Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein–catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.
Lipid
hydroperoxides are key mediators of diseases and cell death.
In this work, the structural and dynamic perturbations induced by
the hydroperoxidized POPC lipid (POPC-OOH) in fluid POPC membranes,
at both 23 and 37 °C, were addressed using advanced small-angle
X-ray scattering (SAXS) and fluorescence methodologies. Notably, SAXS
reveals that the hydroperoxide group decreases the lipid bilayer bending
rigidity. This alteration disfavors the bilayer stacking and increases
the swelling in-between stacked bilayers. We further investigated
the changes in the apolar/polar interface of hydroperoxide-containing
membranes through time-resolved fluorescence/anisotropy experiments
of the probe TMA-DPH and time-dependent fluorescence shifts of Laurdan.
A shorter mean fluorescence lifetime for TMA-DPH was obtained in enriched
POPC-OOH membranes, revealing a higher degree of hydration near the
membrane interface. Moreover, a higher microviscosity near TMA-DPH
and lower order are predicted for these oxidized membranes, at variance
with the usual trend of variation of these two parameters. Finally,
the complex relaxation process of Laurdan in pure POPC-OOH membranes
also indicates a higher membrane hydration and viscosity in the close
vicinity of the −OOH moiety. Altogether, our combined approach
reveals that the hydroperoxide group promotes alterations in the membrane
structure organization, namely, at the level of membrane order, viscosity,
and bending rigidity.
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