Identification and characterization of the red clover necrotic mosaic virus origin of assembly sequence

The Red clover necrotic mosaic virus (RCNMV) genome, consisting of two single-stranded +-sense RNA molecules, are packaged by a single capsid protein (CP) into 30--35 nm icosahedral particles with T = 3 quasi-symmetry. It is unknown whether the genomic RNAs, RNA-1 and RNA-2 are co-packaged into a single virion or packaged individually into separate particles. There are two lines of evidence of evidence to support either claim. Density gradient centrifugation of RCNMV virus particles results in a single sharp band whereas the profile of RNA purified from particles results in 2--3 times more RNA-2 than RNA-1. These results suggest that RCNMV produce more than one type of particle with similar densities. However, it is unknown how both RNAs are encapsidated into particles. It is likely that both RNAs contain packaging signals that are recognized by CP initiating assembly into particles. To identify specific signals on the RNAs we engineered mutations into both RNA-1 and RNA-2 and tested their packaging efficiencies using a plant infection assay. We demonstrated that RNA-1 does not package in the absence of RNA-2. RNA-2 elements were studied for their importance for packaging and a 209-nucleotide region in the movement protein open reading frame was identified. Further deletions to pin point the specific element indicate that a 34-nucleotide bulged stem loop (previously identified as the trans -activator stem loop that directs the synthesis of coat protein subgenomic RNA from RNA-1) is the origin of assembly of RCNMV.