The Preliminary Study Of Autophagy Induced By The Infection Of BmNPV

Bombyx mori nucleopolyhedrovirus?BmNPV?,pathogenic to silkworm,Bombyx mori Linnaeus,is a DNA virus that a large,circular,supercoiled and double-stranded DNA genome within a rod-shaped nucleocapsid.During the course of infection,a large number of viral proteins are synthesized in a relatively short time and a large amount,inducing the host celluler antiviral defense response such as the cellular autophagy.In this study,we mainly targets at the analysis of autophagy against BmNPV infection,in which the viral protein polyhedrin and the autophagic marker proteins,BmGABARAP and BmLC3D,were selected as the research focuses.The results are summarized as follows:1.Phylogenetic analysis and classification of autophagy related gene 8?atg8?of Bombyx moriThe published amino acid sequences of human Atg8s were retrieved and used as queries in BLASTP searches against the SilkDB with default settings.As a result,besides BGIBMGA011783-TA that had been reported as atg8 gene previously,the gene BGIBMGA010251-TA was identified in this study.The BLASTP program on the NCBI website was applied to search human Atg8 orthologs in the database of a specific insect species whose genomes have been partially or completely sequenced.A total of 208 sequences that contain ATG8domain were retrived.Multiple alignments of amino acid sequences were performed with the software MEGA 7.0 using the built-in MUSCLE program at default settings.The poorly aligned positions and variable regions of the alignment were trimmed by the program Gblocks with default parameters,to obtain conserved blocks,and phylogenetic trees were constructed using maximum likelihood?ML?method of the software MEGA 7.0.The reliability of a phylogenetic tree was estimated by the bootstrap method with 1000 replications.The numbers of atg8 genes in insect genomes varied from one to three.Relative to three subfamilies of GATE-16,GABARAP and LC3of human ATG8 family,there were not GATE-16 paralogs that were found to be encoded by insect genomes.So these insect ATG8s identified in this study belong to subfamily GABARAP and LC3.The genes,BGIBMGA011783-TA and BGIBMGA010251-TA of the Bombyx mori genome were classified as GABARAP and LC3 subfamily,respectively,and their corresponding proteins are named BmGABARAP and BmLC3D respectively.The results of Western blot and subcellular localization revealed that BmLC3D co-localized with BmGABARAP,the well-defined autophagic indicator,indicating the BmLC3D is a new member of silkworm ATG8 family protein.Site-directed mutagenesis were used to mutate the residue G116 and G100 of BmGABARAP and BmLC3D respectively,and Western blot analysis confirmed that they were ATG4 cleavage site of BmGABRAP and BmLC3D.Additionally,though residue G100 of BmLC3D was mutated,the fluorescence microscopy analysis showed that the autophagic dots were formed.According to the conserved domain prediction in a BLASTP search,BGIBMGA010251-PA was appeared to be a pre-protein containing tandem head-to-tail repeats of the monomeric ATG8 sequence.At glycine 100,BGIBMGA010251-PA was cleaved by silkworm ATG4a and/or ATG4b to produce two ATG8s.One contained amino acids 1 to 100 which was described above as the fusion protein eGFP-BmLC3D;the other is amino acids 101 to 245 that was designated cleaved BmLC3D?cBmLC3D?in this study.These findings above prompted the possibility that the cBmLC3D was an ATG8 as well,which should be confirmed further.2.BmNPV infection induced autophagy in BmN cellsWestern blot findings suggested that the endogenous BmGABARAP-II band could be detected at 32 h in response to the medium nutrition depletion?MND?stress.Accompanying the progress of MND stress,the amount of Bm GABARAP-II increased slowly.The BmGABARAP punctas were observed in BmN cells,while BmLC3D spots were hardly detected.In infected BmN cells,BmGABARAP-II could be detected at 0 h post-infection?p.i.?,and the the amount of BmGABARAP-II increased rapidly.The fluorescence microscopy analysis also showed that both BmGABARAP and BmLC3D punctas could be formed to be hollow structures,differ from that emerged in MND-Stress.These results indicated that BmNPV infection induced autophagy in BmN cells.3.Preliminary study on the involvement of polyhedrin in autophagyThe co-immunoprecipitation together with Western blot dectction showed that polyhedrin interact with BmLC3D,which was further proved by colocalization of polyhedrin with the eGFP-BmLC3D.These results indicated that polyhedrin was involved in autophagy and contained an ATG8 interaction motif?AIM?.Using the AIM sequence logo,the AIM of polyhedrin was predicted to be ⁴⁷WDLL⁵⁰ and ¹⁰⁷FPIV¹¹⁰.Western blot analysis showed that the ⁴⁷WDLL⁵⁰ was the AIM of BmGABARAP.The AIM of BmLC3D should be confirmed further.