Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets.

The infection and replication cycle of all viruses depend on interactions between viral and host proteins. Each of these protein-protein interactions is therefore a potential drug target. These host-virus interactions often involve a disordered protein region on one side of the interface and a folded protein domain on the other. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind peptides from human proteins. Of 281 high/medium confidence peptides, 23 interactions involving eight SARS-CoV-2 protein domains were tested by fluorescence polarization, and binding was observed with affinities spanning the whole micromolar range. The key specificity determinants were established for six of these domains, two based on ProP-PD and four by alanine scanning SPOT arrays. Finally, two cell-penetrating peptides, targeting Nsp9 and Nsp16, respectively, were shown to function as inhibitors of viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously provide information on potential host-virus interactions and identify ligands with antiviral properties.

A syntenin inhibitor blocks endosomal entry of SARS-CoV-2 and a panel of RNA viruses

Viruses are dependent on interactions with host factors in order to efficiently establish an infection and replicate. Targeting such interactions provides an attractive strategy to develop novel antivirals. Syntenin is a protein known to regulate the architecture of cellular membranes by its involvement in protein trafficking, and has previously been shown to be important for HPV infection. Here we show that a highly potent and metabolically stable peptide inhibitor that binds to the PDZ1 domain of syntenin inhibits SARS-CoV-2 infection by blocking the endosomal entry of the virus. Furthermore, we found that the inhibitor also hampered chikungunya infection, and strongly reduced flavivirus infection, which are completely dependent on receptor mediated endocytosis for their entry. In conclusion, we have identified a novel pan-viral inhibitor that efficiently target a broad range of RNA viruses.

Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, current methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a scalable viral peptide discovery approach covering 229 RNA viruses that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an {Phi}xFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its {Phi}xFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction blocks SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.

Cytoplasmic short linear motifs in ACE2 and integrin beta3 link SARS-CoV-2 host cell receptors to endocytosis and autophagy

The spike protein of the SARS-CoV-2 interacts with angiotensin converting enzyme 2 (ACE2) and enters the host cell by receptor-mediated endocytosis. Concomitantly, evidence is pointing to the involvement of additional host cell receptors, such as integrins. The cytoplasmic tails of ACE2 and integrin {beta}3 contain a plethora of predicted binding motifs. Here, we confirm the functionality of some of these motifs through affinity measurements. The class I PDZ binding motif in the ACE2 cytoplasmic tail binds the first PDZ domain of the scaffold protein NHERF3. The clathrin-adaptor subunit AP2 2 interacts with an endocytic motif in the ACE2 with low affinity and the interaction is abolished by phosphorylation of Tyr781. Furthermore, the C-terminal region of integrin b3 contains a LC3-interacting region, and its interaction with ATG8 domains is enhanced by phosphorylation. Together, our data provides possible molecular links between host cell receptors and endocytosis and autophagy. One sentence summaryAffinity measurements confirmed binding of short linear motifs in the cytoplasmic tails of ACE2 and integrin {beta}3, thereby linking the receptors to endocytosis and autophagy.