Abstract
BackgroundMost retroviruses enter their host cells by fusing the viral envelope with the plasma membrane. Although the protein machinery promoting fusion has been characterized extensively, the dynamics of the process are largely unknown.ResultsWe generated human immunodeficiency virus-1 (HIV-1) particles pseudotyped with the envelope (Env) protein of ecotropic murine leukemia virus eMLV to study retrovirus entry at the plasma membrane using live-cell microscopy. This Env protein mediates highly efficient pH independent fusion at the cell surface and can be functionally tagged with a fluorescent protein. To detect fusion events, double labeled particles carrying one fluorophor in Env and the other in the matrix (MA) domain of Gag were generated and characterized. Fusion events were defined as loss of Env signal after virus-cell contact. Single particle tracking of >20,000 individual traces in two color channels recorded 28 events of color separation, where particles lost the Env protein, with the MA layer remaining stable at least for a short period. Fourty-five events were detected where both colors were lost simultaneously. Importantly, the first type of event was never observed when particles were pseudotyped with a non-fusogenic Env.ConclusionThese results reveal rapid retroviral fusion at the plasma membrane and permit studies of the immediate post-fusion events.
Highlights
Most retroviruses enter their host cells by fusing the viral envelope with the plasma membrane
We have developed a system to study the dynamics of human immunodeficiency virus-1 (HIV-1) entry based on fluorescent live cell microscopy, in which the MA domain of the main structural protein Gag is labeled by fusion to a fluorescent protein [10]
Characterization of double labeled HIV-1 pseudotypes To monitor the fusion of retroviral particles at the plasma membrane of living cells, we established a double labeling strategy in which a fluorescent label in the MA domain of HIV-1 Gag (MA.mCherry) was combined with another fluorescent label fused to eMLV Env (Env.YFP), which was used to pseudotype HIV-1 particles
Summary
Most retroviruses enter their host cells by fusing the viral envelope with the plasma membrane. Enveloped viruses enter host cells by membrane fusion at the plasma membrane or at intracellular membranes. This process is mediated by the interaction of cellular receptors and Env glycoproteins. Previous investigations have employed bulk biochemical assays or cell-cell fusion to study the viral fusion process (for review see [3]). Single particle tracking of fluorescently labeled viruses has become possible and has been successfully applied to characterize the entry of various viruses (for review see [4]). The lipophilic dye DiD was used for labeling the membrane of enveloped virus particles [5,6,7]. As DiD is incorporated into the outer leaflet of the membrane its redistribution after virus-cell contact indicates primarily the lipid mixing of the contacting leaflets (termed hemifusion) and not the formation of the fusion pore [7]
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