Research interests

Viruses are obligate intracellular parasites. Therefore, they completely depend on cellular resources to achieve most efficient replication, but at the same time must counteract innate and adaptive antiviral defenses in order to survive. For this reason, viruses can be used as model systems in cell biology and immunology. In addition, viruses are important human pathogens causing some of the most relevant infectious diseases such as influenza, hepatitis or Dengue fever. My research group is particularly interested in hepatitis C virus (HCV) and dengue virus (DENV) replication and assembly as well as the interaction of these viruses with their host. Viruses are obligate intracellular parasites. Therefore, they completely depend on cellular resources to achieve most efficient replication, but at the same time must counteract innate and adaptive antiviral defenses in order to survive. For this reason, viruses can be used as model systems in cell biology and immunology. In addition, viruses are important human pathogens causing some of the most relevant infectious diseases such as influenza, hepatitis or Dengue fever. My research group is particularly interested in hepatitis C virus (HCV) and dengue virus (DENV) replication and assembly as well as the interaction of these viruses with their host.

 

The hepatitis C virus (HCV)


Development of HCV cell culture systems
Propagation of HCV in cell culture has for a long time not been possible and overcome only gradually by different technical developments. The first achievement was the establishment of the HCV replicon system (Lohmann et al., 1999). Since the first description in 1999, the HCV replicon system became a very important tool that has been used with great success to study viral RNA replication, to identify host cell factors required for HCV replication, to delineate how HCV counteracts the innate immunity and to develop and evaluate antiviral drugs (reviewed in Bartenschlager et al., 2003 Bartenschlager et al., 2004; Bartenschlager 2006).

The HCV replicon system

 

HCV replicons recapitulate only parts of the viral replication cycle, but do not support infectious HCV production. This limitation has recently been overcome. In collaboration with the group of Dr. Wakita in Tokyo we developed full-length HCV genomes that support production of infectious HCV particles in cultured human hepatoma cells (Wakita et al., 2005). This system is based on a particular HCV isolate that was cloned from a Japanese patient with fulminant hepatitis C. Upon transfection of the complete JFH-1 genome or chimeric JFH-1 genomes, HCV particles were produced that are infectious for naive cells as well as for chimpanzees. Moreover, infection can be neutralized with antibodies directed against the envelope protein E2 or the putative (co)receptor CD81 or by immunoglobulins from patient sera. Similar findings have been made by others (Lindenbach et al., 2005; Zhong et al., 2005).

Production of infectious HCV in cell culture

 

Since this original discovery we developed numerous improvements of this cell culture system (Pietschmann et al., 2006; Kaul et al., 2007; Schaller et al., 2007; Steinmann et al., 2008) and characterized viral and cellular proteins contributing to the various steps of the HCV life cycle (Koutsoudakis et al., 2007; Steinmann et al., 2007; Jirasko et al., 2008; Appel et al., 2008).

 

HCV assembly

Model for assembly of infectious HCV particles

 

Host cell factors involved in the HCV replication cycle

By integration of most recent technologies such as high resolution light microscopy, high throughput methods for proteomics and transcriptome analysis and mathematic modeling of biological process, we are aiming to identify cellular networks required for productive replication of HCV. We have initiated RNAi-based systematic screening approaches to identify cellular factors that are involved in HCV entry, replication and release.

An additional main focus of our work is the investigation of interactions of HCV with cellular host cell factors, especially cyclophilins. We demonstrated recently that HCV replication and assembly completely depend on cyclophilin A (CypA) (Kaul et al., 2009). This host factor is required for replicase activity and most likely for NS2 protease activity. We propose a kinetic regulation of CypA dependence (Kaul et al., 2009).

 

Essential role of cyclophilin A for HCV replication (from Kaul et al., 2009)

 

Morphological studies of the HCV replication complex

By using high resolution microscopy approaches, in particular electron tomography combined with 3D image reconstruction we want to dissect the biogenesis and architecture of the viral replication complex.

EM image of Huh7 cells transfected with HCV

 

HCV and its interaction with innate immunity

Chronic HCV infection is a major global health problem with 170 million infected individuals worldwide, which represents 2-3% of the world population. These patients are at a high risk of developing serious liver damage; about 20% of persistently infected individuals develop liver cirrhosis within 20 to 30 years of initial infection and once cirrhosis is established, the rate of HCC development is 1 – 6 % per year. There is growing consensus that persistence results from a complex interplay of viral and cellular factors. Therefore, we are interested to dissect HCV interaction with the innate immune reaction and found that the NS3/4A protease blocks the induction phase by proteolytic cleavage of an essential adaptor protein of the signaling pathway (Meylan et al., 2005). Currently, we are aiming to unravel in molecular detail the viral and immunological mechanisms leading to persistence caused by HCV (see e.g. Binder et al., 2007; Dazert et al., 2009). To this end we have developed a cell culture system. HCV can persist and replicate for several weeks allowing for the first time the analysis of the viral mechanisms of persistence. Viral and host cell factors playing a key role in the establishment and maintenance of the persistence reservoir of HCV, the membrane-associated replication complex, as well as viral RNA itself will be analyzed. Also, the interplay between the type 1 IFN-induced antiviral status and the T-cell response will be investigated (Jo et al., 2009). These studies are performed within a recently granted research network coordinated by myself.

 

The Dengue virus (DENV)

Hypothetical DENV replication cycle

 

Morphological studies of the DENV replication complex and assembly sites

Positive-strand RNA viruses are known to rearrange cellular membranes to facilitate viral genome replication. The biogenesis and three-dimensional organization of these membranes and the link between replication and virus assembly sites is not fully clear. Using microscopic techniques, especially electron tomography we established the first 3D structure of DENV induced replication and assembly sites. Our data show that DENV modifies ER membrane structures to promote replication and efficient encapsidation of the genome into progeny virus. This architecture of DENV replication and assembly sites could explain the coordination of distinct steps of the flavivirus replication cycle and serves as a model to study the more complex organization of HCV replication sites.

3D reconstruction of DENV replication and assembly site (from Welsch et al., 2009)

 

Molecular mechanism of DENV replication

Not much is known about the functions of the different flaviviral non-structural proteins, especially the hydrophobic flavivirusproteins NS2A, NS4A, and NS4B. It has been suggested that theycontribute to the inhibition of the interferon-alpha/beta response. Furthermore, NS2A may target the viral RC tomembrane organelles, and NS4B may modulate viral replicationvia an interaction with NS3. As starting point for detailed investigations of the molecular functions of NS4A and NS4B we developeda topology model for these two proteins (Miller et al., 2006, 2007) and will use it for further molecular and cell biological studies of the DENV replication cycle.
A striking observation made in DENV infected cell lines is that the viral replicase factor NS5 resides predominantly in the nucleus of the infected cell. Therefore, we are currently characterizing NS5 with respect to nuclear localization and the role nuclear NS5 may play for the viral replication cycle.

 

Host cell factor involved in the DENV replication cycle

By using the same siRNA screening platform we aim to identify and characterize host cell factors required for dengue virus infection and replication using screens

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Methods applied

Apart from basic molecular and cell biology techniques, we utilize with increasing intensity high-resolution confocal microscopy, time lapse microscopy and electron microscopy techniques. An additional focus is put on high-throughput siRNA-based screening approaches by using reverse transfection and automated microscopy combined with image analysis. In some projects, especially those related to signaling, mathematical modelling is used to describe virus - host cell interaction.