Optimization of Maize fine streak virus (MFSV) protein expression in Drosophila S2 cells
F. CISNEROS (1), M. Redinbaugh (2)
(1) The Ohio State University, Wooster, OH, U.S.A.; (2) USDA, ARS - The Ohio State University, Wooster, OH, U.S.A.
Phytopathology 101:S37

MFSV is a negative-sense RNA virus in the family Rhabdoviridae that is transmitted by the leafhopper G. nigrifons. The virus replicates in both its maize host and insect vector. In order to determine whether Drosophila S2 cells can be used as a system to study MFSV replication, we tested conditions for optimal expression of MFSV proteins, a replicon construct and the T7 RNA polymerase. Two of the three proteins required for synthesis of the MFSV genomic RNA, MFSV N and P, could be expressed from pMT/V5-His-Topo vector in S2 cells for at least 4 days after induction with CuSO4. Experiments are underway to assess MFSV L protein expression in S2 cells. Preliminary results suggest that the L protein can be detected only when expressed from linear DNA fragments. The replicon construct carrying the MFSV transcriptional initiation and termination sequences flanking an antisense GFP cDNA sequence downstream of a T7 promoter was inserted into the same vector. The transcript accumulated in transfected cells for at least 4 days. As expected, the GFP protein did not accumulate in transfected cells in the absence of the MFSV components and T7 polymerase. Expression of constructs encoding the T7 polymerase indicated that the protein could be detected in S2 cells for at least 4 days when fused to a nuclear localization signal peptide (NLS), but did not accumulate when lacking the NLS. Our results indicate that the proteins and constructs required for MFSV replication can be expressed in S2 cells.