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  • Analysis of individual HIV-1 budding event using fast AFM reveals a multiplexed role for VPS4.

Analysis of individual HIV-1 budding event using fast AFM reveals a multiplexed role for VPS4.

Biophysical journal (2022-09-01)
Shimon Harel, Yarin Altaras, Dikla Nachmias, Noa Rotem-Dai, Inbar Dvilansky, Natalie Elia, Itay Rousso
ABSTRACT

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Although the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (∼100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wild-type (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 38% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.

MATERIALS
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Product Description

Sigma-Aldrich
Anti-VPS4 antibody produced in rabbit, ~1.0 mg/mL, affinity isolated antibody, buffered aqueous solution