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Updated: Jun 17, 2025
Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes
Published on: July 28, 2016
Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy
Frauke Muecksch1,2, Severina Klaus1, Vibor Laketa1,3
1Department of Infectious Diseases, Virology, Heidelberg University Medical Faculty, Center for Infectious Diseases Research (CIID), Heidelberg, Germany.
Insights
Human immunodeficiency virus (HIV)-1 assembly relies on Gag proteins binding to the plasma membrane. This study shows Gag creates a specific membrane environment that recruits envelope (Env) glycoproteins for efficient viral assembly.
Area of Science:
- Virology
- Cell Biology
- Biochemistry
Background:
- Human immunodeficiency virus (HIV)-1 assembly initiates at the plasma membrane, driven by Gag protein interactions.
- Envelope (Env) glycoproteins are recruited to assembly sites, a process influenced by Gag's matrix (MA) domain and Env's cytoplasmic tail.
- The precise mechanisms regulating Env recruitment and incorporation into virions remain incompletely understood.
Purpose of the Study:
- To investigate the real-time dynamics of Env recruitment during HIV-1 assembly.
- To elucidate the role of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in Gag targeting and Env recruitment.
- To understand the interplay between Gag, Env, and host cell membranes during viral assembly.
Main Methods:
- Utilized a chemical dimerizer system to reversibly deplete PI(4,5)P2 and control HIV-1 assembly.
- Employed super-resolution and live-cell microscopy for real-time tracking of viral components.
- Performed single virion tracking to analyze Gag and Env accumulation kinetics.
Main Results:
- Gag and Env proteins accumulated at assembly sites with similar kinetics.
- PI(4,5)P2 depletion inhibited Gag plasma membrane targeting and Env cluster formation, confirming Gag's essential role in Env recruitment.
- Disruption of Gag lattices by PI(4,5)P2 depletion led to the loss of both Gag and Env clusters, indicating a Gag-induced membrane microenvironment.
Conclusions:
- HIV-1 assembly involves a Gag-induced and maintained membrane microenvironment crucial for attracting Env glycoproteins.
- Gag targeting to the plasma membrane, mediated by PI(4,5)P2, is essential for establishing this microenvironment and subsequent Env recruitment.
- These findings provide critical insights into the regulation of viral assembly and Env incorporation.
Abstract:
Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.
