ABSTRACT
The red clover necrotic mosaic virus genome is composed of two single-stranded RNA components, RNA-1 and RNA-2. The viral capsid protein is translated from a subgenomic RNA (sgRNA) that is transcribed from genomic RNA-1. Here, a 34-nucleotide sequence in RNA-2 is shown to be required for transcription of sgRNA. Mutations that prevent base-pairing between the RNA-1 subgenomic promoter and the 34-nucleotide trans-activator prevent expression of a reporter gene. A model is proposed in which direct binding of RNA-2 to RNA-1 trans-activates sgRNA synthesis. This RNA-mediated regulation of transcription is unusual among RNA viruses, which typically rely on protein regulators.
Subject(s)
Mosaic Viruses/genetics , RNA, Viral/genetics , Transcriptional Activation , Base Composition , Base Sequence , DNA, Complementary , Gene Expression , Genes, Reporter , Green Fluorescent Proteins , Luminescent Proteins/genetics , Models, Genetic , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Promoter Regions, Genetic , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Viral/biosynthesis , RNA, Viral/chemistry , Sequence AlignmentABSTRACT
The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two single-stranded RNA species. The polycistronic RNA-1 encodes the viral RNA polymerase and capsid protein (CP) and the monocistronic RNA-2 encodes the 35 kDa cell-to-cell movement protein (MP). Nicotiana benthamiana plants transformed with the RCNMV MP gene were generated. When inoculated onto the MP transgenic plants, cell-to-cell movement of RNA-1 occurred at a rate similar to wild-type virus. However, long-distance (leaf-to-leaf) movement of RNA-1 was not observed. Neither CP nor virions were detected in the inoculated leaves of the MP transgenic plants. When RNA-1 was coinoculated with RNA-2 mutants, which do not express a functional MP, onto MP transgenic plants, CP and virions were readily detected and a systemic infection resulted. These results demonstrate that both RNA-1 and RNA-2 are necessary for the accumulation of both CP and virions. Furthermore, CP accumulation was found to be required for long-distance movement of RCNMV. Therefore, these data provide evidence that CP, in the form of virions, is necessary for the long-distance movement of RCNMV.
Subject(s)
Mosaic Viruses/growth & development , RNA, Viral/physiology , Viral Proteins/genetics , Virus Assembly/physiology , Capsid/analysis , Capsid/physiology , Fabaceae/virology , Genes, Viral/genetics , Mosaic Viruses/physiology , Plant Leaves/virology , Plant Viral Movement Proteins , Plants, Genetically Modified , Plants, Medicinal , Plants, Toxic , RNA, Viral/genetics , RNA, Viral/metabolism , Nicotiana/genetics , Nicotiana/virology , Virion/metabolismABSTRACT
The movement proteins (MPs) of tobacco mosaic tobamovirus (TMV) and red clover necrotic mosaic dianthovirus (RCNMV) enlarge plasmodesmata size exclusion limits, transport RNA from cell to cell, and bind nucleic acids in vitro. Despite these functional similarities, they have no sequence homology. However, they do appear to have similar secondary structures. We have used transgenic plants expressing either the TMV MP or the RCNMV MP, and a chimeric TMV that encodes the RCNMV MP as its only functional MP gene, to demonstrate that the MPs of TMV and RCNMV are functionally homologous. Further, both TMV and RCNMV can act as helper viruses to allow the cell-to-cell movement of the heterologous movement-defective viruses. These data support the conclusion that, despite other differences, such as particle morphology, host range, and sequence, TMV and RCNMV share a common mechanism for cell-to-cell movement.
