Title: Factors Influencing the Production of MFSV Full-Length Clone: Maize Fine Streak Virus Proteins in Drosophila S2 Cells Authors
|Cisneros, Fiorella -|
Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: June 1, 2010
Publication Date: August 1, 2010
Citation: Cisneros, F., Redinbaugh, M.G. 2010. Factors Influencing the Production of MFSV Full-Length Clone: Maize Fine Streak Virus Proteins in Drosophila S2 Cells. Phytopathology. 100:S680. Technical Abstract: Maize fine streak virus (MFSV) is negative-sense RNA virus member of the genus Nucleorhabdovirus. Our goal is to determine whether Drosophila S2 cells can support the production of a full-length clone of MFSV. We have previously demonstrated that the full-length MFSV nucleoprotein (N) and phosphoprotein (P) can be steadily expressed in S2 cells for at least 4 days. In contrast, the expression of the MFSV replicase protein (L) had not been detected in S2 cells under the same conditions. This is not surprising since this protein contains some characteristics that may not be desirable when propagated in bacteria. To avoid these problems, we are testing the expression of the L protein using linear DNA fragments (LDF) instead of circular plasmids. We have generated a LDF containing only eukaryotic regulatory elements for expression of foreign genes in Drosophila S2 cells flanking the MFSV-L gene by means of PCR. Our preliminary results suggest that the MFSV-L protein can be produced in S2 cells. Experiments are underway to optimize the expression of this protein. Finally, the expression of the T7 DdRp needed for the generation of a sense full-length clone of MFSV have also been tested. Our results indicate that T7 DdRp can be steadily expressed in S2 cells only when fused to the Nuclear Localization Signal peptide (NLS) for at least 4 days. These results are important in order to optimize the conditions for the production of an infectious full-length clone of MFSV in S2 cells.