Comovirus Isolation and RNA Extraction

Comoviruses are small, icosahedral viruses with a diameter of approx 28 nm (Fig. 1). Presently the genus Comovirus is comprised of 15 different viruses, of which the type species, cowpea mosaic virus (CPMV), is the most thoroughly studied (,). All viruses are transmitted by beetles and have a rather narrow host range. Most comoviruses have legumes as their natural hosts and usually cause mosaic or mottling symptoms. Comoviruses are mechanically transmissible, and can replicate to high levels in infected cells. Purified preparations of comoviruses consist of two, sometimes three, distinguishable particles, which can be separated by centrifugation on sucrose density gradients. These particles are designated as bottom (B), middle (M), and top (T) component, corresponding to their position in the centrifuge tube. The B- and M-component are nucleoprotein particles, each containing a segment of the single-stranded, bipartite RNA genome (denoted RNA-1 and RNA-2, respectively); T-component consists of empty protein shells. Both B- and M-components, or their RNAs, are necessary for infectivity (). Open image in new window Fig. 1. Genetic organization of the CPMV genome. The icosahedral virus particles consist of two components, denoted B and M, that contain RNA-1 and RNA-2, respectively. The ORFs in the RNA molecules are indicated with open bars and VPg with a black square. The RNAs are translated into polyproteins that are proteolytically processed at six specific sites, indicated below the ORFs, into several stable intermediate and nine final cleavage products. These latter products are indicated in the ORFs. Functions of the different domains in the polyproteins are shown above the ORFs with the following abbreviations: MP, movement protein; CO-PRO, cofactor required for proteinase; HEL?, putative helicase; PRO, proteinase; POL, RNA-dependent RNA polymerase.