Date of Award

8-2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

David A. Brian

Committee Members

Melissa A. Kennedy, Albrecht Von Arnim, Stephen J. Kennel

Abstract

Primary and higher order RNA structures in the 5’ and 3’ untranslated regions of the genomes of positive-strand RNA viruses are known to function as cis-acting elements for genome translation and replication. Four cis-acting replication elements have been identified in the bovine coronavirus (BCoV) 3’ untranslated region that are postulated to recruit viral and cellular proteins for assembly of the replication complex and initiation of negative-strand synthesis. This study had the goal of identifying and establishing the function of the viral proteins that bind BCoV 3’ terminal cis-acting RNA structures. A major surprising discovery was a specific interaction of the BCoV-encoded 2’-O-methyltransferase (2’-O-MT), an enzyme normally involved in 5’-terminal cap methylation, with a 3’-terminal cis-acting octamerassociated bulged stem-loop. This discovery led to detailed analyses of BCoV 2’-O-MT that is summarized as follows: (i) Enzyme probing revealed the higher-order structure of the cis-acting 3’-terminal bulged stem loop. (ii) Specific binding of the 2’-O-MT to both the 5’ cap-0 structure and to the 3’-terminal bulged stem-loop was documented. (iii) Enhanced translation of a viral genomic RNA was demonstrated as a function of 2’-O-MT methylating activity. (iv) A requirement for 2’-O-MT methylating activity for virus replication was demonstrated with the use of the mouse hepatitis coronavirus reverse genetics system. (v) Over-expression of the viral 2’-O-MT in virus-infected cells had no detrimental in-trans effect on viral genome replication, transcription, or translation, but affected DI RNA accumulation in-cis. These results indicate that the coronaviral 2’-O-MT is essential for virus genome translation and replication and, as a multifunctional protein, is an attractive target for development of an anticoronaviral therapeutic drug.

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