Genetic analysis of M94 of murine cytomegalovirus

Maninger, Silke (2010): Genetic analysis of M94 of murine cytomegalovirus. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The M94 gene of murine cytomegalovirus (MCMV) is one of the about 40 core genes of the subfamily of β-Herpesvirinae with unknown function. MCMV is utilised as an in vivo model for studying human cytomegalovirus (HCMV) due to the strict species specifity of HCMV and major homologies to MCMV. HCMV is an important human pathogen with worldwide distribution. Although infection of the immune competent host is usually clinically silent, infection of the foetus and the newborn causes severe organ damage and infection of immunocompromised patients induces life-threatening disease. M94 has not been studied intensely as a single gene before, but studies on its homologues in other Herpesviridae demonstrated expression as true late gene, presence in the virion, interaction of UL94 and UL99 in HCMV and capsid binding in α-Herpesvirinae. While UL94 in HCMV is essential, M94 homologues in α-Herpesvirinae are not essential, indicating an essential function of M94 conserved in β-Herpesvirinae but lost in α-Herpesvirinae. For the identification of this essential function, a library of random mutants was generated by a modified Tn7 transposon mutagenesis. The modifications in the transposon mutagenesis produced a library of high diversity and wide coverage containing 32,000 primary clones including stop and insertion mutants. The analysis of 613 clones by sequencing resulted in 399 unique mutants containing a correct 15 base pair (bp) insertion in M94. 116 stop and insertion mutants were individually reinserted into the viral genome and studied in the viral context. The primary analysis showed the ability of certain mutants to complement the M94 deletion, thereby identifying the essential regions in the M94 protein. Secondly, the M94 mutants were re-inserted as second gene and analysed for their inhibitory capacity of the M94 wild type (wt) functions to identify dominant negative (DN) mutants. The inhibitory mutants found were then verified as DN mutants by regulated expression in the virus context and by analysis of their specific phenotype induced by overexpression. Further analysis of the DN mutant in comparison to a M94 deletion mutant revealed no effect of M94 in viral cleavage-packaging, despite previous publications. Additional analysis showed a block in secondary envelopment for both the DN mutant and the deletion mutant and the determination of the spread deficient phenotype of the M94 deletion virus. Altogether these results constitute a new model for secondary envelopment in β-Herpesvirinae.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Virology, Herpesviridae, MCMV, M94, Transposon, dominant negative mutants, cleavage/packaging
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 29. Juni 2010
• 1. Bericht­erstatter:in: Beckmann, Roland