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

Abstract

AbstractTemperature regulation is a key aspect of homeostasis, and several systems are involved in orchestrating adjustments in gene expression at the cellular level. One such factor is RBM3, a cold-inducible RNA-binding protein implicated in several aspects of mRNA processing and regulation. The upper respiratory tract serves as a unique environment regarding temperature regulation. Physiologically, the lower tract is relatively stable at 37°C, while the upper tract fluctuates at or below 33°C. Adapting to this temperature differential, and the subsequent differences in transcriptome and proteome, is essential for respiratory viruses that infect and cause disease in the lower tract while simultaneously replicating and transmitting from the upper tract. At present, our understanding of the molecular mechanisms underlying influenza virus infection at cooler temperatures is lacking. Unsurprisingly, RBM3 protein levels are highest in nasopharyngeal tissue. Coupled with its known role in positively regulating bound RNA, it is an appealing candidate for manipulation by respiratory viruses. We found siRNA knockdown of RBM3 significantly decreased viral replication. To disentangle the direct effect of RMB3 from the shift in global gene expression at colder temperatures, we generated an A549 cell line constitutively overexpressing RBM3 at 37°C. Overexpression resulted in a significant increase in levels of viral replication. RBM3 was found to readily bind viral NP mRNA during infection and prolong the half-life of these transcripts. In contrast, an RBM3 RNA binding null mutant reverses this phenotype, validating that direct interaction of RBM3 has a stabilising effect on viral transcripts. The proviral nature of increased RBM3 levels was further validated in the more clinically relevant model of well-differentiated primary nasal epithelial cells. These data suggest a role for RBM3 in supporting influenza virus replication in the nasopharyngeal tract. Understanding IAV replication in this environment could prove fundamental to elucidating the mechanisms of influenza transmission and reassortment.Author SummaryTo establish a productive infection, respiratory viruses must overcome or adapt to several systems within the body. One such obstacle is the temperature gradient of the respiratory tract. The upper respiratory tract, which serves as the primary route of viral transmission, provides the virus with a drastically different cellular environment compared to the lower respiratory tract, due to differences in growth temperatures. Here, we detail our investigation into the differences in the RNA binding protein (RBP) landscape between 33°C and 37°C. Our aim was to identify specific RBPs that are upregulated at 33°C and to explore their role in the influenza A virus (IAV) lifecycle, thereby advancing our knowledge of the molecular mechanisms underlying IAV infection in the upper respiratory tract. Through a combination of molecular virology and mass spectrometry, we identified a cold-inducible RBP, RBM3, as an important post-transcriptional regulator of IAV nucleoprotein (NP) mRNA. We show that RBM3 binds specifically to viral NP mRNA, stabilising the mRNA, and ultimately promoting the production of infectious virions, while abolishing the RNA binding capabilities of RBM3 reversed this effect. Overall, we find that enhanced RBM3, as seen in the upper respiratory tract, positively influences viral replication.