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Study of the Role of mutations identified in the M27, M36, m139, m141, and m143 ORFs of the murine cytomegalovirus (MCMV) temperature-sensitive mutant tsm5

Alali, Abdulaziz (2011)
Ph.D. thesis, University of Birmingham.

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Infection with human cytomegalovirus (HCMV), usually asymptomatic in healthy individuals, can cause severe or fatal disease in infants and immunocompromised patients. The generation of a potent protective vaccine is necessary to protect vulnerable people. Because of host restriction, murine cytomegalovirus (MCMV) is used as a model for HCMV. Previously, we have generated a temperature-sensitive mutant, tsm5, which failed to replicate in mice yet protected them against virus challenge. Several mutations have been identified in this mutant by Comparative Genome Sequencing (GCS) (Roche NimbleGen); 10 synonymous and 15 non-synonymous single nucleotide polymorphisms (SNPs). Among these are m139 (Y565X) and m141 (V195M), shown to be essential for replication in macrophages but not in fibroblasts, m143 (M232I), shown to play an important role in the inhibition of the PKR-mediated host antiviral response, M27 (A658S ), involved in interference with interferon- signalling, and M36 (V54I), an anti-apoptotic protein. In the present study, the above mentioned mutations were introduced individually into the MCMV K181 (Perth) variant bacterial artificial chromosome (BAC) using RecE/T homologous recombination. An in vitro phenotypical analysis revealed that only the double (Mt[M27\(^A\)\(^6\)\(^5\)\(^8\)\(^S\)M36\(^V\)\(^5\)\(^4\)\(^I\)]) and the m139 (Mt[m139\(^Y\)\(^5\)\(^6\)\(^5\)\(^X\)]) mutants showed a temperature sensitive phenotype in MEF and/or Raw 264.7 macrophages cells

Type of Work:Ph.D. thesis.
Supervisor(s):Sweet, Clive and Martin, Brian
School/Faculty:Colleges (2008 onwards) > College of Life & Environmental Sciences
Department:School of Biosciences
Subjects:QR Microbiology
Institution:University of Birmingham
ID Code:1703
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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