Structure-inspired design of Nsp8-based protein inhibitors to suppress SARS-CoV-2 replication
- 1Institute of Chemistry, Academia Sinica
Abstract
SARS-CoV-2 relies on a conserved RNA-dependent RNA polymerase (RdRp) complex composed of nsp12 and its cofactors nsp7 and nsp8 to replicate its RNA genome. Whereas most antiviral strategies target viral enzymes or surface proteins directly, an alternative approach is to disrupt the assembly or function of an essential viral molecular machine using a defective component derived from the pathogen itself. Here, guided by structural analyses of the nsp12-nsp7-nsp8 replication complex, we designed truncated nsp8 proteins that retain the ability to associate with nsp12 but are defective in engaging RNA. Using a purified nsp12-nsp7-nsp8 system capable of RNA primer extension, we show that selected truncated nsp8 variants inhibit polymerase activity when introduced into an otherwise functional complex. These results are consistent with a competitive mechanism in which the defective nsp8 variants associate with nsp12 and interfere with incorporation or function of wild-type nsp8, thereby compromising formation of a productive replication complex. To further explore this strategy, we used structure-guided in silico analysis of the nsp8-nsp12 interface to identify interaction hotspots and screened corresponding single-amino-acid substitutions. Several variants exhibited enhanced inhibitory activity in the reconstituted polymerase assay. Together, these findings establish a proof-of-concept strategy in which a structurally engineered, pathogen-derived protein can act as a dominant-negative inhibitor of an essential viral replication machinery. This approach provides a framework for developing protein- or peptide-based inhibitors that target conserved protein-protein interactions within viral replication complexes.
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BibTeX
@misc{sterbova2026structureinspired,
title = {{Structure-inspired design of Nsp8-based protein inhibitors to suppress SARS-CoV-2 replication}},
author = {P. Sterbova and H. H. Lin and I.-P. Tu and W.-h. Chang},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.30.747997},
url = {https://www.biorxiv.org/content/10.64898/2026.08.30.747997v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Structure-inspired design of Nsp8-based protein inhibitors to suppress SARS-CoV-2 replication
AU - P. Sterbova
AU - H. H. Lin
AU - I.-P. Tu
AU - W.-h. Chang
PY - 2026
DA - 2026/08/30
PB - bioRxiv
DO - 10.64898/2026.08.30.747997
UR - https://www.biorxiv.org/content/10.64898/2026.08.30.747997v1
AB - SARS-CoV-2 relies on a conserved RNA-dependent RNA polymerase (RdRp) complex composed of nsp12 and its cofactors nsp7 and nsp8 to replicate its RNA genome. Whereas most antiviral strategies target viral enzymes or surface proteins directly, an alternative approach is to disrupt the assembly or function of an essential viral molecular machine using a defective component derived from the pathogen itself. Here, guided by structural analyses of the nsp12-nsp7-nsp8 replication complex, we designed truncated nsp8 proteins that retain the ability to associate with nsp12 but are defective in engaging RNA. Using a purified nsp12-nsp7-nsp8 system capable of RNA primer extension, we show that selected truncated nsp8 variants inhibit polymerase activity when introduced into an otherwise functional complex. These results are consistent with a competitive mechanism in which the defective nsp8 variants associate with nsp12 and interfere with incorporation or function of wild-type nsp8, thereby compromising formation of a productive replication complex. To further explore this strategy, we used structure-guided in silico analysis of the nsp8-nsp12 interface to identify interaction hotspots and screened corresponding single-amino-acid substitutions. Several variants exhibited enhanced inhibitory activity in the reconstituted polymerase assay. Together, these findings establish a proof-of-concept strategy in which a structurally engineered, pathogen-derived protein can act as a dominant-negative inhibitor of an essential viral replication machinery. This approach provides a framework for developing protein- or peptide-based inhibitors that target conserved protein-protein interactions within viral replication complexes.
ER -