The Interaction Between Two Baculovirus Proteins And Host Proteins

Baculoviruses are arthropod-specific viruses with a double-stranded, circular DNA genome. There are extensive virus-host interactions during the life cycle of the viruses. Interaction between viral and host proteins usually involves in regulation of viral life cycle and control of host machine. ODV-E25and PP31are two important proteins of baculovirus. ODV-E25was first identified as an envelop protein in ODV of AcMNPV. odv-e25gene is one of the baculovirus core genes, and its homologs present in all sequenced baculovirus genomes sequenced to data. PP31is a phosphorylated DNA binding protein, which locolized in the virogenic stroma. pp31gene presents in all sequenced genomes of lepidopteran baculoviruses. The roles of ODV-E25and PP31in virus life cycle are not clear yet. In this study, we did analysis on interactions between ODV-E25/PP31and host proteins..1. A cDNA library of Sf9cell infected by Autographa california multiple nucleopolyhedrovirus (AcMNPV) was constructed. To construct the cDNA library, total RNA was extracted from Sf9cells infected by AcMNPV, and used for synthesis of complementary DNAs using SMART technology. The cDNAs were co-transformed into yeast AH109cells with the yeast expression vector pGADT7to produce a cDNA library, in which the cDNAs were fused with the GAL4activation domain-coding sequence. The cDNA inserts in the library were detected by PCR, showing a size renage of0.25kb to2.1kb. The titer of the library is6.735×108cells/ml.2. The AcMNPV orf94(odv-e25) was cloned into pGBKT7, fused with the GAL4DNA binding domain-coding sequence. The resultant plasmid was used as a bait to screen the cDNA library above by Yeast two-hybrid assays, to identify cDNA clones encoding proteins interacting with the ODV-E25. As a result,121positive clones were identified, which represent ten different cDNA fragments. Two of them have homologs found in data bases. They are separately homologous to a mitochondrial ribosomal protein and a glyceraldehyde-3-phosphate dehydrogenase.3. In a previous study, a cDNA clone from a cDNA library of larva of Plutella xylostella was identified, by yeast two-hybid assays, encoding a protein interacting with PP31of Plutella xylostella granulovirus (PlxyGV). The putative product of the cDNA clone is a homolog of receptor for activated protein C kinase (RACK). In this study, the PlxyGV pp31and rack gene was separately expressed in an E. coli to produce protiens fused with a GST or a6×His tag. It was shown that the rack was expressed as a38kD peptide as prospected. The38kD His-tagged peptide was co-purified with GST-PP31by GST-bind resin in GST Pull-down assays, confirming interaction between the PlxyGV PP31and the RACK protien of P. xylostella.