Roles Of Insect Autophagy Pathway In Acmnpv Infection

In eukaryotic cells,autophagy plays important roles in cellular homeostasis,stress responses,and infected pathogens immunity.Autophagy contributes to innate antiviral immunity by delivering entried viruses to lysosomes for degradation or it can be hijacked by viruses for efficient replication.Recently,roles of autophagy in viral infection received widespread attention.Baculoviruses are important insect pathogens.Presently,however,the relationship between host cellular autophagy pathway and baculoviruses infection is not clear.In this study,the comparative genome analysis indicated that more than 40 autophagy-related genes(ATG)are highly conserved in yeast,human,and sequenced insect genomes.Quantitative real-time PCR analysis of the transcription level of more than 40 autophagy-related genes in AcMNPV-infected Trichoplusia ni cells Tnms42 and Spodoptera frugiperda cells Sf9 showed that the expression level of almost all the ATG genes was changed(down-regulated or up-regulated)during 0 ~ 6 hours post-infection,and gradually down-regulated during 12 ~ 48 hours post-infection.Further analysis revealed that the expression level of 14 ~ 22 ATG genes was up-regulated in the infected cell lines,whereas 11 ~ 13 of them was down-regulated.To evaluate the formation of autophagosome in AcMNPV-infected Sf9 cells,a GFP-tagged ATG8 transient-expression plasmid GFP-SfATG8 pBlue was constructed.In transient transfected and then infected Sf9 cells,the number of the GFP-ATG8 puncta significantly increased at 36 ~ 48 h post-infection.Western blot analysis indicated that ATG8-PE can be detected at 36 ~48h postinfection.Using a dsRNA-based RNA interference,we found that knockdown the expression of ATG14 and ATG101 in Sf9 cells significantly decreased the expression of a reporter gene LacZ,which is controlled by AcMNPV immediately early gene ie1 promoter,whereas the the knockdown did not affect the expression of GUS,which is directed by AcMNPV p6.9 late promoter.These results suggest that ATG14 and ATG101 might be involved in efficient entry of budded virions of AcMNPV.