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.