SARS-CoV-2 NSP8-Derived Peptide Effectively Suppresses the Activity of Helicase NSP13

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 13 (NSP13) is a multifunctional helicase that plays a central role in viral RNA replication and suppression of host innate immunity. Beyond its essential function within the replication-transcription complex (RTC), NSP13 antagonizes type I interferon (IFN-I) signaling through interaction with the host kinase TBK1, making its protein-protein interactions attractive antiviral targets. In this study, we investigated whether fragments derived from the viral cofactor NSP8 can competitively interfere with NSP13 interactions. Using molecular dynamics simulations, interaction mapping, and neural network-based binding free energy prediction, we characterized NSP13 interfaces with (a) full-length NSP8 in 2 RTC binding modes, (b) N- and C-terminal NSP8 fragments, and (c) TBK1. Structural analyses revealed that the N-terminal fragment of NSP8 (NSP8-N, residues 1 to 87) binds NSP13 with high affinity and occupies residues critical for both TBK1 association and canonical NSP8-NSP13 interactions, including Met⁶⁸, Tyr⁹³, Phe⁹⁰, Gly⁶⁷, Ser³⁵⁰, and Thr³⁵¹. Predicted binding free energies indicate that NSP8(N) forms a more stable complex with NSP13 than either the native RTC configuration or the NSP13-TBK1 complex, supporting a competitive binding mechanism. Functionally, coexpression experiments in poly(I:C)-stimulated A549 cells showed that, while NSP13 suppresses IFN-β transcription, coexpression of NSP8 or its N-terminal domain restores IFN-β levels to those of control cells. These findings support a dual inhibitory model in which NSP8-derived peptides sequester NSP13, preventing immune suppression and potentially impairing its recruitment to the RTC, highlighting the NSP8 N-terminal α-helical region as a promising scaffold for antiviral development.