Masters Theses

Date of Award

5-2002

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

David A. Brian

Abstract

A naturally-occurring 2.2kb defective interfering (DI) RNA of bovine coronavirus (BCo V) has been cloned and is being used as a minireplicon to characterize the cis-acting elements in its structure for DI RNA replication. The DI RNA is comprised of the two ends of the virus genome, the 5 '-most 498-nt and the 3 '-1803-nt including a poly(A) tail of 68-nt, and it replicates in the presence of the parent helpervirus. Within the DI RNA is a single open reading frame (ORF) resulting from the fusion of 288-nt of gene 1 a and the entire 1348-nt N gene. A 30-nt in-frame reporter has been inserted at base position 1098 to enable quantitative Northern analyses of DI RNA replication. The hypothesis that cisacting elements for replication lie within the 288-nt partial ORF 1 a region was suggested by (1) the presence of a similar region in all coronavirus DI RNAs described to date, and (2) a previous experiment which showed that DI RNA replication was blocked by conversion of the ORF 1 a start codon to a non-start codon. To test this hypothesis, the study described here was undertaken to map and characterize the cis-acting element( s) within the partial ORF la region. For this, 5 prime-ward in-frame deletions of the 288-nt gene 1 a region were made from its 3' terminus and transcripts were tested for replication. It was learned that 3 '-terminal, in-frame deletions of 102 nt ( encoding aa 63-96) but not 141 nt (encoding aa 50-96), were tolerated for DI RNA replication. Further analysis of the remaining 186-nt revealed the presence of two cis-acting elements. The first is a protein encoded by the ORF. This was learned from frameshift experiments that changed the identity of 12 and 26 amino acid stretches, respectively, while maintaining a fused ORF. These changes resulted in the loss of DI RNA replication. The second cis-acting element is a higher-order RNA structure. This was learned by testing a phylogeneticallyconserved, computer (mfo/d)-predicted 36-nt RNA stem-loop located between nt 308 and 343. Mutations predicted to disrupt the stem while maintaining the wild-type amino acid sequence destroyed replicating ability whereas compensatory mutations restoring the secondary structure also restored replicating ability. This study is the first to identify cisacting elements for coronavirus RNA replication mapping within the ORF 1 a region of the genome. It is also the first study to show any function for coronavirus gene 1 a. It is predicted that the cis-acting elements identified here for DI RNA replication will have the same function in the full-length bovine coronavirus genome.

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