Date of Award

12-2008

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

David A. Brian

Committee Members

Chunlei Su, Igor B. Jouline, Albrecht von Arnim

Abstract

Coronaviruses are a family of positive-sense, single-stranded, 5’-capped and 3’- polyadenylated RNA viruses that replicate entirely in the cell cytoplasm. Replication of the viral genome requires translation to produce proteins used for RNA synthesis and virion assembly. The 5’- and 3’- untranslated regions of the coronavirus genome have been found to contain cis-acting elements that are required for replication of the genome and a defective interfering RNA. Presumably, both viral and cellular proteins interact with these elements and serve as trans-acting factors in genome translation and replication. Of interest is the functional significance of a 5’-proximal cis-acting 397-nucleotide region which includes the 210-nucleotide 5’ untranslated regions and the 5’ proximal 187 nucleotides of the coding region for the first nonstructural protein, p28.

Here, research examining the structural requirements in this region and in the 3’ untranslated region for bovine coronavirus replication is presented. The following features were discovered: (i) A second higher order structure within the coding region of p28 is required for replication but not translation of the defective interfering RNA. Although no viral proteins were found to bind this region, two cellular proteins of ~60 and ~100kDa were found which might prove to be essential trans-acting factors. (ii) p28, the first synthesized protein in the genome is an RNA binding protein that interacts with cis-replication stem-loops in the 5’ and 3’ untranslated regions. p28 may therefore function through these interactions to regulate genome translation or replication. (iii) In constructs with a renilla luciferase reporter containing only the genomic 5’ and 3’ untranslated regions, in vitro translation in the absence of viral proteins revealed a 5’ and 3’ end interaction that mediates a repression of cap-dependent translation. Therefore, translation of the coronavirus genome may require the assistance of viral proteins.

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