Host transcriptional corepressor TLE1 is commonly exploited by RNA viruses for replication.
Viral genomes encode transcriptional regulators that alter the expression of host genes to facilitate virus replication, and the comprehensive regulatory network they form is largely elusive. Utilizing influenza A virus (IAV) as a model, we demonstrate that the viral nucleoprotein (NP) harnesses the host transcriptional corepressor TLE1 to enhance viral polymerase activity while suppressing the cellular innate response. NP-TLE1 recognition was realized via a conserved basic helix-loop-helix (bHLH)-like motif across IAV subtypes, which subsequently recruited another transcription factor, Kruppel-like factor 4 (KLF4), to suppress antiviral signaling. Blockage of the NP-TLE1 interaction by a peptide AH49A provided antiviral protection in vitro and in vivo. Moreover, the NP-TLE1-KLF4 axis was widely observed across various RNA viruses, as evidenced by the presence of a bHLH-like motif in the SARS-CoV-2 nucleocapsid protein. Loss-of-function substitutions in the bHLH-like motif attenuated the replication of both IAV and SARS-CoV-2. Our study reveals how RNA viruses exploit the components of the host transcription machinery to facilitate virus replication.