Probing the Reovirus Infection

This project is entitled Probing the reovirus infection From touchdown to cell entry. Viruses are small parasitic particles that exclusively replicate using the cell machinery of living cells. Because of their simplicity, they depend on a host organism in nearly all stages of the infection cycle. Viruses have acquired the relevant molecular passwords or entrance tickets and thus, are able to control and utilize cellular functions due to million years of evolution and adaption to their cellular hosts. This makes them species-specific and only infect a narrow range of organisms. The infection cycle of a virus particle consists of a series of consecutive steps, beginning with landing or touchdown of the virus to the cell surface via preliminary interactions between the virion and the exposed cell surface molecules (cell surface receptors). Host cell penetration (viral entry) follows attachment and further by uncoating, virus replication, assembly and finally the virus release from the host cell. We will look at the first touchdown of a single virus on living cells and see how the virus binds to cell surfaces molecules, since these processes are poorly understood up to now and offer novel potential therapeutic strategies. We will focus on reoviruses, which are involved in e.g. lethal encephalitis and own oncolytic properties, encouraging the development of reovirus-based therapies for cancer treatment. We will study how a single virus binds to a cell surface, how the cell surface receptors contribute to the binding and decipher the dynamics of the first virus entry step. Moreover, we aim to specifically deliver a single virus to a defined cell, which is important for specific applications, such as e.g. delivery of genetic material to fight a tumor. To this end, we will use the atomic force microscope (AFM). The microscope is used like a nanoscopic sensor, which means that the tool is able to measure interactions at the single-molecule level, e.g. unimaginable low forces (trillionth Newton) between surface cell receptors and viruses. For this purpose we can hang a virus to a kind of fishing line (so called cantilever equipped with a cross linker). Moreover, the AFM can also visualize biological structures in the size of a few millionths of a millimeter, which makes it possible to localize a single virus on a cell. By hanging a virus to the measurement needle (cantilever), we can also specifically deliver a single virus to the cell of interest by cutting the cross linker at the right time and place. This interdisciplinary project combines aspects of bio-nanotechnology, virology, chemistry, medicine and biophysics and will push forward our knowledge in describing quantitatively virus infection. Moreover, it will shed light into the complex topic of reoviruses role in its suitability as cancer-killing reagents.

Glycans pull the trigger but JAM-A initiates reovirus entry into living cells: Solving the mystery behind the molecular and mechanistic basis of reovirus binding to the cell surface using high-throughput nanoscopy methods. While not recognized as a common pathogen, it has been shown that reovirus infection contributes to the development of celiac disease by breaking immunological tolerance to orally ingested gluten. Moreover, reovirus efficiently lyses tumor cells and has shown efficacy in clinical trials for refractory human cancers. However, the molecular and mechanistic basis of reovirus binding to the cell surface has remained mysterious. Current knowledge of virus entry relies mainly on ensemble studies that provide an average of a population, but virus infection is a multistep process in which the dynamics of each individual step are crucial, and the propensity of virions to establish polyvalent interactions complicate the full picture. Feeling a little bit like FBI-Special Agents Mulder and Scully, we aimed to solve this mystery in identifying all the perps involved in this complex mechanism and trying to uncover any possible obscure conspiracy. Armed with an advanced weapon, consisting in an atomic force microscope combined with a confocal microscope, we guard mammalian cells preserved in tissue-culture conditions and observe the attempted break-in of individual viruses. Using nanoscopic cantilevers as nano-fishing rods decorated with single virions as baits, we recorded the virions interaction with the cell surface, allowing us to uncover a major breakthrough in the reovirus' binding mechanism. We showed that reovirus early binding to cell surface is regulated by glycans and that the attachment factors (sialic acid for reoviruses) are more than 'simple tethers or unspecific steps' as previously thought. Our study highlights a physiologically relevant interplay between attachment factors (-linked sialic acid glycans [-SA]) and a specific entry receptor (junctional adhesion molecule A [JAM-A]). It is the first time that such a direct coalition between virus, attachment factors and specific entry receptors is observed during viral infection. Our in vitro and cellular experiments reveal indeed an up-to-now unknown consolidation: Binding to -SA, which is engaged with low affinity, serves as the initial attachment event and triggers a conformational change in the viral 1 attachment protein that enhances further specific interactions with the high-affinity JAM-A receptor. This two-step adhesion strengthening mechanism has only been hinted at and not been shown directly for viruses and provides evidence for glycan-mediated cell targeting influencing viral tropism. Using this approach, we were able to provide the community with unique opportunities to manipulate reovirus binding efficiency and infectivity for vaccine and oncolytic applications. In this context, we nailed down that short specific glycans could be used to enhance virion binding potential, which offers an exciting application for both applications.