Positional effect of deletions on viability, especially on encapsidation, of Brome mosaic virus D-RNA in barley protoplasts.

Brome mosaic virus (BMV), a tripartite RNA plant virus, accumulates RNA3-derived defective RNAs (D-RNAs) in which 477-500 nucleotides (nt) are deleted in the central region of the 3a protein open reading frame (ORF), after prolonged infection in barley. In the present study, six artificial D-RNAs (AD-RNAs), having deletions of the same size as the naturally occurring D-RNA but at different positions in the 3a ORF, were constructed and tested for their amplification and encapsidation in barley protoplasts by coinoculation with BMV RNA1 and 2, or RNA1, 2, and 3. Northern blot analysis of RNA accumulation in total and virion fractions showed that deletions of 492 nt in the 3'-proximal and the 5'-proximal regions of the 3a ORF decreased encapsidation efficiency of the AD-RNAs compared with that of RNA3, whereas deletions in the central region enhanced encapsidation efficiency. The present results also show that deletion positions affect competition with RNA3 in the amplification and encapsidation of AD-RNAs.