Molecular Biology and Application of Recombinant Foamy Viruses

Overview

Viruses have proven to be versatile tools for modern molecular medicine and serve as versatile models to study basic mechanisms in almost any area of biology. For instance, the interaction of host cells with exogenous and endogenous damage in the form of virus infections or malignant transformation, resp., has been shown to often utilize common pathways. In addition, manipulating the control, regulation and interplay of adaptive, innate and intrinsic immunity are essential for both, virus replication as well as all phases of cancer development.

The goal of the research group “Molecular Biology and Application of Recombinant Foamy Viruses” is thus to exploit the apathogenic foamy viruses (FV), a distinct and ancient group of retroviruses with a long-lasting coevolution with their hosts, for use in molecular medicine as a vaccination scaffold and gene transfer and to unravel their host-virus interactions to identify pathways also relevant in cancer. Current studies focus on chromatin-targeting functions in Gag while future studies will also address the importance of FV Pol proteins for this essential process. In addition, the dual role of the cell-encoded APOBEC3 cytidine deaminases as antiviral restriction factors and their inactivation by the FV Bet protein as well as the strong contribution of the APOBEC3A and –B isoforms to genome hypermutation in defined epithelial cancer entities are the other focus of our studies. Since APOBEC3 cytidine deaminases are often induced by inflammatory signaling and infections, this newly identified pathway likely contributes to infection and inflammation-driven oncogenesis. In future studies, we will analyze the correlation between APOBEC3A and –B expression, infection and inflammation in additional human cancer types.

Additional interests of the group are related to in vitro evolution of viruses, virus- host co-adaptation and viral zoonoses.

Current Research Projects

The role of APOBEC3 genome hyper-mutation in human cancers

M.Sc. Juliane Hafermann, PhD project
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We recently showed in an in vitro engineered tumor cell culture system based on the inducible and isogenic expression of human APOBEC3 proteins that the APOBEC3A isoform induces two distinct mutational signatures. These mutational signatures have been previously detected at high prevalence in different human cancers of epithelial origin leading to the mutagenesis of relevant cancer driver or suppressor genes.

In order to analyze the effects of untimely and/or prolonged over-expression of APOBEC3A, corresponding cell clones and controls have been extensively characterized. Importantly, expression of functional APOBEC3A but not an enzymatically inactive variant displayed severe cyto- and genotoxicity. Reduced viability under chronic APOBEC3A expression led to the selection of cells that have lost APOBEC3A expression by different means (deletion of the expression cassette and acquisition of inactivating mutations in the APOBEC3A gene). This finding may explain why in human tumors, APOBEC3 expression does not strongly correlate with tumor stage since they have lost functional APOBEC3 or APOBEC3 expression. As also supported by published data, we assume that APOBEC3 genome mutagenesis does occur during different phases of tumor development. In line with this, profiling of head and neck cancers revealed complex pattern of APOBEC3A and -B expression and the presence of the corresponding mutational signatures.

Analysis of the engineered tumors cell clones that had expressed wt or inactive APOBEC3A revealed substantial variation with respect to cell growth and migration between individual clones. Selected cell clones were also injected into immune-deficient mice, the analysis of the resulting tumors is currently ongoing. These studies were conducted in close cooperation with Prof. Dr. P. Lichter, Dr. Zapatka, Prof. Dr. L. Alexandrov, Prof. Dr. J. Hess, Dr. K. Müller-Decker, PD. Dr. O. Popanda and Dr. A. Szabowski.

Characterization of the nuclear accumulation of foamy virus proteins and genomes

M.Sc. Guochao Wei, PhD project
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Foamy viruses (FV) are retroviruses with a unique molecular biology and several features that make them attractive candidates for gene delivery, protein transfer and vaccine antigen delivery and expression. For instance, FVs are considered apathogenic, have a high physical stability and an advantageous genome integration profile. During the last years, corresponding vectors based on feline FV (FFV) have been established and functionally characterized. Application of selected vectors in different animal models revealed their applicability and the induction of immune responses also in the fully immune-competent authentic host (in cooperation with Prof. Dr. J. Denner, Prof. Dr. Sue VandeWoude and Dr. M. Mühle).

In the current project, the chromatin binding site (CBS) of FFV Gag and functionally relevant residues within this motif were identified and characterized. The integrity of the CBS is not required for proper particle assembly, genome packaging, reverse transcription and release, as well as entry into new target cells. In contrast, mutagenesis of critical residues in the CBS turned out to impair chromatin association and productive infection and spread in newly infected target cells. We propose a molecular switch mechanism for the CBS function to preferentially induce chromatin binding upon entry of intact particles or partially disassembled capsids but absent with soluble Gag protein or capsomeres.

In addition, the capacity to specifically translocate heterologous proteins via fusion to full-length and truncated Gag proteins is evaluated. For this purpose, the GFP protein is used as cargo. The present data show that almost the full length Gag is needed for GFP transfer which may limit the potential of such a protein transfer system.

Function and relevance of foamy virus miRNAs in host – virus interactions

M.Sc. Wenhu Cao, PhD project
© dkfz.de

The importance of micro RNAs (miRNAs) as regulators of almost all cell-encoded functions and proteins as well as their relevance for virus replication and pathogenicity has been established over the last years. Together with the groups of Prof. Dr. B Cullen and Prof. Dr. J. Kuzmak we recently showed that the bovine FV (BFV) genome encodes a unique RNA polymerase III-driven miRNA cluster that is contained within a novel dumbbell-shaped primary miRNA. The primary miRNA consists of two closely spaced hairpin-bulge elements with imperfect base pairing. Three of the mature BFV miRNAs make up more than 2/3 of the miRNA pool in chronically BFV-infected bovine cells. The miRNA cassette located in the long terminal repeats is highly conserved among known BFV isolates and also active in BFV-infected cows.

In order to determine the biological function of the BFV miRNAs and the potential use of the novel RNA Pol III-driven miRNA cassette as a novel miRNA expression tool, we currently study the function of the miRNA cassette in sub-genomic reporter assays. The data reveal a substantial activity of the cassette and the inter-dependency of the two stem-loop structures for proper function. By use of bioinformatics and mRNA and miRNA sequencing of BFV-infected cells, potential molecular targets of the BFV miRNAs have been identified. Selected bovine target genes are currently subjected to in vitro target identification with respect to RNA target binding and quantification of target mRNA and protein levels. In parallel, BFV genomes have been constructed that lack the miRNA cassette completely. These miRNA deletion mutants have an attenuated replication phenotype in bovine cell cultures and we are underway to compare them also in cattle to the wt counterpart. Functional studies using the wt and miRNA-deficient genomes will be used to complement the animal experiments.

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