Asymmetry in Icosahedral Viruses

Viruses reprogram the host cell to preferentially produce progeny virus. They do so by transferring their genetic information, encoded in nucleic acids, from a protective protein shell through a hostile environment into the cell. Infection begins with binding to suitable structures on the cell surface, followed by uptake within vesicles that bud from the inner side of the plasma membrane into the cytosol. Once the conditions are favourable for safely opening this container and discharge its genetic cargo, the virion converts into a subviral particle with holes for exit of the nucleic acid. It is still enigmatic how the highly concentrated and tightly folded nucleic acids exit the viral shell, pass through a lipid membrane, and finally arrive unharmed at their destination where they force the host cells synthesis machinery to produce new viruses. Rhinoviruses, the main cause of recurrent common colds, possess a single stranded RNA genome of about 7,200 nucleotides. This corresponds to a total length of about 2 m. This single strand is exceedingly folded into short double stranded regions because of many stretches with local complementarity. In this way it can be confined within a volume of roughly 4 x 10-21 litres giving rise to concentrations in the range of kg per litre! It is currently ignored how this RNA molecule can exit the shell through one of the small holes that open in the capsid when triggered by the acidic pH and other factors prevailing in the membrane vesicles mentioned above, called endosomes. We here address the question of how the RNA is arranged within the icosahedral capsid allowing it to start exiting with one of its ends. We shall use various complementary techniques, in particular cryogenic electron microscopy, to identify local asymmetric features of the RNA molecule that might play a role in orienting the icosahedral particle with respect to the endosomal membrane allowing efficient and unencumbered transit of the viral genome through the lipid membrane. To this end we shall develop novel approaches for attaching metal to the RNA tails and investigate the processes immediately preceding and following the structural changes of the virion within endosomes including the assessment of novel means of their inhibition.

The main causative agent of the Common Cold are rhinoviruses (RVs). Because of the infection being mostly limited to the nasal mucosa only poor immunity is being generated, which does not extend onto the other numerous RV strains. Therefore, one can become repeatedly infected by a novel RV variant. RVs are icosaedrons with a diameter of roughly 30 nm. The do not have a lipid envelope and are composed of 60 copies each of only four different proteins and a single stranded RNA genome. There are two burning questions in the research on RV: First, how does the asymmetric RNA fold within the viral shell and, second, how does the RNA leave the capsid and passes through at least one lipid membrane to arrive in the cytosol where the replication takes place. Employing different techniques we were able to show how the virion changes its three-D structure to release the RNA and, with novel classification methodology it became possible to demonstrate deviations of the otherwise strictly icoahedral symmetry by the interaction of the different RNA segments with the identical copies of the viral proteins. There are a number of low-molecular mass compounds that inhibit infection but so far none could be developed into a medication. These compounds are directed against various viral protein such as the proteinases, the RNA polymerase etc. or they stabilize the capsid against the above structural modifications as to prevent that the genome arrives in the cytosol. For one of the newer compounds we were able to identify the binding site on the capsid, which is important for its further development. Additionally, we were able to stabilize particular folding patterns of the viral RNA with specifically binding compounds, which identified the RNA as potential target for antiviral therapy.