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Updated: Sep 10, 2025
Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions
Published on: June 24, 2013
Time-Resolved Fluorescence Imaging and Correlative Cryo-Electron Tomography to Study Structural Changes of the HIV-1
Zaida K Rodriguez1,2, Jonathan R Andino-Moncada3, Sergey A Buth4
1The Salk Institute for Biological Sciences, La Jolla, San Diego, California 92037, United States.
Abstract:
The conical HIV-1 capsid protects the internal viral genome and facilitates the infection of target cells. Highly potent antivirals, such as the clinically approved drug Lenacapavir (LEN), block HIV-1 replication by changing the capsid structure and modulating its function. However, structural studies of the HIV-1 capsid, its disassembly, or stabilization by antivirals have been challenging. Here, we developed a correlative light and cryo-electron microscopy (CLEM) workflow to characterize HIV-1 capsid morphology, starting from a small volume of viral particles harvested from cellular supernatants. We report two critical improvements in sample preparation, namely, (1) affinity capture and retention of fluorescent HIV-1 particles on cryo-EM grids to enable mapping virus/capsid location prior to sample vitrification and (2) streamlined alignment protocols to subsequently identify and correlate regions of interest in fluorescence and cryo-EM images. These improvements enable a reproducible CLEM workflow to accurately locate capsids for cryo-electron tomography (cryo-ET) studies. Using this approach, we resolved ultrastructures of HIV-1 capsids treated with LEN and the cellular metabolite inositol hexaphosphate (IP6), revealing distinct modes of capsid lattice stabilization. Finally, using our CLEM workflow, we demonstrate the feasibility of correlating time-resolved fluorescence imaging of capsid disassembly to end point cryo-ET structures. These advances will facilitate in vitro structural studies to define the mechanisms of HIV-1 capsid stabilization and disassembly. The CLEM workflow developed here can also be extended to studying structural changes in other viruses in response to diverse stimuli.
Insights
A new correlative light and cryo-electron microscopy (CLEM) workflow enables detailed structural studies of human immunodeficiency virus type 1 (HIV-1) capsids. This method visualizes how antivirals like Lenacapavir stabilize HIV-1 capsid structures.
Area of Science:
- Structural Biology
- Virology
- Microscopy
Background:
- The human immunodeficiency virus type 1 (HIV-1) capsid is crucial for viral genome protection and cell infection.
- Antivirals, including Lenacapavir (LEN), target HIV-1 replication by altering capsid structure and function.
- Structural characterization of HIV-1 capsids, their disassembly, and antiviral-induced stabilization presents significant challenges.
Purpose of the Study:
- To develop an improved correlative light and cryo-electron microscopy (CLEM) workflow for characterizing HIV-1 capsid morphology.
- To enable precise structural analysis of HIV-1 capsids, including those treated with antivirals and cellular metabolites.
- To facilitate in vitro studies on HIV-1 capsid stabilization and disassembly mechanisms.
Main Methods:
- Development of a novel CLEM workflow integrating affinity capture of fluorescent HIV-1 particles on cryo-EM grids.
- Implementation of streamlined alignment protocols for correlating fluorescence and cryo-electron tomography (cryo-ET) images.
- Application of the CLEM workflow to analyze HIV-1 capsids treated with Lenacapavir (LEN) and inositol hexaphosphate (IP6).
Main Results:
- A reproducible CLEM workflow was established for accurate capsid localization for cryo-ET.
- Distinct modes of HIV-1 capsid lattice stabilization were resolved upon treatment with LEN and IP6.
- The workflow demonstrated feasibility in correlating time-resolved fluorescence imaging of capsid disassembly with end-point cryo-ET structures.
Conclusions:
- The developed CLEM workflow significantly advances in vitro structural studies of HIV-1 capsid stabilization and disassembly.
- This methodology provides insights into the mechanisms of antiviral action on HIV-1 capsid structure.
- The CLEM approach is adaptable for studying structural dynamics in other viruses under various conditions.
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