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Viruses are small parasites, which are strictly dependent on a plethora of cellular functions and activities for their successful replication and propagation. Many of these virus-host cell interactions are still unknown or not yet fully defined. Our group is dedicated to filling this knowledge gap by detailed structural and functional analysis of processes ranging from invasion of the host cell and intracellular growth to virion assembly and release, using picornaviruses as model systems. These are small ribonucleic acid (RNA)-containing particles, which can cause serious morbidities and even death in infected species. Well know examples are polio-, rhino-, coxsackie- and foot-and-mouth disease virus. For many representatives vaccines are not available; therapeutic targeting of host interactions is thus an emerging alternative for treating picornaviral infections. We expect that our work will also result in the discovery of new anti-viral drug targets for combatting these pathogens.
We use a combination of biochemical, biophysical and molecular biological tools to study the pathobiology of a various picornaviruses, mostly in 2-D tissue culture models. Gradient rate zonal centrifugation, electron microscopy (EM) including single particle cryo-EM, and fluorescence spectroscopy methods are employed to qualitatively and quantitatively assess virus morphogenesis and uncoating. Recently we have introduced bio-orthogonal (“click”) chemistry to investigate the role of a fatty acid modification of a picornaviral structural protein. Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) analysis has been implemented to elucidate the secondary structure organization of the viral RNA genome and its relevance for virus infectivity. By using pharmacological inhibitors, siRNA, CRISPR/Cas9 and RNA cross-linking/mass spectrometry we plan to identify novel host factors essential for replication of various members of this large family of small RNA viruses.
Heinrich Kowalski obtained a PhD in Biochemistry from the University of Vienna, Austria. He then went to the USA for postdoctoral research at the Stanford Medical School. Heinrich subsequently joined the Medical University of Vienna, Department of Pathology followed by his moving to the Department for Medical Biochemistry. Since 2014 he runs a virology research group at the Max Perutz Labs.
In tight collaboration with the Blaas group at the Max Perutz Labs we have found that the RNA genome of human rhinovirus 2, a prototypic picornavirus, in vivo exits its protective capsid shell presumably through a pore at the two-fold axis with the 3´ end first. This sheds new light on the viral uncoating process and the still rather elusive spatial organization of the RNA genome in the virion.
We recently demonstrated the importance of the fatty myristic acid modification of the capsid protein VP4 in multiple stages of the coxsackievirus B3 infection cycle. Using a highly specific inhibitor of the host enzyme N-myristoyltransferase (NMT) responsible for this modification, we extended the finding to a number of other picornaviruses, uncovering NMT as a potential new drug target.
In a concerted effort with other research teams we have developed a new procedure for producing rhinovirus at high purity. The process incorporates a monolithic ion exchange chromatography as a final polishing step to successfully eliminate contaminants from virions as prerequisite for their use in ultrasensitive biophysical analysis such as single molecule fluorescence correlation spectroscopy.
The Rhinovirus Subviral A-Particle Exposes 3'-terminal Sequences of its Genomic RNA.
Harutyunyan, Shushan; Kowalski, Heinrich; Blaas, Dieter
Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End.
Harutyunyan S, Kumar M, Sedivy A, Subirats X, Kowalski H, Koehler G, Blaas D
Capillary Electrophoresis, Gas-Phase Electrophoretic Mobility Molecular Analysis, and Electron Microscopy: Effective Tools for Quality Assessment and Basic Rhinovirus Research.
Victor U. Weiss, Xavier Subirats, Mohit Kumar, Shushan Harutyunyan, Irene Gösler, Heinrich Kowalski, Dieter Blaas
This research is funded by the FWF Grant (P27196-B13);
Project title: "Uncoating of rhinovirus RNA: Structure & Protein Interactions"
and the FWF Grant (P 23308-B13);
Project title: "Identification and characterization of the human rhinovirus type C receptor(s)" shared with Dr. Blaas.