Summary Project description 2nd funding period
This project aims at a quantitative understanding of dynamic events in HIV-1 replication, specifically focusing on the currently ill-understood post-entry phase. Capsid uncoating, reverse transcription of the viral RNA, cytoskeletal transport and nuclear import of subviral complexes are temporally and functionally intertwined, and their coordination appears critical for productive infection. Interactions of host factors with the HIV-1 capsid protein (CA) affect these processes in a complex, cell type-dependent manner. Also, the mode of infection (via cell free virus or cell-cell contact) may influence the course of events. Since this convolute is difficult to unravel by ensemble measurements, we want to apply single virus tracking (SVT) for direct observation and analysis of individual viral entry events with high temporal resolution upon cell-free (Aim1) and synaptic (Aim2) transmission. The project is linked to high spatial resolution studies of HIV-1 entry and replication complexes (Kräusslich). We collaborate with Kräusslich in studies of productive post-entry complexes in fixed cells. Dynamic analyses, however, are hindered by lack of live-cell compatible labels for HIV-1 structural proteins. A main part of the first funding period was therefore devoted to establishment of minimally invasive labeling of HIV-1 envelope (Env) and CA by genetic code expansion/click chemistry (with Lemke and Kräusslich). Efficient site-specific labeling of HIV-1 Env was accomplished, permitting investigation of the architecture and motility of Env clusters on the plasma membrane. Positions in CA tolerant to modification were also identified, opening the possibility to observe the fate of incoming capsids by SVT. Building on these achievements, we will follow the fate of incoming capsids during transition to the nuclear pore in different cell types by SVT, integrating host factors implicated in capsid uncoating and intracellular transport. 4D multi-color tracking algorithms are developed in collaboration with Hamprecht/Rohr. Labeled Env protein will be used for dynamic and structural analyses of HIV-1 transmission through virological synapses (VS). To detect virions transmitted through VS, we are developing a robustly replicating, stably fluorescently labeled HIV-1 derivative, suitable for studying viral spread in complex systems. A candidate has been identified, whose molecular characterization, refinement and additional adaptation will continue in the next funding period. Replication competent virus derivatives will be used to study dynamics of HIV cell-cell transmission in collaboration with Kräusslich and Fackler. We will also participate in a concerted effort with Lemke, Johnsson and Urban/Grimm to further develop minimal invasive labeling strategies into a standard tool for virological applications. Finally, we established the basis for complementary biochemical analysis of dynamic HIV-cell interactions in cell entry by proximity labeling, which will be pursued together with Lusic/Beck.
Sandra Schifferdecker, PhD