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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - HERPES (Herpesvirus Effectors of RNA synthesis, Processing, Export and Stability)

Teaser

Summary

Herpes simplex virus 1 (HSV-1) is an important human pathogen, which intensively interacts with the cellular transcriptional machinery at multiple levels during lytic infection. Employing next-generation sequencing to study RNA synthesis, processing and translation in short intervals throughout lytic HSV-1 infection, my laboratory made the surprising observation that HSV-1 triggers widespread disruption of transcription termination of cellular but not viral genes. Transcription commonly extends for tens-of-thousands of nucleotides beyond poly(A)-sites and into downstream genes. In contrast to textbook knowledge, HSV-1 infection does not inhibit splicing but induces a broad range of aberrant splicing events associated with disruption of transcription termination. Exploring these fascinating phenomena will provide fundamental insights into RNA biology of human cells.
The proposed work combines both hypothesis-driven and innovative unbiased screening approaches. I will utilise cutting-edge methodology ranging from high-throughput studies to advanced single molecule imaging. Thereby, I will detail the molecular mechanisms responsible for disruption of transcription termination and aberrant splicing. I will identify novel cellular proteins governing transcription termination using a genome-wide Cas9-knockout screen. I will develop RNA aptamer technology to visualise and track single RNA molecules suffering from poly(A) read-through. I will elucidate why transcription termination of some cellular and all viral genes remains unaltered throughout infection. I hypothesize that the alterations in RNA processing are depicted by specific changes in RNA Polymerase II CTD phosphorylation and in the associated proteins. I will characterise these dynamic changes using mNET-seq and quantitative proteomics. Finally, data-driven quantitative bioinformatic modelling will detail how the coupling of RNA synthesis, processing, export, stability and translation is orchestrated by HSV-1.

Work performed

We discovered that HSV-1 disrupts transcription termination of cellular genes by at least two different mechanisms. In collaboration with Prof. Yongsheng Shi from Irvine, CA, USA, we identified the major viral transactivator ICP27 to interfere with transcription termination by directly targeting the termination machinery. Moreover, binding of ICP27 to GC-rich RNA sequences rescues transcription termination (manuscript in preparation) (WP1). The second mechanism presumably stems from a virus-induced cellular stress response.

We identified disruption of transcription termination and transcription into downstream genomic regions to result in a dramatic increase in chromatin accessibility selectively downstream of genes. We hypothesize this to result from a defect in histone repositioning.

We successfully established mNET-seq and ChIPmentation to characterize the molecular machinery involved in RNA processing during the course of lytic HSV-1 infection (WP2). Data analysis is ongoing and we are about to include mutant viruses and phosphospecific antibodies.

We developed a novel approach termed single cell thiol-(SH)-linked nucleotide conversion sequencing (sc2RNA-seq) as well as new computational tools (GRAND-SLAM) to decipher real-time changes in transcriptional activity at single cell level (Erhard et al., resubmitted). We filed a patent on our computational approach to dissect new from old RNA in sc2RNA-seq data.

We extensively tested the usefulness of the RNA aptamer Broccoli for live-cell RNA imaging and found it to be not sufficiently stable and bright even upon concatemerization. We are now developing new reporter assays to visualize disruption of transcription termination using fluorescent proteins (WP3). These will also be suitable to schreen for novel RNA viral RNA elements governing RNA processing and export (WP4).

We recruited an excellent PhD student in bioinformatics who is analysing the large amounts of data we are obtaining.

Final results

The goal of this ERC Consolidator research program is to elucidate the molecular mechanisms responsible for the profound alterations in RNA processing I observed in HSV-1 infection and utilise this model to decipher the regulation of the transcriptional machinery in human cells. The key objectives are:

O1: To identify the viral gene product(s) and the molecular mechanism(s), which are responsible for the disruption of transcription termination in HSV-1 infection.

O2: To detail the molecular mechanisms by which RNA processing is coordinated in human cells.
RNA polymerase II (Pol II) transcriptional activity as well as coupled pre-mRNA processing are orchestrated by complex changes in the phosphorylation of the 52 heptad repeats in its C-terminal domain (CTD) (10). I hypothesize that the alterations in RNA processing in HSV-1 infection are depicted by both specific changes in Pol II CTD phosphorylation and in the associated proteins. I will characterise these dynamic changes in the HSV-1 model using mNET-seq (11) and quantitative proteomics (12). This will clarify whether HSV-1 affects the â€œwritersâ€ or the â€œreadersâ€ of the Pol II CTD molecular code and will comprehensively detail the functional role of the complex regulatory mechanisms in RNA processing.

O3: To identify novel host factors/RNA elements governing transcription termination in human cells and develop live cell imaging of single RNA molecules suffering from poly(A) read-through. I will pioneer RNA aptamer technology to visualise poly(A) read-through in living cells. I will then set up a genome-wide Cas9 knockout screen and identify novel cellular genes governing transcription termination.

O4: To identify and characterise the viral RNA elements and mechanisms, which maintain transcription termination of viral genes, using a novel high throughput screen. I hypothesize that HSV-1 employs multiple mechanisms to maintain normal transcription termination of the viral genes and mediate efficient export and translation of the intronless viral transcripts. I will identify the viral RNA elements mediating both ICP27-dependent and -independent effects and detail the underlying molecular mechanisms.

O5: To elucidate how the coupling of RNA synthesis, processing, export, stability and translation is modulated throughout HSV-1 infection using data-driven quantitative bioinformatic modelling.