The Functional Study On Bmmpv Orf60 And The Host Protein Sqstm1/p62 In Autophagy

Sequestosome 1(SQSTM1,namely p62,SilkDB: BGIBMGA002620-PA)of Bombyx mori is an adaptor as well as a cargo of autophagy-lysosome pathway(ALP).The research presented interaction of p62 with polyhedrin and Bm60 coded by orf60 of Bombyx mori nucleopolyhedrovirus(Bm NPV).The main results were showed as follows:1.Bm60 played a role in formation of ODV envelope.Using Red/ET recombination system the orf60 was disrupted,and then the polyhedrin was transposed to construct wild type(WT)and orf60 lacking(WT-60null)bacmids.The polyhedra were present in BmN cells infected with WT-60 null under a light microscopy.The ODV envelope produced by WT-60 null was not observed by a transmission electron microscopy;however,these virions were also embedded into the polyhedra.Polyhedra of WT-60 null were purified,treated with an alkaline solution.The ODVs were pelleted by sucrose gradient centrifugation.Western blot analysis of WT-60 null ODV observed the nucleocapsid protein VP39,and some per os infectivity factors(PIFs)such as PIF1,PIF2,PIF3 and PIF5,but not P74/PIF0 and ODV-E66.These results indicated that lacking of Bm60 led to termination of envelope shaping of ODV.2.The autophagic analysis of Bm60.Previous study found that many proteins of BmNPV formed aggregate/aggresome in the cytoplasm.The p62,as an adaptor in the ALP,plays an important role in aggrephagy.This study found that Bm60 could be degraded by autophagy through the interaction with p62.Bm60 could be modified by cellular ubiquitin in a rare form.3.The interaction of p62 with polyhedrin.Over-expression of p62 significantly inhibited occlusion body production,suggesting protective activity of p62 against infection of BmNPV.In infected BmN cells,p62 was co-localized with polyhedrin.Co-immunoprecipitation(Co-IP)showed that p62 interacted with polyhedrin.These results indicated that p62 mediated degradation of polyhedrin by ALP.4.A study of p62.Protein p62 fused to N-and C-terminus of EGFP was expressed using BmNPV bacmidsystem.Western blot analysis with a mouse monoclonal anti-GFP antibody showed great difference of band profiles of EGFP-p62 and p62-EGFP,suggesting that p62 may be cleaveged or translationally modified.Truncated-p62 proteins fused with mCherry were expressed.The fluorescence microscopy results suggested that aa 1-200 and 530-612 of p62 played an important role in formation of p62 inclusion bodies.In infected BmN cells,p62 was co-localized with some well-studied cellular proteins,such as ubiquitin,LC3,HSC/HSP70 and NEDD8.These results indicated that p62 acted as an adaptor to promote the degradation of viral proteins by ALP.