AG Bartenschlager

Our original interests focused on the delineation of the HCV genome organization, polyprotein processing and characterization of the NS3/4A proteinase complex. Another topic was the biochemical characterization of the NS5B RNA-dependent RNA polymerase. In the last few years we concentrated on the development of HCV cell culture systems and their application to study viral replication and virus - host interaction.

Research Team Members

Research Projects

I. Improvement of the HCV replicon system and establishment of the first system for production of infectious HCV in cell culture

Since the first description of the HCV replicon system (Lohmann et al., 1999) we contributed substantially to improvements of this system. For instance, we described a comprehensive analysis of cell culture adaptive mutations and demonstrated that host cell permissiveness is of high importance in efficiency HCV RNA replication (Lohmann et al., 2001, 2003; Appel et al., 2001). In addition, we developed a number of novel and innovative replicon systems including transient replication assays, high throughput testing formats (reviewed in Bartenschlager et al., 2004) and novel replicon cell lines (Windisch et al., 2005).

In close collaboration with the group of Dr. Takaji Wakita at the university in Tokyo we established the first system for production of infectious HCV (Wakita et al., 2005). Current research aims at improving the yields of this culture system, on the construction of intra- and intergenotypic HCV chimeras and the generation of reporter viruses that facilitate studies on viral RNA replication and infection. Moreover, the virus system is now used to study various aspects of the HCV life cycle.

II. Role of cis-acting RNA sequences for HCV RNA replication

We mapped sequences in the HCV genome that are essential for RNA replication. Thus far our analyses concentrate on the 5' and 3' NTRs and their role in RNA translation and replication. We found that the complete 5' NTR as well as the highly conserved X-tail sequence at the 3' end are essential for replication (Friebe and Bartenschlager, 2001; 2002).

More recently, we characterized a novel cis-acting RNA element in the 3' coding region of NS5B. This element designated 5BSL3.2 forms a kissing-loop interaction with a hairpin structure in the 3' terminal X-tail. Genetic studies suggest that this interaction is essential for HCV RNA replication (Friebe et al., 2005).

III. Role of viral proteins for HCV RNA replication and virus production

The phospho protein NS5A plays an important role in viral RNA replication. It is a hot spot for cell culture adaptive mutations that enhance RNA replication. However, these mutations at the same time impair virus production suggesting that NS5A is involved in regulating a switch from RNA replication to assembly. In addition, NS5A is the only protein that can be complemented in trans (Appel et al., 2005) arguing that this protein plays multiple roles in the viral life cycle. Another protein for which no clear function in the viral life cycle has been described thus far is NS2. Finally, we are interested in the biogenesis of the HCV replication complex and the mechanism how NS4B contributes to formation of the replication complex.

IV. Studies of viral morphogenesis

Taking advantage of the novel HCV cell culture sytem and systems we established for the DV, we are interested in studying several aspects in morphogenesis including the role of viral and host cell factors in assembly and release of infectious particles.

V. HCV and innate immunity

We have shown that translation/replication of the HCV replicon is highly sensitive to the cellular antiviral defense induced by interferon-alpha and -gamma (Frese et al., 2001; 2002). In case of interferon-alpha, this inhibition is mediated by an MxA-independent pathway. In this project we attempt to identify the cellular effector(s) mediating the antiviral effect of interferon.

More recently, we have contributed to the discovery that the signal transducer Cardif that is required for the activation of IRF-3 is proteolytically cleaved by the HCV NS3/4A protease (Meylan et al., 2005). Thus, HCV actively counteracts the early induction of the antiviral defense which may facilitate the establishment of a persistent infection.

VI. Dengue virus replication and pathogenesis

Work in the past two years led to the establishment of a large panel of tools in the laboratory that allows us to study DV replication and virus - host interaction. To this end we performed a first characterization of the topology of DV NS4B and the role of this protein for viral RNA replication and virus production.

VII. Development of antiviral drugs and drug resistance assays

The only therapy currently available for chronic hepatitis C is the combination of (pegylated) interferon-alpha and ribavirin but only ca. 50% of infected patients develop a sustained response. Therefore, more efficient therapeutics with less side-effects are required. In this project cell-based assay systems are used to define novel targets for antiviral intervention. Moreover, since causal therapeutics are expected soon, we will develop cell-based systems that allow the phenotypic and genotypic determination of therapy resistance.