Deletion and point mutation analyses were employed to determine gene p
roducts and cia-acting signals involved in translation, replication, a
nd encapsidation of barley yellow dwarf virus (PAV serotype) RNA in oa
t 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 reveal
ed that sequence upstream of the shifty heptanucleotide was required f
or ribosomal frameshifting, and that a 3'-translational enhancer stimu
lated translation more efficiently when located in closer proximity to
the translated ORFs. Deletion of the coat protein gene reduced the ac
cumulation of genomic but not subgenomic RNA. The carboxy-terminally e
xtended form of the coat protein, produced by readthrough of its stop
codon, was not required for encapsidation. Although the ORF6 product w
as not necessary, cis-acting RNA signals in and around ORF6 were requi
red for RNA replication. Defective RNAs harboring various deletions we
re not replicated in trans by the co-inoculated wild-type helper genom
e, suggesting that replication of PAV RNA may be coupled to translatio
n. (C) 1995 Academic Press, Inc.