Nuclear Localization Of The P17 Protein Of Avian Reovirus Is Correlated With Autophagy Induction And An Increase In Viral Replication

Avian reovirus (ARV) is an important poultry pathogen that belongs to Reoviridae family, Orthoreovirus genus. The genome of ARV is composed of 10 double-stranded RNA segments that can be divided into the large (L), medium (M), and small (S) groups and encode 10 structural proteins and four non-structural proteins. The p17 is encoded a 17 kDa non-structural protein by the S1 gene of ARV and contains 146 amino acids. Previous studies have shown that p17 is a nucleocytoplasmic shuttling protein, which have a basic region of NLS. The nucleocytoplasmic distribution of p17 is coupled to the transcriptional activity of the cell and p17 is a CRM1-independent nucleocytoplasmic shuttling protein. It was proved that the p17 triggers autophagy through activation of p53/PTEN, AMPK, and PKR signling pathway but does not induce cellular apoptosis. In addition, p17 plays an important role in regulating cell cycle and host cellular translation. The p17 expression could retard the growth of cells thourgh the activation of p53 pathway. Thus, the p17 is an essential multifunctional viral protein that plays significant parts in regulating autophagy, cell cycle and protein translation. However, there are still many unknown factors about the functions of p17. In this study, we characterized the NLS and NES of p17 and investigated the mechanism by which p17 regulates autophagy and the effect on virus production of mutating the p17 NLS.1. Characterization of signal sequences determining the NLS and NES of the ARV p17In this study, GFP-labeled assay was used to investigate the subcellular localization of p17. The localizations of p17 fused to the C-terminal end of GFP were examined by fluorescence microscopy. It was showed that the results of GFP-labeled assay were consistent with the localization of p17 in ARV-infected cells detected by indirect immunofluorescence assay. To examine the subcellular distribution of p17 protein, pEGFP-p17 was transfected into both Vero and DF1 cells. At different time points after transfection, p17 subcellular distribution was observed under an inverted fluorescence microscope. In addition, we compared the subcellular localization of p17 with another two non-structural proteins of ARV.According to the previously studies, we mutated several consecutive basic amino acids in p17 sequence and found that the mutation of lysine (K) at position 122 and arginine (R) at position 123 to alanine (A) localized the majority of p17 to the cytoplasm.The p17 is a nucleocytoplasmic shuttling protein and this protein exits the nucleus via a CRM1-independent pathway. By using software prediction and sequence alignment, several candidate NES motifs with striking similarity to the NES sequences reported in other proteins were probed in the p17 of ARV. To characterize these sequences, GFP-labeled assay was used. The results were showed that the sequence at position 19 to 26 of p17 localized the GFP to the cytoplasm via a CRM1-independent pathway.2. Nuclear localization of the p17 of avian reovirus contributes to autophagy induction and viral productionp17 is known to trigger autophagy, but the molecular mechanism of p17 regulation of cell autophagy remains to be further studied. This study investigated the effect on p17 NLS causes autophagy and viral replication. To confirm whether p17 nuclear transport affects autophagy mediated by p17, we constructed the eukaryotic expression plasmids of p17 with a mutated nuclear localization signal. The expressing p17 with a mutant NLS had a significantly lower level of autophagy at 24 hpt than cells expressing wild type p17, but there was no significant difference in autophagy at 36 hpt.We further examined the effect of p17 nuclear localization on viral amplification. We assessed the yield of virus in transfected Vero and DF1 cells with wild type p17 and NLS-mutated p17 following ARV infection. When cells were transfected with wild type p17, the level of viral products increased with time of infection increasing. This observation indicates that p17 promotes virus production while also promoting autophagy. When cells were transfected with wild type p17, the virus yield was significantly enhanced compared with transfection of NLS-mutated p17.