Summary of Research and current Research program

Prof. Dr. Sven-Erik Behrens

"Replication and pathogenesis of hepatitis C virus and related viruses Worldwide, about 200 million people are infected with the hepatitis C virus (HCV). In ca. 85% of the patients the infection becomes persistent and often culminates to chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). In many countries hepatitis C is the leading cause for liver transplantations, and this situation is expected to considerably aggravate in the next 20 years. Moreover, HCV infections are associated with a number of extra-hepatic diseases such as mixed cryoglobulinemia and glomerulonephritis. An important issue along the way to develop urgently needed novel antiviral therapies and vaccines is to gain further insights into the molecular mechanisms that govern the HCV life cycle and that are responsible for the persistency of HCV infections.

In the last ca. 10 years, studies performed in my laboratory, several of which were performed with bovine viral diarrhea virus (BVDV) as a model system for HCV, led to important new insights into the function of virus-encoded and host-encoded factors during the viral replication process. About four years ago we initiated to explore novel types of vaccination procedures; more recently, we started investigating potential mechanisms of viral pathogenesis and persistence. Several of these accomplishments have been patented and some of them are currently applied for the development of novel antiviral substances and vaccines. Our work was/is supported by the DFG, the BMBF, the Fonds der chemischen Industrie (Germany), the NIH, the state of Pennsylvania (US) and several pharmaceutical companies (see Curriculum vitae).

1. As an initial effort in our HCV program, we identified the nonstructural protein 5B (NS5B) as the RNA-dependent RNA polymerase (RdRp) of HCV (EMBO J. 15: 12-22). NS5B is currently explored by major pharmaceutical companies as one of the most promising targets for antiviral approaches. Following a break of several years, NS5B turned back into the focus of our interest; currently, we explore the conditions that enable this important enzyme for a specific initiation of viral RNA synthesis.

2. The second major achievement concerned the development and characterization of a subgenomic RNA replicon system of the closely HCV-related pestivirus BVDV (Bovine viral diarrhea virus; J.Virol. 72: 2364-72). This discovery paved the way for the subsequent construction of a similarly organized HCV replicon by Prof. Bartenschlager and colleagues. Until 2005, the HCV replicon remained the only available experimental system that allowed functional studies of HCV replication in cell culture. We apply the considerably more robust BVDV system, often in direct comparison with the HCV replicon, to characterize the role of viral proteins and to analyze the structure and function of individual replication- and translation signals encoded by the viral RNA (see, for example: J.Virol. 73: 3638-48; J.Virol. 74: 5825-35; J.Virol. 75: 7791-7802; J.Virol. 77: 5352-59; Virology 333: 349-66).

3. In collaboration with Dr. Rehermann at the National Institute of Health (NIDDK, Bethesda) we currently evaluate BVDV replicons that express foreign antigens for a potential use as T-cell inducing/tolerance-breaking RNA vaccines. Expressing the HCV NS3 protein as a T-cell antigen in dendritic cells, we obtained encouraging data in mouse which suggest that BVDV replicons may be applied as therapeutic vaccines for the treatment of chronic HCV (Immunity 20: 47-58). We will next apply the BVDV replicon/dendritic cell approach to try to break the immune-tolerance to antigens of other microbial pathogens and tumor-associated antigens. Moreover, we evaluate novel types of procedures that should allow a straight-forward application of RNA vaccines in the clinics.

4. Another important discovery of our laboratory concerns the fact that members of the so-called "NF/NFAR group of proteins are involved in the replication process of BVDV and HCV. Specifically, the cellular proteins were shown to mediate a circular conformation of the viral genome, which is important for the regulation of translation and replication of the viral RNA genomes (EMBO J. 22: 5655-5665; RNA 10: 1637-52; RNA13 in press). Some NF/NFAR-proteins encode double-strand RNA-binding motifs (dsRBM) and thus belong to a protein family many members are in the current focus of interest due to their emerging roles in RNA metabolism (prominent examples are the RNA-dependent RNA kinase PKR, and the RNase III-like proteins Dicer and Drosha). Several lines of evidence suggest that the NF/NFAR-proteins, which in naïve cells are mainly nuclear, regulate gene expression on the transcriptional and post-transcriptional level and that the proteins are involved in the nuclear export of RNA. For example, the prototype NF/NFAR protein, NF90/NFAR-1 (DRBP76; ILF3) acts as an anti-decay factor of the mRNA of IL-2 and of urokinase plasminogen activator that are major players in immunity and inflammation. Moreover, NF90/NFAR-1 was shown to be part of the Exportin V complex involved in the nuclear export of small, helical RNAs. Finally, the entire protein group was indicated to be part of the so-called APOBEC complex in the cytoplasm and to be in a yet undefined manner part of the cellular response to a viral infection. Hence, a major focus of present work aims at a further biochemical characterization of the NF/NFAR proteins and at identifying cellular interaction partners that also participate in viral replication. Thus, we hope to understand how the interplay of these cellular factors with viral components supports viral replication and affects viral pathogenesis.

5. In a very recent project, we initiated investigating the replication process of an RNA plant virus, namely the Tombusvirus TBSV, which resembles HCV in several aspects. The replication of TBSV is particularly robust and occurs even in yeast cells. Using cytoplasmic extracts of plant host cells, we were recently able to establish an in vitro translation/replication system with TBSV RNA, which will allow us to characterize the function of host factors by depletion/complementation studies. Thus, we hope to gain important information on plant homologues of the NF/NFAR proteins and to apply experience obtained with this system to HCV, where in vitro replication is not yet conceivable.

Gezieltes Screening von Inhibitoren gegen verschiedene Mitglieder der Flaviviridae Familie wie HCV, BVDV, West-Nil. Erstellung von Gutachten zu neuen Diagnoseverfahren, Vakzinierungsverfahren und Screeningverfahren antiviraler Substanzen gerichtet gegen RNA Viren. Expertise bei der Entwicklung neuer Vakzinierungsverfahren. Expertise mit Pflanzenviren.