SARS-CoV-2 Recombinant Proteins

According to latest research findings, Angiotensin-converting enzyme 2 (ACE2) found in the lower respiratory tract of humans, is probably the cell receptor through which SARS-CoV-2 enters cells, the same with SARS-CoV [1].

Virus-host interactions affect viral entry and replication. S glycoprotein of SARS-CoV-2 binds to host cell receptors, ACE2, which is a critical step for virus entry [2]. S glycoprotein includes two subunits, S1 and S2 [3]. S1 determines the virus-host range and cellular tropism with the key function domain, receptor-binding domain ( RBD), while S2 mediates virus-cell membrane fusion by two tandem domains, heptad repeats 1 (HR1) [4] and HR2 [5]. After membrane fusion, the viral genome RNA is released into the cytoplasm, and the uncoated RNA translates two polyproteins, pp1a and pp1ab [6], which encode non-structural proteins, and form replication-transcription complex (RTC) in double-membrane vesicle [7]. Continuously RTC replicate and synthesize a nested set of subgenomic RNAs [8], which encode accessory proteins and structural proteins, mediating endoplasmic reticulum (ER) and Golgi, newly formed genomic RNA, nucleocapsid proteins and envelope glycoproteins assemble and form viral particle buds [9]. Lastly, the virion-containing vesicles fuse with the plasma membrane to release the virus.

However, the transmission routine of this virus among hosts, the potential mechanisms for human-to-human transmission and pathogenic mechanisms of the SARS-CoV-2 are still unclear. Elabscience® provides the superior recombinant proteins for SARS-CoV-2 research.

Click on the links below to learn about important SARS-CoV-2 proteins:

Life Cycle of Highly Pathogenic Human Coronaviruses (CoVs)

Jiang S, Hillyer C, Du L. Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses[J].Trends in immunology, 2020.

SARS-CoV-2 Antigens

SARS-CoV-2 structural proteins have important functions in pathogenesis as well as infectious virus assembly. These include Spike protein (S protein), Envelope protein (E protein), Membrane protein (M protein) and Nucleocapsid protein (N protein), which are all encoded by the 3’-end of the virus genome [10].

The S protein of Cov is an essential component for infection of the host cell, responsible for both binding to cellular receptors and subsequent fusion of viral and cellular membranes. The spike protein is a large type I transmembrane protein containing two subunits, S1 and S2. S1 mainly contains a Receptor Binding Domain (RBD), which is responsible for recognizing the cell surface receptor. Elabscience® provides SARS-CoV-2 high-purity spike protein and a variety of mutants with point mutation in the RBD domain (Spike RBD mutant protein) for your related research. S2 contains basic elements needed for the membrane fusion. The S protein plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity [11].

All coronaviruses express the E protein, a multifunctional short polypeptide involved in virus morphogenesis and virulence. SARS-CoV E protein contains an alpha-helical transmembrane (TM) domain that spans the lipid membrane, and it oligomerizes forming a pentameric structure that displays ion channel activity, a remarkable function for this protein that may affect virus host interaction [12].

 The primary role of CoV N protein is to package the genomic viral genome into long, flexible, helical ribonucleoprotein (RNP) complexes called nucleocapsids or capsids. The nucleocapsid protects the genome and ensures its timely replication and reliable transmission [13]. CoV N proteins have three distinct and highly conserved domains: the N-terminal domain (NTD/domain 1) and C-terminal domain (CTD/domain 3), which are separated by a intrinsically disordered central region (RNA-binding domain/domain 2) [14].

The recently sequenced genomes of SARS-CoV-2 strains combined with the comparative analysis of the SARS-CoV genome organization and transcription allowed us to construct a tentative list of gene products [15]. It was suggested that SARS-CoV-2 had 16 predicted non-structural proteins (referred to nsp1-nsp16) constituting polyproteins pp1a and pp1ab, which were translated from ORF1a and ORF1ab of the virus genome.

Based on previous research on SARS-CoV, these non-structural proteins form a large Replication/transcription complex with a double membrane structure, which is responsible for the replication and transcription of the virus genome and subgenome. In this process, the non-structural proteins play individual roles and cooperate with each other as well [16].

