Researcher at Sheffield University has developed a new test that can be used to assess the attachment of viruses to host cells and to test the potential inhibitors of viral infections.
Using the test, the team was able to demonstrate the binding of a spike protein in acute coronavirus 2 (SARS-CoV-2) respiratory syndrome to human cells expressing the angiotensin 2 converting enzyme (ACE2).
Spike proteins are the main structure used by SARS-CoV-2 to bind ACE2 receptors expressed on target cells, before infecting them and potentially causing coronavirus 2019 (COVID-19).
The SARS-CoV-2 virus binds to ACE-2 receptors in human cells, the initial stage of COVID-19 infection. Illustrated credit: Kateryna Kon / Shutterstock
The researchers also found that incubating cells with unfracted heparin stopped the surge of proteins that bind them.
The pre-printed version of paper can be accessed on the server bioRxiv*, while this paper underwent peer review.
SARS-CoV-2 infection mechanism
On binding to ACE2, the spike protein undergoes proteolytic division of the host cell into two subunits: S1, which contains receptor binding domains (RBD) and S2, which allow fusion with the host cell membrane and virus infusion.
“Serine host cell surface protein, TMPRSS2 [transmembrane serine proteinase 2]”It was also thought to be involved in virus entry and it was proposed to split S1 and S2, which led to the activation of the fusion machine,” wrote Peter Monk and colleagues.
The new test uses cells that express ACE2 and TMPRSS2
To investigate SARS-CoV-2 that binds to host cells, the team developed a new test using a transitional urinary bladder RT4 carcinoma cell line, which expresses ACE2 and TMPRSS2.
They found that the intact recombinant form of the viral surge protein containing both S1 and S2 (S1S2), but not only in the S1 domain, binds strongly to RT4 cells in a temperature-dependent manner.
The binding activity increased sharply at 37 ° C, indicating that proteolytic cleavage might be involved, the team said.
Are there other mechanisms for virus entry?
Monk and colleagues say that most cell types only express ACE2 levels that are low enough, suggesting that the surge protein might also interact with other receptor sites to get virus entry.
Certain viruses such as herpes simplex are known to bind to the host glycosaminoglycan called sulfuric deposits, the team said.
In addition, a study by one group suggested that soluble glycosaminoglycan heparin could inhibit the entry of SARS CoV-2 into “Vero” cells – cell lines derived from monkey kidney epithelium.
“These authors also demonstrated that heparin can interact with recombinant S1 RBD and cause conformational changes, leading to the suggestion that SARS-CoV-2 might use sulfate liver hosts as an additional site for attachment during infection,” the researchers wrote.
Unfracted heparin completely stops the bond
Given that the new test seems to mimic some features of SARS-CoV-2 infection, the researchers used it to test the effect of incubating RT4 cells with heparin at 37 ° C.
The team reports that unfrracted heparin (UFH) actually inhibits the binding of S1S2 cells to RT4.
Treating cells with two low molecular weight heparin (LMWHs) that has been used clinically also inhibits binding, but only partially and not as strongly.
“This shows that heparin, especially the non-diffracted form, can be considered to reduce the clinical manifestations of COVID-19 by inhibiting ongoing viral infections,” wrote Monk and the team.
Can spike proteins also bind to the host cell’s sulfate supply?
The authors say the interaction they observed between heparin and protein spike suggests that it might also bind to sulfate liver cells.
To test this hypothesis, they treated RT4 cells with a mixture of heparinase I and III, enzymes that degrade sulfate molecules, before testing the binding of S1S2.
Treatment did not result in a significant reduction in RT4 cell binding, indicating that sulfate exposure did not play a significant role in the attachment of the SARS-CoV-2 surge protein to host cells:
“Although our data support UFH inhibitory activity, it does not support the notion that sulfate deposits are very important for viral infections,” the team wrote.
What are the implications of this research?
The researchers say that LMWHs, which have been used to treat COVID-19 patients and have been shown to improve results, are much smaller than UFH and have a more predictable pharmacokinetics.
Monks and colleagues argue that their research shows that previous use of heparin should be considered when a viral infection is still an important factor in influencing the severity of the disease.
“The use of UFH rather than LMWH must also be considered, although we note that the administration and safety profile of UFH might prevent this in some cases,” they added.
Finally, the researchers said their newly developed flow cytometric test to assess the binding of SARS-CoV-2 spike protein to the host cell supports the previous findings that heparin can inhibit viral attachment to monkey kidney epithelial cells.
“Our new test could be the first screen to be useful for new inhibitors of coronavirus infection,” concluded the team.
* Important Notification
bioRxiv publishing initial scientific reports that are not reviewed by colleagues and, therefore, should not be considered conclusive, guide clinical practice / health-related behaviors, or be treated as pre-existing information.