Decoding murine cytomegalovirus and characterizing the antigenicity of viral small ORF-derived peptides ; Entschlüsselung des murinen Cytomegalovirus und Charakterisierung der Antigenität von kleinen viralen ORF-Peptiden

The human cytomegalovirus (HCMV) is a ubiquitous pathogen that establishes a life- long infection upon primary infection. While primary infection is mostly asymptomatic, HCMV is responsible for significant morbidity and mortality in immunocompromised patients and neonates. There is currently no vaccine. The strict species specificity of this large double-stranded DNA virus poses a major challenge in understanding cytomegalovirus (CMV) pathogenesis. Murine cytomegalovirus (MCMV) exhibits significant similarity to HCMV and represents a widely used model to study CMV pathogenesis. The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbour ~ 170 open reading frames (ORFs). More recently, RNA-seq based omics approaches revealed HCMV gene expression to be substantially more complex, comprising several hundred viral ORFs, similarly observed for other herpesviruses including KSHV, EBV and our own work on HSV-1, where an integrative analysis was further extended to provide a unified nomenclature for all gene products. The aim of my PhD project was to generate and utilize available multi-omics datasets to provide a state-of-the-art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. By developing a novel nomenclature strategy, I annotated 365 viral transcription start sites (TiSS) giving rise to 380 and 454 viral transcripts and ORFs, respectively. The latter included >200 small ORFs, some of which represented the most highly expressed MCMV gene products. I further developed a novel time-resolved TiSS profiling approach (dSLAM-seq) to accurately annotate TiSS and determine the temporal kinetics of viral TiSS usage by metabolic labelling of newly-synthesized RNA. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed three classes of viral ...