Characterization of Barley Yellow Dwarf Virus Subgenomic Rnas.

Transcription of subgenomic RNAs (sgRNAs) is a common strategy used by many positive strand RNA viruses of plants and animals to regulate viral gene expression. Barley yellow dwarf virus (BYDV) produces three nested 3'-coterminal subgenomic RNAs in infected cells. SgRNA1 serves as the messenger for the structural and movement proteins. sgRNA2 encodes a small open reading frame (ORF6), that appears not to be translated. The role of sgRNA3 is unknown, as it encodes no ORFs. Neither sgRNA2 nor sgRNA3 is needed for BYDV RNA replication in oat protoplasts. However, sgRNA2 does function as a riboregulator of viral translation in virus-infected protoplasts. This dissertation focuses on the control of synthesis of sgRNA2, and the biological roles of sgRNAs 2 and 3 in plants. The first aim is to determinine the primary and secondary structures in BYDV RNA required for synthesis of subgenomic RNA2. The minimal promoter for sgRNA2 was previously mapped to a 143 nt region (nt 4810-4952) just downstream of its putative transcription start site at nt 4809. This region encompasses the 3' BYDV cap-independent translation element (BTE, nts 4814-4918) that is essential for virus replication. Deletion of the entire 3' BTE from within a duplicated copy of the sgRNA2 promoter did not affect sgRNA2 synthesis from this promoter, confirming the functional independence of the sgRNA2 promoter from the 3' BTE. I also found that a small stem-loop containing the conserved hexanucleotide sequence GUGAAG at its 5' end supports basal levels of sgRNA2 synthesis. All functional sgRNA2 promoter constructs retained potential base pairing between sequences flanking the BTE. This reveals that the sgRNA2 promoter is split by the embedded 3' BTE and that both the primary and secondary structures are required for sgRNA synthesis. Such an overlapping arrangement of translational and transcriptional control signals has not been observed in other viruses. The second aim is to examine the roles of sgRNA2 and sgRNA3 in whole plant infections. Infectivity of viral RNA containing mutations that knock out synthesis of one or both of these RNAs was tested. All sgRNA knockout mutants infected oat plants and usually showed normal levels of viral RNA accumulation, disease onset, and symptoms. ELISA revealed that coat protein levels in plants infected with the mutants deficient in sgRNA2 alone, or sgRNA2 and sgRNA3 were about double that observed in plants infected with wild-type virus. SgRNA3 knockout mutant virus gave wild-type levels of coat protein. SgRNA2 was found to modestly inhibit virion accumulation, while the absence of sgRNA3 in virus infections had little effect on virus accumulation and disease development in infected oats. These surprising results are the first example of subgenomic RNAs that are apparently dispensible for virus infection. They indicate that sgRNA2 must play only a minor role as a riboregulator of viral gene expression. However, absence of sgRNAs 2 and/or 3 may reduce virus fitness in subtle ways that were not detected in my experiments.