Research Program of SFB 535
Invasion and Replication Strategies of Pathogens
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Summary

Infectious agents still pose a major health threat to humans and animals. For this reason the focus of CRC 535 has remained of great interest since its planning and first approval in 1996.

The CRC and all its projects (P) focus on the molecular research of pathogens. In this context the interaction of pathogenic agents with the host organism and the utilization of host cell factors play important roles, but of main interest are the characteristics of the pathogens themselves. The CRC studies two major steps in the life cycle of pathogens:
A) What are the mechanisms that have evolved to allow a pathogen to identify its victim and to invade its host, e.g. what are the invasion mechanisms and surface structures used by the pathogen?
B) The pathogen must be capable of replication within the host organism, which usually requires more or less significant utilization of host components or at least demands a Pecific adaptation of the pathogen to its environment within the host. Therefore the pathogen must follow a specific replication strategy and control its gene expression accordingly.
In fact, both aspects A and B of the pathogen are tightly interconnected. Thus, it is logical that certain pathogens or pathogen groups are analyzed in part A as well as in part B.


 
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Organization of the research topics in CRC 535

Positive strand RNA viruses. This group of viruses represents an important topic within the CRC. Project (P) A18 studies the entry of pestiviruses into the host cell and P B1 the following event of the IRES-regulated genome translation, which is used by hepatitis C virus,and  members of the genus pestivirus, and the family picornaviridae. Another common property of these viruses is the processing of a large polyprotein by cell- and virus-encoded proteases. The elucidation of this process for pestiviruses of different pathogenicity led to the identification of a previously unknown host factor (Jiv) by P B4. Jiv has been shown to play a role in the life cycle of many positive strand RNA viruses. The generation of pestivirus variants with different pathogenicities is often based on RNA recombination between viruses or with cellular mRNAs. P B8 was able to convincingly demonstrate, that this process does not only take place during RNA synthesis as commonly accepted, but also in the absence of viral RNA replication. The stunning observation that the viral core protein is dispensable for viral replication and morphogenesis led to the new P B13. These interesting findings in turn raise new questions. In the current funding period answers will be obtained and provide a more complete picture.

P A18 (previously P A1, T. Rümenapf, T. Krey) has identified CD46 as a receptor for pestiviruses, yet at least one other essential receptor must exist and remains to be identified. Furthermore, surprising details of viral entry have been described and will be investigated by cell biological methods.

P B1 (M. Niepmann) has identified and characterized several cellular factors that bind to the picornavirus 5’-terminal Internal Ribosomal Entry Site (IRES) during the cap-independent translation initiation and are involved in the attenuation of a poliovirus vaccine strain. The enhancement of translation initiation by the 3’-terminal region of hepatitis C virus RNA is of special interest. Results from these studies will lead to new information on the regulation of viral RNA transcription and replication, which will be further investigated in detail.

P B4 (N. Tautz) has identified the chaperone Jiv as the cofactor of the viral autoprotease NS2, providing an explanation of the mechanism by which the generation of the viral nonstructural protein NS3 is regulated. This proteinase is important for the pathogenicity of the Bovine Viral Diarrhea Virus (BVDV). As the amount of Jiv is limited the uncleaved precursor protein NS2-3 prevails in late phase of the replication cycle. Jiv seems to be important also for other plus strand RNA viruses. The role of this special regulatory mechanism together with viral and cellular factors for the viral life cycle and the structures involved is currently being investigated. P B4 is an outstanding example to demonstrate that very complex biological questions can be solved by long-term support/funding.

P B8 (P. Becher) has very convincingly investigated by use of molecular genetics a phenomenon, that could not be solved for a long time: the RNA recombination leading to the numerous naturally occurring variants of BVDV. Surprisingly, recombination between two defective BVDV genomes can take place without RNA replication. The viral and cellular factors which make this possible must now be identified.

Pestiviruses belong to the enveloped viruses. Current opinion holds that the viral envelope of pestiviruses and other members of the family Flaviviridae enwraps an isometric capsid derived from the core protein and that this capsid is essential for viral morphogenesis. P B13 (T. Rümenapf, H.-J. Thiel) overturns this dogma. The applicants have generated a core-negative variant that is able to produce virus particles and have defined a region of unknown function in the non-structural protein NS3, which seems to be essential for viral morphogenesis. The function of the core protein and the role of NS3 for morphogenesis are some of the most interesting questions of this last funding period.
 
