Duck hepatitis A virus 3CD and 3D proteins are key facilitators of hnRNP K expression and cytoplasmic entry, and hijack hnRNP K to facilitate viral translation and replication.

Picornaviruses, constituting a diverse family of viral pathogens, cause numerous important diseases in humans and animals. The pathogenesis of these viruses is associated with many host factors. Studying host‒virus interactions is crucial for obtaining an in-depth understanding of viral pathogenesis. Here, we found that the picornavirus duck hepatitis A virus type 1 (DHAV-1) induces the expression of cellular heterogeneous nuclear ribonucleoprotein K (hnRNP K) and facilitates its nucleocytoplasmic shuttling to interact with DHAV-1 genomic RNA. hnRNP K positively regulates DHAV-1 translation and replication. hnRNP K utilizes its nucleic acid binding activity to interact directly with the DHAV-1 IRES and 3'UTR. The finding of this interaction between hnRNP K and the viral 3'UTR is important because it has not been reported in previous studies on RNA viruses. Furthermore, screening of DHAV-1 proteins identified the nonstructural proteins 3CD and 3D as key facilitators of hnRNP K expression and cytoplasmic entry. Meanwhile, hnRNP K interacts with 3CD and 3D through its KH1 and KI domains, with the KI domain being the most critical. 3CD and 3D also bind to DHAV-1 untranslated regions. These interactions help hnRNP K facilitate DHAV-1 translation and replication. In summary, hnRNP K forms complexes with 3CD and 3D, which binds to untranslated regions, promoting DHAV-1 translation and replication. These findings reveal a novel host‒virus interaction mechanism supporting viral translation and replication, offering new insights into picornavirus pathogenesis.