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Cellular Polarity and Viral Infection

Steeve Boulant

Department of Infectious Diseases, Virology, and DKFZ

German Cancer Research Center

Im Neuenheimer Feld 581

69120 Heidelberg, Germany

The laboratory is interested in how intestinal epithelial cells that line the gut surface can tolerate the presence of the commensal microflora and at the same time recognize and fight enteric pathogens.

As obligate intracellular parasites, viral pathogens must breach the protective cellular membrane and deliver their genetic material to establish a productive infection in the cell. They have evolved to exploit normal cellular processes, hijacking a variety of endocytosis mechanisms to reach intracellular sites permissive to membrane penetration. Similarly viruses have developed strategies to bypass the innate immune response generated by the cell to promote their efficient replication and spread.

Viral entry constitutes the first step in viral life cycle. First, viruses bind cell-surface receptors allowing viral particle internalization. Then, vesicular transport delivers the particles to specific cellular compartments where viruses penetrate the endosomal membrane and enter the cytosol of the infected cell. Although much information is available concerning the biochemistry of virus/receptors interactions, little is known about the dynamics of these interactions in living cells. It remains unclear whether internalization of the viral particle is active and induced upon binding to the receptor or whether it is a passive process where viruses and receptors are hitchhiking on existing endocytic structures. Similarly, our understanding of the intracellular trafficking of endocytic compartment containing viral particles from the plasma membrane to site of membrane penetration is even less well understood.

After entry and membrane translocation, the viral particle or viral genetic material is located in the cytosol of the host where it could be recognized by the cell innate immune response system. Due to technical limitations, the spatio-temporal location within the cell where viral material is recognized, in regard to site of membrane translocation, remains to be determined. Its characterization will provide invaluable information, as it may constitute a simple strategy used by viruses to avoid the innate immune system.

Our laboratory aims to characterize, with live-cells, the spatio-temporal aspects of virus fate, from receptor binding to induction of the innate immune response.

Our viral models

In the lab, we use viruses as a model system to understand cellular mechanisms, from intracellular trafficking to antiviral innate immune response. We mainly use Reovirus, a small non-enveloped virus prototype for the Reoviridae family which contains the medically important virus Rotavirus. We have developed and are developing tools that will allow us to track individual viral particles within a living cell from entry to translocation into the cytosol of the host.

Virus Entry

Reovirus enter cells by clathrin-mediated endocytosis after binding to the tight junction protein Jam-A and ?1 integrin. We are interested in both the events that immediately precede virus entry and the events that follow the entry step. Our goal is to characterize how viruses attach to their receptors and whether this interaction promotes their active uptake by the cell. We are particularly interested in the molecular aspects that allow the coupling of the viral particles to the endocytic machinery.

Anti-viral innate immune response

The primary site of infection of the Reovirus family is the epithelium cells from the respiratory and digestive tracts. Our laboratory studies the innate immune response of polarized intestinal epithelium cells (IECs) induced during viral infection. Results show that IECs can generate a different qualitative/quantitative innate immune response upon viral infection, depending where the infection originates from (apical vs basolateral). As such, polarized cells constitute a unique system to study how cells compartmentalize and spatio-temporally regulate signal transduction pathways.

Our goal is to elucidate the molecular mechanisms that differently regulate innate immune response in polarized epithelial cells infected by viruses. Ultimately, we aim to understand how the innate immune response in the intestine is regulated to maintain colonic homeostasis. Indeed, misregulation of the signaling pathways or inappropriate response to the commensal flora might result in local inflammation and this is highly suspected to be responsible for inflammatory bowel diseases (e.g. Crohn’s Disease).

Approaches

We use multi-disciplinary approaches that consider the structure/function of a protein together with its spatio/temporal regulation/localization within living cells. We study virus receptor interactions in live-cells using single particle and molecule imaging. We combine high spatio-temporal resolution and three-dimensional imaging of live polarized epithelium cells together with confocal and TIRF microscopy to follow individual virus particles during their journey to the cytoplasm of the host cell.