Jul 2026· International Journal of Molecular Sciences· Vol 27, pp. 6022· 0 citations· 169 references
Medicine
TL;DR
Thermodynamic association profiles and verified interactomic data from BioGRID reveal a strong propensity for ORF7b to participate in liquid–liquid phase transitions alongside human and viral partners, and a robust, physically grounded framework that identifies specific molecular priorities for future in vitro and in vivo validation.
Abstract
Accessory proteins of SARS-CoV-2 play crucial roles in viral pathogenesis, yet their structural properties remain elusive. ORF7b, a small accessory protein comprising only 43 amino acids, is widely assumed to parallel the structure–function relationships of its SARS-CoV ortholog based solely on sequence homology. In this study, we challenge this paradigm through direct physicochemical and structural characterization. Sequence analysis and electrostatic profiling reveal that the SARS-CoV-2 protein is a macromolecular polyanion with a net charge of −4 at neutral pH, featuring a diffuse negative surface that is highly responsive to pH changes. Complete 3D structures generated via ab initio modeling display a helical core flanked by two highly fluctuating, disordered termini. Residue Interaction Network (RIN) topology and Normal Mode Analysis (NMA) identified specific hinges governing these flexible extremities. Furthermore, the calculated dipole moment vector is tilted outward by 24°, misaligning with the central axis. Molecular dynamics simulations suggest that while the soluble structure is highly stable in water, it undergoes severe distortions and insufficient solvation within a membrane-mimetic environment. Thermodynamic association profiles and verified interactomic data from BioGRID reveal a strong propensity for ORF7b to participate in liquid–liquid phase transitions alongside human and viral partners. Taken together, these unique properties suggest that ORF7b operates as a dynamic peripheral membrane protein rather than a sedentary transmembrane component, providing a fresh framework for future therapeutic targeting. Overall, these in silico findings shift the current paradigm on ORF7b2 topology and provide a robust, physically grounded framework that identifies specific molecular priorities for future in vitro and in vivo validation.
Mapping transient protein-protein interactions remain a major challenge in studying viral host-pathogen interfaces. While some virus-host interactions are stable and readily captured, the majority are highly dynamic, reflecting the need for a small number of viral proteins to engage distinct host factors at different stages of the life cycle. Here, we employ a protein engineering strategy based on the site-specific incorporation of the non-canonical amino acid p-azido-L-phenylalanine (AzF) to enable photo-crosslinking proteomic analysis of the SARS-CoV-2 accessory protein Orf3a in live cells. Genetic installation of AzF at residue K198 of Orf3a permitted UV-induced covalent capture of proximal host interacting proteins, overcoming challenges associated with membrane localization and limited protein abundance. A total of 248 high-confidence Orf3a-interacting proteins were reproducibly identified and subjected to gene ontology analysis, revealing enrichment in innate immune signaling, antiviral defense, RNA processing, and viral replication-associated pathways. Orf3a is an accessory protein that functions as a viroporin and traffics across multiple cellular compartments, and was found to interact with host RNA helicases, RNA-binding proteins, immune regulators, and metabolic enzymes implicated in SARS-CoV-2 infection. Together, these results demonstrate that genetically encoded, site-specific photo-crosslinking enables selective capture of transient interactions that are often missed by nonspecific 254nm UV crosslinking approaches and highlights Orf3a as a multifunctional protein that engages diverse host pathways. More broadly, this study establishes a generalizable framework for leveraging non-canonical amino acid-based protein engineering approaches to interrogate dynamic host-pathogen interactions.
E. Lundrigan, Noreen Ahmed, J. P. Pezacki· Protein engineering, design...· 0 citations
Structural and biochemical studies have elucidated the architecture of the nsp14 N7-methyltransferase domain, revealing an S-adenosyl-L-methionine (SAM)-dependent fold with a defined cofactor-binding site and an adjacent cap-binding pocket that orients the RNA substrate for methyl transfer.
Yifan Zhao, Rhea Guo, Yang Yang et al.· Microorganisms· 0 citations
Protein aggregation is increasingly recognized as a biologically relevant process in viral proteins, yet the molecular determinants governing such phenomena remain poorly understood. Intrinsically disordered proteins (IDPs) are ubiquitous in viral proteomes, where conformational plasticity not only enables functional diversity but also permits aberrant self‐assembly. Members of the Paramyxoviridae family, including henipaviruses such as the Nipah and Hendra viruses (two biosafety level‐4 pathogens), encode the P, V, and W proteins that share a common intrinsically disordered N‐terminal domain (NTD). V and W are virulence factors that undergo fibrillation. Here, we explored the aggregation landscape of seven homologous PNT1 subdomains from the NTD that harbors a conserved cryptic amyloidogenic region (CAR). By integrating Taylor dispersion analysis (TDA), Raman spectroscopy, and all‐atom molecular dynamics (MD) simulations, we unraveled both early assembly kinetics and structural outcomes. Despite the conserved CAR motif, the PNT1 homologs exhibit strikingly divergent aggregation behaviors. TDA revealed distinct oligomerization pathways with variations in nucleation, growth kinetics, and monomer recruitment, indicating virus‐specific pathways. Raman spectroscopy unveiled substantial variations in β‐sheet content, side‐chain packing, and aromatic residue environments, while MD simulations showed that conformational heterogeneity in the CAR flanking regions modulates hydrogen‐bond networks and structural stability. These findings indicate that the sequence context beyond a conserved amyloidogenic core governs the aggregation landscape of Henipavirus P, V, and W proteins. Evolutionary diversification modulates conformational heterogeneity and assembly pathways, giving rise to distinct structural outcomes. This work sheds light on the molecular grammar underlying viral protein fibrillation with potential implications for viral pathogenicity.
Harshita Sawdekar, Julien Mignon, Frank Gondelaud et al.· Protein Science· 0 citations
The phylogenetic relationship of the A222V substitution in the S protein relative to various global isolates is investigated, indicating high mutation rates in the S gene, characterised by diverse point mutations.
Kiky Martha, Ariesaka, M. M. Nuryady et al.· 0 citations
The utility of the LW25.13 peptide as a scaffold that can be adapted to different virus variants, which may prove useful for the development of peptides against new coronavirus variants of concern in the future, is illustrated.
Nina Raasch, L. Weissenborn, Elie Richel et al.· Journal of Medicinal Chemist...· 0 citations
