Examination of Host Factors to Identify Novel AntiInfluenza Targets

SMSU Event Center BC

Influenza virus readily undergoes genetic changes so that antivirals and vaccines become ineffective. A novel approach used to treat viral infection centers on targeting host proteins integral for viral replication. I aimed to establish the importance of two host nuclear export factors on influenza RNA expression; XpoT, which exports cellular tRNAs, and Xpo5, responsible for cellular pre-microRNA nuclear export. To do this, I employed siRNA-mediated knockdown. I performed triplicate trials, transfecting siRNA SMARTpools or non-target control for 48 hours on two cell types, A549 and VERO cells. Cells were then infected with influenza A at a high multiplicity of infection for four hours when protein and RNA was isolated. Total RNA concentration was determined using optical density and integrity confirmed using agarose gel electrophoresis. Reverse transcription with oligo dT primers and equal concentrations of total RNA provides cDNA for qPCR analysis with gene specific primers to reveal the relative abundance of specific RNAs. Our results show each siRNA resulted in specific target knockdown, as expected. We discovered there is no significant change in influenza PA or NP total mRNA expression when Xpo5 is downregulated in either cells type. In VERO cells, there was also no significant change when XpoT was downregulated. In A549 cells there was a slight but significant inhibition of NP with XpoT down regulation but no significant change in PA expression. Our results conclude that neither host factor is essential for influenza total RNA expression. Future directions aim to use this approach to examine additional host factors.