Expression of Tomato spotted wilt virus RNA and protein in yeast from cDNA clones
Thompson AN1, Kainz M2, German TL1
1Department of Entomology, University of Wisconsin-Madison, Madison, WI, USA.
2Department of Biology, Colgate University, Hamilton, NY, USA
Correspondence: ant@entomology.wisc.edu
Tomato spotted wilt virus (TSWV) is a plant infecting virus in the family Bunyaviridae that replicates in its insect vector, Frankliniella occidentalis. Our long term goal is to produce infectious Tomato spotted wilt virus entirely from cloned components expressed from plasmids in yeast cells. We are using yeast cells because (1) they provide number of selectable markers to facilitate the delivery of all components required for the system from plasmids; (2) the system has been used successfully for positive sense RNA viruses and (3) the ability to use well established yeast genetic approaches to help understand the host genes involved in TSWV replication. We are using an approach similar to that used for animal viruses to establish the replicon system. We produced a product that was a faithful copy of the TSWV S RNA by reverse transcription PCR using primers that added a modified phage T7 promoter to generate the desired 5¨ end. We then added a modified Hepatitis Delta Virus (HDV) ribozyme to generate the proper 3¨ end and cloned the fragment into the yeast expression vector pRS426. Finally, we marked our construct by changing a single nucleotide in the NSs ORF to create a silent mutation that added a PmlI restriction site to the S sequences. This generated the plasmid TSWVREP, the basis of our replicon system. The TSWV N open reading frame was amplified from the plasmid pACT2-N, inserted behind the Gal1/Gal10 promoter, and cloned into yeast plasmid pLDB138 to create pTSWVN. The T7 RNA polymerase gene was amplified by PCR, modified to include the SV40 T antigen nuclear localization sequence, and inserted downstream from the ADH promoter to create pT7RNAP. Yeast cells (strain L2612) transformed with TSWVREP and pT7RNAP express the TSWV S RNA. Northern analysis verifies that S RNA extracted from viral RNPs isolated from infected plants and replicon RNA isolated from yeast co-migrate RT-PCR products using RNA from plant RNPs are not cleaved with PmII whereas RT-PCR products from the replicon S RNA extracted from yeast are digested with PmII as expected. Western analysis of yeast cells transformed with pTSWVN demonstrated that TSWV N protein is efficiently and stably expressed. Finally we have isolated an RNP-like structure from yeast transformed with all three plasmids that bands in a CsCl isopycnic gradient at the same density (1.32g/cc) as TSWV RNPs isolated from plants. Our data suggests that we are producing authentic, traceable, TSWV RNPs in yeast cells.
