Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

Alphaviruses are single-stranded, enveloped, positive-sense RNA viruses within the family Togaviridae that cause significant human and veterinary disease worldwide. Venezuelan equine encephalitis virus (VEEV) is regarded as a prototypic encephalitic alphavirus, serving as a valuable model to study neuroinvasive alphaviruses, for which no FDA-approved therapeutics or vaccines are currently available. Targeting host factors required for viral pathogenesis represents a promising antiviral strategy with a reduced likelihood of resistance development. Nuclear transport receptors, including importins, are exploited by viruses to support infection and evade host antiviral responses. In addition to its structural role in virion assembly, VEEV capsid is a key virulence determinant that blocks host nucleocytoplasmic trafficking and mediates cytopathogenicity. Although the capsid protein is known to utilize the importin α/β1 pathway, the specific importin α isoform involved and its functional relevance remain unclear. Here, the role of importin α1 (KPNA2) in VEEV capsid subcellular localization, nucleocytoplasmic trafficking blockage, viral replication, and virus-induced cell death using complementary genetic and pharmacological approaches was investigated. Using recombinant VEEV expressing a V5-tagged capsid, full-length capsid was shown to associate with importin α1 and CRM1 in infected cells. Genetic ablation or pharmacological disruption (with compounds I2 or 1564) of importin α1 significantly reduced capsid nuclear localization, which was reversed following KPNA2 complementation, identifying importin α1 as a key mediator of capsid nuclear import. However, the loss or inhibition of importin α1 was insufficient to relieve capsid-inhibition of nucleocytoplasmic trafficking, suggesting functional redundancy as we found that the capsid also interacts with both importin α4 and importin α3. Importantly, loss or inhibition of importin α1 function partly protected cells from VEEV-induced cell death. While viral replication was unaffected by importin α1 loss, treatment with compounds 1564 or I2 reduced VEEV replication. Collectively, these findings demonstrate that importin α1 facilitates VEEV capsid nuclear import and contributes to virus-induced cytopathogenicity but is dispensable for nucleocytoplasmic trafficking inhibition and viral replication, further highlighting nuclear transport pathways as attractive targets for host-directed antiviral intervention.