Studying Viral Entry into Cells

Researchers combine confocal and atomic force microscopy to better understand this critical step of viral infections

Viruses cannot reproduce on their own. They require the internal machinery of host cells for replication and propagation. One of the challenges that successful viruses have overcome is evolving complex, multi-step mechanisms to bind to host cell surfaces and then transport themselves inside the cell.

Prof. Dr. David Alsteens, Research Associate of the FNRS

Dr. David Alsteens and his group at the Louvain Institute of Biomolecular Science and Technology, Belgium, along with colleagues at the University of Pittsburgh, USA, have recently published work in Nature Communications focused on better understanding how one type of virus binds to the cell surface. Their work is particularly interesting as they were able to study this for the first time in living cells by using a combination of confocal and atomic force microscopy (AFM).

Dr. Alsteens was kind enough to answer some questions about his use of confocal microscopy, AFM and his research.

Please broadly describe the research goals of your lab. How did this publication fit into your overall research?

The ‘NanoBiophysics lab’ aims at better understanding the complex biological processes that take place on the cell surface under relevant physiological conditions. To this end we combine AFM and point scanning confocal microscopy to localize molecules, receptors and cells while mapping their nanomechanical properties or biophysical interactions between the AFM probe and the biological sample. We have been using force-distance based-AFM for several years already to study a variety of biological samples at both molecular and cellular levels to better understand how single-molecule interactions can drive biological processes.

Some key applications of my lab include, among others, imaging G-protein coupled receptors while quantifying their ligand-binding free-energy landscape, studying the first entry steps of viruses to animal cells (Reovirus, Rotavirus, Herpesvirus, Ebola virus-like particles) with a focus on the understanding of the dynamics of the interactions established at the surface of living cells.

Our recent publication perfectly fits into the main research’s axes of my lab. Since my postdoc stay at ETH Zürich (Basel, Switzerland), we further developed the combination of the latest generation of Bio-AFM with a high-resolution optical microscope. The ambition is to develop a new methodology in biophysics and virology to study virus entry at high-resolution, directly on living cells, and to provide quantitative information on the molecular details underlying the early steps of cell infection.

In our recent work, we combine the latest generations of confocal laser scanning microscope (CLSM) and AFM to follow the early steps of single virus entry. We also succeeded to describe these first steps in a quantitative manner and provided the kinetic and energetic parameters of the virus-receptors interactions.

The Alsteens Lab in front of their confocal – AFM equipment

Could you describe in layman’s terms how your combination of confocal and AFM technologies contributed to the findings in this publication?

Thanks to an atomic force microscope coupled to a confocal microscope, we investigated how reoviruses interact with mammalian cell surfaces in cell culture conditions. A very sharp tip is functionalized with a single virus and approached to the cell surface until soft contact, enabling it to interact with the cell surface component including glycans and receptors (Figure 1).

Figure 1: Confocal microscopy image of an AFM tip bearing a single virus (green spot) on a layer of CHO cells.

The combination with the confocal microscope permits us to follow in real time the position of the AFM tip on the cell and the localization of some fluorescently labelled receptors. Upon retraction of the AFM tip, the bounded virus starts to detach from the receptors and the deflection of the tip during the retraction movement is monitored. The deflection is directly proportional to the force acting between the virus and the receptors, thus enabling us to quantitate the binding strength. The measured binding forces are then analyzed in detail and gives access to parameters that describe the interaction on a kinetic and energetic manner. We can also determine the number of bonds that have been formed between the virus and the cell (Figure 2).

Figure 2: Probing reovirus binding to CHO cells expressing JAM-A. (a) Cartoon of the experiment highlighting that CHO cells (having no JAM-A) are fluorescently labeled. (b) Confocal image showing an AFM tip on the top of two adjacent cells expressing or not JAM-A receptors. (c) FD-based AFM height image and corresponding adhesion channels. The adhesion map reveals that most adhesion (bright pixels) are localized on the cells expressing JAM-A receptors.

Altogether, this study allowed us to make a major breakthrough in understanding the binding mechanism of the reovirus to cells. We showed for the first time that reovirus early binding to cell surface is regulated by glycans, known as attachment factors (sialic acid for reoviruses). Until now, those interactions were often neglected and seen as unspecific interactions, the role of which was to concentrate the virus at the cell surface. However, these links are much more than ‘simple tethers or unspecific step’ as previously thought. Our study highlights a physiologically relevant interplay between the attachment factors (α-linked sialic acid glycans [α-SA]) and the specific entry receptor (junctional adhesion molecule A [JAM-A]). Our in vitro and cellular experiments revealed a cooperative effect. The α-SA binding to the viral glycoprotein, which is engaged with low affinity, serves as the initial attachment event and further triggers a conformational change into the viral glycoprotein. The glycoprotein adopts a more extended conformation that facilitate its specific interactions with the high-affinity JAM-A receptor. Moreover, we discovered that short sialylated glycans induce an enhancement of reovirus receptor binding, due to a conformational change in the glycoprotein. This leads to an increased binding avidity and ultimately infectivity, which can be applied for future vaccine and oncolytic treatments.

You mention in the publication that these insights could lead to the use of reoviruses as oncolytic agents – could you elaborate on that?

The main means of cancer treatment such as chemotherapy, radiotherapy, and even targeted kinase inhibitors and mAbs are limited by lack of efficacy, cellular resistance, and toxicity. Oncolytic viral therapy offers a novel therapeutic strategy that has the potential to dramatically improve clinical outcomes by having a wide spectrum of anticancer activity with minimal human toxicity.

Reovirus is a leading candidate for therapeutic development and currently in phase III trials. Reovirus selectively targets transformed cells with activated Ras signaling pathways. Ras genes are some of the most frequently mutated oncogenes in human cancer and it is estimated that at least 30% of all human tumors exhibit aberrant Ras signaling. By targeting Ras-activated cells, reovirus can directly lyse cancer cells, disrupt tumor immunosuppressive mechanisms, reestablish multicellular immune surveillance, and generate robust antitumor responses. Reovirus phase I clinical trials have shown indications of efficacy, and several phase II/III trials are ongoing at present. Reovirus’ extensive preclinical efficacy, replication competency, and low toxicity profile in humans have placed it as an attractive anticancer therapeutic for ongoing clinical testing. Despite advances in our understanding of the host and viral determinants that underlie reovirus replication and killing of transformed cells, many gaps in knowledge remain. With all these developments but also open questions in mind, our study provides unique opportunities to manipulate reovirus binding efficiency and infectivity for vaccine and oncolytic applications.

Learn More

Read the full article “Glycan-mediated enhancement of reovirus receptor binding” in Nature Communications

Learn about the ZEISS technology used in this publication:

Read Next

Leave a Reply

Your email address will not be published. Required fields are marked *

Visits on this Page:6244