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
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.
Conformational dynamics of single HIV Env molecules
Single molecule imaging is inherently mechanistic as it describes the behavior of hundreds or thousands of single molecules free from assemble averaging, can identify non-accumulating intermediates and thus describes with great detail the molecular mechanism by which biomolecular machines function. We are therefore applying single molecule fluorescence resonance energy transfer (smFRET) to directly visualize conformational changes in single HIV Env molecules. These studies allow insights into immune evasion and how HIV Env is activated by receptor and coreceptor for fusion. This approach was introduced into our lab by James Munro and represents a collaborative effort together with the laboratories of Scott Blanchard, Peter Kwong and the program project group led by Irwin Chaiken.
By introducing two dyes into a single HIV Env molecule into an otherwise native virus, we can monitor the conformational changes of single HIV Env molecules in the context of a native trimer on the surface of a viral particle. A first report appeared in Science (Munro 2014). Our results demonstrate the ability of the unliganded HIV Env to sample three conformational states. The low-FRET conformation most likely corresponds to the closed ground state of the HIV trimer (Pancera 2014). The structure of the two more open conformations are less well understood. Below we present “The Human Trimer” movie by James Munro, Xiaochu Ma and Walther Mothes to illustrate how an individual FRET traces can likely be interpreted. For a full description of how our findings reveal the behavior of the HIV Env and how binding of receptor, co-receptor, antibodies and small molecules affects the conformational landscape, see Munro 2014.
Video 3 The Human Trimer