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Rhinoviruses are the main causative agent of the common cold. They are small (30 nm in diameter) icosahedral particles containing a single stranded RNA genome of about 7,200 bases. For infection, they bind to plasma membrane proteins of the host cell and enter by exploiting several endocytosis pathways. Once exposed to the acidic pH in endosomes they undergo conformational changes that result in a slight expansion of the protein shell with concomitant opening of channels at the two-fold axes of symmetry. It is currently assumed that the RNA exits through one of these pores and crosses the endosomal membrane to arrive in the cytosol where it becomes translated and replicated, finally culminating in the synthesis of progeny virions.
However, this simplistic model cannot explain how the highly concentrated and intricately structured RNA molecule with its numerous double stranded regions should rapidly unravel to fit through such a small pore. Neither do we understand how it is protected against the hydrolytic enzymes present in late endosomes and what host cell factors, in addition to the acidic milieu, are required for it to be transferred into the cytosol. How does the RNA leave the viral shell to arrive unscathed in the cytosol of the host cell? Could it be that a pentamer temporarily opens like a lid to let the RNA exit in bulk?
Electron microscopy and X-ray crystallography has shown that the attachment points of the viral genomic RNA to the inner wall of the protein shell change on expansion of the particle in vitro. To find out whether the same happens in vivo we shall isolate intermediates of the process from cells shortly after infection and analyse their 3D-structure by cryo-electron microscopy image reconstruction. As we already saw particles lacking a pentamer in negative stain we shall attempt isolating and analysing such particles from infected cells. In parallel, we shall compare the secondary structure of the RNA inside the virion, in the expanded particle, and in refolded RNA by selective chemical modification followed by deep sequencing. Finally, we shall explore the possibility to inhibit RNA exit with compounds binding defined structural elements of the RNA; such compounds might be useful as novel antivirals.
Doctorate at the Université Montpellier, France, Venia Legendi at the Medical Faculty, University of Vienna (Biochemistry), Fellowship of the French Governement, Fellowship of the Ligue Française de la Lutte contre le Cancer, Höchst Preis, Th. Körner Preis, sabbatical in Structural Biology at the IBMB-CSIC, Barcelona, Spain, 176 peer reviewed publications in scientific journals.
On binding to the respective cellular receptor the virus is taken up by exploiting various endocytosis pathways. Exposure to the acidic pH in endosomes leads to expansion and opening of pores in the viral shell. Concomitantly, the sites of interaction between the RNA genome and the inner face of the protein shell change. RNA exit begins with the 3'-poly-(A). In vitro, the process stops after about 700 bases have left the virion. In vivo, the entire RNA exits within a few minutes. We ask what might aid RNA exit inside the cell and what role the ionic environment might play in the process.
Over the years many compounds attaching to a hydrophobic pocket in the viral capsid accessible from outside have been identified and their binding geometry has been determined by X-ray crystallography. These inhibitors mostly act via preventing the conformational changes of the shell necessary for subsequent RNA exit. By using cryo-electron microscopy we determine the binding geometry of novel antivirals.
So far only viral proteins, in particular the protein shell, were explored as possible drug targets. This also includes viral proteinases and polymerases that are not part of the virion but are being produced under the direction of the viral RNA inside the cell. We study the possibility of targeting the viral RNA with compounds recognizing structural elements that might be important for RNA exit, translation, replication, and/or encapsidation.
Insights into minor group rhinovirus uncoating: the X-ray structure of the HRV2 empty capsid.
Garriga, Damià; Pickl-Herk, Angela; Luque, Daniel; Wruss, Jürgen; Castón, José R; Blaas, Dieter; Verdaguer, Núria
Productive entry pathways of human rhinoviruses.
Fuchs, Renate; Blaas, Dieter
Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas-phase electrophoretic mobility molecular analysis: Part I.
Weiss, Victor U; Subirats, Xavier; Pickl-Herk, Angela; Bilek, Gerhard; Winkler, Wolfgang; Kumar, Mohit; Allmaier, Günter; Blaas, Dieter; Kenndler, Ernst
The Group Blaas participates in the special Doctoral Program "Structure and Interaction of Biological Macromolecules" reviewed and funded by the Austrian Science Fund FWF.