Mechanism Of Newcastle Disease Virus-mediated Amino Acid Metabolic Reprogramming In Host Cells

Newcastle disease virus?NDV?is a member of the Paramyxoviridae family and a single-stranded,negative-sense RNA virus.NDV infects many kinds of chickens and birds and results in high morbidity and mortality,which cause huge economic losses and seriously threatening the development of poultry industry.The virus,a strict intracellular parasite,needs to manipulate the metabolism and resources of host cells to create favorable conditions for its own survival and proliferation.In this study,metabolomics and molecular biology were used to study metabolic reprogramming in NDV-infected cells,and then to understand the metabolomic profiling and clarify the effect of amino acid metabolism on NDV replication and its mechanism.The main research contents are as follows:1.DF-1 cells infected with the vNDV strain Herts/33 and the lungs from Herts/33-infected specific pathogen-free?SPF?chickens were analyzed via UHPLC-QTOF-MS.These results showed that NDV infection broke the metabolic homeostasis of DF-1 cells and chicken lung,promoted their metabolic reprogramming,and significantly affected the metabolism of amino acids and nucleotides.The identification of these different metabolites and metabolic pathways will provide considerable important information for further understanding of NDV replication needs and pathogenesis.2.Through the target metabolomics analysis of UHPLC-MS,the changes of amino acid metabolism in DF-1 and A549 cells after NDV infection and glutamate and aspartate were significantly increased.The amino acid composition analyses of purified-NDV,DF-1 and A549 cells showed that NDV had certain preference for glutamate and aspartate.These results provide a possible explanation for the efficient and rapid replication of NDV on DF-1 and A549 cells from the perspective of amino acid metabolism.3.By analyzing the changes of amino acid transporter in NDV-infected cells,we found that the mRNA levels and expression levels of SLC1A3 were significantly up-regulated after NDV infection.After down-regulating SLC1A3 by siRNA,inhibitor,and knock-down cell line construction,the replication level of NDV was significantly decreased.These results suggest that NDV may facilitate self-replication by increasing the expression level of SLC1A3 in infected cells.4.NDV replication required glutamine and there was in a dose-dependent manner.Through the detection and analysis of glutamine transporters and related enzymes during glutamine metabolism,we found that GOT1 and GPT were closely related to NDV replication.The results of ¹³C labeled glutamine tracer experiment showed that after NDV infection,glutamine metabolism was changed,glutamine catabolism and glutamate production were promoted.5.Through immunofluorescence and Western blot analysis,we found that SLC1A3 was located on cell membrane,mitochondrion,and lysosome.And SLC1A3 may be regulated by activation of p53 and NF-?B pathway,which contributed to the transport,production of glutamate,and the maintenance of TCA cycle.In conclusion,this study found that amino acid metabolism was significantly changed after NDV infection.And it was found for the first time that NDV may facilitate self-replication by increasing the expression level of SLC1A3 in infected cells.In addition,we also found that glutamine is closely related to NDV replication.Glutamine catabolism,glutamate transportation and production were promoted in NDV-infected cells.These results will help us to understand the demand of amino acids and how to regulate the host cell amino acid metabolism during NDV replication,which provide some clues and directions for future research.