The localization of virus-specific double-stranded RNA of cowpea mosaic virus in subcellular fractions of infected Vigna leaves

The fraction of a homogenate of cowpea mosaic virus (CPMV) infected leaves sedimenting at 1000g for 15 min was further divided by centrifugation on a discontinuous gradient consisting of layers of 60, 45, and 20% sucrose. It was possible to separate the nuclei and nuclear fragments from most of the chloroplasts.Hybridization with ³H]uridine labeled CPMV RNA and electron microscopy were used to detect CPMV-specific double-stranded RNA in different fractions of the gradient. By hybridization 70–90% of the double-stranded RNA was found in the so called chloroplast-fraction. By electron microscopy it was confirmed that double-stranded RNAs with lengths characteristic for double-stranded viral RNAs occurred predominantly in this chloroplast-fraction. The chloroplast-fraction also contained vesiculated membrane structures and amorphous electron-dense material, which are characteristic structures from CPMV-infected cells, suggesting that CPMV-specific double-stranded RNA might be associated with either or both of these structures rather than with the chloroplasts.