Deletion and point mutation analyses were employed to determine gene products and cis-acting signals involved in translation, replication, and encapsidation of barley yellow dwarf virus (PAV serotype) RNA in oat protoplasts. Of the six open reading frames (ORFs), only ORFs 1 and 2, which include the putative RNA-dependent RNA polymerase gene, were required for replication. In vitro translation of these mutants revealed that sequence upstream of the shifty heptanucleotide was required for ribosomal frameshifting, and that a 3'-translational enhancer stimulated translation more efficiently when located in closer proximity to the translated ORFs. Deletion of the coat protein gene reduced the accumulation of genomic but not subgenomic RNA. The carboxy-terminally extended form of the coat protein, produced by readthrough of its stop codon, was not required for encapsidation. Although the ORF6 product was not necessary, cis-acting RNA signals in and around ORF6 were required for RNA replication. Defective RNAs harboring various deletions were not replicated in trans by the co-inoculated wild-type helper genome, suggesting that replication of PAV RNA may be coupled to translation.