Sub-Projects of SFB 535
Invasion and Replication Strategies of Pathogenes
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SP A18

Internalization and membrane fusion of BVDV


Prof. Dr. Till Rümenapf

Dr. Thomas Krey
Institut für Virologie, Justus-Liebig-Universität, Gießen



Summary

Understanding of BVDV (bovine viral diarrhea virus) entry into the host cell has recently been substantially enlarged by identification of bovine CD46 as cellular receptor and the characterization of the penetration mechanism of BVDV. At first glance the virus uses known receptor structures as well as a mechanism of penetration similar to those used by other enveloped RNA viruses. However, pestiviral invasion into the host cell exhibits additional twists compared to other members of the family Flaviviridae. Recent studies suggest, that virus binding to bovine CD46 is not sufficient to successfully mediate an infection. At least one additional cellular factor, likely a coreceptor, is required for BVDV infection. Remarkably, CD46 is constitutively recycled from the plasma membrane by clathrin dependent endocytosis, using the same pathway that applies to internalization of BVD virions. However, BVDV is not internalized as a cargo of its cellular receptor, bovine CD46. The project A18 particularly investigates the internalization defect of a bovine cell line (CRIB cells) that is completely resistent against BVDV infection using biochemical and genetic methods. The main approach will be a functional complementation assay of this cell line by retroviral transfer of a cDNA library from susceptible bovine cells into CRIB cells. In addition, the viral interaction partner of the coreceptor will be identified.

The second part of the project investigates the BVDV glycoprotein E2, which binds to the cellular receptor and is likely involved in fusion of viral envelope with the endosomal membrane during endocytosis. Compared to other members of the Flaviviridae fundamental differences are observed. Pestiviruses are resistant to environmental acidification and require an additional activiation step during entry process to facilitate pH-dependent fusion. This activation step was suggested to occur by breakage of disulfide bonds in the viral glycoproteins by an unknown mechanism. To understand the function of E2 and the E1-E2 heterodimer the three-dimensional structure of the viral gylcoproteins will be determined. The structural model will be supported by biochemical and functional analyses including mapping of the disulfide bridges, identification of the fusion peptide and localization of the receptor binding domain in E2. In addition, in cooperation with Félix Rey, Paris, crystallization of BVDV E2 and X-ray analysis of the resulting crystals is planned.

This project provides important advances in understanding of the entry mechanism of pestiviruses. Insights into the molecular mechanisms of BVDV invasion may help to elucidate the pathogenesis of the reportable veterinary diseases "Classical swine fever" and "Bovine viral diarrhea". Moreover pestiviruses serve as a model for other members of the family Flaviviridae (particularly the closely related hepatitis C virus).
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