Focussing the scientific potential of one institute on pestiviruses over many years was only possible by the funding of the CRC and has led to an outstanding position worldwide in the field of pestiviruses.

Hepatitis B virus (HBV). The topic HBV (virus family hepadnaviridae) has been funded since the beginning of CRC 535 as well as by the predecessor programs.

The absence of a feasible cell culture system has led M. Kann (P B5) to develop an innovative approach for the investigation of intracellular transport of the HBV capsid with permeabilized cells and recently also with protein-transporting lipids and intact cells. He discovered that the transport depends on genome maturation within the capsid and ends in the nuclear baskets of the nuclear pore complex (NPC) at the inside of the nuclear membrane. Here the capsid disassembles spontaneously, releases the genome and surprisingly reassembles. As a side finding he observed that HBV capsids enter the NPC with a diameter of about 35 nm, which is larger than the 27 nm described in textbooks. The cellular partners of the capsids during assembly, transport, and disassembly are the focus of the current funding period.

P A2 (W. Gerlich, D. Glebe) has experienced a breakthrough in the last 3 years with a functional cell culture system that has already delivered many important results. It was surprising that the small envelope protein of HBV as the major component of the viral envelope (and of the HBV vaccine) does not participate in the initial binding of the virus to its target cell, but instead only the preS1 domain. Similarities of the small HBV envelope protein to envelope proteins of other viruses (retroviruses and also pestiviruses, P A16) suggest that it play a role during endocytosis and release of the capsid, which shall now be investigated. With the identification of a high affinity preS1 envelope protein element the search for the HBV binding receptor has reached a promising new level.

Negative strand RNA viruses. These viruses show many similarities in the structure of their envelope proteins and invasion mechanisms, despite the seemingly large diversity of their replication mechanisms. Likewise measles virus (P A17, A. Maisner), the Borna disease virus (BDV) (P A3, W. Garten), members of the filoviridae (P A13, S. Becker) and influenza viruses (P B12, S. Pleschka) bud from the surface of the infected cell and fuse their viral membrane with the cellular membrane (plasma- or endosomal membrane) after binding to a new target cell. Budding, binding and membrane fusion are the result of highly regulated interactions between viral membrane and cellular components (proteins, lipids).

P A17 (A. Maisner) studies a phenomenon of measles virus, that like several other enveloped viruses, induces fusion between the infected cell and neighboring uninfected cells, thereby allowing the virus to spread from cell to cell without release of free viral particles. Nevertheless, this mechanism is a dead end as these syncytia are not viable and can therefore not serve as a virus reservoir. The new P A17 that is based on extensive previous work are viral factors which regulate the balance between cell fusion and virion release.

The topic of. P A3 (W. Garten) that concentrates on BDV has been part of the CRC from the beginning. While at first only ORFs were known as possible candidates of the BDV membrane proteins, the work of P A3 has meanwhile elucidated function, modifications, and proteolytic processing of these proteins and has now begun to look at cellular partners and their role during viral invasion of the cell.

P A13 (S. Becker) follows the process of virus maturation with Marburg Virus. In the last years new structural elements of the cell, multivesicular bodies, and viral peptide sequence motives (late domains) have been identified. Both bring together viral matrix proteins located inside the cell and viral surface proteins on the outside and finally induce budding of the virions from the cell membrane.

P B9 (E. Mühlberger) also deals with Filoviridae - a special focus in Marburg – particularly  with the aspect of viral genome replication. In 2001, E. Mühlberger already provided a notable contribution to the research on these highly dangerous viruses by establishing a method by which it is possible to generate infectious Ebola Virus directly and exclusively from cDNA. Recently this was also achieved for Marburg Virus. P B9 concentrates on viral transcription and the switch to RNA replication, as well as the replication efficiency as a pathogenicity determinant.

P B12 (S. Pleschka) investigates the transition from viral genome replication to maturation of influenza virus particles. The starting point of this project was the observation that an inhibitor of the virus induced Raf/MEK/ERK signaling cascade impairs virus production. Nuclear RNP export was determined as one of the steps blocked in viral replication. The activation mechanisms of the cellular signal transduction by the virus and the viral target molecules of the signal cascade are the principle aspects of the project.

