Two abundant subgenomic RNAs, designated RNA 3B and RNA 5, were found to be associated with the V strain of tomato aspermy cucumovirus (V-TAV). Sequence determination showed that the 3'-terminal 323 nucleotides (nt) of RNA 3B was identical to RNA 5, whereas its 5'-terminal 163 nt was a direct repeat (one nt difference) of the 5'-half of RNA 5, and that both RNAs are completely homologous to the 3'-terminal untranslated region of TAV RNA 3. TAV RNAs 3B and 5 were also detected in the infection of a pseudorecombinant virus consisting of TAV RNA 3 and RNAs 1 and 2 from cucumber mosaic virus. Furthermore, only RNA 5, not RNA 3B, was detected in a TAV mutant in which one of the repeats was deleted from RNA 3. These genetic studies clearly show that both RNA species are derived from TAV RNA 3. However, in contrast to TAV RNAs 4 and 4A, which encode coat protein and 2b protein, respectively, RNAs 3B and 5 represent a novel class of subgenomic RNAs from TAV that do not function as mRNAs. Possible functional roles for such a class of viral subgenomic RNAs are discussed.


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