Date of Award


Degree Type


Degree Name

Doctor of Philosophy


The potexvirus clover yellow mosaic virus (CYMV) is a 540 nm long flexible rod-shaped virus which contains as its genetic material a messenger-sense single-stranded RNA. We have cloned a cDNA to the genomic RNA (gRNA) of CYMV and, through collaboration, have determined its entire nucleotide sequence. The 7015 nucleotide gRNA encodes five major open reading frames (ORFs) which are arranged in a similar manner to those in other potexvirus gRNAs. A variety of in vitro and in vivo techniques were used to examine how viral proteins encoded by CYMV are expressed. Expression of ORFs encoded 3{dollar}\sp\prime{dollar} in the gRNA appear to be controlled primarily through the production of subgenomic messenger RNAs.;Members of a population of 1.2-kb viral RNAs present in some CYMV infections were cloned and sequences, and were found to represent gRNA mutants with large internal deletions. These small viral RNAs (termed defective RNAs (D RNAs)) were shown to be dependent on the parental virus for replication and not to interfere with symptom development in infected plants. The prototype CYMV D RNA is 1172 nucleotides in length and is composed the 5{dollar}\sp\prime{dollar} and 3{dollar}\sp\prime{dollar} termini of the CYMV gRNA. A unique feature of all sequenced CYMV D RNAs is that they encode a hybrid ORF representing the in-frame fusion of portions of the 5{dollar}\sp\prime{dollar} and 3{dollar}\sp\prime{dollar} terminal ORFs of the gRNA. Biologically active transcripts synthesized in vitro from a cloned cDNA of the prototype D RNA were used to investigate the importance of this fusion ORF. The fusion ORF was essential for viability of the prototype D RNA since mutations introduced into the prototype D RNA which disrupted large portions of the fusion ORF prevented its accumulation in planta. We suggest that translation of the D RNA may be coupled to its replication and/or encapsidation and/or stability. Modified in vitro transcripts of the D RNA were also used to characterize a hexanucleotide sequence (5{dollar}\sp\prime{dollar}..ACUUAA..) conserved in potexvirus gRNAs. It was determined that deletion or substitution of the entire six-base sequence in the D RNA rendered it nonviable in vivo. We propose that this hexamer motif represents a cis element involved in viral RNA replication.



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