The Silkworm Autophagy Adaptor Protein P62/Ref(2)p Contains Four Nuclear Localization Signals And Was Cleaved Duing BmNPV Infection

p62 protein,also the best known sequentsome1/（SQSTM1）,is an autophagy-specific adaptor involved in regulating various signaling pathways including autophagy.The p62 protein has multiple functional domains.In the silkworm genome,the p62 gene expresses two protein products,p62 and Ref（2）p.Protein sequence alignment analysis showed that,compared with p62,Ref（2）p lacks PB1 domain,whereas the other sequences were identical,indicating that there were biological similarities between Ref（2）p and p62.Our previous studies have demonstrated that:⑴over-expression of p62 in cells followed by infection with BmNPV T3 will lead to a remarkable decrease in polyhedrin expression and polyhedra production in the nucleus of infected cells,suggesting a restriction activity of p62 against infection of BmNPV.⑵The silkworm p62 and Ref（2）p are CRM1-dependent nucleoplasmic shuttling proteins.⑶The expression profiles of endogenous p62 and Ref（2）p change greatly upon infection with BmNPV T3.It is speculated that p62 and Ref（2）p may be cleaved,thus losing the restriction effect on propagation of viral progeny.Based on these studies,in this thesis we further investigated the nuclear localization signals in sequences of silkworm p62and Ref（2）p,and identified a cleavage site in p62 and Ref（2）p upon BmNPV infection.The main results are showed as follows.1.Determination of the nuclear localization signals of p62 and Ref（2）pThe Ref（2）p protein,as the paralog of the silkworm p62,was used as the research subject.According to the rules of amino acid sequence of nuclear localization signal,a series of Ref（2）p truncated mutants were constructed and analyzed using a Leica TCS SP7 confocal laser scanning microscope.It is found that the silkworm Ref（2）p protein contains four nuclear localization signals,namely NLS1（aa 97-101）、NLS2（aa 127-130）、NLS3（aa 78-81）and NLS4（aa 108-110）,which was further confirmed by a cellular fractionation experiment.The strength of four nuclear localization signals was determined by laser confocal microscope and protein expression analysis of nuclear/cytoplasm fractionation to be NLS3>NLS1a>NLS1b>NLS2.2.Identification of the cleavage site of the silkworm host protein p62 and Ref（2）p upon BmNPV T3 infection⑴The previous results have inferred that the cleavage site of Ref（2）p upon BmNPV T3 infection is located between aa 246～286.Based on these results,a series of deletion mutants were constructed to be fused with e GFP for transient expression.At48 h post transfection,Bm N cells were infected with BmNPV T3,and Western blot analysis was performed to determine that the cleavage site was located in aa 272-276.The point mutants in this region were constructed.The expression profile of e GFP-Ref（2）p-L₂₇₆E showed the band with a molecular weight about 60 k Da disappeared,indicating that the Ref（2）p cleavage site was the L₂₇₆,i.e.the L₃₈₀ of p62.⑵Previously,we used the BmNPV bacmid system to express the fusion proteins p62-e GFP and e GFP-p62 and found that the expression profiles of two protein were quite different.The L₃₈₀ of p62 was mutated to E₃₈₀,and the fusion proteins e GFP-p62(L₃₈₀E)and p62(L₃₈₀E)-e GFP were expressed using the BmNPV bacmid system.It was showed that a specific band disappeared,indicating that L₃₈₀ is the cleavage site of p62.3.An attempt study to determine the protein（s）responsible for cleavage of p62/Ref（2）p during BmNPV infectionAccording to the time course of virus infection,totally 88 viral genes to express some late and structural viral components were cloned to be fused with m Cherry.These proteins were individually co-expressed with wild-type Ref（2）p.Protein samples were harvested for Western blot analysis,thus further analyzed the proteins for cleavage of Ref（2）p.The BmNPV bacmid system was used to express the fusion proteins e GFP-p62and e GFP-Ref（2）p respectively.Co-IP was performed by the GFP-Trap（?）M kit,and then LC-MS/MS was performed to preliminarily analyze the viral proteins that might interact with Ref（2）p and p62.Totally 21 for Ref（2）p and 82 viral proteins for p62 were observed.These results laid the foundation for further investigation to determine the protein（s）to cleave Ref（2）p and p62.