Negative Staining Transmission Electron Microscopy of HIV Viral Particles Permeabilized with PFO and Capsid Stabilized with IP6

Derrick Lau; Chantal L Márquez; Michael W Parker; And Till Böcking

doi:10.21769/BioProtoc.4536

Negative Staining Transmission Electron Microscopy of HIV Viral Particles Permeabilized with PFO and Capsid Stabilized with IP6

Bio Protoc. 2022 Oct 20;12(20):e4536. doi: 10.21769/BioProtoc.4536.

Authors

Derrick Lau¹, Chantal L Márquez^{1

2}, Michael W Parker³, And Till Böcking¹

Affiliations

¹ EMBL Australia Node in Single Molecule Science and ARC Centre of Excellence in Advanced Molecular Imaging, School of Medical Sciences, University of New South Wales, Sydney, Australia.
² Laboratory of Molecular and Cellular Virology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile.
³ St. Vincent's Institute of Medical Research, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Australia.

Abstract

The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM. Graphical abstract.

Keywords: Capsid; Electron microscopy; HIV; Inositol-6-phosphate; Negative staining; Pore-forming proteins; Transmission electron microscopy; Ultrastructure; Virus.