Helenius Projects

Project 1: “How influenza virus hijacks the endocytic machinery”

Abstract: Influenza viruses (Orthomyxovirus) are spherical or filamentous enveloped particles. The helically symmetric nucleocapsid consists of a nucleoprotein and a multipartite genome in eight segments of single-stranded antisense RNA containing up to 10 genes. The envelope carries a hemagglutinin attachment protein and a neuraminidase. Both hemagglutinin and neuraminidase antigens undergo genetic variation, which is the basis for the emergence of new strains. Epidemics typically occur in the winter and cause considerable morbidity in all age groups. Notable pandemic outbreaks with associated mortality have occurred in 1918, 1957, 1968 which caused about 20 million deaths worldwide. Recent pandemic threats even made it more important to study the entry mechanism.

Experimental approach: The influenza virus infection system is well established in the lab and most of the tools needed for the in depth analysis of virus entry pathways are available and running in the lab. Generally, all work will be carried out under S2 condition with living virus and will comprise of eukaryotic cell culture, transfection of cells, over-expression of dominant negative constructs, classical virology toolbox etc. Most of the readout systems used will strongly depend on immuno-fluorescence microscopy as well as bioinformatic downstream analysis (depending on the applicant’s preferences).

Project 2: “How the human cytomegalovirus enters host cells”

Abstract: Human cytomegalovirus (HCMV) belongs to the Herpesviridae family. The central core harbours double-stranded DNA which encodes more than 200 genes. Cytomegalovirus infection can result in mainly three distinct clinical syndromes: one per cent of all live births acquire a congenital cytomegalovirus infection, another manifestation of cytomegalovirus infection is a mononucleosis syndrome and finally cytomegalovirus infection leads to severe complications in immuno-compromised individuals. The ability of HCMV to enter and infect a wide range of different cell types suggests that HCMV utilizes multiple receptors or its initial binding partners are ubiquitously exposed on the cell surface. Therefore it is not surprising that several host cell molecules have been suggested to act as receptor for HCMV.

Experimental approach: The human cytomegalovirus project will be carried out in concert with a collaboration partner who is providing the virology tools for the analysis. First of all a GFP dependent quantitative readout system has to be re-established. Once achieved GFP based infection assays have to be performed to reproduce the activation of different signalling pathways post infection. Further downstream analysis of the activated signalling pathways will be carried out using techniques like over-expression of dominant negative constructs, application of specific inhibitor drugs, siRNA treatment, immuno-fluorescence microscopy and molecular biology in general.

Project 3: “Differentiation between different endocytosis routes by RNAi mediated knockdown”

Abstract: The entry of viruses from different virus families has already been studied for several years. Specific knockdown of key factors of individual pathways allowed more in detail analysis of single pathways. But if viruses like influenza utilize several entry pathways at the same time with similar kinetics this approach will show it’s limitations. Therefore new approaches to isolate distinct endocytic uptake routes have to be taken. One approach is the targeted knockdown of key proteins of different pathways by RNA interference. Initial evidence shows that several of these pathways seem to be essential for the survival of host cells which make it necessary to apply more sophisticated knockdown systems for this approach.

Experimental approach: Starting point of the project is the reapplication of a virial shRNA mediated knockdown system of several target genes. After directional high throughput cloning of theses constructs, recombinant viruses will created and target cells infected under different conditions. Once the stable cell lines are established several biochemical as well as different in the lab established virus systems will be utilized to investigate the endocytic profile of these recombinant cell lines.

Contact for Projects 1-3:
Stefan Moese
Tel: +41-44-632 3149
E-Mail: stefan.moese@ethz.ch

Project 4: The role of the endoplasmic reticulum in Simian Virus 40 entry

Simian Virus 40 (SV40) is a non-enveloped DNA tumour virus that enters cells by caveolar endocytosis and is delivered to the endoplasmic reticulum (ER) in intact form. Some evidence suggests that from there it transported to the cytosol before entering the nucleus through nuclear pore complexes. Replication of the virus occurs in the nucleus. We are interested in why SV40 enters the endoplasmic reticulum (ER), (i.e. whether ER entry is essential to infection and if so why), how SV40 penetrates through the membrane of the ER, and how it is transported to the nucleus.

Experimental work will involve isolated viruses and mammalian cell culture. The methods used will incude biochemical approaches (such as cell fractionation, in vitro assays with purified viruses), electron microscopy, cell biological perturbations, and molecular biology approaches (the construction of recombinant viruses, transfection of cells, and expression of proteins).

