osteoporosis (Duong et al. 2016). It is a potent CTSK inhibitor but also shows activity against CTSL (Kumar et al. 2007) so could potentially be investigated for Covid-19 patients. The antileprotic drug clofazimine and the antituberculous drugs rifampicin and isoniazid have been shown to inhibit cathepsins B, H and L from purified goat and bovine brains (Kamboj et al. 2003).
Within the host cell endocytic vesicle, SARS-CoV-1 Spike (S) protein is cleaved between residues 797 and 798 by cathepsin L1 (CTSL) (Huang et al. 2006). The roles of S protein in viral binding to the host cell membrane and fusion of viral and host cell membranes and thus the central role of S protein in determining the host range and tissue tropisms of the virus are reviewed by Belouzard et al. (2012).Spike protein S2: mediates fusion of the virion and cellular membranes by acting as a class I viral fusion protein. Under the current model, the protein has at least three conformational states: pre-fusion native state, pre-hairpin intermediate state, and post-fusion hairpin state. During viral and target cell membrane fusion, the coiled coil regions (heptad repeats) assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes.
It is generally believed that inhibition of ER glucosidase I and/or II prevents the removal of the terminal glucose moieties on N-linked glycans and results in misfolding and retention of glycoproteins in the ER and ultimate degradation via the ER-associated degradation (ERAD) pathway (Simsek et al. 2005, Alonzi et al. 2013). As a consequence of the abnormal trafficking and degradation of viral glycoproteins, virion assembly and secretion are inhibited (Chang et al. 2009, Taylor et al. 1998).
Long-term suppression of ER glucosidases I and/or II with more potent inhibitors may cause significant side effects, particularly in nerve and immune systems (Sadat et al. 2014).
EIDD-2801, is an isopropylester prodrug of the ribonucleoside analogue N4-hydroxycytidine (NHC, EIDD-1931) that shows broad spectrum antiviral activity against various RNA viruses including Ebola, Influenza and CoV (Toots et al. 2019). NHC acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. NHC was shown to inhibit multiple genetically-distinct Bat-CoV viruses in human primary epithelial cells without affecting cell viability. Prophylactic/therapeutic oral administration of NHC reduced lung titers and prevented acute lung failure in C57B/6 mice infected with CoV. The potency of NHC against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses (Sheahan et al. 2020).
EIDD-2801, is an isopropylester prodrug of the ribonucleoside analogue N4-hydroxycytidine (NHC, EIDD-1931) that shows broad spectrum antiviral activity against various RNA viruses including Ebola, Influenza and CoV (Toots et al. 2019). NHC acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. NHC was shown to inhibit multiple genetically-distinct Bat-CoV viruses in human primary epithelial cells without affecting cell viability. Prophylactic/therapeutic oral administration of NHC reduced lung titers and prevented acute lung failure in C57B/6 mice infected with CoV. The potency of NHC against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses (Sheahan et al. 2020).
Li is a selective ATP competitive inhibitor of GSK-3 (Ryves and Harwood, 2001). Lithium carbonate has been and continously is in clinical trials with bipolar disorder patients (Moore et al, 2009). LY2090314 has been in clinical trials for metastatic pancreatic cancer and acute leukemia ([NCT01632306], [NCT01287520], [NCT01214603]). Clinical trials of GSKi for Alzheimer's disease were unsuccessful.
The use of GSKi remains controversial because of their possibly oncogenic properties. Evaluation of GSKi in clinical trials has been hampered by the fear that inhibition of GSK-3 may stimulate or aid in malignant transformation as GSK-3 can phosphorylate pro-oncogenic factors such as beta-catenin, c-Jun and c-Myc which targets them for degradation (Patel & Woodgett, 2008). However, no studies have been reported suggesting that treatment of mice with GSKi resulted in an increase in cancer incidence. In fact, many patients with bi-polar disorder have been treated with lithium for prolonged periods of time. There does not appear to be any evidence that these patients have increased incidences of cancer (McCubrey et al, 2014).
The GSKi kenpaullone and lithium chloride were found to reduce viral Nucleoprotein phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic symptoms. Effect of GSK-3 inhibition were reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus (Wu et al, 2009).
Fan et al. constructed a pangolin coronavirus model to screen 2406 approved drugs for their ability to inhibit cytopathic effects and thereby identify candidates for treating Covid-19 infection (Fan et al. 2020). Three drugs, cepharanthine, selamectin and mefloquine hydrochloride, exhibited complete inhibition of cytopathic effects in cell culture. Selamectin is excluded from inclusion here as it is a vetinary drug not approved for human use.
Using Human Pluripotent Stem Cell-derived Colonic Organoids (hPSC-COs) and humanized mouse models, Duan et al. 2020 screened 1280 FDA-approved drugs, which uncovered mycophenolic acid and quinacrine dihydrochloride as promising candidates for SARS-CoV-2 entry inhibition, with greater efficacy than drugs currently being investigated for therapeutic use in COVID-19 (preprint https://www.biorxiv.org/content/10.1101/2020.05.02.073320v1.full).
Molecular dynamic simulations of SARS-CoV-2 spike protein and human ACE2 receptor complexes with stilbenoid analogues potentially having activities against these targets revealed resveratrol to have good affinity for the spike:ACE2 complex. Resveratrol could be a promising anti-COVID-19 drug candidate acting through disruption of the spike protein (Wahedi et al. 2020).
