Researchers have identified a second receptor that might be important in SARS-CoV-2 infectivity, according to a new report published in Science on October 20. This cofactor helps facilitate virus-host cell interactions in cells with low levels of angiotensin-converting enzyme 2 (ACE2), the primary receptor on host cells, and might explain the increased pathogenicity of SARS-CoV-2.

Why is SARS-CoV-2 more pathogenic than previous coronaviruses?

Both SARS-CoV and SARS-CoV-2 uptake is mediated by ACE2 cellular receptors. But unlike the 2003 coronavirus, SARS-CoV-2 has a polybasic furin-type cleavage site at the junction of its S1 and S2 subunits in the spike protein. Similar sites are found on other pathogenic viruses including Ebola, HIV-1, and highly virulent strains of avian flu.

“That SARS-CoV-2 uses the receptor ACE2 to infect our cells was known, but viruses often use multiple factors to maximize their infectious potential,” explained author Giuseppe Balistreri, PhD, of the University of Helsinki, in a statement.

The presence of the polybasic furin cleavage site can enhance pathogenicity of the virus by priming its fusion activity to create additional cell surface receptor-binding sites, including a carboxyterminal motif in the spike protein that has been observed from structures of SARS-CoV-2 captured via cyroelectron microscopy.

These sequences are known to bind and activate neuropilin (NRP1 and NRP2) receptors at cell surfaces. NRP1 and homolog NRP2 are abundantly expressed in almost all pulmonary and olfactory cells.

To determine if SARS-CoV-2 could indeed use NRP1 for viral entry and infectivity, researchers from the Technical University of Munich and the University of Helsinki pseudotyped lentiviral particles with SARS-CoV-2 spike proteins. Cells that have almost no detectable ACE2 or NRP1 were then transfected with ACE2 and transmembrane protease, serine 2 (TMPRSS2), or NRP1.

The University of Helsinki team found that while NRP1 alone hardly promoted infection in cells, when coexpressed with ACE2 and TMPRSS2, there was a noticeably enhanced infection. In Caco-2 cells, which endogenously express ACE2, NRP1 expression increased infection, suggesting that it can potentiate infection in the presence of other host factors.

“If you think of ACE2 as a door lock to enter the cell, then neuropilin-1 could be a factor that directs the virus to the door,” said Balistreri. “ACE2 is expressed at very low levels in most cells. Thus, it is not easy for the virus to find doors to enter. Other factors such as neuropilin-1 might help the virus finding its door.”

Next, the researchers explored the role of NRP1 in cell culture experiments with wild-type SARS-CoV-2 and a mutant that accumulates mutations in the furin cleavage site of the spike protein. They found that infection by wild-type viruses, but not the mutant virus, increased in the presence of NRP1, suggesting that NPR1 requires a furin-cleaved substrate to be effective.

They also tested to see if the specific furin cleavage site sequences could be taken up in vivo by attaching them to silver nanoparticles and administering them into the nose of anesthetized mice. They observed increased uptake into the olfactory epithelium with this treatment.

Investigating a new antiviral strategy

The researchers developed monoclonal antibodies that were designed to functionally block the extracellular b1b2 domain of NRP1, which is known to mediate the binding of sequences on the spike protein. They confirmed that the antibodies did bind to the specified domain in vitro. Cell culture treatment with the antibodies significantly reduced infection by SARS-CoV-2 pseudotyped viruses in cells expressing ACE2, TMPRSS2, and NRP1, but not in cells expressing only ACE2 and TMPRSS2.

“It is currently too early to speculate whether blocking directly neuropilin could be a viable therapeutic approach, as this could lead to side effects,” Balistreri concluded. “This will have to be looked at in future studies. Currently our laboratory is testing the effect of new molecules that we have specifically designed to interrupt the connection between the virus and neuropilin. Preliminary results are very promising and we hope to obtain validations in vivo in the near future.”