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

Xingxing Ren,Ming Liao,Huanan Li,Pingsheng Hu,Aobaixue Zhou,Yuandi Yu,Jinyu Huang,Wenbao Qi,Shukai Liu,Bo Li

doi:10.3389/fmicb.2019.01625

Xingxing Ren, Ming Liao + Show 8 more

Open Access

https://doi.org/10.3389/fmicb.2019.01625

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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.

Highlights

Influenza A virus is a single-stranded, segmented, negative-sense RNA virus in the Orthomyxoviridae family that causes a highly contagious respiratory disease in humans and animals (Nelson and Holmes, 2007)
To identify host factors associated with PB2 that are essential for efficient AIV replication in human cells, we constructed a recombinant influenza A/Chicken/Guangdong/V/2008 (H9N2) PB2-Flag virus with the PB2 C-terminal fusing three tandem Flag epitopes by reverse genetics technology (Figure 1A)
To globally identify host cellular factors that interact with the PB2 protein, we infected the HEK-293T cells with the rVPB2−Flag virus and purified reconstituted viral PB2 using the Flag antibodies coupled with affinity gel and identified the co-purified factors by liquid chromatographymass spectrometry (LC-MS) (Figure 1C)

Summary

Introduction

Influenza A virus is a single-stranded, segmented, negative-sense RNA virus in the Orthomyxoviridae family that causes a highly contagious respiratory disease in humans and animals (Nelson and Holmes, 2007). Pathogenic strains of influenza A viruses have led to several serious pandemics and caused high mortality rates, for example, the pandemics of 1918, 1957, 1968. High mutation rates of influenza virus can escape host immune response. For this reason, vaccines and drugs that act directly on viruses are potentially ineffective (Osterholm et al, 2012). There is a pressing need for a better understanding of the influenza virus replication mechanism to find novel potential antiviral targets

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