Newcastle Disease Virus Infection Upregulates The Assembly Of Eukaryotic Translation Initiation Factor4F To Benefit Viral Translation And Replication

Newcastle disease virus (NDV), the member of the Paramyxoviridae family, is a single-stranded,nonsegmented, negative-sense RNA virus that infects most species of birds, resulting in substantial lossto the poultry industry. The genome contains six major genes that encode the structural proteins in theorder3’-NP-P-M-F-HN-L-5’, as well as two non-structural proteins, W and V via mRNA editing. NDVis dependent on the cellular translational machinery to synthesize viral proteins, and the viral mRNAsare capped and polyadenylated by L protein during synthesis. The structure of the NDV mRNA cap ism7G(5’)ppp(5’)GpPyp, which lacks2’-O-methylation and belongs to the cap0type. All viralreplication events occur within the host cell cytoplasm, and the positive-sense RNA intermediates areformed, which act as mRNA using the host cell translation machinery to translate proteins. However, theregulation of translational machinery in NDV-infected cells and the detailed cellular pathways has notbeen studied.Translation initiation is considered to be a rate limiting process for overall protein synthesis ineukaryotes. Most eukaryotic mRNAs present a5’7-methyl GTP terminal cap structure as well as apoly(A) tail at3’ end, and translated by cap-dependent translation. Cap-dependent translation beginswith7-methyl-GTP cap recognition by eukaryotic translation initiaton factor4F (eIF4F) whichcomposed of eIF4E, eIF4G and eIF4A. eIF4F functions are regulated by cellular signal pathways ineukaryotic cells by means of phosphorylation cascade reactions. A major pathway that regulates eIF4Fcomplex assembly is the PI3K/Akt/mTOR pathway. Another pathway that regulates the translationinitiation is MAP kinase-interacting serine/threonine-protein kinase1(Mnk1). A number of viruses canalso stimulate various host cell signalling pathways to phosphorylate translational control proteins,enhancing the activity of eIF4F components and inactivating translational repressor proteins. In thepresent work, we tried to illustrate the regulation of host translational machinery by Newcastle diseasevirus in host protein translation initiation.To determine whether eIF4F is modified by NDV infection, the phosphorylation of eIF4G and eIF4Ewas analyzed by Western blotting. The results showed that both phosphorylation was significantlyincreased at6–12hpi, correlating with robust synthesis of viral proteins. Treating HeLa cells withUV-inactivated NDV Herts/33did not alter eIF4F phosphorylation or NDV NP protein expression. Thebinding between eIF4G and eIF4E was investigated, significantly increased eIF4G and eIF4E wasdetected in the eIF4F complex from NDV infected cells. In addition, eIF4E is redistributed post NDVinfection and depleting of eIF4E or/and eIF4G reduces viral protein synthesis. These data indicate thatNDV infection stimulated the phosphorylation of eIF4F and also promoted the assembly of eIF4Fcomplexes. Both eIF4E and eIF4G are important host factors for NDV protein synthesis and growth.To determine the host intracellular pathways involved in NDV infection, we first examined thePI3K/Akt pathway by determining the level of phosphorylated downstream proteins. We also analysised the effect of PI3K/Akt pathway on the formation of eIF4F complexes and viral protein synthesis inNDV-infected cells. The results showed that Akt and mTOR phosphorylation was enhanced at6to12hpi and phosphorylation of p70S6K and4E-BP1, two downsteam effectors of Akt/mTOR signaling,were also elevated. In addition, the role of PI3K in Akt and downstream proteins phosphorylation afterNDV infection was confirmed by LY294002. We also found that NDV-induced eIF4G phosphorylationrequires mTOR activity. Inhibition of upstream PI3K signaling with LY294002resulted in a reduction inthe assembly of eIF4F. In addition,4E-BP1phosphorylation induced by NDV infection is resistant torapamycin treatment. In that case, rapamycin treatment did not affect the assembly of eIF4F andsynthesis of viral proteins. These data suggest that NDV infection induced phosphorylation of eIF4Gand4E-BP1as well as enhaced assembly of eIF4F through PI3K/Akt pathway.We also examined the MAPK pathway involved in NDV infection and eIF4F activation. We show herethat Mnk1is strongly phosphorylated for NDV infection, correlating with the phosphorylation of eIF4Eat Ser209. Both Erk and p38are activated in NDV-infected cells, however, only p38activation isdirectly and correlates with eIF4E activation. The role of eIF4E phosphorylation in eIF4F assembly inNDV-infected cells, was examined using specific inhibitors. Phosphorylation of eIF4E contributedneither to NDV mediated eIF4F complex assembly, nor to NDV infection. While p38MAPK/Mnk1pathway is not essential for translation, they might impart a level of control that governs the efficiencywith which translation initiates on viral mRNAs.To further identify the direct interaction between NDV and host protein translation machinery, weperformed m7GTP pull down and co-immunoprecipitation to confirm the direct interaction betweenNDV NP protein and eIF4F complex. Mapping of the eIF4F-binding domain on the NP protein usingNDV NP truncation mutants showed that the N-terminal391amino acids are necessary for NP tointeract with eIF4F, and the length of C-terminal portion of NP may play a role in regulating thecontaction capacity of NP protein and eIF4F complex.Overall, our study provides insight into how NDV manipulates host signaling pathways to regulateprotein translation initiation. NDV infection activates PI3K/Akt/mTOR and p38MAPK/Mnk1pathwaysto upregulate phosphorylation of eIF4E and eIF4G, as well as assembly of eIF4F complex to stimulatecap-dependent translation. To favor viral gene translation and replication, the translation initiationmachinery is redistributed and interacts with NP. Further understanding of the mechanisms employed byNDV to recruit and interact with eIF4F complexs will provide additional insight into how NDVinterferes with host translation and optimizes viral gene expression. Our studies also provide insightsinto NDV-host interaction and may provide host cell targets for therapeutic intervention against NDVinfection.