A single amino acid substitution in the capsid protein of Zika virus contributes to the African lineage-specific virulence phenotypes
The re-emerged Zika virus (ZIKV) can cause severe neurological complications termed congenital Zika syndrome (CZS) in infants born to ZIKV-infected mothers. However, increasing evidence shows the ancestral African lineage ZIKV displays more virulent phenotype than the contemporary Asian ZIKV in multiple cell and animal models. However, the viral determinants and underlying mechanism of this lineage specific virulence phenotype remain largely unknown. In the present study, phylogenetic analysis and sequence alignment identified a panel of lineage specific amino acid substitutions between African and Asian ZIKVs. Then, these amino acid substitutions were introduced into an infectious clone of Asian ZIKV by standard reverse genetic technology. Further characterization in vitro demonstrated that the recovered mutant virus with a lysine to arginine substitution at position 101 of capsid (C) protein (termed K101R) produced larger plaques in BHK-21 cells, and replicated more efficiently and induced more severe cytopathic effects (CPE) in multiple cells including human neuronal precursor cells (NPCs). More importantly, the K101R mutant virus replicated more efficiently in mouse brain tissues, and induced stronger inflammatory responses, leading to higher mortality than the wild type (WT) virus in neonatal mice. Despite no effect on RNA binding affinity of recombinant C protein to viral RNA, structural modeling and biochemical assays showed that the K101R substitution increased the viral protease cleavage efficiency of full C protein. Taken together, our study identified a single amino acid substitution K101R in the capsid protein responsible for the well-characterized virulence enhancement phenotypes, highlighting the importance of viral determinants for the co-circulating of two lineages of ZIKV. Overall design: Retinal mRNA profiles of infected WT or K101R mutant virus mice (11 d.p.i.)were generated by deep sequencing ,in triplicate.