Elabsicnece® offers key non-structural proteins of SARS-CoV-2 which are essential to virus life cycles. Nsp1 was proved to be able to suppress host gene expression by promoting host mRNA degradation and was involved in cellular chemokine deregulation. Nsp2 plays an important role in viral transcription and replication, and is an attractive target for anti-virus drug development. Nsp3 contains a papain-like protease (PLpro) domain, which is required to process the viral polyprotein into functional, mature subunits.Nsp5 is a proteolytic enzyme, known as a 3c-like proteinase because of its recognition sites are very similar to those of the viral 3C proteases of micrornas virus. Nsp7 forms a hexadecamer with Nsp8, they may work together and participate in viral replication by acting as a primase. Nsp10 plays a pivotal role in viral transcription by stimulating both nsp14 3'-5' exoribonuclease and nsp16 2'-O-methyltransferase activities, therefore plays an essential role in viral mRNAs cap methylation. Nsp10 is a critical regulator of coronavirus RNA synthesis and may be important in polyprotein processing. Nsp13 is a multifunctional protein with nucleoside hydrolase and helicase activity, it enables the unwinding of double-stranded DNA and RNA from the 5 '-3' end, powered by hydrolyzed deoxynucleotides and nucleotide triphosphate glands. Nsp14 has two different functional domains: an exoribonuclease domain acting on both ssRNA and dsRNA in a 3' to 5' direction and a N7-guanine methyltransferase domain. It may be involved in the proof-reading during the viral RNA replication and transcription [17].

Envelope protein  >> Papain-pke protease >> 3c-like proteinase >> Spike protein >> Nucleocapsid protein >>

Genome-wide Structure and Function Modeling of SARS-COV-2
https://zhanglab.ccmb.med.umich.edu/COVID-19/

SARS-CoV-2 Entry Receptors

ACE2>>

Receptors Help SARS-CoV-2 Invading The Human Body
2020 THE UNIVERSITY OF TOKYO

The invasion of host cells is the most important part of coronavirus (CoV) infection. The envelope spike (S) glycoprotein is responsible for CoV cell entry and host-to-host transmission. For productive entry into host cells, viruses attach to specific cell surface receptor molecules. This is the case of CoV, whose use of distinct entry receptor molecules is responsible for their broad host range and tissue tropism [18].

Several CoV of the genus alpha, including TGEV and hCoV-229E, use aminopeptidase N (APN) for cell entry [19], whereas hCoV-NL63 binds to the ACE2 [20]. In the beta-CoV, the SARS-CoV and the MERS-CoV use ACE2 and dipeptidyl peptidase 4 (DPP4) receptors, respectively [21]. APN, ACE2 and DPP4 are membrane-bound ectoenzymes with multiple functions such as angiogenesis, cell adhesion and blood pressure regulation. These three proteins catalyze peptide-bond hydrolysis of short peptides [22].

Similarly to SARS-CoV, SARS-CoV-2 employs ACE2 as a receptor for cellular entry, but the detailed mechanism of host cell entry is not yet clear [23]. Viral entry may also depends on TMPRSS2 protease activity and cathepsin B/cathepsin L activity may be able to substitute for TMPRSS2 [24].CD147 and CD299 may be involved in the virus invasion process as well [25].

SARS-CoV-2 Cytokine Storm

IL1β IL18 IFNγ TNFα GM-CSF IL6 CRP IL8

SARS-CoV-2 Infection Disables Cross-talk Between Immune Cells
Agarwal S, June C H. Harnessing CAR T-cell Insights to Develop Treatments for Hyperinflammatory Responses in Patients with COVID-19[J]. AACR, 2020.

Cytokine storm is a phenomenon of immune system disorder caused by the rapid increase of pro-inflammatory cytokine level after being stimulated by microorganism or medicine. The SARS-CoV-2 recognizes ACE2 to infect the body through S glycoprotein, which can replicate in large numbers [26], thus rapidly activating CD4+ T cells to proliferate and differentiate into Th1 cells and secreting proinflammatory cytokines such as IL-6、γ interferon and granulocyte-macrophage colony stimulating factor (GM-CSF). Among which, GM-CSF can activate monocytes to further release IL-6 and other factors, leading to cytokine storms that develop patients' conditions into acute respiratory distress syndrome (ARDS), multiple organ failure (MOF) and even death. Therefore, the IL-6 and GM-CSF that released by T lymphocytes and monocytes may be the key link in cytokine storms induces by SARS-CoV-2 [27]. And monocytes, as non-specific immune cells, which suggests that the mechanism of cytokine storms induces by SARS-CoV-2 may be closely related to the balance disruption of specific and nonspecific immune responses.

Elabscience® provides high-quality recombinant proteins of key inflammatory factors in the course of SARS-CoV-2 infection.

