Screening And Identification Of Host Proteins Interacting With Three AcMNPV Proteins

Baculoviruses are the most beneficial viruses to humans in all known types of viruses. They are potentially important pollution-free biological pesticides, and are wieldly used, as eukaryotic expression vectors, for production of bio-active drugs and vaccines. The interaction between virus and host cells is a focus in the research fields of virology. Studing interactions between baculovirus proteins and host cell proteins would helprevealing the movement of the virus in the cells, functions of viral proteins and mechanism of virus replication.AcMNPV is a representative species of baculovirus.The pk-1 gene of AcMNPV encodes a serine/threonine kinase that is required for very late expression such genes as polh and p10. Homologs of the pkl gene present in all the genomes of lepidopteran baculoviruses. AcMNPV vubi, encodes a ubiquitin. Many viruses have been reported to evolve different strategies to utilize the ubiquitin-proteasome pathway for their own benefits. The viral ubiquitin plays an importent role in virus budding and release, but the mechanism is not clear. The P6.9, rich in basic amno acid residues, is associated with viral DNA and required for assembly of nucleocapsid. The holologs of p6.9 gene present in all baculovirus genomes sequenced to data. In the study, yeast two-hybrid experiments were performed to screen genes encoding proteins interacting with AcMNPV PK1, ubiquitine and P6.9 respectively, from a cDNA library of Sf9 cells.Two-hybrid screening identified several independent cDNA clones interacting with PK1. Sequence analysis revealed that some cloned have homolog in the protein databases. These are RNA-binding protein, CG6831 gene product from transcript CG6831-RA, a dehydrogenase, a RNA-dependent DNA polymerase, and a protein tyrosine phosphatase. To further characterize interaction between the viral PK1 and the 10-5 protein, homolog of RNA-binding protein, the pkl of AcMNPV and the 10-5 gene were cloned and expressed in E. coli for production of the proteins. Polyclonal antibodies specific to AcMNPV PK1 and to the 10-5 protein respectively were obtained from rabbits immunized with the purified proteins. Immunoprecipitation assays were performed using the PK1-specific antibodied or 10-5-specific antibodies which were mixed with lysate of the Sf9 cells infected with AcMNPV. GST-pulldown assays were performed to further validate the interaction between PK1 and the 10-5 protein. In addition, the Sf9 cells were transfected by a transient expression vector of pk1 to test effect of PK1. Apoptosis was observed in the cell culture following transfection. It suggests that AcMNPV may be involved in cell apoptosis.Yeast two-hybrid screening identified two independent cDNA clones of Sf9 cells, which encode proteins interacting with AcMNPV ubiquitin. These two host proteins are homologous to a RNA polymerase-associated protein and a polyubiquitin respectively. Similarly, two genes encoding proteins interacting with AcMNPV P6.9 were identified from the cDNA library of Sf9 cells. These host proteins are homologous to a ribosomal protein and a mitochondrial matrix protein respectively.