Analysis of viral RNA uncoating with spatiotemporal resolution
One of the earliest steps following viral entry of its host cell is a conformational alteration of the particle leading to release of the viral RNA, an event termed "uncoating", which makes the viral genome available for translation and replication. We recently found that this event is highly coordinated at the level of rearrangement of the genomic RNA inside the capsid. In concert with the strict 3´ to 5´ polarity of its ejection this appears to be required for the ordered and timely transfer to the cytosol. The primary aim of the project carried out in close collaboration with the Blaas group at MFPL and experts of on-campus facilities is to determine the conformation of the viral RNA in- and outside the virion, to dissect essential interactions with the capsid and to elucidate their dynamic changes during uncoating. In addition, we noticed that the entire process is promoted by host factor(s), which will be identified using novel experimental setups. Techniques span biochemical analysis, SHAPE chemistry, UV cross-linking, mass spectrometry, RNAseq, cryo-EM, and fluorescence correlation spectroscopy, paired with standard methods in molecular biology. The results will considerably advance our understanding of the rules governing the ordered disassembly of virus particles for successful invasion of their host cell.
Strategies towards analysis of non-cultivatable viruses
In the wake of large-scale (next generation) sequencing efforts an ever-increasing number of new viruses have been discovered for which no cell culture system exists. This seriously hampers their structural and functional characterization. Focusing on C-type rhinoviruses, which do not grow in standard tissue culture cells due to lack of the cognate receptor we have started to generate virus-like particles as surrogates for structural analysis by cryo-electron microscopy (cyro-EM). In addition, we conduct reverse genetics for targeting these viruses towards other receptors such as integrin family members expressed on many cells in conventional tissue culture. By means of deliberately changing their host tropism, we hope to expedite and facilitate exploration of such fastidious viruses.
Role of N-Myristyltransferase family members in the covalent modification of rhinovirus proteins
Replication of picornaviruses requires a battery of host factors. In mammalian cells, two N-myristyltransferase isozymes (NMT1 and NMT2) are expressed from different genes with only partially overlapping function. We strive to understand the contribution of the individual NMTs to the myristoylation of certain picornaviral proteins using biochemical and molecular biological tools in combination with bio-orthogonal chemistry. Employing a novel bacterial demyristylase, we further seek to uncover the still unclear function of the myristate moiety in the viruses’ penetration of the host cell.
