SPODOPTERA-FRUGIPERDA SF-9 CELLS NUCLEAR FACTOR BINDS TO A SPECIFIC SEQUENCE ON THE BACULOVIRUS POLYHEDRIN PROMOTER

The promoter of the baculovirus polyhedrin gene is used for the heterologous expression of proteins in the baculovirus expression system. The polyhedrin gene of baculovirus is a very late gene that is hyper-expressed 18-24 h post-infection (P.I). We analyzed the DNA-binding activity of the polyhedrin promoter using gel mobility shift analysis, DNaseI footprint analysis and UV cross-linking experiments. Specific DNA-binding activity was found in uninfected cells. Specific binding activity was also found in cells during the early stages of viral infection and it decreased 18-24 h P.I. The DNA-binding activity localized to positions -72 to -86 on the polyhedrin promoter relative to the initial ATG. The molecular mass of the protein interacting with the DNA was in the range of 200 kDa. The data implies the presence of a host-encoded DNA-binding protein that acts as a negative regulator and is involved in the delayed expression of polyhedrin. In an initial attempt to test this hypothesis, transient cotransfection assays were performed in infected cells. These experiments were designed to assess the in vivo competition of proteins that interact with the polyhedrin promoter. The results support the concept of negative regulation. Thus it is possible that baculovirus utilizes an insect cell-derived nuclear factor to control the differential expression of late versus very late genes. A model that can account for our observations is discussed.