We continue our longstanding cooperation with the group of Prof. Wolfgang Holter and PD Dr. Manfred Lehner, St.Anna Children’s Hospital and Children’s Cancer Research Institute, Vienna. Here we develop novel antiviral strategies using recombinant chimeric antigen receptors (CAR) directed to CMV viral glycoprotein B or to cellular NKG2D. These transgenes are delivered by lentiviral vectors and as synthetic mRNA (Full et al., 2010); this research led us to the discovery that CMV infected cells are resistant to CAR-mediated T-cell killing in an HLA-recognition independent manner, with possible involvement of the CMV effectors UL36 and UL37x1, beyond their known anti-apoptotic functions (Proff et al., 2016, 2018). In a close cooperation with the biopharmaceutical company Amgen we investigate the feasibility of related BiTE®-based immunotherapies for CMV infection (Brey et al., 2018).
Lymphocyte growth transformation by Herpesvirus saimiri (HVS) is a useful tool for immunologist as well as a model that allows us to study episomal viral genomes. Our analysis of the viral episomal chromatin structure in human T-cells, together with a seminal characterization of episomal DNA replication (Alberter et al., 2011, Vogel et al., 2010), can serve to identify factors that regulate chromatin permissiveness within the vector genome and will allow sustained transgene expression from rhadinoviral vectors. CTCF is the chromatin organizer in eukaryotic cells and also important for the viral chromatin. CTCF protein binding to the HVS genome was studied in virus transformed human T lymphocytes (Zielke et al., 2012), revealing that a single CTCF binding site was crucial for the maintenance of viral episomes and the transformed state.
The viral effectors that HVS uses to transform human and monkey T-cells to antigen independent growth are studied using virus-genetic approaches in the context of the viral genome, which we manipulate by standard molecular techniques and homologous recombination. These viral mutants are then studied in transformation assays in vitro with marmoset and human primary lymphocytes. We have shown that constitutive STAT3 activation by the Tip oncogene is not required for human T-cell transformation by HVS and that the Tip-Lck interaction is necessary for transformation and that IL2-dependence of transformation is coupled to a specific tyrosine residue – a finding that directed our research to the T-cellular IL2 signaling pathway and the role of STAT5.
KSHV/HHV8 is a human herpesvirus with close relationship to HVS, Rhesus Rhadinovirus (RRV) is the corresponding homolog from Old World Primates. We are investigating the potential of rhadinoviral proteins to disrupt PML nuclear bodies and other cellular functions, again employing recombinant viruses. This showed that gamma-herpesviral effectors antagonize nuclear domain 10 instituted intrinsic immunity in different ways. The HVS ORF3 was able to mediate the selective degradation of the cellular protein Sp100 (Full et al., 2012). The related ORF75 of KSHV was identified as an essential viral protein as it mediates disappearance of ATRX and dispersal of Daxx from ND10 (Full et al., 2014). Notably, the viral ORF75 protein RRV, despite more closely related to KSHV, resembles the HVS ORF3 in its predominant targeting of the major ND10 component SP100 in a proteasome dependent manner (Hahn et al., 2016). In a further search for antiviral restriction factors, Dr. Florian Full in cooperation with Prof. Michaela Gack (Chicago) demonstrated that the centrosomal protein TRIM43 restricts herpesvirus infection by regulating nuclear lamina integrity (Full et al., 2018) and, in cooperation with the group of Prof. Jean-Laurent Casanova, we showed that human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses (de Jong et al., 2018).
Using next-generation sequencing (NGS), we attempt unbiased pathogen identification from diagnostic samples of patients with presumed diseases of probable infectious origin. Here we recently detected Borna disease virus 1 (mammalian 1 orthobornavirus, BoDV-1) in brain tissue of a patient with fatal encephalitis of unknown origin (Korn et al., 2018), demonstrating that BoDV-1 is indeed pathogenic in humans. We further develop applications and use NGS in genome wide CRISPR/Cas9 knockout screens, in particular as a platform to search for cellular factors restricting the replication of Herpesviruses and other pathogens.