Translation of nervous necrosis virus involves eIF4E but not RPS6 phosphorylation and viral particle assembly in remodeled microtubule-organizing center

Background Nervous necrosis virus (NNV) is a bipartite positive-sense RNA virus that causes piscine viral nervous necrosis disease in larvae and juvenile marine fish. Although NNV has been studied for decades, the translational mechanisms underlying NNV propagation are still unknown. Methods The molecular mechanism of NNV translation was investigated using grouper brain (GB) cell line infected with giant grouper (GG) NNV strain. Bromouridine and puromycin labelling were used to detect newly synthesized viral RNAs and proteins in viral factories (VFs), respectively. In addition, immunocytochemical staining, Western blotting and RNA fluorescence in situ hybridization (FISH) following densitometry and statistical analyses were employed to determine the involvement of cellular components during viral mRNA translation in infected control/inhibitor treated cells. Viral particles in remodeled microtubule-organizing center (MTOC) were analyzed using transmission electron microcopy. Results In this study, we found that upon infection, newly synthesized NNV RNAs and replication intermediates colocalized with mitochondria. Meanwhile, the positive-sense mRNAs of RNA1 and RNA2 produced in mitochondria gradually moved toward the perinuclear area. Colocalization of the neo-synthesized proteins with RNA2 and coat protein suggested that viral translation proceeded at VFs within particular compartments in the cytoplasm. The phosphorylated eukaryotic initiation factor, p-eIF4E and its binding protein, p-eIF4E-BP, also colocalized with RNA2 and coat protein in VFs, suggesting that free eIF4E was recruited by the 5'-cap of RNA2 to initiate coat protein translation. Moreover, the initiation of coat protein translation by phosphorylation of eIF4E at Ser209 was further confirmed by upregulation of p-MNK1 and its upstream kinases, p-p38 and p-ERK. We also found that ribosomal protein RPS6 strongly colocalized with coat protein in VFs, but its phosphorylated form, p-RPS6, and its catalytic kinase (p-p70S6K) were gradually degraded after infection. A remodeled MTOC constructed by γ-tubulin, Vimentin, Plectin and trans-Golgi was observed and acts as compartment for coat protein synthesis and virion assembly. Conclusion Our results suggest that NNV coat protein translation is initiated by p-eIF4E in VFs for 5'-cap viral RNA2 recognition, binding and later virion assembly by coat protein in the remodeled MTOC.