Seeing is believing – visualizing individual steps of the retroviral lifecycle
Our laboratory is interested in the cell biology and pathogenesis of retroviral infections. Acknowledging the persuasive power of directly visualizing events as they happen in real time, we apply various imaging techniques to provide insights into the dynamics of pathogenic processes. In the past years, our laboratory contributed to the understanding of the spreading of retroviruses in tissue culture cells. More recently, we have expanded our efforts into both, macroscopic and nanoscopic dimensions. Using two-photon microscopy we visualize the spread of retroviruses at the macroscopic level of tissues directly within a living animal. In addition, we apply single molecule imaging technologies to understand the molecular mechanism by which the HIV envelope glycoprotein (Env) mediates entry into cells. Some of the first advances that we have made along these new directions in our laboratory are presented below.
- In vivo imaging of virological synapses
- Conformational dynamics of single HIV Env molecules
In vivo imaging of virological synapses
The concept of virological synapses describes the ability of virus-infected cells to establish contact with uninfected cells and transfer virus to neighboring cells. This process is dependent on the expression of the viral Env protein in the infected cell that functions as an adhesion protein to establish cell-cell contact with receptor-expressing cells (Sherer 07). Once the contact is established, virus assembly is redirected towards the site of cell-cell contact and viral particles are transferred to neighboring cells (Jin 09). Importantly, this concept was developed entirely based on in vitro evidence and if this was true in vivo remained unknown. To solve this question, Xaver Sewald performed a visual screen to identify primary mouse leukocytes that when infected with the Friend murine leukemia virus (MLV) would form long-lived virological synapses in a living animal. Surprisingly, Xaver Sewald discovered that MLV-infected B cells establish long-lived virological synapses in vivo (Sewald 12). Intravital imaging of primary mouse B cells infected with MLV expressing a fluorescent capsid protein (Gag-GFP, green) were found to be more immobile than the uninfected B cells (red) (video 1).
When we zoomed into these immobile infected B cells, we often observed that viral capsids visualized with Gag-GFP would polarize to one side of the cell where it must be in contact with an unlabeled mouse cell within in the tissue (video 2).
The polarization of Gag-GFP to one side of the cell was entirely dependent on the expression of the viral Env protein. Thus, these in vivo structures exhibited the very same Env-dependent polarization of viral capsid previously described for in vitro virological synapses and thus represent the first documentation of virological synapses in vivo (Sewald 12). Infected B cells form virological synapses with uninfected CD4+ T cells as well as uninfected B cells and are required for the early spread in mice. We are currently using these imaging methods in combination with the utilization of knock-out mice to understand the early steps that lead to the establishment of a retroviral infection in mice. We are also expanding these technologies to study the dissemination of HIV in humanized mice.