Research On The Prion Aggregation Of AcMNPV LEF-10

The Autographa californica Multiple Nucleopolyhedrovirus（Ac MNPV）late expression factor LEF-10 is required for baculovirus late gene expression.Previously study has found that over-expression of LEF-10 formed high molecular weight complexes in both E.coli and Sf9 cells,which has strong SDS-resistance character.The same aggregation character has been reported in some prion.Study on this subject contains two parts.Part one,observe and detect the aggregation of LEF-10 with cell biology technique and immunological technique.Part two,study the aggregation character of LEF-10 with yeast red/white assay,especially identifying the Prion Domain（Pr D）and Prion Site（Pr S）,which using a series of truncated or mutational LEF-10.To investigate the aggregation character of natural LEF-10,we constructed recombinant baculovirus v Ac/P（lef-10）-LEF-10-His.Our data suggested that the natural LEF-10 could aggregate to form punctate spots in virus infected Sf9 cells.The similar aggregation phenomenon also had been observed in virus infected Sf9 cells which overexpress LEF-10-GFP.Following western blot showed that the natural LEF-10 has never express beyond 8 hour past infection.The interaction between full-length LEF-10 and LEF-101–65 was confirmed in HEK 293 T cells by bimolecular fluorescence complementation（Bi FC）assays,indicating that the full-length LEF-10 was more prone to aggregation.Molecular weight mass spectrometry suggested that LEF-10_27-78 exists in monomer,dimmer,trimer,tetramer.This aggregation type help us understand and remind us that LEF-10 maybe is a prion.Prion character research on LEF-10 is conducted by yeast red/white assay,which confirming their [PSI+] or [psi-]phenotype is a standard of prion or non-prion.The assay system is composed of a reconstructied plasmid named p Sup35N1-5MC and a yeast strain LJ14 deleting sup35 gene from its chromosome genomes.Employing the yeast red/white assay system,we confirmed that LEF-10_1-48、LEF-10_1-65、LEF-10 are prion,and their [PSI+] phenotype could be cured to [psi-] phenotype.Then,we conducted amino acid sequence alignment of the LEF-10 with CLUSTALW.Proceeding from the conserved amino acids situation,we identified three relatively conserved regions（C1,C2 and C3）and constructed 5 truncated LEF-10(LEF-10_1-41 、 LEF-10_35-62、LEF-10_54-78、LEF-10_1-62、LEF-10_35-78).LEF-10_1-41 showed [PSI+] phenotype causing it has prion domain.To investigate the precise prion domain（Pr D）,the following 19 truncated LEF-10(LEF-10_1-34、LEF-10_1-33、LEF-10_12-41、LEF-10_12-34、LEF-10_12-33、LEF-10_13-41、LEF-10_13-34、LEF-10_13-33、LEF-10_16-41、LEF-10_16-34、LEF-10_16-33、LEF-10_14-32、LEF-10_14-31、LEF-10_14-30、LEF-10_14-29、LEF-10_15-32、LEF-10_15-31、LEF-10_15-30、LEF-10_15-29was constructed.The yeast assay system showed that LEF-10_14-32 is the prion domain of LEF-10.To investigate the prion site（Pr S）,the 8 single amino acid mutations was separately constructed in both full-length LEF-10 and truncated LEF-10_1–41 by overlap PCR..The yeast assay showed that 16 mutational LEF-10(LEF-10_1-41^L12A、LEF-10_1-41^I13A、LEF-10_1-41V16A、LEF-10_1-41^N20A、LEF-10_1-41^L21A、LEF-10_1-41^I24A、LEF-10_1-41^I29A、LEF-10_1-41^V33A、LEF-10^L12A、LEF-10^I13A、LEF-10^V16A、LEF-10^N20A、LEF-10^L21A、LEF-10^I24A、LEF-10^I29A、LEF-10^V33Ashowed [psi-] phenotype,and the conclusion is that these 8 hydrophobic amino acids are prion site（Pr S）.In conclusion,our study found that LEF-10 is a prion-like protein.As prion-like protein has only been reported in mammal animal and fungus,our finding extends the current knowledge of the existence range of prion-like proteins,and provides a new evidence for the hypothesis that prion is a common phenomenon in biological world.The found of prion domain and prion sites of LEF-10 could explain the strong aggregation tendency to some extent.It is helpful to investigate the function of LEF-10 on Ac MNPV life process.