The bipartite genome of pea enation mosaic virus (PEMV) is often accompanied by a non-essential third RNA ( 0.3 × 10) of unknown origin and function. Although the Wisconsin strains of PEMV originally lacked this RNA, we have monitored the appearance of a putative replicative form of this species in PEMV-infected tissue. In later generations encapsidated single-stranded RNA 3 appeared. We have used a 750 bp clone generated against the ds replicative form of RNA 3 to probe viral and host-derived nucleic acids to establish the relationship of this RNA to PEMV infection. Northern blot analysis showed that RNA 3 is distinct from viral genomic RNA and from host RNA. Similarly, Southern blot analysis showed that RNA 3 is distinct from the host genome. Infectivity analysis of fractionated viral RNAs coupled with Northern blot analysis confirmed that RNA 3 is both non-essential for PEMV infection, and non-infectious when inoculated on its own. RNA 3 does not influence symptom expression, aphid transmission or particle morphology. We conclude that RNA 3 of PEMV is a satellite RNA.


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