The Role of Crm1 in Nuclear Export of Influenza mRNA in A549 Cells

Event Center A&B

Influenza is a negative strand RNA virus that is segmented. The influenza replicates by transcribing viral mRNA in the nucleus, then using host pathways to splice viral mRNAs and export them out of the nucleus. Studies of retroviral mRNA nuclear export reveal viral mRNAs utilize the Crm1 and Nxf1 export pathways. Published studies of influenza mRNA nuclear export reveal no dependence on Crm1 while select mRNAs export via Nxf1. Previous experiments in the Newcomb Lab revealed a cell type difference regarding influenza NP mRNA nuclear export. In human lung adenocarcinoma epithelial cells (A549), NP mRNA nuclear export is Nxf1-mediated. However, NP mRNA nuclear export is independent of Nxf1 in human embryonic kidney cells (293T). Because of this cell type difference, the role of Crm1 in influenza mRNA nuclear export needed to be readdressed. Published data did not address the role of Crm1 in A549 cells. In this study, we specifically examined the role of Crm1 in influenza mRNA nuclear export in A549 cells. Crm1 nuclear export was inhibited using Leptomyocin B, a Crm1 inhibitor, during infection with influenza A/Udorn/307/1972(H3H2) virus. At 3.5 hours post infection, cells were collected and fractionated. RNA was isolated from the cytoplasmic fractions and subject to reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to assess mRNA nuclear export by analysis of cytoplasmic mRNA. Crm1 inhibition resulted in no difference in PA, PB1, and PB2 mRNA nuclear export in A549 cells. Our results support use of an as yet undefined nuclear export pathway for influenza polymerase encoding mRNAs.