Pathways and kinetics of foamy virus uptake and its glycoprotein-mediated fusion process

• Applicants: Professor Dr. Don C. Lamb; Professor Dr. Dirk Lindemann
• Subject Area: Virology ; Biochemistry ; Biophysics
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 264854711

Lipid membrane enveloped viruses have evolved a variety of elegant strategies to deliver their genetic information into susceptible host, on which they depend for replication. The first barriers to overcome are cellular membranes, either at the cell surface or internal organelles. Enveloped viruses use glycoproteins embedded in their viral membrane for fusion with cellular membranes, thereby providing access of their inner protein shell (capsid) containing the viral genome to the host cell cytoplasm. The overall aim of this proposal is to gain insight into the entry mechanisms of enveloped viruses and retroviruses in particular, using foamy viruses (FVs) as a model system. FVs are one of the most ancient retroviruses having co-evolved with their natural hosts and are endemic to non-human primates, cats, cattle and horses. A hallmark of FVs, which are promising candidates as gene transfer vehicles in gene therapeutic strategies, is their apparent apathogenicity in natural hosts and also in zoonotically infected humans. We have developed fluorescently tagged, infectious FV particles allowing us to study viral replication and tracing the fate of single viral particles by time-lapse microscopy techniques in the living cells. Using this new tool, we could demonstrate that FVs enter host cells predominantly by endocytic mechanisms, deliver their genetic information to the host cell nucleus in a strictly cell cycle dependent manner and require cellular mitosis for viral genome integration into host cell chromatin. Furthermore, we developed a method for single viral particle tracing of capsid and glycoprotein tagged fluorescent FVs in living cells. Using this method, we frequently observed productive fusion events resulting in release of the viral capsid into the cytoplasm. Most strikingly, we detected a previously unknown intermediated entry step (tethering state), characterized by an increased distance between viral capsid and glycoprotein signals that precedes subsequent independent intracellular movement of both structures. The focus of this proposal is to elucidate the details of initial steps of FV uptake with a special focus on the fusion process using a combination of genetic, biochemical and imaging techniques. We will develop new viral tools for quantitation and visualization of other previously characterized intermediate steps of FV fusion such as mixing of viral and cellular lipids or exchange of small molecules between virus and host cell cytoplasm prior to release of the viral capsid. Combining these new tools and the established single viral particle tracing methodologies of fluorescently tagged FV particles, we will characterize the uptake pathways of FVs including identification of the types of endocytic pathways exploited, determine the kinetics and the order of the individual uptake steps and investigate how they depend on different viral components by analyzing chimeric viruses containing components of different retrovirus species.

• DFG Programme: Research Grants