Research Group of A. Ensser

Virology

Head of Institute:
Prof. Dr. med. Klaus Überla

Oncogenic Rhadinoviruses and Vectors for Gene Therapy

Antiviral Immunotherapy against the human Cytomegalovirus

We continue our 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 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).

T and NK cell growth transformation by Herpesvirus saimiri (HVS)

T and NK cells growth transformed by Herpesvirus saimiri (HVS) are a model 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). This revealed that a single CTCF binding site was crucial for the maintenance of viral episomes and also to maintain 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 for the generation of mutants. This allows studies on the function of selected viral genes in the context of the viral genome, which we manipulate by standard molecular techniques and homologous recombination. The early phase of growth transformation, immediately after viral infection, can only be studied by recombinant viruses carrying deletions or mutations of candidate transformation-associated genes. 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. We also found that STAT6 is differentially regulated by Tip (Mazumder et al., 2012). This collaboration with Prof. Sticht yielded important insights in the STAT-SH2 domain interaction to the residues neighboring the phosphotyrosines Y114 and Y127 of Tip. A novel marker virus expressing murine H-2KK was used to study the spectrum of cells that can be infected with HVS. This led to our discovery that HVS is able to infect and transform human natural killer (NK) cells Vogel et al., 2014). While few, mostly tumor derived human NK cell lines are available to date, this finding can facilitate the generation of patient specific, functional NK cell lines.

Cellular restriction of the Kaposi sarcoma associated human herpesvirus 8 and related Rhadinoviruses

Figure 1. RRV infection depletes SP100 and PML from human SLK cells.
CRISPR-Cas9 knockout of the indicated ND10 components further demonstrates that the respective other proteins are not necessary for RRV ORF75 to effect this depletion.

KSHV/HHV8 is a human herpesvirus with close relationship to HVS, Rhesus Rhadinovirus (RRV) is the corresponding homolog from Old World Primates. In collaboration with Prof. Stamminger, Erlangen, and Prof. Jung, Univ. of Southern California, we are investigating the potential of rhadinoviral proteins to disrupt PML nuclear bodies and other cellular functions, again employing recombinant viruses (Brulois et al., 2012, 2014, 2015). 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. This depletion was independent of respective other ND10 components, and of the presence of an intact ND10 structure (Figure 1).

Using the CRISPR-CAS9 knockout of ND10 genes revealed a restriction of RRV by the ND10 component DAXX in human cells (Hahn et al., 2016). We have further established a genome wide CRISPR-CAS9 knockout platform to search for cellular factors restricting the replication of Herpesviruses (Figure 2).

Figure 2. Genome wide knockout screen of rKSHV.219 infected SLK cells transduced with a genome wide lentiviral CRISPR-Cas9.
rKSHV.219 infected, GFP bright cells (1%) were enriched by FACS and then sgRNAs, corresponding to Genes knocked out, were identified by next generation sequencing on our MiSeq system. The volcano plot was generated with CLCbio Genome Workbench and shows sgRNAs enriched (left) or depleted (right) in the GFP bright population. This pilot experiment confirms that ND10 proteins are restricting rhadinoviral infection and demonstrates the power of this technology (funded by IZKF Erlangen).
 
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Summary