|LEWANDOWSKI, DENNIS - UNIVERSITY OF FL
Submitted to: American Society for Virology Meeting
Publication Type: Proceedings
Publication Acceptance Date: 6/1/2003
Publication Date: N/A
Technical Abstract: Tomato spotted wilt virus (TSWV) has one negative sense (L) and two ambisense (M) and (S) RNAs. The M RNA encodes the membrane glycoproteins (G1 and G2) in the viral-complementary sense and a non-structural protein (NSm) in the viral sense. NSm has been presumed for several years to be the TSWV movement protein (MP) based upon several lines of indirect evidence, although the lack of a tractable TSWV reverse genetics system has impeded direct demonstration. We determined that a Florida field isolate of TSWV could complement local, and to a lesser extent, systemic movement of a movement-defective Tobacco mosaic virus (TMV) expression vector in Nicotiana benthamiana. To determine if NSm was responsible for this complementation, we cloned the NSm open reading frame (ORF)from this isolate and constructed two TMV hybrids, one with the NSm ORF inserted downstream of the TMV MP subgenomic (sg) promoter and a second with the NSm ORF inserted downstream of the TMV coat protein sg promoter. Both TMV/TSWV hybrids replicated in tobacco suspension cells and directed expression of NSm as determined by Northern and western blots, respectively. These free-RNA hybrids formed local lesions in N. tabacum cv. Xanthi nc and moved into upper leaves of N. benthamiana, directly demonstrating that NSm is indeed the TSWV MP. To monitor movement in plants, the ORF for the jellyfish green fluorescent protein (GFP) was inserted behind an additional sg promoter downstream of NSm. These TMV/TSWV hybrids moved and expressed both NSm and GFP in plants. These results demonstrate the usefulness of a well-characterized viral genetic system to dissect gene functions for a virus for which a genetic system is currently unavailable.