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

Q Science > QR Microbiology

Alali, Abdulaziz ; Sweet, Clive,Martin, Brian

University:University of Birmingham

Department:School of Biosciences

关键词: Q Science;    QR Microbiology;   

Others  :  http://etheses.bham.ac.uk//id/eprint/1703/3/Alali_11_Phd.pdf

来源: University of Birmingham eTheses Repository

【 摘 要 】

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