The genome of coronaviruses encodes for the production of around 30 viral proteins that perform specific essential functions contributing to the proliferation and escape of the virus from the host cell. Disrupting these proteins and their interaction with the host cellular machinery, therefore, is an attractive broad-spectrum antiviral drug strategy. Such a therapy works by preventing the proliferation of the virus while being unlikely to induce the development of resistance against the therapy in the virus.
Viral short linear interaction motifs (SLiMs) are peptide sequences that mediate protein-protein interactions, providing ligand binding and modification sites. Some viruses, including SARS-CoV-2, utilize SLiMs to disrupt protein signaling in the host cell, lessening the immune response. In a new study recently uploaded to the preprint server bioRxiv*, proteomic peptide-phage display is utilized to profile the novel SLiM-based interactions that mediate host factor interactions in 229 RNA viruses, with a special focus on SARS-CoV-2, to reveal mechanistic insights and identify drug targets.
How was the study performed?
A phage display library that contained 19,549 unique sixteen amino acid long peptides sourced from the unstructured regions of over a thousand proteins from RNA viruses was assembled, which included over 20 types of coronavirus. One hundred thirty-nine host factor proteins that are thought to interact with SARS-CoV-2 were also recombinantly produced, and next-generation sequencing was employed to identify the viral peptides that bound with these proteins.
SARS-CoV-2, SARS-CoV-1, and MERS-CoV exhibited a number of both common and unique interactions with human host factors, three of which were selected for further investigation as competitive antiviral agents. Vectors expressing green fluorescent protein fused with four copies of each SLiM were produced, with controls in the form of SLiMs with mutated binding motifs also being synthesized.
The peptides selected targeted host proteins G3BPs, Ezrin and Radixin, and MAP1LC3s, with the first being selected in particular for further investigation. The SARS-CoV-2-N (nucleocapsid) peptide that interacts with G3BP is involved in viral replication and packaging of viral RNA, while G3BP is known to play a role in cellular immune signaling and the assembly of cytosolic stress granules, large protein-RNA assemblies that form in response to infection. Several other viruses are known to inhibit G3BP, or to recruit G3BP to support viral replication, and so a highly efficacious inhibitor could make an excellent wide spectrum drug target.
Antiviral G3BP inhibitors
Cells were transduced with the Lentivirus vector and later infected with SARS-CoV-2, then the viral titer was determined after 16 hours. The presence of the labeled G3BP binding peptide in the infected cells reduced viral titer by around 3.4 times compared to untreated cells, suggesting that the peptides had competitively inhibited the function of the SARS-CoV-2 sourced SLiMs, and therefore overall viral replication rate, with high specificity. In search of a more potent inhibitor of G3BP, the group tested a 25 amino acid residue sourced from Semliki Forest virus nsp3, known to bind around ten times more strongly to the protein than the SARS-CoV-2-N sourced peptide. Virus proliferation was significantly more potently inhibited when this peptide was present in cells, further indicating that blocking the access of SARS-CoV-2 peptides to G3BP could make an appealing drug strategy.
In investigating how these peptides interact with G3BP, the group found that an FxFG motif belonging to the SARS-CoV-2-N G3BP binding peptide was responsible for G3BP inhibition and the resulting stress granule disassembly observed in SARS-CoV-2 infection, with wild-type better inhibiting formation than one bearing a mutated FxFG motif.
VeroE6 cells were infected with SARS-CoV-2, and at six hours post-infection, the cells exhibited varied concentrations of the N peptide, so were categorized between early-stage (low) and late-stage (high). The peptide was found to localize with G3BP around stress granules at early stages, while they localized less acutely in later stages. The authors propose that this suggests that during the early stages of infection, N peptide levels are insufficient to disrupt stress granule formation, localizing within these structures until concentrations are high enough to disrupt the granules, or indicating that the virus takes advantage of stress granule RNA machinery. This work potentially demonstrates that a peptide-based inhibitor that disrupts G3BP interaction with SARS-CoV-2 could make an efficacious and broad-spectrum antiviral.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
- Thomas Kruse, Caroline Benz, Dimitriya H. Garvanska, Richard Lindqvist, Filip Mihalic, Fabian Coscia, Ravi Teja Inturi, Ahmed Sayadi, Leandro Simonetti, Emma Nilsson, Muhammad Ali, Johanna Kliche, Ainhoa Moliner Morro, Andreas Mund, Eva Andersson, Gerald McInerney, Matthias Mann, Per Jemth, Norman E Davey, Anna K Överby, Jakob Nilsson, Ylva Ivarsson. Large-scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities. bioRxiv. doi: https://doi.org/10.1101/2021.04.19.440086, https://www.biorxiv.org/content/10.1101/2021.04.19.440086v1