Remodelling of the cellular environment in Epstein-Barr virus infection

Abstract

For successful infection viruses modify the host cell environment by transcriptional, posttranscriptional or post-translational regulation of cellular signaling pathways. In the work described in this thesis bioinformatics analysis coupled with biochemical validations were used to dissect critical features of these viral strategies in the context of infection by Epstein–Barr virus (EBV), a human herpesvirus associated with lymphoid and epithelial cell tumors.

The EBV nuclear antigen EBNA1 is the only viral protein ubiquitously expressed in all EBV infected cells and virus-associated neoplasms but its contribution to oncogenesis is not fully understood. EBNA1 binds to cellular chromatin via a bipartite Gly-Arg repeats (GRs) domain that resembles the AT-hook of the High Mobility Group-A (HMGA) architectural transcription factors. By microarray gene expression analysis of stable or inducible EBNA1 expressing cells, we found that EBNA1 orchestrates a broad and pleotropic rearrangement of cellular transcription, which resembles the one induced by architectural transcription factors and chromatin remodelers. Similar to HMGAs, EBNA1 is highly mobile in the nucleus and promotes large-scale chromatin de-condensation without recruitment of ATP-dependent remodelers, probably by displacing linker histone H1. The GR domain was sufficient for this effect. Furthermore, expression of the chromatin-binding GR domain was shown to regulate transcription. Thus, through its capacity to remodel the organization of cellular chromatin EBNA1 may reset the cellular transcriptional profile and prime the infected cells for malignant transformation.

MicroRNAs (miRNAs) are powerful prost-transcriptional regulators of complex signaling networks. In order to assess the possible role of EBV-encoded miRNAs in the regulation of SUMO-dependent signaling cascades, a meta-predictor of miRNAs targets was developed and tested against a comprehensive database of the SUMO interactome that includes components of the SUMOconjugation machinery, their interacting partners and substrates, and other signal transducers and regulators of the system. This prediction strategy suggests that EBV miRNAs may target the expression of key SUMO-regulated cellular proteins involved in immune defense, DNA damage response, apoptosis, chromatin remodeling and TGF-beta signalling. Regulation of essential members of canonical TGF-beta/Smad signaling by EBV miRNAs was confirmed in EBV positive cells entering the productive virus cycle. Thus, EBV miRNAs are likely to regulate key aspects of viral replication and pathogenesis.

Post-translational modifications by ubiquitin (Ub) and ubiquitin-like molecules (UbLs) regulate the stability and function of signal transducers and are often targeted by viruses in different phases of the infection. The large tegument protein of EBV, BPLF1, is an early viral product that plays a key role in virus assembly and maturation. The catalytic N-terminal domain of BPLF1 acts as a deneddylase that inactivates nuclear cullin-based Ub ligases (CRLs) to induce an S-phase-like environment that is required for efficient viral DNA replication. Structural bioinformatics methods were applied to predict the structure of BPLF1 and the site of interaction with cullins. A novel mechanism by which BPLF1 induces the proteasomal degradation of cullins by competing for binding of the CRL sequestering factor CAND1 was proposed and successfully validated. The work described in this thesis illustrates the power of gene expression analysis and bioinformatics tools for the characterization of different strategies adopted by viruses to remodel the host cellular environment in order to favor their own replication and transmission.