SARS-CoV-2, which causes the COVID-19 disease, is a novel coronavirus which results in an acute respiratory syndrome. Studies have shown that SARS-CoV-2 virus enters the host cells via its spike (S) protein, which has a distinct furin cleavage site, by binding to the human cell surface ACE2 protein (1,2). The binding triggers receptor-induced conformational changes to expose the protease cleavage site in the spike protein to serine protease TMPRSS2. TMPRSS2 then cleaves and activates the spike protein, causing a viral infection.
Coronavirus spike protein and nucleocapsid protein and SARS-CoV-2
The Receptor Binding Domain (RBD) on the S protein plays a critical role in binding to the host cell membrane. Nucleocapsid protein is the most abundant protein in SARS-CoV-2. Research suggests that its function includes enhancing virion assembly and viral transcription through formation of complexes with genomic RNA (3). The study of SARS-CoV-2 spike protein and nucleocapsid protein and their functions in viral infection may aid target selection for diagnostic research and vaccine development.
Immunohistochemistry analysis shows detection of differential expression levels of SARS-CoV-2 Coronavirus Nucleocapsid and demonstrates antibody specificity. Immunohistochemical analysis of SARS-CoV-2 Coronavirus NP using SARS/SARS-CoV-2 Coronavirus NP Monoclonal Antibody (B46F) (Cat. No. MA1-7404) shows significant staining in human lung and placenta tissues infected with SARS-CoV-2 when compared to the control tissues without SARS-CoV-2.
SARS-CoV-2 Spike Protein RBD Chimeric Recombinant Rabbit Monoclonal Antibody verified by neutralization to ensure that the antibody binds to stated antigen. ELISA based SARS-CoV-2 Inhibitor Screening Assay shows that the binding of SARS-CoV-2 Spike Protein RBD to Human ACE2 was inhibited by SARS-CoV-2 Spike Protein RBD Chimeric Recombinant Rabbit Monoclonal Antibody (Cat. No. 703974). Recombinant SARS-CoV-2 Spike Protein RBD was coated at a concentration of 0.5 µg/mL onto a plate. This was pre-incubated with Anti-SARS- CoV-2 Spike Protein RBD Chimeric Recombinant Rabbit Monoclonal Antibody at a concentration range of 0.1 to 10 µg/mL and a binding signal of biotinylated Human ACE2 was detected using Streptavidin-HRP conjugate. The plate was developed using TMB Stabilized Chromogen (Cat. No. SB02) and read at 450 nm with Thermo Scientific Varioskan LUX multimode microplate reader (Cat. No. VLBLATD2). The X axis represents antibody concentrations and the Y axis represents percent binding signal of Human ACE2 to SARS-CoV-2 Spike Protein RBD. Dotted line represents 50% inhibition.
SARS-CoV-2 Spike Protein RBD Chimeric Recombinant Rabbit Monoclonal Antibody verified by relative expression to ensure that the antibody binds to stated antigen. Antibody specificity was demonstrated by Luminex bead-based immunoassay showing differential detection of the target antigen across a panel of related viral proteins. Relative detection of spike protein S1 subunit was observed with SARS-CoV-2 but not with S1 proteins from other members of Coronavirus family using SARS-CoV-2 Spike Protein RBD Chimeric Recombinant Rabbit Monoclonal Antibody (Cat. No. 703974).
ACE2 and SARS-CoV-2
Angiotensin-converting enzyme 2 (ACE2), a homolog of ACE, is a glycoprotein that is mainly expressed in the heart, kidneys, blood vessels, and testes. It has been demonstrated that the ACE2 protein plays a role in hypertension, cardiac function, heart function, and diabetes. In addition, studies have indicated that the entry of SARS-CoV and HCoV-NL63 viruses into host cells both involve their binding to the human cell surface ACE2 protein, causing human respiratory diseases (8).
