Viral RNA Genome Uncoating: The Where´s and When´s

Enteroviruses, a large genus within the picornavirus family of important animal and human pathogens, undergo conformational changes during cell entry preparing the release of their RNA genomes into the cytosol for replication. In recent work with HRV-A2, a prototype common cold virus belonging to the genus rhinovirus A, we found that after conversion of the native virion into the subviral A-particle, triggered by the acidic endosomal milieu, it takes about 10 min until exit of the genome starts from the 3-poly-(A) tract. This raises the question of what is happening during this lag time; it is possible that the viral shell and/or the nucleic acid need additional conformational changes and/or interactions with cellular factors for initiation of genome egress. It is also possible that the subviral particle must proceed to a particular cellular compartment, dissociate from the receptor, and/or establish an intimate contact with a lipid bilayer of particular composition and fluidity. We want to explore the first possibility by using cryo-electron microscopy image reconstruction of particles recovered at different times after acidification in vitro or after infection in vivo. The second possibility will be addressed by determining, in the host cell at different times after infection, where the viral capsid protein VP4, N-terminal sequences of VP1, the viral genomic poly-(A) tail and, completing the uncoating process, sequences close to the 5- end are exiting from the virion. These investigations will be complemented by single molecule microscopy experiments aimed at characterizing the putative membrane pore and to identify an eventual oligomeric state of the viral proteins involved. Knowledge gained from these studies is indispensable for a full understanding of the infection process, the physicochemical background of RNA exit, and the development of novel viral inhibitors.

Rhinoviruses (RV) are the cause of about 50% of all mild respiratory infections summarized as the well-known common cold. They are part of the large family of Picornaviruses that infect animals and man. Among them are agents causing foot-and-mouth disease, poliomyelitis, and hepatitis A. Rhinoviruses can also exacerbate asthma and in combination with this and other conditions become life threatening. Because of the close similarity between rhinoviruses and the above-mentioned other members of the picornavirus family they are often considered a less harmful model, since many results obtained with RVs can be extrapolated onto more dangerous picornaviruses. The first step in infection is the docking onto a suitable protein at the surface of the host cell. Subsequently, the virus is being taken up into the cell and, following various pathways, being transferred into different compartments. The decisive step is the release of the RNA genome into the cytosol. Based on this blueprint the cellular machinery produces new viruses that get to the outside by destroying the host cell. The more than 179 different RV types, there are three very differently constructed receptors, one of them we identified many years ago. In the course of the present project we could demonstrated that RVs that bind the identical receptor nevertheless follow different internalization pathways. This means that not only the receptor alone is responsible for the choice but most probably, differences in stability and charge of the viral capsid are contributing. In the frame of various international collaborations we developed methods for the preparation of highly pure virus und novel analytics. We were involved in the identification of a cellular phospholipase that is essential for infection; when absent, infection is strongly reduced. Similarly we found that blocking another host cell protein, a myristoyltransferase, results in inhibition of the virus. These are beautiful examples for the fact that interventions on the basis of the cell are very well suited to inhibit viral infection without running the risk that they become resistant by mutation. Finally, we could identify the attachment site of a novel substance that neutralizes the few pleconaril-resistant RV types. Pleconaril is an antiviral that has been known for many years but never made it to approval as a drug because of secondary effects. Interestingly, the novel binding site only marginally overlaps with that of almost all currently known capsid-binding antiviral substances, including pleconaril.