🏆Molecular Oncology Journal Best Poster Awards🏆

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Gyuli Kim, Leibniz Institute of Virology (Left above)

P9 Cell Cycle-Dependent Acquisition of Latent Heterochromatin by de novo-Infecting KSHV Episomes

Gyuli Kim1, Thomas Günther1, Martin Hamann1, Kay Grünewald2, Adam Grundhoff1

1Leibniz Institute of Virology, Hamburg, Germany. 2Center for Structural Systems Biology, Hamburg, Germany

Introduction: Kaposi sarcoma-associated herpesvirus (KSHV) is responsible for the development of several neoplastic diseases. After de novo infection, the KSHV genome is chromatinized and establishes latency through polycomb repressive complexes (PRCs)-mediated repressive histone marks, including H3K27me3. Acquisition of PRC-associated marks can be readily observed in vitro infection of proliferating cell lines. However, non-cycling cells represent the likely primary targets of KSHV infection in vivo, and the influence of the cell cycle on polycomb repression of the viral epigenome remains poorly understood.

Objectives: We aimed to investigate how primary acquisition and subsequent maturation of latent viral chromatin differs between proliferating and resting host cells.

Materials & methods: To elucidate chromatin composition of viral episomes in the early stage of latency, we investigated histone variant deposition and histone modifications after KSHV infection of conditionally immortalized human endothelial cells (HuARLT) using Chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag).

Results: Our ChIP-seq and CUT&Tag based genome-wide profiling revealed that H3K27me3 was greatly enriched on viral episomes in proliferating compared to resting cells, whereas host loci showed no differential H3K27me3 enrichment. We observed that H3K27me3 accumulation and reduction of relative H3.3 levels on KSHV episomes occur simultaneously, indicating that licensed replication of viral episomes augments episome-wide replacement of non-canonical H3.3 with canonical H3 variants. Interestingly, when the cell cycle was arrested for extended periods of time, slow establishment of H3K27me3 marks was observed, presumably due to either residual cellular proliferation activity or proliferation-independent incorporation of canonical H3.

Conclusions: Our data shows differential epigenetic modification of the KSHV genome according to the host cell cycle. We are currently investigating phenotypic consequences of delayed H3K27me3 acquisition in resting cells.

Saskia Stein, Medical University Hannover (Right above)

P5 Targeting LANA with a small molecule diminishes proliferation of KSHV infected B-cells

Saskia C. Stein1,2,3, Philine Kirsch4,2,5, Aylin Berwanger4,2,5, Martin Empting4,2,5,3, Thomas F. Schulz1,2,3

1Institute of Virology, Medizinische Hochschule Hannover, Hannover, Germany. 2Deutsches Zentrum für Infektionsforschung (DZIF), Braunschweig, Germany. 3Cluster of Excellence RESIST (EXC 2155), Hannover, Germany. 4Helmholzt-Institute for Pharmaceutical Research Saarland, Department of Antiviral and Antivirulence Drugs, Saarbrücken, Germany. 5Department of Pharmacy, Saarland University, Saarbrücken, Germany

The Latency Associated Nuclear Antigen (LANA) of the Kaposi’s sarcoma-associated herpesvirus (KSHV) is a protein that fulfills various important functions during the viral lifecycle. It is essential for tethering the viral episome to the host chromosomes and latent replication of the viral DNA and thereby ensures maintenance of the viral DNA in the host cells during cell division in the latent phase of the viral infection. This vital function makes it a desirable target for therapeutic intervention. Using fragment-based drug design, we developed small molecules with the potential to disrupt the interaction of LANA with the viral episome.1,2 One of the top compounds (#20) disrupted the interaction of the LANA DNA binding domain with the LANA binding site of the viral genome and caused a dose-dependent reduction of viral replication in a transient replication assay. 

Maintenance of infection in iSLK cells de-novo infected with recombinant KSHV (Bac16 and rKSHV.219) was not affected by treatment with the compound. The ratio of GFP positive (infected cells) decreased at a similar rate in compound treated versus DMSO treated cells. However, this compound inhibited the proliferation of KSHV infected PEL cell lines, but not of KSHV-negative B-cell lines. We are currently exploring whether this compound interferes with the known ability of LANA to sustain the survival and proliferation of PEL cells by recruiting cellular proteins. 

(1) Kirsch et al., 2020. https://doi.org/10.1016/j.ejmech.2020.112525. 

(2) Berwanger et al., 2023. https://doi.org/10.1021/acs.jmedchem.3c00990.Â