SARS-CoV-2 requires acidic pH to infect cells Kreutzberger, Alex J. B.; Sanyal, Anwesha; Saminathan, Anand ...
Proceedings of the National Academy of Sciences - PNAS,
09/2022, Letnik:
119, Številka:
38
Journal Article
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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.
The role of membrane potential as a source of energy and as a signaling cue remains unexplored in most intracellular organelles. Here, we introduce DNA based voltage sensors, (i) Voltair, a ...fluorescent nanodevice that quantitatively reports the absolute membrane potential and (ii) mVivo, a photoactivable nanodevice that reports changes in membrane potential, across intracellular membrane. DNA based voltage probes consists of a voltage-sensitive fluorophore, a reference fluorophore as ratiometric and fiducial marker, and a targeting moiety that localizes the probe to specific organellar membranes. Using Voltair, we could quantify the resting membrane potential of different organelles in situ. DNA based voltage probes can potentially guide the rational design of biocompatible electronics and further enhance our understanding of how intracellular organelles use membrane potential as a battery source and as a transmission signal.
Lysosomal calcium (Ca
) release is critical to cell signaling and is mediated by well-known lysosomal Ca
channels. Yet, how lysosomes refill their Ca
remains hitherto undescribed. Here, from an RNA ...interference screen in
, we identify an evolutionarily conserved gene,
, that facilitates lysosomal Ca
entry in
and mammalian cells. We found that its human homolog TMEM165, previously designated as a Ca
/H
exchanger, imports Ca
pH dependently into lysosomes. Using two-ion mapping and electrophysiology, we show that TMEM165, hereafter referred to as human LCI, acts as a proton-activated, lysosomal Ca
importer. Defects in lysosomal Ca
channels cause several neurodegenerative diseases, and knowledge of lysosomal Ca
importers may provide previously unidentified avenues to explore the physiology of Ca
channels.
SARS-CoV-2 Omicron subvariants have generated a worldwide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of vaccination-induced antibodies. To manage ...Omicron subvariants and prepare for new ones, additional means of isolating broad and potent humanized SARS-CoV-2 neutralizing antibodies are desirable. Here, we describe a mouse model in which the primary B cell receptor (BCR) repertoire is generated solely through V(D)J recombination of a human V
1-2 heavy chain (HC) and, substantially, a human Vκ1-33 light chain (LC). Thus, primary humanized BCR repertoire diversity in these mice derives from immensely diverse HC and LC antigen-contact CDR3 sequences generated by nontemplated junctional modifications during V(D)J recombination. Immunizing this mouse model with SARS-CoV-2 (Wuhan-Hu-1) spike protein immunogens elicited several V
1-2/Vκ1-33-based neutralizing antibodies that bound RBD in a different mode from each other and from those of many prior patient-derived V
1-2-based neutralizing antibodies. Of these, SP1-77 potently and broadly neutralized all SARS-CoV-2 variants through BA.5. Cryo-EM studies revealed that SP1-77 bound RBD away from the receptor-binding motif via a CDR3-dominated recognition mode. Lattice light-sheet microscopy-based studies showed that SP1-77 did not block ACE2-mediated viral attachment or endocytosis but rather blocked viral-host membrane fusion. The broad and potent SP1-77 neutralization activity and nontraditional mechanism of action suggest that it might have therapeutic potential. Likewise, the SP1-77 binding epitope may inform vaccine strategies. Last, the type of humanized mouse models that we have described may contribute to identifying therapeutic antibodies against future SARS-CoV-2 variants and other pathogens.
Trojanisches Pferd: Ein DNA‐Ikosaeder (schwarz, siehe Schema), das durch Aptamere (rot) zusammengehalten wird, kann molekulare Frachten wie fluoreszierendes Dextran (grün) einschließen. In Gegenwart ...eines molekularen Auslösers (graue Sechsecke) weichen die Aptamere zurück, und das Ikosaeder öffnet sich unter Freisetzung seines Inhalts.
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