Researchers reported that SARS-CoV-2 may use the same receptor ACE2 for host cell entry as SARS-CoV (1,2), yet SARS-CoV-2 exhibits a significantly higher binding affinity to ACE2 (9). Like SARS-CoV and HCoV-NL63, SARS-CoV-2 attaches to host cells through the binding of RBD (Receptor Binding Domain) within the spike (S) protein to ACE2 receptor protein. Not surprisingly, in vitro studies using antiserum against human ACE2 blocks the pseudo-typed entry of both SARS-CoV and SARS-CoV-2 into Vero cells (8). Furthermore, by introducing a recombinant human ACE2 protein into engineered human tissues, researchers have shown that it significantly reduces SARS-CoV-2 infections at early stages possibly due to blocking the attachment of SARS-CoV-2 to the endogenous ACE2 protein on human cell membranes (10). This preliminary result may suggest a new direction for current and future drug development.
IHC and western blot analysis of ACE2 in human kidney and lung tissue using ACE2 antibodies (A) Immunohistochemical analysis of ACE2 in paraffin-embedded human kidney tissue using an ACE2 Recombinant Rabbit Monoclonal antibody (SN0754) (Cat. No. MA5-32307).(B) Western blot analysis of ACE2 in human kidney tissue by an ACE2 monoclonal antibody (CL4013) (Cat. No. MA5-31394). (C) Immunohistochemical analysis of ACE2 in paraffin-embedded carcinoma of human lung tissue with an ACE2 monoclonal antibody (OTI4D2) (Cat. No. MA5-26628).
TMPRSS2 and SARS-CoV-2
TMPRSS2 belongs to the serine protease family, and it contains a type II transmembrane domain, a receptor class A domain, a scavenger receptor cysteine-rich domain, and a protease domain. Human TMPRSS2 is expressed in the prostate, colon, stomach, and salivary glands (4). In 2009, it was first published that TMPRSS2 activates influenza virus by cleaving hemagglutinin (5). Since then, accumulating evidence indicates TMPRSS2 is involved in activating different types and strains of viruses, such as influenza A virus, influenza B virus, MERS coronavirus, and SARS coronavirus. It was shown that TMPRSS2 proteolytically cleaves and activates the spike protein of SARS-CoV (6). Spike protein first attaches to the receptor protein ACE2 on the host cell membrane, and the binding triggers receptor-induced conformational changes. This conformational change is thought to expose the protease cleavage site in spike protein to serine protease TMPRSS2. TMPRSS2 then cleaves and activates spike protein, causing viral infection (7). After the recent SARS-CoV-2 global crisis, Hoffmann et al confirmed TMPRSS2 also plays a critical role in the priming of the SARS-CoV-2 spike protein (1), similar to previous findings for SARS-CoV. The same study also shows that an inhibitor of TMPRSS2 significantly reduced SARS-CoV-2 infection in a cell model, and it suggests TMPRSS2 is a potential target for treatment of
IHC and western blot analysis of prostate, endometrium tissues, and 22Rv1 cells using TMPRSS2 antibodies (A) Immunohistochemical analysis of TMPRSS2 in human prostate and endometrium tissues using a TMPRSS2 polyclonal antibody (Cat. No. PA5-83286). Corresponding RNA-seq data are presented for the same tissues. (B) Western blot analysis of extracts of 22Rv1 cells, using a TMPRSS2 Polyclonal antibody (Cat. No. PA5-96019).
Furin and SARS-CoV-2
Furin belongs to the proprotein convertase family, and it is a type I transmembrane protein. It is expressed in various types of eukaryotic tissues and cells. Studies have shown furin cleaves a wide spectrum of proproteins at their multibasic motifs. Furin also activates fusion proteins of many types of viruses, such as Ebola and Yellow Fever virus. The recent global outbreak of SARS-CoV-2 is different from the previous SARS-CoV in terms of its higher infectivity yet lower immune responses reported (11). By comparing the spike protein sequences of these two viruses, researchers found that SARS-CoV-2 spike protein contains a distinct furin cleavage site within its receptor binding domain (RBD) which is not present in SARS-CoV spike protein (11,12). This finding points to the possible role of furin in the activation of SARS-CoV-2 and could explain its higher infectivity. An in vitro study using pseudoviruses, packed with SARS-CoV-2 spike protein, proved that TMPRSS2 and furin proteases both cleave SARS-CoV-2 spike protein (11). The study also shows furin pre-activates spike protein at the S1/S2 site, and TMPRSS2 activates spike protein at the S2’ site (12,13). TMPRSS2 and furin may play different roles in the activation of the spike protein to facilitate cell entry of the virus. Further study of furin’s mechanism in the viral activation may provide a target for therapy to reduce SARS-CoV-2’s infectivity.
