Date of Award:

5-1-2000

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

Joseph K.-K. Li

Committee

Joseph K.-K. Li

Committee

Gregory J. Podgorski

Committee

Anne J. Anderson

Committee

William H. Scouten

Committee

John D. Morrey

Abstract

Bluetongue virus (BTV) is an arthropod-borne pathogen that causes bluetongue disease (BD) throughout the world, with economic losses primarily coming from infection of domestic livestock. Although BTV has been well studied at the molecular level since 1969, the mechanism of viral replication has not yet been fully determined. The virus is composed of seven structural and four nonstructural proteins, and the role of each protein has been investigated but has not been conclusively established within the viral life cycle. This study focused on the nonstructural protein NS2 in an attempt to examine its functions as part of the viral replication and assembly process. NS2 has been successfully cloned and expressed in a prior study, and three single-stranded RNA (ssRNA) binding regions were mapped within the protein. To further investigate the functions of NS2 in interacting with ssRNA, a prokaryotic expression system was utilized to generate a series of seven deletion mutants for the determination of proposed ssRNA-binding sites. These seven mutants were then expressed in large scale. Each truncated protein was purified to near homogeneity, and then examined for the ability to bind to ssRNA by electrophoretic mobility shift assays (EMSA). As a result, the three ssRNA-binding regions of BTV nonstructural protein NS2 were conclusively localized. NS2 was expressed in mammalian cells as a fusion to a yellow fluorescent protein (YFP), under the control of a cytomegalovirus promoter. The cellular location of the expressed, YFP-tagged, NS2 protein was then observed by laser scanning confocal microscopy. The NS2/YFP co-localized with assembling virions in the cytoplasm. The expressed NS2/YFP was found to have ssRNA-binding ability when extracted from cells, and this ability was independent of BTV infection. Plaque assays were performed to determine the effect of the NS2/YFP protein upon the production of viral progeny when the transfected cells were infected by BTV. The same procedures were also carried out for a NS2 deletion mutant which had all three proposed ssRNA-binding sites removed. Overexpression of NS2 was found to inhibit viral replication, while the NS2 mutant had no effect upon the formation of viral plaques. Normal and NS2-expressing mammalian cells, with and without BTV infection, were also examined using transmission electron microscopy. Viral inclusion bodies and progeny virions were observed within the cytoplasm of the normal cells, but far fewer virions were found in the cells expressing NS2/YFP. The presence of the overabundant NS2 inhibited the completion of the viral replication process, most likely by sequestering the viral mRNAs away from the assembling virions. These findings are consistent with the proposed role of NS2 as a carrier of nascent viral mRNAs into assembling viral cores within intracellular viral inclusion bodies. NS2 thus has a vital role in bringing BTV ssRNA segments to the sites of progeny virion assembly for packaging into the viral inner cores where the double-stranded RNA viral genome is produced.

Download

Share

COinS

Copyright for this work is retained by the student. If you have any questions regarding the inclusion of this work in the Digital Commons, please email us at .