Direct Visualization of HIV-1 with Correlative Live-Cell Microscopy and Cryo-Electron Tomography

Abstract

SUMMARY Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state, and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, direct observation of structural details of infecting HIV-1 has not been realized due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by developing a correlative high-speed 3D live-cell imaging and cryoET method. Using this methodology, we showed, for the first time under near-native conditions, that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host-cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantage of our correlative live-cell and cryoET approach to image dynamic processes, such as viral infection.

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

“…Here it would be advantageous to identify and locate regions of interest by cryofluorescence microscopy and use their coordinates for guiding the micromachining process. This strategy is used with great success in correlative cryofluorescence light microscopy and electron tomography (20,21). However, current implementations lack resolution to precisely target subcellular features in 3D and thus, advanced optical techniques; e.g., confocal microscopy, need to be integrated into the workflow, either in the form of integrated solutions (e.g., optical microscopy integrated into the EM), or in easier to implement modular approaches.…”

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

“…Cryo-CLEM experiments on mammalian cells have been previously performed using organic fluorescent dyes or proteins tagged with fluorophores (Kaufmann et al, 2014, Liu et al, 2015, Schorb and Briggs, 2014, Schorb et al, 2016, Bykov et al, 2016, Schellenberger et al, 2014, Jun et al, 2011). Though organic dyes are very bright, it can be challenging to use these compounds to efficiently label specific proteins inside cells.…”

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

“…Cryo-CLEM methods have been used to identify viruses in virus-infected cells to understand mechanisms associated with HIV-1 entry and herpes-simplex virus type 1 (HSV-1) transport in neurons, and to develop improved correlation workflows 24–26 . In addition, (cryo-)CLEM procedures were used to determine the arrangement of HIV-1 particles anchored to cell plasma membranes via tetherin, a host cellular restriction factor that inhibits enveloped virus release 14 .…”

“…Cryo-CLEM of prokaryotes has also been described 22,27–29 and sections of this protocol that describe imaging and analysis parameters are also relevant to studies of these systems. With further development of cryo-CLEM technologies 13,24,26 and macromolecular-labeling strategies 30–32 , we anticipate that CLEM and cryo-CLEM methods will be routinely used for investigations of virus replication and broader topics in structural cell biology.…”