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

Abstract

Temperature 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.