Targeting conserved interaction surfaces of alphavirus nsP4 supports the rational design of live-attenuated vaccine candidates
5 affiliations
- 1Institute of Basic Medicine, North Sichuan Medical College, Nanchong, People’s Republic of China
- 2Institute of Bioengineering, University of Tartu, Tartu, Estonia
- 3Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
- 4School of Pharmacy and Medical Sciences, Griffith University, Gold Coast, Australia
- 5Institute for Biomedicine and Glycomics, Griffith University, Gold Coast, Australia
Abstract
Alphaviruses are emerging arboviruses that pose a growing threat to global public health; however, effective interventions remain limited. Recent structural studies of the chikungunya virus (CHIKV) replicase core revealed that the viral RNA-dependent RNA polymerase nsP4 interacts with both nsP1 and nsP2 and is predicted to engage viral RNA. The conservation and multifunctionality of nsP4 make it a challenging but promising target for rational attenuation. Here, we evaluated 58 structure-guided mutations in nsP4 predicted to disrupt interactions with nsP1, nsP2, or viral RNA using a trans-replicase system. Sixteen selected mutations were further analyzed in a CHIKV-o’nyong-nyong virus chimera and Semliki Forest virus (SFV). Most mutants were viable but showed varying degrees of attenuation in mammalian and mosquito cells. Viruses carrying mutations in predicted RNA-interaction sites frequently acquired pseudoreversions and/or second-site compensatory changes, whereas those harboring mutations affecting nsP1 or nsP2 interfaces were more genetically stable. Three substitutions representing defects in nsP1 interaction (F164A), predicted RNA interaction (K174S), and nsP2 interaction (D393A) were further characterized in authentic CHIKV and displayed attenuation consistent with that observed in SFV. In vivo, all three variants showed reduced pathogenicity in the SFV mouse model, with F164A being avirulent, immunogenic, and conferring complete protection against lethal challenge. In a CHIKV mouse model, these mutations attenuated disease and reduced serum virus titers and inflammatory cell infiltration. Together, these findings provide a molecular framework for the rational design of live-attenuated alphavirus vaccine candidates and support broader evaluation of nsP4-based attenuation.
IMPORTANCE: Alphaviruses are an increasing global health concern, with hundreds of thousands of chikungunya cases reported worldwide in 2025; however, vaccine options remain limited, and no specific antiviral treatment is available. Recent structural studies have revealed conserved functional interfaces in the viral RNA polymerase nsP4, but their value for attenuation had not been systematically explored. Here, using structure-guided mutagenesis, validation in multiple viral systems, and in vivo analysis, we show that selected nsP4 mutations can strongly attenuate replication and pathogenicity while preserving immunogenicity. These findings identify conserved nsP4 interfaces as a shared vulnerability of alphaviruses and provide a molecular framework for the rational design of broadly applicable live-attenuated vaccines while also highlighting nsP4 as a functionally constrained target relevant to antiviral development.
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BibTeX
@article{yang2026targeting,
title = {{Targeting conserved interaction surfaces of alphavirus nsP4 supports the rational design of live-attenuated vaccine candidates}},
author = {Xiaoyao Yang and Evelin Väljaots and Kaiyu Li and Krystyna Naumenko and Sandra Koit and Yaw Bia Tan and Xiang Liu and Victor Rodrigues de Melo Costa and Suresh Mahalingam and Dahai Luo and Andres Merits and Jian Yang},
journal = {mBio},
year = {2026},
month = sep,
doi = {10.1128/mbio.01638-26},
url = {https://doi.org/10.1128/mbio.01638-26},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Targeting conserved interaction surfaces of alphavirus nsP4 supports the rational design of live-attenuated vaccine candidates
AU - Xiaoyao Yang
AU - Evelin Väljaots
AU - Kaiyu Li
AU - Krystyna Naumenko
AU - Sandra Koit
AU - Yaw Bia Tan
AU - Xiang Liu
AU - Victor Rodrigues de Melo Costa
AU - Suresh Mahalingam
AU - Dahai Luo
AU - Andres Merits
AU - Jian Yang
PY - 2026
DA - 2026/09/10
JO - mBio
DO - 10.1128/mbio.01638-26
UR - https://doi.org/10.1128/mbio.01638-26
AB - Alphaviruses are emerging arboviruses that pose a growing threat to global public health; however, effective interventions remain limited. Recent structural studies of the chikungunya virus (CHIKV) replicase core revealed that the viral RNA-dependent RNA polymerase nsP4 interacts with both nsP1 and nsP2 and is predicted to engage viral RNA. The conservation and multifunctionality of nsP4 make it a challenging but promising target for rational attenuation. Here, we evaluated 58 structure-guided mutations in nsP4 predicted to disrupt interactions with nsP1, nsP2, or viral RNA using a trans-replicase system. Sixteen selected mutations were further analyzed in a CHIKV-o’nyong-nyong virus chimera and Semliki Forest virus (SFV). Most mutants were viable but showed varying degrees of attenuation in mammalian and mosquito cells. Viruses carrying mutations in predicted RNA-interaction sites frequently acquired pseudoreversions and/or second-site compensatory changes, whereas those harboring mutations affecting nsP1 or nsP2 interfaces were more genetically stable. Three substitutions representing defects in nsP1 interaction (F164A), predicted RNA interaction (K174S), and nsP2 interaction (D393A) were further characterized in authentic CHIKV and displayed attenuation consistent with that observed in SFV. In vivo, all three variants showed reduced pathogenicity in the SFV mouse model, with F164A being avirulent, immunogenic, and conferring complete protection against lethal challenge. In a CHIKV mouse model, these mutations attenuated disease and reduced serum virus titers and inflammatory cell infiltration. Together, these findings provide a molecular framework for the rational design of live-attenuated alphavirus vaccine candidates and support broader evaluation of nsP4-based attenuation. IMPORTANCE: Alphaviruses are an increasing global health concern, with hundreds of thousands of chikungunya cases reported worldwide in 2025; however, vaccine options remain limited, and no specific antiviral treatment is available. Recent structural studies have revealed conserved functional interfaces in the viral RNA polymerase nsP4, but their value for attenuation had not been systematically explored. Here, using structure-guided mutagenesis, validation in multiple viral systems, and in vivo analysis, we show that selected nsP4 mutations can strongly attenuate replication and pathogenicity while preserving immunogenicity. These findings identify conserved nsP4 interfaces as a shared vulnerability of alphaviruses and provide a molecular framework for the rational design of broadly applicable live-attenuated vaccines while also highlighting nsP4 as a functionally constrained target relevant to antiviral development.
SN - 2150-7511
ER -