Avian Influenza A Virus Polymerase Recruits Cellular RNA Helicase eIF4A3 to Promote Viral mRNA Splicing and Spliced mRNA Nuclear Export

Abstract

The influenza A virus replicates in a broad range of avian and mammalian species by hijacking cellular factors and processes. Avian influenza A viruses (AIVs) generally propagated poorly in mammalian cells, but some mutants of virus-encoded RNA polymerase components, especially PB2 subunit, can overcome host restriction. Host factors associated with PB2 may be essential for efficient AIV replication in mammalian cells. Here, we infected human cells with the PB2 Flag-tagged replication-competent recombinant AIV and identified cellular proteins that coprecipitate with PB2 protein by mass spectrometry. We confirmed one of the coprecipitating host factors, DEAD-box protein eIF4A3, that interacts with viral PB2, PB1, and NP proteins. Depletion of endogenous eIF4A3 significantly reduced virus replication. Later studies showed that eIF4A3 is essential for viral RNA polymerase activity and viral RNAs synthesis. Upon systematic dissection of the influenza virus progeny mRNA generation, from pre-mRNA processing to nuclear export, we found that the depletion of eIF4A3 resulted in significant defects in the ratio of M2 to M1 and NS2 to NS1, and the proportion of viral spliced mRNA in the nucleus increased, indicating that eIF4A3 plays a significant function in viral nascent intron mRNA splicing and spliced mRNA (M2 and NS2) nuclear export. Additionally, we confirmed that in specific deletion of eIF4A3, the synthesis of reduced NS2 can significantly impair neo-synthetized viral ribonucleoprotein (vRNP) nuclear export. Taken together, our findings revealed that eIF4A3 is a key mediator of AIV polymerase activity, mRNA splicing, and spliced mRNA nuclear export.

Nuclear export of plant pararetrovirus mRNAs involves the TREX complex, two viral proteins and the highly structured 5′ leader region

“…However, the involvement of viral RNA-dependent RNA polymerases was demonstrated for influenza A virus (IAV), the heterotrimeric polymerase of which recruits cellular helicase DDX19 (also known as DBP5) on viral mRNPs to enhance their nuclear export ( 110 ). It was also recently shown that the same polymerase, especially its PB1 and PB2 subunits, recruit cellular RNA helicase eIF4A3, which is part of the EJC, to promote viral mRNA splicing and nuclear export of the spliced mRNAs ( 111 ).…”

Real-Time Visualization of the Infection and Replication of a Mouse-Lethal Recombinant H9N2 Avian Influenza Virus

“…MDCK cells grown in six-well plates were infected at a multiplicity of infection (MOI) of 0.001 at 37°C, as described previously ( 36 ). One hour later, cells were washed with PBS, and then incubated with DMEM containing 1 μg/ml TPCK trypsin at 37°C with 5% CO 2 .…”

Influenza A virus segments five and six can harbor artificial introns allowing expanded coding capacity

“…Splicing machinery is generally conserved among vertebrate species; however, splicing is also a recognized host determinant for avian-and mammalian-derived influenza viruses [9][10][11][12][13]. Most notably, avian-adapted influenza viruses have been reported to replicate poorly in mammalian cells due to excessive M splicing [24,25].…”

“…In contrast, as IAV infection progresses mRNAs produced from the M segment are spliced more often, increasing the amount of spliced M2 relative to unspliced M1 over time [7]; after contributing to viral entry the M2 ion channel is thought to primarily be required late in replication during viral assembly [8]. Furthermore, splicing dysregulation in different host environments reduces viral replication efficiency, likely as a result of alterations to viral protein ratios [9][10][11][12][13]. These observations together demonstrate the importance of splicing in optimizing the influenza viral replication processes.…”