Project 5: Entry of tumorigenic human papillomavirus type 16 (HPV-16)

HPV-16 is a non-enveloped DNA virus that causes warts, cervical carcinomas, and anogenital tumours. Cervical cancer is the third most common cancer among women worldwide, and it is the most common cancer among women in developing countries. To develop new anti-viral therapies, it will be important to understand HPV entry into ist host cells. HPV-16 enters cells by endocytosis, and it seems likely that it penetrates into the cytosol from classical endosomes. Replication occurs in the nucleus.

The goal of this project is to analyze the cell signalling pathways activated by the virus, to identify the endocytic pathway, and to characterise the route to the nucleus. A multitude of experimental approaches will be used including electron microscopy, live cell light microscopy, immunofluorescence microscopy, pharmacological inhibition of endocytic mechanisms, systematic siRNA mediated silencing of cellular components, bioinformatics, and/or biochemical assessment of the activation of signalling pathways. All studies are performed with so called pseudoviruses that are not infectious. Given the size and complexity of the project, the actual study to be performed by a student in collaboration with me, will be limited to a sub project of closest interest to the student.

Contact for Projects 4 and 5:
Mario Schelhaas
+41-(0)44-632 6901
mario.schelhaas@bc.biol.ethz.ch

Project 6: Tracking viruses on the surface of and within living cells.

For productive infection, a virus must enter a cell and deliver its genetic information to the appropriate intracellular compartment to initiate replication and progeny production (Smith, A.E. and Ari Helenius. How viruses enter animal cells. Science. 2004). The earliest step in this process requires a virus to bind to the surface of a cell, move laterally within the plane of the plasma membrane, and induce a signal within the cell to trigger endocytosis. All these steps occur when the virus is extra-cellular; From the outside, intracellular processes are stimulated by the membrane-bound virus to prepare the cell for its internalization. To study this process, we have been tracking the movement of viral particles as they bind to the plasma membrane of a live cell and as they move laterally along its surface (Ewers, H., Smith, A.E., Sbalzarini, I., Lilie, H., Koumoutsakos, P., and Helenius, A. Single particle tracking of murine polyomavirus-like particles on live cells and artificial membranes. PNAS. 2005). Our analysis revealed that the differing modes of virus motion observed on the cell surface required intracellular factors. We are interested in defining the cellular factors that promote the changes for particle internalization, and in the details of how a virus induces these intracellular changes from the outside of a cell.

This project combines cell biological, biochemical, biophysical and computational methods, and a student’s preference of technique will direct the exact nature of his or her own research. Typically, infectious virus or recombinant, non-infectious virus-like-particles are studied in mammalian cells and in prepared membrane bilayers or vesicles. Methods will include live and fixed cell fluorescence microscopy (confocal, total internal reflection and epiflourecence) including single particle tracking of viruses at near video rate, as well as electron microscopy. Biochemical and biophysical studies will be used to define viral binding and lateral diffusion more precisely. Computational-oriented individuals would have the opportunity to work at the interface of cell biology and technology with our computational collaborators here at ETH.

Project 7: Defining novel lipid raft endocytic pathways used by viruses

Like many animal viruses, the non-enveloped DNA tumor viruses of the Polyomavirdae family hijack cellular endocytic pathways for productive entry and infection of mammalian cells (Smith, A.E. and Ari Helenius. How viruses enter animal cells. Science. 2004). Polyomaviruses start the infectious process by binding to glyco-lipids called gangliosides on the surface of the cell (Smith, A.E., et. al. Ganglioside-dependent cell attachment and endocytosis of murine polyomavirus-like particles. FEBS Lett. 2003). For internalization, these viruses follow an entry pathway that uses lipid-raft mediated endoytosis. Polyomavirus entry has been intensively studied in the lab, and a picture of the endocytic trafficking routes used by the virus has emerged, but remains incomplete. Current studies focus on identification of the signaling and regulatory components of these lipid-raft pathways and on quantifying the dynamics of endocytosis and intracellular trafficking.

This project combines techniques in cell biology, molecular biology, and biochemistry that are well established in the lab, with the ultimate research focus determined by student interest. Electron microscopy, general molecular biology techniques, immuno-fluorescence and direct observation of fluorescently-tagged expressed proteins will complement the live cell fluorescence microscopy and quantitative live cell image analysis to investigate the dynamics and properties of the virus-containing intracellular vesicles.

Contact for projects 6 and 7:
Alicia Smith
Tel: + 41-44-623-6901
Email: alicia.smith@bc.biol.ethz.ch