hnRNP K is a novel ITAF that negatively regulates foot-and-mouth disease virus translation and replication and is antagonized by viral 3C protease.

Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region. The regulation of internal initiation requires the interaction of IRES-transacting factors (ITAFs) with the IRES. In this study, we identified a novel ITAF, heterogeneous nuclear ribonucleoprotein K (hnRNP K), which negatively regulates foot-and-mouth disease virus (FMDV) translation and viral replication. Further investigation revealed that the KH2 and KH3 domains of hnRNP K directly bind to domains II, III, and IV of the FMDV IRES, resulting in the inhibition of IRES-mediated translation by interfering with the recognition of another positive ITAF, polypyrimidine tract-binding protein (PTB). Conversely, hnRNP K-mediated inhibition was antagonized by the viral 3C protease through the cleavage of hnRNP K at the Glu-364 residue during FMDV infection. Interestingly, the N-terminal cleavage product, hnRNP K-1-(364) retained partial inhibitory effects on IRES activity, whereas the C-terminal cleavage product, hnRNP K364-465, became a positive regulator of FMDV replication. Our findings expand the current understanding of virus-host interactions concerning viral recruitment and the modulation of ITAFs, providing new insights into translational control during viral infection. IMPORTANCE The translation of picornaviral genome RNA mediated by the internal ribosomal entry site (IRES) is a crucial step for virus infections. Virus-host interactions play a critical role in the regulation of IRES-dependent translation, but the regulatory mechanism remains largely unknown. In this study, we identified an ITAF, hnRNP K, that negatively regulates FMDV replication by inhibiting viral IRES-mediated translation. In addition, we describe a novel translational regulation mechanism involving the proteolytic cleavage of hnRNP K by FMDV protease 3C. The cleavage of hnRNP K yields two cleavage products with opposite functions: the cleavage product hnRNP K1-364 retains a partial inhibitory effect on IRES activity, and the cleavage product hnRNP K-364(-)465 becomes a positive regulator of FMDV replication. Our findings shed light on the effect of a novel ITAF on the translational regulation of picornavirus and provide new insights into translational control during viral infection.