Using a virtual screen of the main targets involved in Covid-19 infection with 7922 FDA-approved drugs, Durdagi et al. 2020 ranked compounds based on their docking scores. Promising ACE2 receptor-binding domain inhibitors included denopamine and rotigaptide amongst the top 5 hits. These compounds could be clinically tested to check whether they may be considered to be use for the treatment of COVID-19 patients (preprint https://chemrxiv.org/articles/preprint/Screening_of_Clinically_Approved_and_Investigation_Drugs_as_Potential_Inhibitors_of_COVID-19_Main_Protease_A_Virtual_Drug_Repurposing_Study/12032712).
Using an in-silico structure-based virtual screening approach, Choudhary et al. 2020 found the FDA-approved drug eptifibatide acetate bound to the virus binding motifs of the ACE2 receptor (preprint https://chemrxiv.org/articles/preprint/Identification_of_SARS-CoV-2_Cell_Entry_Inhibitors_by_Drug_Repurposing_Using_in_Silico_Structure-Based_Virtual_Screening_Approach/12005988).
Redka et al. 2020 utilised a deep learning drug design platform to interrogate the polypharmacological profiles of FDA-approved small molecule drugs or going through clinical trials, with the goal of identifying molecules predicted to modulate targets relevant for COVID-19 treatment. Top drug hits predicted to bind to the ACE2 receptor included a number of broad-spectrum antibiotics such as latamoxef, cefazolin, cefoxitin, enoxacin and pheneticillin, amongst others. This study may identify and prioritise candidates for testing in Covid-19 patients (preprint https://chemrxiv.org/articles/preprint/PolypharmDB_a_Deep_Learning-Based_Resource_Quickly_Identifies_Repurposed_Drug_Candidates_for_COVID-19/12071271).
7a:O-glycosyl 3a
tetramerSARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramer:glycosylated-ACE2SARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramer:glycosylated-ACE2SARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramer:glycosylated-ACE2SARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramer:glycosylated-ACE2SARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramer:glycosylated-ACE2SARS coronavirus genomic RNA:7a:O-glycosyl
3a tetramerSARS coronavirus genomic RNA:7a:O-glycosyl
3a tetramerSARS coronavirus genomic RNA:7a:O-glycosyl
3a tetramerSARS coronavirus genomic RNA:7a:O-glycosyl
3a tetramerSARS coronavirus genomic RNA:O-glycosyl 3a
tetramerSARS coronavirus genomic RNA:O-glycosyl 3a
tetramerSARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramerSARS coronavirus genomic RNA: 7a:O-glycosyl 3a
tetramercoronavirus gRNA with secondary
structure:RTC:nascent RNA minus strandgRNA with secondary
structure:RTCgRNA with secondary
structure:RTCgRNA:RTC:RNA primer:RTC
inhibitorsgRNA:RTC:RNA primer:RTC
inhibitorsgRNA:RTC:RNA primer:RTC
inhibitorsgRNA:RTC:nascent RNA minus strand with mismatched
nucleotidegRNA:RTC:nascent RNA minus strand with mismatched
nucleotidegRNA:RTC:nascent RNA minus strand with mismatched
nucleotidegRNA:RTC:nascent RNA minus strand:RTC
inhibitorsgRNA:RTC:nascent RNA minus strand:RTC
inhibitorsgRNA:RTC:nascent
RNA minus strandgRNA:RTC:nascent
RNA minus strandgRNA:RTC:nascent
RNA minus strandgRNA:RTC:nascent
RNA minus strandgRNA:RTC:nascent
RNA minus strandgRNA:RTC:nascent
RNA minus strandcomplement (minus strand):RTC:RTC
inhibitorscomplement (minus strand):RTC:RTC
inhibitorscomplement (minus
strand):RTCcomplement (minus
strand):RTCcomplement (minus
strand):RTCcomplement (minus
strand):RTCRNA complement
(minus strand)RNA complement
(minus strand)strand subgenomic
mRNAsstrand subgenomic
mRNAsstrand subgenomic
mRNAsstrand subgenomic
mRNAsdimer:SARS coronavirus genomic
RNAdimer:SARS coronavirus genomic
RNAgenomic RNA (plus
strand)N-glycan-PALM-Spike
trimerN-glycan-PALM-Spike
trimercoronavirus genomic
RNA (plus strand)coronavirus genomic
RNA (plus strand)coronavirus genomic
RNAcoronavirus genomic
RNASARS-CoV-1 genomic
RNA (plus strand)SARS-CoV-1 genomic
RNA (plus strand)SARS-CoV-1 genomic RNA complement
(minus strand)SARS-CoV-1 genomic RNA complement
(minus strand)SARS-CoV-1 genomic RNA complement
(minus strand)polyadenylated SARS-CoV-1 subgenomic mRNAs
(plus strand)polyadenylated SARS-CoV-1 genomic
RNA (plus strand)polyadenylated SARS-CoV-1 genomic
RNA (plus strand)polyadenylated SARS-CoV-1 genomic
RNA (plus strand)polyadenylated SARS-CoV-1 genomic
RNA (plus strand)polyadenylated SARS-CoV-1 genomic
RNA (plus strand)plus strand
subgenomic mRNAsSARS-CoV-1 genomic
RNA (plus strand)SARS-CoV-1 genomic
RNA (plus strand)SARS-CoV-1 genomic
RNA (plus strand)SARS-CoV-1 genomic
RNA (plus strand)SARS-CoV-1 genomic RNA complement
(minus strand)SARS-CoV-1 genomic RNA complement
(minus strand)N-glycan-PALM-Spike
trimerN-glycan-PALM-Spike
trimerN-glycan-PALM-Spike
trimerN-glycan-PALM-Spike
trimer