Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy

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.