Abstract Following the entry into the host cell, SARS‐CoV‐2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B‐cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID‐19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild‐type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication. New nanobodies targeting Nsp9, a nonstructural protein required for Sars‐CoV‐2 replication, are generated. These nanobodies specifically recognize Nsp9 in saliva samples from COVID‐19 patients and stabilize a tetrameric form of Nsp9, not compatible with its role in facilitating viral replication. These results potentially identify novel diagnostic and therapeutic antiviral strategies to combat Sar‐CoV‐2 and, therefore, COVID‐19.
【저자키워드】 COVID‐19, SARS‐CoV‐2, nanobodies, NMR spectroscopy, Nsp9, 【초록키워드】 coronavirus disease, Saliva, coronavirus, Transcription, diagnostic, molecular dynamics, COVID‐19, SARS‐CoV‐2, Replication, immunoassays, Protein, nonstructural proteins, immunoassay, Epitopes, Viral, nanobody, viral replication, therapeutic, Patient, Lineage, Nsp9, nonstructural protein, molecular, nuclear magnetic resonance, oligomerization, epitope, antiviral strategy, Nsps, host cell, COVID‐19 patients, complex, saliva samples, B‐cell, implication, Result, identify, required, applied, supported, characterized, unique, recognize, expressed, analysis, purified, RTC, New, COVID‐19 patient, monomeric, 【제목키워드】 Strategy, COVID‐19, SARS‐CoV‐2, Nsp9, Against,