Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

Sean Larsen,Steven Bui,Laura L Newcomb,Hilario Medina-Ramirez,Adeba Mohammad,Veronica Perez

doi:10.1186/1743-422x-11-154

Abstract

BackgroundInfluenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1.MethodsHere we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription - quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA.ResultsInhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export.ConclusionsThese results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we conclude that in both A549 and 293 T cells, PA, PB1, and PB2 mRNA nuclear export is Nxf1 and Crm1 independent. Our data support the hypothesis that PA, PB1, and PB2 mRNAs, encoding the influenza RdRP, utilize atypical mRNA nuclear export.

Highlights

Influenza is a segmented negative strand RNA virus
We find influenza mRNA nuclear export is Nxf1-mediated with the exception of the influenza RNA dependent RNA polymerase encoding mRNAs; PA, PB1, and PB2
We detect less virus production in cells inhibited for Nxf1-mediated nuclear export via expression of dominant negative Nxf1 than control (Figure 1A). 48 hours post transfection with DNA plasmids to express dominant negative Nxf1, A549 cells were infected with influenza A Udorn at MOI of 2.5 to assay single cycle infection

Summary

Introduction

Influenza is a segmented negative strand RNA virus. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. The rapidly evolving nature of this segmented RNA virus leads to the emergence of new, unseen subtypes, which have potential to cause a pandemic. The 2009 novel H1N1 influenza virus proved not as pathogenic as initially expected, resulting in fewer deaths than predicted. Findings that the highly pathogenic H5N1 avian influenza is able to evolve increased transmissibility in the ferret model [3,4], has again emphasized the real possibility that a mutation or recombination event could result in the emergence of a highly pathogenic and transmissible influenza virus that will cause a severe and deadly pandemic. Greater understanding of the molecular mechanisms of influenza replication will facilitate the ability to identify novel antiviral targets and develop effective antiviral therapies

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