While not recognized as a common pathogen, a recent study from the Dermody lab published in Science in 2017 supports a role for reovirus infection in the development of celiac disease by breaking immunological tolerance to orally ingested gluten.1In addition, reovirus efficiently lyses tumor cells and has shown efficacy in clinical trials for refractory human cancers.2However, 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 coalition of 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.3,4Using nanoscopic cantilevers derivatized with single virions, we spent hours in the dark running experiments recording the virions rod bites. Altogether, we analyzed thousands of force-distance-curves and maps allowing us to uncover a major breakthrough in the reovirus’ binding mechanism. We showed for the first time that reovirus early binding to cell surface is regulated by glycans and therefore that the attachment factors (sialic acid for reoviruses) are more than ‘simple tethers or unspecific step’ as previously thought.5Our study highlights a physiologically relevant interplay between attachment factors (α-linked sialic acid glycans [α-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 α-SA, which is engaged with low affinity, serves as the initial attachment event and triggers a conformational change in the viral σ1 attachment protein that enhances further specific interactions with the high-affinity JAM-A receptor (see Fig 1). 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.
Figure 1: Glycan-mediated enhancement of reovirus receptor binding.Upon binding of α-SA, the σ1 outer capsid glycoproteins undergo a conformational change leading to a more extended conformation. This results in an increased affinity for JAM-A.
The final touch of this study took place in the middle of nowhere in the Belgian countryside during a secret meeting with our collaborators from the Dermody lab. This time, armed with Belgian beer and fries, we pondered over the final scope of this study and ultimately, 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. In summary, our findings are of crucial interest since this is the first time that anyone has assayed, at the single virus level, the cooperative interplay between attachment factor and entry receptor in virus infection.
1. Bouziat, R.et al.Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease. Science356, 44-50 (2017).
2. Coffey, M. C., Strong, J. E., Forsyth, P. A. & Lee, P. W. Reovirus therapy of tumors with activated Ras pathway. Science282, 1332-1334 (1998).
3. Alsteens, D.et al.Nanomechanical mapping of first binding steps of a virus to animal cells. Nat. Nanotechnol12, 177-183 (2017).
4. Newton, R.et al.Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces. Nat. Protoc.12, 2275 (2017).
5 .Marsh, M. & Helenius, A. Virus entry: open sesame. Cell124, 729-740 (2006).