Viral particles work as effective molecular devices in the transfer of the viral genome and accessory proteins from infected to non-infected cells. Viral structures have evolved not only to secure stability of the particle during passage, but also to allow efficient and specific assembly and disassembly. For entry into host cells, viral particles must possess built-in systems for receptor binding, membrane penetration, intracellular targeting, and genome uncoating. During entry into host cells, viruses typically undergo a process of stepwise destabilization and uncoating often linked spatio-temporally with the movement of the particle from compartment to compartment in the cell (Marsh and Helenius, 2006). Cellular ’cues’ are needed to trigger changes in the particle, and numerous cellular factors help to guide the incoming virus to its final location in the cytosol or the nucleus.
Structural analysis of functional virus–host–interactions in the course of entry provides a means to understand this dynamic process and to elucidate basic cellular mechanisms exploited by the virus. As model systems we as use primarily herpesviruses, retroviruses and selected other viruses well suited for the particular entry aspect under scrutiny. To address selected steps in the viral entry pathway we successfully established a number of cellular and subcellular investigation systems. This allows a systematic analysis of the macromolecular dynamics during viral entry. The native structural information will be combined with existing biochemical and crystallographic data. In the project we will use the established systems in a hybrid approach with other methods to analyze the structure, dynamics and interplay of viral glycoproteins during entry and to further analyze directed intracellular transport.
The multi-disciplinary project will provide training in a variety of methods. This will encompass biochemical and biophysical analyses, correlation microscopy spanning numerous imaging means and ranges of resolution (from advanced life cell light and fluorescent microscopy to cryo electron microscopy and tomography) and the use of state of the art image reconstruction and processing techniques for structure determination and analysis. There will also be the opportunity to perform molecular biology experiments, cell culture techniques, virus production and purification and immunogold-labelling.
Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology
Project reference number: 238
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Kay Grunewald | Structural Biology | Oxford University | UK | kay@strubi.ox.ac.uk |
2006. Virus entry: open sesame. Cell, 124 (4), pp. 729-40. Read abstract | Read more
Detailed information about the replication cycle of viruses and their interactions with host organisms is required to develop strategies to stop them. Cell biology studies, live-cell imaging, and systems biology have started to illuminate the multiple and subtly different pathways that animal viruses use to enter host cells. These insights are revolutionizing our understanding of endocytosis and the movement of vesicles within cells. In addition, such insights reveal new targets for attacking viruses before they can usurp the host-cell machinery for replication. Hide abstract
2008. Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry. Proc. Natl. Acad. Sci. U.S.A., 105 (30), pp. 10559-64. Read abstract | Read more
The concerted action of four viral glycoproteins and at least one cellular receptor is required to catalyze herpes simplex virus 1 entry into host cells either by fusion at the plasma membrane or intracellularly after internalization by endocytosis. Here, we applied cryo electron tomography to capture 3D intermediates from Herpes simplex virus 1 fusion at the plasma membrane in their native environment by using two model systems: adherent cells and synaptosomes. The fusion process was delineated as a series of structurally different steps. The incoming capsid separated from the tegument and was closely surrounded by the cortical cytoskeleton. After entry, the viral membrane curvature changed concomitantly with a reorganization of the envelope glycoprotein spikes. Individual glycoprotein complexes in transitional conformations during pore formation and dilation revealed the complex viral fusion mechanism in action. Snapshots of the fusion intermediates provide unprecedented details concerning the overall structural changes occurring during herpesvirus entry. Moreover, our data suggest that there are two functional "poles" of the asymmetric herpesvirion: one related to cell entry, and the other formed during virus assembly. Hide abstract
2007. Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells. Cell, 131 (3), pp. 516-29. Read abstract | Read more
Cell entry of Simian Virus 40 (SV40) involves caveolar/lipid raft-mediated endocytosis, vesicular transport to the endoplasmic reticulum (ER), translocation into the cytosol, and import into the nucleus. We analyzed the effects of ER-associated processes and factors on infection and on isolated viruses and found that SV40 makes use of the thiol-disulfide oxidoreductases, ERp57 and PDI, as well as the retrotranslocation proteins Derlin-1 and Sel1L. ERp57 isomerizes specific interchain disulfides connecting the major capsid protein, VP1, to a crosslinked network of neighbors, thus uncoupling about 12 of 72 VP1 pentamers. Cryo-electron tomography indicated that loss of interchain disulfides coupled with calcium depletion induces selective dissociation of the 12 vertex pentamers, a step likely to mimic uncoating of the virus in the cytosol. Thus, the virus utilizes the protein folding machinery for initial uncoating before exploiting the ER-associated degradation machinery presumably to escape from the ER lumen into the cytosol. Hide abstract