Project Details
Projekt
Pervasive transcription in human cytomegalovirus infection
Applicant
Professor Dr. Lars Dölken
Subject Area
Virology
Term
since 2021
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 443644894
In recent years, systems biology approaches revealed herpesvirus gene expression to be much more promiscuous and complex than initially thought. As such, it is now generally recognized that human cytomegalovirus (HCMV) expresses 700 - 1,000 transcripts as well as an equal number of large and small open reading frames (ORFs). However, precision nuclear run-on sequencing (PRO-seq) has revealed that transcription initiates from the HCMV genome at >7,000 transcription start sites (TSS). Employing comprehensive TSS profiling and integrative computational analysis, we found that the vast majority of these 7,000 TSS result in highly unstable viral transcripts, i.e. promoter or enhancer RNAs, and thus represent non-productive transcription that does not give rise to functional viral proteins. To directly validate highly unstable viral transcription, we engineered HeLa-MTR4-degron cells to rapidly degrade the nuclear RNA degradation machinery, the nuclear exosome. RNA-seq analysis of these cells infected with wild-type HCMV in the presence of cycloheximide resulted in identifying two so far unknown viral immediate early (IE) genes, UL20 and UL22 and a novel IE promoter RNA UL21A. Based on our extensive preliminary data, this highly collaborative project aims to understand the molecular mechanisms of HCMV transcription more comprehensively. The project has four primary objectives: (i) comprehensively mapping viral pervasive transcription during lytic infection, (ii) analyzing the functional roles of IE2 and UL69 in viral early gene expression, and (iii) improving HCMV latency models by engineering viruses for conditional IE2 and UL69 expression using protein-degradation technology (degron-tags). (iv) Moreover, our project will substantially contribute to the DEEP-DV Transcription Atlas. Taken together, this project has the potential to substantially advance our understanding of HCMV biology of lytic infection, latency and reactivation.
DFG Programme
Research Units
