|Lewandowski, Dennis - , CREC|
Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 24, 2005
Publication Date: November 10, 2005
Citation: Lewandowski, D.J., Adkins, S.T. 2005. The tubule-forming nsm protein from tomato spotted wilt virus complements cell-to-cell and long-distance movement of tobacco mosaic virus hybrids. Virology. 342:26-37. Interpretive Summary: Demonstrating viral protein function in the absence of a reverse genetics system is complicated because of the inability to do even simple mutagenesis experiments. It has proven difficult to create an infectious clone of viruses with negative-strand or ambisense genomes, such as those within the genus Tospovirus. Despite several lines of evidence showing that the Tomato spotted wilt virus (TSWV) NSm protein has characteristics of plant virus movement proteins, including sequence similarity, expression during the early stages of infection, RNA-binding activity and the ability to induce the formation of tubules, a movement function has never been proven. Using a combination of approaches that included in planta complementation experiments and cloning and mutagenesis of the putative MP, the lack of a reverse genetics system for TSWV was overcome allowing demonstration that NSm is indeed the TSWV movement protein. This report continues a cooperative virology research effort between ARS and the University of Florida Citrus Research and Education Center.
Technical Abstract: A Florida isolate of Tomato spotted wilt virus (TSWV) was able to complement cell-to-cell movement of a movement-defective Tobacco mosaic virus (TMV) vector. To test for complementation of movement in the absence of other TSWV proteins, the open reading frame for the NSm protein was expressed from TMV constructs encoding only the TMV replicase proteins. NSm was expressed from either the coat protein or movement protein subgenomic promoter, yielding viruses that moved cell to cell in inoculated leaves of tobacco, providing the first functional demonstration that NSm is the TSWV movement protein. Furthermore, these viruses moved into upper leaves of Nicotiana benthamiana, demonstrating that NSm can support long-distance movement of viral RNAs. Tubules, characteristic of the NSm protein, were also formed in tobacco protoplasts infected with these viruses. The C-terminus of the NSm protein was shown to be required for movement. The combination of single-cell and intact plant experiments to examine multiple functions of a heterologous viral protein provides a generalized strategy with wider application to other viruses also lacking a reverse genetic system.