A Critical Role for Nonviral Protein Cages in Unraveling Viral Polybasic Cleavage Site Functions
Polybasic cleavage sites (PCSs) embedded within viral spike proteins constitute a pivotal factor in virus biology, modulating infectivity, tissue tropism, and interspecies transmission. These short amino acid sequences, rich in positively charged residues such as arginine and lysine, serve as specific substrates for host cell proteases. Cleavage at these sites is a prerequisite for activating viral fusion machinery, facilitating cellular entry, and promoting replication competence in target tissues. The presence of PCSs in diverse viral families, spanning phylogenetically distant representatives such as coronaviruses and avian influenza viruses, marks them as convergent evolutionary adaptations critical for viral success in mammalian hosts.
Among coronaviruses, the prototypical example of PCSs’ biological impact is found in SARS-CoV-2 and MERS-CoV. In these pathogens, the acquisition of polybasic cleavage motifs at the spike glycoprotein significantly enhances proteolytic processing by furin-like enzymes within the human respiratory tract. This biochemical modification increases the efficiency of viral entry and augments transmissibility between individuals. Multiple studies have delineated how furin-mediated cleavage exposes fusion peptides, enabling viral membrane fusion at the cell surface or within endosomal compartments. The functional consequences of such processing extend beyond mere infectivity, encompassing expanded cellular tropism and possibly increased pathogenicity.
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The influence of polybasic cleavage sites is not confined to coronaviruses. In highly pathogenic avian influenza virus subtypes, specifically H5 and H7, the hemagglutinin (HA) protein harbors similar PCS motifs. These sites permit cleavage by a broader spectrum of proteases beyond the limited trypsin-like enzymes targeting low-pathogenic strains. As a result, viruses bearing multibasic HA cleavage sites attain systemic dissemination capabilities, infecting multiple organs rather than restricting replication to the respiratory or intestinal epithelium. This viral attribute is strongly linked to increased virulence and zoonotic potential, underscoring the PCS as a molecular marker for pathogenicity shifts and pandemic risk.
At the molecular level, proteolytic cleavage of PCSs often exposes a C-terminal sequence motif characterized by a basic amino acid-rich pattern, commonly referred to as the C-end rule (CendR) motif (R/KXXR/K). This motif mediates high-affinity interactions with neuropilin (NRP) receptors, primarily NRP1 and NRP2, which are broadly expressed transmembrane proteins involved in a variety of physiological processes such as angiogenesis, immune modulation, and neuronal guidance. The binding of viral proteins containing CendR motifs to NRPs has emerged as a mechanism facilitating viral internalization, further enhancing infectivity and potentially influencing intracellular trafficking.
Neuropilins’ role as viral entry factors extends beyond coronaviruses, with evidence implicating them in the cellular uptake of Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus, among others. In SARS-CoV-2, NRP1 engagement has been shown to augment infectious entry, especially in cell types with low ACE2 receptor availability. However, a comprehensive understanding of whether all viruses harboring PCSs exploit NRPs for entry remains unresolved. It is unclear if neuropilins act alone or as part of a co-receptor complex, and to what extent auxiliary host factors modulate the internalization and subsequent intracellular fate of these viruses following PCS cleavage.
The evolutionary emergence of PCSs raises pressing questions around their role in zoonotic spillover and viral adaptation. Are polybasic motifs representative of convergent evolutionary pressures favoring enhanced protease susceptibility? Do PCS sequences alter viral fitness in the natural reservoir hosts or only upon transmission to humans? Experimental dissection of PCSs’ functions traditionally relies on reverse genetics techniques that introduce or delete cleavage sites within viral genomes. Yet, such manipulations are fraught with challenges due to potential lethality from impaired replication or unexpected gain-of-function phenotypes, amplifying biosafety concerns and ethical debates surrounding pathogen research.
In response, innovative methodologies have been proposed to circumvent these experimental limitations, among which nonviral protein cages (NVPCs) emerge as compelling platforms. These self-assembling proteinaceous nanostructures mimic the size and, to an extent, the geometry of viral capsids while lacking infectious material. By engineering NVPCs to display spike proteins containing native or modified PCSs or presenting isolated CendR motifs, researchers can probe the molecular mechanisms of host receptor binding, protease susceptibility, and internalization pathways in a controlled, biosafe environment. This strategy enables the decoupling of structural and functional viral studies from the risks associated with live pathogen manipulation.
