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    Institut für Virologie und Immunbiologie

    Systems biology of herpesvirus infections

    Herpesvirus are large DNA viruses, which co-evolved with their human and animal hosts for millions of years. Humans are infected with eight different herpesviruses causing a broad spectrum of disease ranging from the common cold sores to cancer.

    During these millions of years of co-evolution hosts, herpesviruses learned to both comprehensively modulate their host cell environment and efficiently evade the immune system. Besides representing important pathogens to human health, they thus also represent interesting tools to study fundamental aspects of cell biology and immunology.

    Within their large DNA genomes, herpesviruses encode hundreds of viral proteins and peptides, many of which do not only regulate a single gene, DNA or protein but interfere with complex cellular signal networks. To see beyond the tip of the iceberg of this regulation a systems level approach is required. Our lab employs a broad range of system biology methodology and analysis tools including microarrays, next-generation sequencing and quantitative proteomics to study host cell modulation and immune evasion in various herpesvirus models.

     

    Analysis and modelling of transcriptional, translational and post-translational regulation

    During lytic viral infection, cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. Standard approaches quantifying changes in both total RNA and protein levels do not provide the necessary temporal resolution to elucidate the underlying molecular mechanisms. Our lab pioneered metabolic labelling of newly transcribed RNA using 4-thiouridine (4sU-tagging) to analyse short-term changes in RNA synthesis, processing and decay with superior resolution (Dölken et al., RNA 2008; Friedel et al., NAR 2009; Windhager et al., Genome Research 2012; Rutkowski et al., Nature Communications 2015).

     

    To study short-term changes in translational activity, we employ ribosome profiling. This method is based on large-scale sequencing of ribosome-protected RNA fragments and provides both a quantitative estimate of translational activity as well as a detailed picture of the translated RNA sequences at any given time of interest.

     

    Fig. 1: Principle of 4sU-tagging and ribosome profiling

    Principle of 4sU-tagging:
    4-thiouridine (4sU) is added to cell culture medium to start metabolic labelling of newly transcribed (nascent) RNA. 5 to 60 min later total RNA is isolated. Following thiol-specific biotinylation, total RNA is separated into labelled nascent and unlabelled pre-existing RNA. All three RNA fractions are suitable for down-stream analyses including qRT-PCR, microarrays and RNA-seq.

    Principle of ribosome profiling:
    Following a mild cell lysis, a stringent RNAse digest is performed. The short fragment of RNA molecules actively being translated at the time of cell lysis is protected from RNAse treatment within the ribosome and can then be recovered, cloned and sequenced. Thereby, a detailed picture of translational activity at the time of cell lysis is obtained.

     

    Combining data obtained by 4sU-tagging, ribosome profiling and quantitative proteomics, we can now comprehensively record changes in RNA synthesis, processing and decay as well as their impact on protein production in a single experimental setting (Rutkowski et al., Nature communications 2015). Combined with cell-based and biochemical assays as well as additional high-throughput assays, we are aiming to understand the coordinated regulation of gene expression by viral proteins, microRNAs and RNA-binding proteins as well as their functional relevance in infection.

     

    This work requires intensive bioinformatics analysis. These are performed in a long-standing collaboration by the groups of Prof. Ralf Zimmer and Prof. Caroline Friedel at the Ludwig-Maximilians-University Munich.

     

    Recent and current projects

    Applying 4sU-tagging and ribosome profiling to lytic herpes simplex virus 1 (HSV-1) infection of primary fibroblasts revealed HSV-1 to trigger widespread, host-specific disruption of transcription termination (Rutkowski et al., Nature Communications 2015). This results in extensive transcriptional activity for tens-of-thousands of nucleotides and into downstream genes. This gives the impression that the virus also activates a large number of genes in the cell and can result in misinterpretation of experimental data. It also explains why hundreds of cellular genes seemingly activated by the viruses are not translated into proteins at all. Work is ongoing to elucidate the underlying molecular mechanism.

     

    Fig. 2: HSV-1 disrupts transcription termination

    Transcriptional activity measured by RNA-seq of newly transcribed RNA purified from uninfected and HSV-1 infected cells (4sU applied for 60 min prior to and 7-8 h post infection). Reads mapping to a ≈400kb region of chromosome 19 encoding the SLC12A2 gene are shown (sense strand only). In HSV-1 infection, the SLC12A2 poly(A) site is missed by the polymerase resulting in massive transcriptional activity downstream of the gene’s 3’-end extending for >100.000 nucleotides.

     

     

    Functional role of cytomegalovirus miRNAs

    Although usually asymptomatic in healthy individuals, human cytomegalovirus (HCMV) is the major cause of morbidity in immunocompromised patients and allogeneic bone-marrow or organ-transplant recipients. As such, it poses an important risk factor for graft failure following heart and kidney transplantation, resulting in patient death or the need for re-transplantation. In addition, it is the leading agent of birth defects among congenitally transmitted infections affecting about 1:1,000 new-borns.

    During primary infection and reactivation, HCMV encounters an array of innate and adaptive immune responses. MicroRNAs (miRNAs) represent a novel entity of viral factors counteracting these defences requiring no protein expression to exert their function. This makes them ideal, non-immunogenic tools for these viruses to regulate their own as well as host gene expression during latency and reactivation thereof. To date, more than 80 miRNAs have been identified in six human herpesviruses – at least 11 pre-miRNAs are expressed by HCMV. We established its murine model to study the biology and function of cytomegalovirus miRNAs (Dölken et al., J Virol 2007) and identified two MCMV miRNAs to be required for efficient virus persistence and host to host spread (Dölken et al., PLoS Pathogens 2010).

    Employing a broad range of technologies including reverse virus genetics and high-throughput technologies (RIP-Chip and PAR-CLIP) we explore the function of herpesvirus miRNAs to answer whether they may serve as readily accessible targets for novel antiviral agents.

     

    Fig. 3: Accumulation of murine CMV miRNAs during lytic infection

    NIH-3T3 murine fibroblasts were infected with wild-type MCMV at an MOI of 10. At different times of infection small RNA libraries were prepared and sequences. The contribution of viral miRNAs to the total cellular pool is shown.
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