Proteomic Analysis Of Bombyx Mori Nucleopolyhedrovirus Polyhedra Matrix And Studies Of Components Polyhedrin And Product Encoded By Open Reading Frame 134

Bombyx mori nucleopolyhedrovirus(Bm NPV), pathogenic for Bombyx mori Linnaeus, is a double stranded-DNA virus with rod-shaped envelope virions. The genome of Bm NPV was 128413 nucleotides long and encoded 136 open reading frames(ORFs). At the late stage of infection, the polyhedrin was highly expressed and a large amount of polyhedra were produced within the nucleus of infected cell. The baculovirus polyhedra is a kind of natural protein crystal, in which some occlusion-derived viruses(ODVs) were embedded. Though progress was made for decades for polyhedrin and polyhedra, the detailed component proteins in baculovirus polyhedra matrix currently remaine obscure. In this study, the Bm NPV polyhedra matrix fraction was subjected to LC-ESI-MS/MS analysis based on Q Exactive, and a total of 91 viral and 32 host proteins were identified. Furthermore, two of these component proteins, namely polyhedrin and the product encoded by Bm NPV O RF134(Bm134), were investigated further. The results are showed as follows:1. Determination of component proteins in Bm NPV polyhedra matrixBm NPV polyhedra matrix proteins were obtained by discontinuous sucrose gradient centrifugation, subjected to SDS-PAGE separation and then LC-ESI-MS/MS analysis based on Q Exactive. Totally 91 viral and 32 host proteins were identified. O f 91 viral proteins, most may be not essential for polyhedra formation as evidenced in the literature, suggesting the powerful ability of polyhedra to encapsulate some proteins. These 32 host components were grouped into one o f six categories, i.e. chaperones, ubiquitin and related proteins, host helicases, cytoskeleton-related proteins, RNA-binding proteins and others. The roles of these host proteins in baculovirus replication and their effects on the polyhedra packaging will be of significant interest in future studies.2. The ubiquitination and autophagic analysis of polyhedrinThe results of Western blot analysis showed that polyhedrin of polyhedra matrix fraction presented polymerization and/or post-translational modification and partial degradation. The immunoprecipitation together with Western blot observations showed that polyhedrin was ubiquitinated, which was confirmed further by co localization of polyhedrin with the host ubiquitin(B.mori Ubi) and the ubiquitin encoded by Bm NPV(Bm Ubi) in infected Bm N cell; moreover, polyhedrin interacted and was co- localized with the microtubule-associated protein 1- light chain 3(Bm LC3, Silk DB: BGIBMGA011783-PA) on the isolation membrane of autophagosome, suggesting polyhedrin was modified by ubiquitins and functioned as a autophagic adaptor to be involved in cel ular autophagy.3. The autophagic analysis of Bm134The Bm134 gene-targeted disruption was performed by using Red/ET recombination system, and a Bm134-deleted,-rescued and wildtype viruses were constructed.Western blot founding suggested that Bm134 was also patially degraded. It was observed that Bm134 were co- localized with the host Bm LC3, sequestosome-1(p62, Silk DB: BGIBMGA002620-PA), ubiquitin(B.mori Ubi and Bm Ubi) by subcellular colocalization analysis. These results indicated that Bm134 was targeted with p62 into the cytoplasmic inclusion bodies then degraded by celluar autophagy. The relationship of Bm134 with Bm LC3, p62 and ubiquitin should be further studied.4. The preliminary study of the engineered polyhedra micro-crystal devicesSome recombinant Bm NPVs to express enhanced green fluorescent protein(EGFP)-fused polyhedrin truncations and Bm134 were constructed to co-infect Bm N cells, and then the polyhedra were purified. After 4 months storage at 4℃, the fluorescence intensity of polyhedra displayed indistinguishable changes, indicating the potential application of polyhedra as a framework to stabilize some proteins.