Bacteria

Many pathogenic bacteria interact with structures on the cell surface that are similar to those used by viruses to stabilize their environment and to manipulate the host organism to their advantage. Different pathogenic E. coli strains possess a transport system, which enables them to bind to intestinal epithelia cells and to inject bacterial proteins. In addition bacteria make use of numerous toxins to preserve and enlarge their living space. The Shigatoxin of the enterohemorrhagic E. coli strains is a factor which is mainly responsible for suppression of the innate immunity. P A11 (C. Menge, G. Baljer) could demonstrate the impact of this toxin on the activation of immune cells in different in vivo systems including animal experiments.

Listeria monocytogenes is a highly versatile bacterium that shows its pathogenicity as an intracellular replicating agent. P B14 (T. Hain, T. Chakraborty) has identified in previous experiments several inducible or repressible gene groups that are involved in the ability of the pathogen to grow intracellularly and to spread from cell to cell. By employing modern methods of DNA microarrays, transcription patterns of these gene groups in different cell compartments of mammalian cells and in cells of Drosophila melanogaster – a know host of L. monocytogenes - shall be analyzed extensively.

Parasites. The spectrum of parasites under investigation in the CRC includes protozoa and helminthes.

Phosphocholin-modified glycoconjungates are main components for the developement of free-living helminthes, but have also immune modulating effects on the hosts of parasitic worms. Enzymes involved in their biosynthesis are important target structures for ?anthelminthica. P A8 (G. Lochnit) is involved in the identification of these enzymes and their genes by biochemical methods and an siRNA approach, using the model organism Caenorhabditis elegans. Moreover cytokine patterns of immune cells that are induced by phosphocholin carrying antigens are analyzed.

P A15 (R. Geyer) aims at the identification of glycans of humans, snails and worms, which are utilized by parasitic helminthes for survival in a strategy called molecular mimicry, whereby carbohydrate epitopes expressed in the host are also expressed on the surface of the parasites. Schistosoma mansoni utilizes this strategy in its temporary host, the freshwater snail Biompholaria glabrata, as well as in humans. In addition, interactions between these structures and DC-SIGN and Toll-like receptors are subjects of investigation.

P B15 (C. Grevelding), another new project, investigates the female maturation of schistosomes, that live as paired adult worms (male and female) and release their eggs in great numbers, which can lead to inflammation. The project searches, on the level of signal molecules, signal transduction and gene induction, for factors released by the male that will induce maturation of the female organism. This could lead to the discovery of target structures for a therapy against bilharziosis.

P B10 (A. Bindereif) focuses on the processing of polycistronic primary transcripts by trans-splicing, a common characteristic of many parasites including protozoans. Recently, cis-splicing was also discovered and described for the causative agent of sleeping sickness, Trypanosoma brucei. The project analyzes the components of different small nuclear ribonucleoprotein complexes (snRNPs) that are required for cis- and trans-splicing by T. brucei, and pursues the goal to identify further cis-introns in the T. brucei genome.

P A12 (K Becker-Brandenburg)? investigates peroxiredoxines as survival factors of the probably most important worldwide pathogen, the intracellularly replicating protozoon Plasmodium falciparum that uses  them to protect itself against reactive oxygen species,. These enzymes, which have already been identified and are available in sufficient quantities shall now be characterized for their 3D-structure and physiological functions.

Facilities and central projects of the CRC
The spectrum of available laboratory space of the CRC ranges from normal biochemical/molecular biological labs of the security level S1 to more advanced S2 for work with infectious agents as well as to high security level labs S3 (Giessen) and BSL4 (Marburg, later also S4).

The confocal laser-scanning microscope is an important shared instrument of the CRC 535 that has been funded in a new more advanced and powerful version within the framework of P B5 (M. Kann).

Microarrays are available from P B14 (T. Hain, T. Chakraborty).

P Z1 (R. Geyer, G. Lochnit), the central project, has ever since provided peptide and glycan analysis of high quality with increasing help of MS techniques.

P Z4 (M. Hardt) has established a high performance electron microscope for cryo-analysis under security level S2. It is now available for structural analysis at high resolution and native immunoelectron microscopy.

P Z3 (W.H. Gerlich, S. Pleschka) administrates (i) all travel and publication costs, (ii) in collaboration with the financial office of the Medical Faculty all funded budgets of the projects and (iii) organizes the internal and external seminars of the CRC 535 with the help of the secretary E. Kaiser.
 

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