Screening And Identification Of Sf9Cellular Proteins Interacting With AcMNPV ODV-E18

Baculoviruses are a family of enveloped viruses with double-standed DNA genome, which specifically infect arthropods. They are harmless to human and vetebrates and are used for biological control of crop pests, and baculovirus expression system is widely used for production of vaccines and other bioactive proteins.During infection baculoviruses produce two types of virions, occlusion derived virion (ODV) and budded virion(BV). ODV is the viral form responsible for primary infection. After released from occlusion bodies in midgut of host insects, ODV particles pass through the peritrophic matrix and enter into the midugut epithelial cells where replication occurs. Since envelop proteins of the ODV play an important role in the invasion process, studying the function of ODV envelop proteins has important theory significance for understanding the molecular mechanisms infection, Knowleges about function of ODV envelop proteins.would provide basis for modifications of the viruses for more efficient application.Autograph californica multiple nucleopolyhedrovirus (AcMNPV) is the type species of baculoviruses and is most widely studied. AcMNPV orf143encodes ODV envelope protein ODV-E18(E18). The homologs of the gene exists in all the sequenced baculovirus genomes, and is one of the31core genes baculoviruses. This study is focused on interaction between AcMNPV E18and host cellular proteins.i) Screening of Sf9proteins interacting with AcMNPV E18.AcMNPV orf143was cloned into a yeast plasmid, fused with the GAL4binding domain-coding sequence, and was used as a bait to screen a cDNA library of Sf9cells, by yeast two-hybrid assays. As a result,278positive clones were identified.48of them were selelected for further examination, of which,42remained to be positive in retesting experiments. Sequencing results show that the42cDNA clones contain35different sequences.15of them encode proteins which have homologs found in data bases. These inclide7mitochondria-related proteins,6proteins related to protein glycosylation and transportation, and2regulatory factors involved in gene expression,ii) Subcellular localization analysis of AcMNPV E18and three Sf9proteinsSubcellular distribution of three Sf9proteins identified, by Yeast two-hybrid assays, to interact with AcMNPV E18were analyzed by fluoresence-labeling and conforcal microscopy, and compared with the E18. These Sf9proteins including homologs of two mitochondrial ribosomal proteins (MRPS17and RPL41) and a myosin light chain9peptide. The coding sequences of the Sf9proteins were individualy fused with a copy of the red fluresent protein gene, and inserted into the AcMNPV genome, together with the e18gene fused with the green fluresent protein gene, by using the Bac-to Bac system. In the cells separately infected by the recombinant viruses, E18-EGFP was seen in cytoplasm around the nuclear membrane; the MRPS17-RFP, RPL41-RFP and MRPS17-RFP are also found in cytoplasm. Co-locolization between E18-EGFP and RPL41-RFP was observed around nuclear membrane, indicating that El8and RPL41may interact in nuclear membrane area.