Protein cages endowed with fluorescent or contrasting agents further facilitate high-resolution imaging studies of viral entry and intracellular trafficking routes. Site-specific incorporation of fluorophores or encapsulation of fluorescent proteins within cages permits real-time visualization using confocal or super-resolution microscopy techniques. These visualizations allow kinetic mapping of endocytic pathways, vesicular sorting, and the identification of subcellular compartments involved in the processing of PCS-bearing particles. Such insights are invaluable for understanding the spatiotemporal coordination of viral entry and subsequent steps defining the infectious cycle.
In complement to imaging, proteomics approaches leveraging laser microdissection enable the isolation and molecular profiling of cells that engage with virus-mimicking cages. This permits the identification of host proteins interacting with the virus-like particles during endocytosis or trafficking. Further layer-specific fractionation techniques provide detailed maps of protein distribution across cellular compartments, elucidating potential host receptors, adaptor molecules, or signaling components mediating PCS-driven viral uptake. Subsequent functional investigation utilizing small-molecule inhibitors, RNA interference, or CRISPR-mediated gene editing can validate the roles of candidate host factors in modulating viral internalization.
Structurally, NVPCs offer advantages for cryo-electron microscopy (cryo-EM) investigations due to their uniform size and inherent symmetry. These properties greatly simplify image reconstruction and improve resolution, crucial for dissecting the conformational impacts of PCS insertions or mutations within viral glycoproteins. For example, NVPCs engineered to present mutated influenza HA proteins bearing introduced polybasic sites can reveal structural alterations correlating with enhanced proteolytic accessibility and virulence. Notwithstanding, the lack of a lipid envelope and smaller size compared to native enveloped viruses like influenza may restrict the full recapitulation of native virus-host interactions.
To overcome these inherent limitations, efforts to pseudotype protein cages with viral envelopes are underway. This approach combines the structural precision of proteinaceous scaffolds with the biological complexity of a membrane bilayer, yielding more faithful virus mimics. Such pseudotyped particles could revolutionize the study of PCSs by enabling assays that simultaneously reflect the structural, functional, and phenotypic consequences of cleavage site variants in a safe laboratory context. Integrative analyses combining structural biology, cell biology, and phenotypic assays promise to elucidate the genotype-to-phenotype continuum governing PCS-mediated viral virulence.
Continued exploration of PCS functions facilitated by nonviral protein cage platforms not only enhances fundamental virology but also informs therapeutic strategies. Understanding how PCSs influence neuropilin-mediated entry or protease susceptibility can guide the development of inhibitors targeting critical protease interactions or host receptor binding. Moreover, protein cage technologies may serve as scaffolds for vaccine antigen display or as delivery vehicles for antiviral compounds, leveraging their modularity and safety profile. Thus, the convergence of protein nanotechnology with viral pathogenesis research heralds innovative avenues to combat emerging viral threats.
In essence, polybasic cleavage sites represent molecular fulcrums within viral glycoproteins that modulate host adaptation, transmission dynamics, and pathogenic potential. The strategic employment of nonviral protein cage systems offers unprecedented opportunities to dissect these multifaceted roles without the risks inherent in handling live pathogenic viruses. Through multidisciplinary integration encompassing structural biology, cell imaging, proteomics, and genetic tools, this emerging approach stands to illuminate key viral mechanisms and accelerate the development of countermeasures against current and future viral pandemics.
Subject of Research:
The investigation and characterization of polybasic cleavage sites in viral glycoproteins using nonviral protein cages as biosafe experimental tools to elucidate mechanisms of viral entry, host interactions, and pathogenicity.
Article Title:
Nonviral Protein Cages as Tools to Decipher and Combat Viral Threats
Article References:
Levasseur, M.D. Nonviral protein cages as tools to decipher and combat viral threats.
npj Viruses 3, 45 (2025). https://doi.org/10.1038/s44298-025-00127-8
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Tags: convergent evolution in virusesfurin-like enzymes in viruseshost cell proteases in viral biologyMERS-CoV proteolytic processingnonviral protein cagesprotein engineering for viral defenseSARS-CoV-2 spike proteintissue tropism in virusesviral entry mechanismsviral polybasic cleavage sitesviral replication competenceviral transmission dynamics
