Chikungunya Virus E2 N218K Enhances Heparan Sulfate-Dependent Attachment and Viral Internalization While Conferring Partial Attenuation in A129 Mice

The E2 glycoprotein of chikungunya virus (CHIKV) binds negatively charged cell-surface heparan sulfate (HS) to facilitate viral attachment and modulate pathogenicity, yet the E2 determinants governing HS-dependent attachment and the effects of altered HS utilization on subsequent infection remain incompletely defined. From a low-passage clinical isolate obtained during the 2010 Guangdong outbreak, we identified a previously unreported E2 substitution, N218K, which introduces a basic residue. In vitro, this mutation enhanced early infection and attachment to mammalian cells and reduced the RNA/PFU ratio; in A129 mice, however, it reduced early serum viral RNA levels, was associated with lower tissue viral RNA burdens at 5 dpi, and delayed mortality, consistent with partial attenuation. Soluble HS competition assays showed that N218K increased HS dependence during viral attachment, whereas DEAE-dextran assays supported a role for charge-dependent interactions in restricting local viral spread. N218K also enhanced viral internalization after normalization of initial attachment, identifying an additional post-attachment phenotype associated with this substitution. Our findings identify N218K as a determinant of HS-dependent attachment and partial attenuation in A129 mice, reveal a “high attachment–low dissemination” phenotype, and further show that this charge-altering E2 substitution enhances post-attachment internalization.