Pushing HIV out the door
Viruses are like pirates: they board a suitable cell and alter its course to suit their own purpose. More specifically, they smuggle their own genetic material into a host cell and reprogram the cell to produce new virus particles. For release of the newly synthesized viruses, HIV exploits cellular proteins involved in the loading, sorting and budding of cellular vesicles known as ESCRT proteins. During budding, HIV makes use of ESCRT to cut the last connection between the virion coat and the cell surface, allowing it to exit the cell. The enzyme VPS4A forms part of the ESCRT machinery and is known to be necessary for the disassembly of the complex after use, allowing its components to be recycled.
The results from ultrasensitive live-cell imaging experiments showed that VPS4A also acts at an earlier stage in the budding process. In the new work, the researchers labeled the enzyme by fusing it with the Green Fluorescent Protein (GFP). This allowed them to track the protein in living cells. By recording the fluorescent signals, they observed how several VPS4A molecules came together to form larger complexes. “In this case, we were able to count how many enzyme molecules assembled at the HIV budding site during its interaction with the nascent virion” says Müller.
Complexes made up of about three dodecamers of VPS4A were observed to undergo transient activation (for about a minute) at a budding site. Shortly thereafter, the virions were observed to emerge from the cell at these locations. Because virion release does not follow immediately upon activation of the enzyme, the investigators believe that at least one further intermediate step is required for budding.
Perhaps this postulated step can be pinned down in a later project. “Our current methodology allows us to monitor the assembly of individual virions, and we are working on further refinements that will allow us to follow the complete life cycle of HIV,” says Lamb. “We can already visualize some steps of the life cycle at the level of a single virus, observe interactions and determine the kinetics of different processes. Of course, this means that we can also label therapeutic agents and observe what effects they have in infected cells. This can help us to optimize the currently available drugs and even allow us to develop new ones.” (göd)
The study was carried out in the context of three Clusters of Excellence, the Center for Integrated Protein Science Munich (CIPSM), the Nanosystems Initiative Munich (NIM) and CellNetworks Heidelberg. The work was also supported by the German Research Foundation (DFG) as part of Priority Program 1175 Dynamics of Cellular Membranes and Their Exploitation by Viruses.
Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT component.
V. Baumgärtel, S. Ivanchenko, A. Dupont, M. Sergeev, P.W. Wiseman, H.-G. Kräusslich, C. Bräuchle, B. Müller, D.C. Lamb.
Nature Cell Biology; doi: 10.1038/ncb2215
Professor Don C. Lamb
Department of Chemistry and Biochemistry, LMU Munich
Phone: +49 (0) 89 / 2180 - 77564
Fax: +49 (0) 89 / 2180 - 77548
PD Dr. Barbara Müller
Department of Infectiology, Virology
Heidelberg University Hospital
Phone: +49 (0) 6221 / 561325