NRP-1 receptor enhances SARS-CoV-2 infectivity

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The COVID-19 pandemic remains active in many parts of the world, with dual efforts to develop antivirals and vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Is causing. Recent studies have investigated the role of a second type of cell surface receptor, called neuropilin, in the invasion of viruses into host cells and their subsequent replication.

Wind chime cleavage presents CendR peptide

SARS-CoV-2 belongs to the same family as SARS-CoV and MERS-CoV and caused early development of SARS and MERS. It is particularly similar to SARS-CoV, both of which bind to the same host cell receptor, angiotensin converting enzyme 2 (ACE2). But it spreads much more extensively and rapidly. SARS-CoV spreads primarily through the lower respiratory tract system, while the other is transmitted through active pharyngeal shedding of upper respiratory tract secretions.

One of the most plausible reasons for SARS-CoV-2 to be highly contagious is the multibasic furin cleavage site at the interface between the two characteristic subunits S1 and S2. Spike protein.. This site is not found in SARS-CoV, but is found in several other pathogens, especially Ebola, the toxic avian influenza strain, and the HIV-1 spike protein.

This new cleavage site may increase the ability of the virus to cause clinical disease by setting the stage at which the virus fuses with the cell membrane, thereby allowing the virus to enter the cell and establish an infection. ..

In addition, it may generate more receptor binding sites on the spike protein. When this site is cleaved by furin, a conserved C-terminal sequence containing arginine (and possibly lysine) is displayed on the spike protein. This array conforms to the C-end rule (CendR). That is, it can be involved and activated by another type of cell surface receptor, neuropyrin 1 and 2.

Cryogenic electron microscopy confirms that the S1 / S2 interface can be used to interact with the receptor.

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Spike-NRP1 binding promotes viral cell invasion

Researchers tested the ability of pseudoviruses expressing the SARS-CoV-2 S protein to be involved in NRP1 and invade and infect host cells in the absence of ACE2.Advantage of Pseudovirus In this situation, you can test for virus invasion separately from the rest of the life cycle. They used plasmid-transfected ACE2 and NRP1-negative cells to allow expression of ACE2 with either TMPRSS2, a transmembrane serine protease, or NRP1.

When only ACE2 was expressed, the cells were more susceptible to infection. When NRP1 was expressed alone, the level of infection was very low. However, expression of ACE2, TMPRSS2, and NRP1 together has been shown to significantly enhance infection, with the latter enhancing virus invasion and infection.

NRP1 inhibition reduces viral invasion

Next, we developed a monoclonal antibody (mAb) that functionally blocks the b1b2 domain of NRP1 that binds to the CendR peptide. They found that one of them could block the binding and internal migration of silver nanoparticles carrying the classic CendR peptide showing NRP1 binding.

This antibody was used to treat cells carrying ACE2, TMPRSS2, and NRP1 and significantly reduced infection. This was not seen in cells that lack NRP1 but have ACE2 and TMPRSS2. In addition, when the pseudovirus was pre-incubated with the recombinant soluble b1b2 domain of NRP1-only, the level of infection was significantly reduced compared to the above results before infecting the above cells.

Using a mutant form of SARS-CoV-2 that has not undergone cleavage, unlike wild-type virus, the mutant virus carries ACE2 / TMPRSS2 / NRP1 compared to cells expressing ACE2 or. Found that it does not cause high levels of infection. ACE2 / TMPRSS2. This supports the need for furin cleavage to produce a CendR peptide that can bind to the NRP1 receptor. Preincubation with NRP1-mAb reduced wild-type infection by 40%, but not mutant virus.

NRP1 expressed in infected cells

Researchers also showed that specific C-terminal sequences generated by spike protein cleavage at the S1 / S2 junction were taken up at high levels by NRP1-expressing cells and that NRP1 expression was high in the mouse olfactory epithelium. It was. In fact, in the latter case, the peptide was also detected in neurons and blood vessels in the tested mouse brain. This indicates that it is a CendR peptide.

They then decided to look at already published data on proteins found in infected cells from two sources. One is human bronchial epithelial cells (HBEC) and the other is cells recovered from the bronchoalveolar lavage fluid (BALF) of severely ill COVID-19 patients. Both showed elevated levels of NRP1. BALF-infected cells also had higher levels of NRP1 and NRP2 RNA transcripts compared to non-infected cells.

NRP1 expressed in olfactory neurons

Comparing published data on the expression of ACE2 and NRP1 in human lung and olfactory tissues, we found that ACE2 levels were very low or almost undetectable. Nevertheless, both NRP1 and NRP2 are expressed at high levels in all cells of these tissues, especially endothelial cells. NRP1 was also detected in developing and immature olfactory neurons.

Because anosmia was found in many COVID-19 patients, they searched for evidence of SARS-CoV-2 infection in the olfactory epithelium and found it in 5/6 cases. In all five, infected cells also express high levels of NRP1 and can be traced especially to newly developing olfactory neurons.

Implications

Current research sheds new light on one of the interesting facts about SARS-CoV-2. Tissues that are preferentially infected with the virus are not tissues that express high levels of ACE2. This indicates that, like other viruses, it relies on other factors to achieve cell invasion and infection. This role appears to be compatible with NRP1 as an enhancer or even as an ACE2-independent entry route in the presence of high viral titers.

This function of NRP may be due to high levels of expression in externally exposed epithelial cells and the ability to transport bound molecules across vascular boundaries, across cell membranes, and into tissues.

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