Cloning Of LIP5 From Spodotera Frugiperda And The Effect Of Overexpression Of LIP5 On The Infectious AcMNPV Production

In eukaryotic cells, the endosomal sorting complex required for transport(ESCRT) is composed of ESCRT-0,-I,-II,-III, VPS4 and some accessory proteins, such as Alix. The ESCRT machinery is required for multivesicular bodies(MVB) formation, cytokinesis, autophagy, and plasma membrane repair. Additionally, ESCRT is also involved in some enveloped viruses entry and/or budding and release. VPS4 is an AAA ATPase. Recently, it was found that, in yeast and human cells, VPS4 forms complex with Vta1(in humans termed as LIP5). Vta1/LIP5 is required for oligomerization and activation of VPS4 and promoting the hydrolysis of ESCRT-III complex.In this study, the coding sequence of LIP5 was isolated from Spodoptera frugiperda Sf9 cells. Sequence analysis indicated that the length of S. frugiperda LIP5 is 969 bp, encoding a protein with predicted molecular weight of 35.35 kDa and isoelectric point of 5.24. Multiple sequence alignment revealed that S. frugiperda LIP5 is closely related with the homologous proteins of yeast, humans, and other insects. The amino acid identities of S. frugiperda LIP5 with that of yeast and humans are 20% and 48.8%, respectively, and with that of the other insects are 45.9-78.6%. The essential domains of MIT1, MIT2 and VSL, which mediate the interaction of LIP5 with ESCRT-III or VPS4, are also highly conserved in insect LIP5. In addition, in insects LIP5, two proline-rich motifs were found within the linker region which connects MIT and VSL domains.By using overlap PCR, we made three truncation mutant of S. frugiperda LIP5 including deletion of the N-terminal MIT1, MIT2, or the C-terminal VSL domain from LIP5(constructs termed as nDF, MD, and cDR, respectively), and six point mutations M63 A, W146 D, K287 A, K290 A, K287A/K290 A, and L315 E. The mutated LIP5 fragments were N-terminally fused with GFP and transiently expressed in Sf9 cells. Overexpression of most of the LIP5 mutant did not alter the morphology of Sf9 cells. However, two mutated constructs K287A/K290 A and MD, which containing the alanine substitution of two charged residues within VSL domain or deletion of the N-terminal MIT2 domain, caused the class E phenotype(K287A/K290A) or changed the morphology of Sf9 cells from round to spindle. Biomolecular fluorescent complementary(BIFC) assay revealed that deletion of the C-terminal VSL domain or double alanine substitution of K287 and K290 disrupted the interaction of LIP5 with VPS4, while deletion of the N-terminal MIT1 or MIT2 domain disrupted the interaction of LIP5 with the ESCRT-III components VPS46 or VPS60.Finally, using the viral complementation system, we found that overexpression of the mutated LIP5 nDF, MD, cDR, M63 A, or K287A/K290 A, significantly reduced the infectious AcMNPV production. These results suggest that LIP5 is involved in AcMNPV infection. However, the detailed mechanism related with this is still need further investigation.