Representative Invitrogen Furin antibody testing data (A) Immunofluorescent analysis of Furin Convertase was performed on HeLa cells using Furin Polyclonal Antibody (Cat. No. PA1-062).(B) Western Blot analysis of Furin performed on various cell lines using a Furin Monoclonal Antibody (ARC1221) (Cat. No. MA5-35627). (C) Immunohistochemistry analysis of Furin in paraffin-embedded human colon tissue. Samples were incubated with Furin Recombinant Rabbit Monoclonal Antibody (Cat. No. MA5-34677).
Neuropilin 1 and SARS-CoV-2
Many studies have focused on ACE2 when it comes to SARS-CoV-2 entry. However, it is expressed at very low levels in respiratory and olfactory epithelial cells. The low levels of ACE2 could mean that there are cofactors that are required to enable the virus. Neuropilin 1 could serve as one of those cofactors. (15)
Neuropilin 1 is a cell surface receptor that belongs to a family of signaling and catalytic proteins. It plays a role in angiogenesis, tumor progression, viral entry, axonal guidance, and immune function. (14) High neuropilin 1 expression has been seen in the epithelial surface layer of respiratory and gastrointestinal tracts–both are known to be affected by SARS-CoV-2. (15) Recent studies have shown neuropilin 1 binds furin-cleaved substrates and increases the chance of infection. The furin-cleaved S1 fragment of the spike protein binds directly to cell surface of neuropilin 1. (14) Other studies have also suggested that neuropilin 1 is responsible for the spread of the virus through the olfactory bulb into the nervous system and increased expression in the lungs of severe SARS-CoV-2 cases. (14) Further research around neuropilin 1 may provide insight to a new SARS-CoV-2 therapeutic target. (16)
Immunohistochemical and western blot analysis of Neuropilin 1. (A) Neuropilin 1 was detected in perfusion fixed frozen sections of rat spinal cord using Neuropilin 1 Polyclonal Antibody (Cat. No. PA5-47027) at 15 µg/mL overnight at 4°C. Tissue was stained using the 557-conjugated Anti-Goat IgG Secondary Antibody and counterstained with DAPI (blue). Specific staining was localized to the dorsal horn. (B) Antibody specificity was demonstrated by detection of differential basal expression of the target across tissues owing to their inherent genetic constitution. Relative expression of Neuropilin 1 was observed in higher levels in mouse heart and rat heart, and in lower levels in mouse brain, rat brain, mouse kidney, rat kidney, and mouse liver using Neuropilin 1 Polyclonal Antibody (Cat. No. PA5-47027) in western blot.
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- Zhou P, Yang XL, Wang XG, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020 Mar;579(7798):270-273. doi: 10.1038/s41586-020-2012-7. Epub 2020 Feb 3.
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- Tai W, He L, Zhang X1, et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol. 2020 Mar 19. doi: 10.1038/s41423-020-0400-4.
- Monteil V, Kwon H, Prado P, et al. Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2. Cell. Pre-Proof. DOI: 10.1016/j.cell.2020.04.004
- Shang J, Wan Y, Luo C, et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A. 2020 May 6. pii: 202003138. doi: 10.1073/pnas.2003138117.
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- Bestle D, Heindl MR , Limburg H, et al. TMPRSS2 and furin are both essential for proteolytic activation 1 and spread of SARS2 CoV-2 in human airway epithelial cells and provide promising drug targets.
- Mayi BS, Leibowitz JA, Woods At, et al. The role of Neurophilin-1 in COVID-19. PLOS Pathogens. 2021 Jan 4. doi: 10.1371/journal.ppat.1009153
- Cantuti-Castelvetri L, Ojha R, Pedro LD, et al. Neurophilin-1 facilitates SARS-CoV-2 cell entry and infectivity. Science. 2020 Nov 13. doi:10.1126/science.abd2985
- Daly JL, Simonetti B, Klein K, et al. Neuropilin-1 is a host factor for SARS-CoV-2 infection. Science. 2020 Nov 13. doi: 10.1126/science.abd3072
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