The cold-inducible RNA-binding protein RBM3 stabilises viral mRNA at the cooler temperatures of the upper respiratory tract.

Abstract

Temperature is a critical determinant of host-pathogen interactions in the respiratory tract, where influenza A virus (IAV) has adapted to the cooler environment of the upper respiratory tract (URT) to enable efficient replication and transmission. The cold-inducible RNA-binding protein RBM3 is highly expressed in nasopharyngeal tissue and is known to stabilise mRNAs under hypothermic conditions; however, its role during viral infection has not been defined. Here, we identify RBM3 as a key host factor that facilitates IAV replication at sub-physiological temperatures. siRNA-mediated knockdown of RBM3 significantly impaired viral replication, while its constitutive overexpression at 37{degrees}C restored replication to levels typically observed at 33{degrees}C. Mechanistically, RBM3 binds directly to viral nucleoprotein (NP) mRNA, prolonging mRNA half-life and inevitably leading to increased viral production. This effect was abolished in an RNA-binding-deficient RBM3 mutant, confirming the requirement for direct RNA interaction, visualised by smiFISH-PLA. Crucially, this positive regulation of NP mRNA and interaction was validated in well-differentiated primary nasal epithelial cells, highlighting the physiological relevance of RBM3 in the human URT. These results reveal a temperature-sensitive host-virus interaction that promotes IAV replication in the cooler URT, a key site for viral shedding and transmission. By linking environmental temperature, host RNA-binding proteins, and viral mRNA stability, this study uncovers a novel mechanism of respiratory virus adaptation and identifies RBM3 as a potential therapeutic target for limiting early-stage viral replication and transmission. Author SummaryTo establish productive infection and ensure transmission, respiratory viruses such as influenza A virus (IAV) must adapt to the diverse environments of the human respiratory tract. One key challenge is the temperature gradient that exists between the upper and lower respiratory tract. The upper respiratory tract (URT), the main site of viral entry and transmission, maintains a lower physiological temperature ([~]33{degrees}C) than the lower respiratory tract ([~]37{degrees}C, LRT), creating a distinct cellular environment. In this study, we investigated how this cooler URT temperature alters the landscape of RNA-binding proteins (RBPs), which play a central role in regulating gene expression after transcription. Using mass spectrometry-based profiling and molecular virology approaches, we identified a cold-inducible RBP, RBM3, as significantly enriched at 33{degrees}C and acting as a key proviral host factor during IAV infection. We show that RBM3 binds directly to viral nucleoprotein (NP) mRNA, mainly in the cytoplasm, in both immortalised cell models and primary nasal epithelial cells. This interaction promotes the stability of NP mRNA thereby enhancing the overall production of infectious virions. Importantly, disrupting RBM3s RNA-binding ability abolished this proviral effect. These findings reveal that RBM3, elevated in the cooler URT environment, directly supports IAV replication. This work highlights how subtle physiological differences in host tissue can reshape post-transcriptional regulation and influence the outcome of respiratory virus infection.