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Molecular characterization of rubella virus nonstructural proteins and viral RNA synthesis Liang, Yuying
Abstract
Rubella virus (RV) is a single-strand positive RNA virus. Its 9762-nucleotide (nt) genomic RNA contains two large open readings frames (ORFs) at the 5'- and 3'-termini, encoding nonstructural and structural proteins, respectively. The nonstructural proteins (NSPs) are first translated as a 200-kDa polyprotein (p200), which undergoes a single proteolytic cleavage into pl50 and p90 by an intrinsic protease, the RV nonstructural protease (NS-pro). The purpose of this thesis work is to characterize RV NSPs and viral RNA synthesis at the molecular level, including the characterization of the RV NS-pro domains, the roles of NSPs in viral RNA synthesis, and the regulatory mechanism for viral RNA synthesis. RV NS-pro is an M-group papain-like cysteine protease (PCP) with both trans- and cis-cleavage activities. Employing an in vitro translation system, I mapped the regions of NS-pro required for trans- and cis- activities, and demonstrated the importance of an X domain in rrarcs-cleavage activity. RV NS-pro catalytic region was also predicted to have a papain-like folding from primary and secondary structure analyses. The time course of RV RNA synthesis was precisely determined. Synthesis of negative-strand RNA stops after 10 h postinfection (hpi), and subsequently switches to highly efficient synthesis of positive-strand RNAs (genomic and subgenomic RNA). The roles of respective NSPs and the underlying regulatory mechanism for RV RNA synthesis were investigated by mutational analysis and complementation experiments. Processing of p200 was found to be crucial for virus replication. Uncleaved p200 was shown to be functional in negative-strand RNA synthesis but not for positive-strand RNA. In contrast, the complex formed by the cleavage products p150/p90 is not an active replicase for negative-strand RNA, but is required for the efficient generation of positive-strand RNAs. Syntheses of both negative- and positive-strand RV RNAs were found to be cis-preferential from complementation experiments. A mechanism for RV NSP translation, processing and RV RNA replication was proposed. Newly translated p200 functions in cis to synthesize a full-length negative-strand RNA. The subsequent processing of p200 into p150/p90 converts this replicase into one with positive-strand RNA specificity, which functions in cis to produce genomic and subgenomic RNA efficiently.
Item Metadata
Title |
Molecular characterization of rubella virus nonstructural proteins and viral RNA synthesis
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2000
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Description |
Rubella virus (RV) is a single-strand positive RNA virus. Its 9762-nucleotide (nt) genomic
RNA contains two large open readings frames (ORFs) at the 5'- and 3'-termini, encoding
nonstructural and structural proteins, respectively. The nonstructural proteins (NSPs) are first
translated as a 200-kDa polyprotein (p200), which undergoes a single proteolytic cleavage into
pl50 and p90 by an intrinsic protease, the RV nonstructural protease (NS-pro).
The purpose of this thesis work is to characterize RV NSPs and viral RNA synthesis at the
molecular level, including the characterization of the RV NS-pro domains, the roles of NSPs in
viral RNA synthesis, and the regulatory mechanism for viral RNA synthesis.
RV NS-pro is an M-group papain-like cysteine protease (PCP) with both trans- and cis-cleavage
activities. Employing an in vitro translation system, I mapped the regions of NS-pro
required for trans- and cis- activities, and demonstrated the importance of an X domain in
rrarcs-cleavage activity. RV NS-pro catalytic region was also predicted to have a papain-like
folding from primary and secondary structure analyses.
The time course of RV RNA synthesis was precisely determined. Synthesis of negative-strand
RNA stops after 10 h postinfection (hpi), and subsequently switches to highly efficient
synthesis of positive-strand RNAs (genomic and subgenomic RNA). The roles of respective
NSPs and the underlying regulatory mechanism for RV RNA synthesis were investigated by
mutational analysis and complementation experiments. Processing of p200 was found to be
crucial for virus replication. Uncleaved p200 was shown to be functional in negative-strand
RNA synthesis but not for positive-strand RNA. In contrast, the complex formed by the
cleavage products p150/p90 is not an active replicase for negative-strand RNA, but is required
for the efficient generation of positive-strand RNAs. Syntheses of both negative- and positive-strand
RV RNAs were found to be cis-preferential from complementation experiments.
A mechanism for RV NSP translation, processing and RV RNA replication was proposed.
Newly translated p200 functions in cis to synthesize a full-length negative-strand RNA. The
subsequent processing of p200 into p150/p90 converts this replicase into one with positive-strand
RNA specificity, which functions in cis to produce genomic and subgenomic RNA
efficiently.
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Extent |
11266510 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-07-24
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0099576
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2000-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.