RESUMO
A previous functional analysis of the genome of grapevine virus A (GVA) was not conclusive as to the role of open reading frame 5 (ORF 5). This ORF encodes a 10-kDa protein (p10) carrying two distinct domains: a basic, arginine-rich domain and a zinc-finger domain. P10 was cloned and expressed in Escherichia coli, and was shown by northwestern assays to interact with nucleic acids. In-frame deletion of the basic region abolished P10's nucleic acid-binding capability, whereas substitution of cysteine residues by serine in the zinc-finger domain did not affect binding. These mutations were inserted into the full-length infectious clone. It has been shown that ORF 5 mutations do not affect replication of GVA-RNA. However, plants inoculated with the aforementioned mutations did not develop symptoms, and Western blot analysis revealed markedly reduced expression of the movement protein (the product of ORF 3).
