The coronavirus disease 2019 (COVID-19) pandemic has lead to millions of deaths and a worldwide economic crisis. Initial attempts to stall the disease focused on social distancing, compulsory mask-wearing, and lockdowns that closed public places. Once mass vaccination schemes began to reduce the number of cases, many governments began to reduce these restrictions and allow travel between nations once again. This led to many local variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, spreading worldwide.

Study: Mutating novel interaction sites in NRP1 reduces SARS-CoV-2 spike protein internalization. Image Credit: CROCOTHERY/ Shutterstock

Many of these variants can evade both vaccine-induced and natural immunity to the wild-type strain initially found in Wuhan. Of particular concern is the Delta strain, which is currently the most common cause of new infections worldwide. Researchers from the Oak Ridge National Laboratory have been investigating altered protein conformations in variants of concern (VOCs) to provide further insight for future therapies against COVID-19.

A preprint version of the study is available on the bioRxiv* server while the article undergoes peer review.

Background

The COVID-19 spike protein is essential for pathogenesis and the target for many vaccines against the disease. The receptor-binding domain (RBD) of the S1 subunit can bind to angiotensin-converting enzyme 2 (ACE2) to permit viral cell entry, and the N-terminal domain of the S2 subunit is responsible for membrane fusion. The spike protein can also use neuropilin 1 (NRP1) as a binding site. NRP1 is a single membrane-spanning non-tyrosine kinase receptor protein. NRP2 is a homolog with 44% identical sequences. Both contain two CUB domains, two coagulation factors, a MAM, and a domain that contains a transmembrane and a short cytoplasmic region. NRP1 is a receptor for class II semaphorins, certain endothelial growth factors, and several other extracellular ligands.

It is essential in angiogenesis and semaphorin dependant axon guidance. The semaphorin is synthesized as an inactive form and requires cleavage to become active. This cleavage can be performed by furin. The SARS-CoV-2 spike protein also requires furin cleavage for activation. The researchers attempted to discover the mechanism by which the spike protein is internalized through NRP1.

The study

Many orthologs of NRP carry a Plasminogen-Apple-Nematode (PAN) domain that contains four to six conserved cysteine residues as a core. Mutations of these amino acids lead to significant immune changes. The researchers identified four cysteine residues in human NRP1 that could suggest the formation of a vestigial PAN domain and theorized that these could allow SARS-CoV-2 binding.

They mutated each of the cysteine residues to alanine and then mutated all four cysteine residues to alanine. Any mutation increased protein stability of NRP1 in HEK cells, with more mutations showing a larger effect. They then assessed the impact of these mutations on the ability of SARS-CoV-2 S1 binding. If any of the first three cysteine residues were mutated, the spike protein showed a significantly reduced ability to bind.

Following confirmation of these results with immunofluorescence data, the researchers discovered that the ability of the S1 subunit to bind to the mutated cells was reduced up to four times. The full-length protein showed also showed significantly reduced binding. As VOCs are currently the most prevalent COVID-related threat, the scientists also examined the ability of the alpha and beta variants to bind to the mutated NRP1, showing equally hindered ability.

Conclusion

The authors highlight their discovery's importance as potential therapeutic targets against COVID-19, even across different variants with different spike protein conformations. SARS-CoV-2 S1 showed massively reduced ability to bind to cells that contained NRP1 with four mutated cysteine residues, even if a fully intact ACE2 receptor was present. Targeting these four cysteine residues could show significant promise in creating anti-covid therapies, which are sorely needed as VOCs continue to emerge with different spike protein conformations.

Multiple studies have shown significantly lower neutralizing antibody effectivity, antibody binding, and immune response to several of these VOCs, especially in immunocompromised individuals. Creating a therapy that could work across variants could be critical in preventing avoidable deaths as restrictions loosen, and cases in many areas begin to rise again. The aforementioned therapy could also be of great use to countries worldwide that are struggling to vaccinate their population due to bottlenecks in production and transport and the extensive logistic requirements needed to provide a cold chain necessary for long-distance vaccine stability.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behaviour, or treated as established information

Written by

Sam Hancock

Sam completed his MSci in Genetics at the University of Nottingham in 2019, fuelled initially by an interest in genetic ageing. As part of his degree, he also investigated the role of rnh genes in originless replication in archaea.