Reference

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  • 2.Tortorici MA, Veesler D. Structural insights into coronavirus entry. Adv Virus Res. 2019; 105: 93–116.
  • 3.Zhang N, Jiang S, Du L. Current advancements and potential strategies in the development of MERS-CoV vaccines. Expert Rev Vaccines. 2014;13(6):761–74.
  • 4.Xia S, Zhu Y, Liu M, Lan Q, Xu W, Wu Y, et al. Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell Mol Immunol. 2020. https://doi.org/10.1038/s41423-020-0374-2.
  • 5.Yu F, Du L, Ojcius DM, Pan C, Jiang S. Measures for diagnosing and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in Wuhan, China. Microbes Infect. 2020.
  • 6.de Wilde AH, Snijder EJ, Kikkert M, van Hemert MJ. Host factors in coronavirus replication. Curr Top Microbiol Immunol. 2018;419:1–42.
  • 7. Sawicki SG, Sawicki DL. Coronavirus transcription: a perspective. Curr Top Microbiol Immunol. 2005;287:31–55.
  • 8.Hussain S, Pan J, Chen Y, Yang Y, Xu J, Peng Y, et al. Identification of novel subgenomic RNAs and noncanonical transcription initiation signals of severe acute respiratory syndrome coronavirus. J Virol. 2005;79(9):5288–95.
  • 9.Perrier A, Bonnin A, Desmarets L, Danneels A, Goffard A, Rouille Y, et al. The C-terminal domain of the MERS coronavirus M protein contains a trans- Golgi network localization signal. J Biol Chem. 2019;294(39):14406–21.
  • 10.Pasternak, A. O., Spaan, W. J. & Snijder, E. J. (2006).Nidovirus transcription: how to make sense...? The Journal of general virology 87, 1403-1421.
  • 11.Wong, S. K., Li, W., Moore, M. J., Choe, H. & Farzan, M. (2004). A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J. Biol. Chem. 279, 3197–3201.
  • 12.Pervushin K., Tan E., Parthasarathy K., Lin X., Jiang F.L. Yu, Vararattanavech D.A., Soong T.W., Liu D.X., Torres J. Structure and inhibition of the SARS coronavirus envelope protein ion channel. PLoS Pathog. 2009;5:e1000511.
  • 13.De Haan C.A., Rottier P.J. Molecular interactions in the assembly of coronaviruses. Adv. Virus Res. 2005;64:165–230.
  • 14. Huang Q., Yu L., Petros A.M., et al. Structure of the N-terminal RNA-binding domain of the SARS CoV nucleocapsid protein. Biochemistry. 2004;43:6059–6063.
  • 15.Sawicki, S.G.; Sawicki, D.L.; Siddell, S.G. A contemporary view of coronavirus transcription. J. Virol. 2007,81, 20–29.
  • 16.Pasternak, A. O., Spaan, W. J. & Snijder, E. J. (2006).Nidovirus transcription: how to make sense...? The Journal of general virology 87, 1403-1421.
  • 17.Perlman, S. & Netland, J. (2009).Coronaviruses post-SARS: update on replication and pathogenesis. Nature reviews Microbiology 7, 439-450.
  • 18.P.S. Masters., The molecular biology of coronaviruses Adv. Virus Res., 66 (2006), 193-292.
  • 19.C.L. Yeager, R.A. Ashmun, R.K. Williams, et al. Holmes Human aminopeptidase N is a receptor for human coronavirus 229E. Nature, 357 (6377) (1992), pp. 420-422.
  • 20.K. Wu, W. Li, G. Peng, F. LiCrystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor. Proc. Natl. Acad. Sci. U.S.A., 106 (47) (2009), 19970-19974.
  • 21.V.S. Raj, H. Mou, S.L. Smits, et al. Haagmans Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.Nature, 495 (7440) (2013), 251-254.
  • 22.P. Mina-Osorio.The moonlighting enzyme CD13: old and new functions to target.Trends Mol. Med., 14 (8) (2008), 361-371.
  • 23.Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020).
  • 24.Hoffmann, M. et al. (2020). SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280.e8 .
  • 25.Zhinan Chen. et al. Function of HAb18G/CD147 in Invasion of Host Cells by Severe Acute Respiratory Syndrome Coronavirus. The Journal of Infectious Diseases, 191(5) (2005), Pages 755–760.
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  • 27.Zhou YG, Fu BQ, Zheng XH, et al. Aberrant pathogenic GM-CSF+ T cells and inflammatory CD14+CD16+ monocytes in severe pulmonary syndrome patients of a new coronavirus. Biorxiv, 2020.

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