Artistic rendition of virus binding cell surface receptors based on scanning electron micrographs

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.

Western blot analysis shows detection of spike proteins in SARS-CoV and SARS-CoV2

Western blot analysis shows detection of spike proteins for both SARS-CoV and SARS-CoV-2. Proteins were loaded on a reducing SDS-PAGE. Lane A: SARS-CoV Spike S1, 5 ng; Lane B: SARS-CoV-2 Spike S1, 30 ng; Lane C: SARS-CoV-2 Spike RBD, 30 ng. Separated proteins were transferred to a membrane and probed with a SARS/SARS-CoV-2 Coronavirus Spike Protein Rabbit Polyclonal Antibody (Cat. No. PA5-81795) at 1:2,000 dilution. The membrane was then probed with goat anti-rabbit IgG (H+L), HRP conjugated secondary antibody diluted 1:10,000. The proteins were visualized using ECL.

ACE2 and SARS-CoV-2

ACE2 Antibodies

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 analysis of ACE2 in human kidney tissue.
A
IHC analysis of ACE2 in carcinoma of human lung tissue.
C

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 Antibodies

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 SARS-CoV-2.

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).

Western blot analysis of TMPRSS2 in 22Rv1 cells.
B

Furin and SARS-CoV-2

Furin Antibodies

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).


References

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