The unprecedented public health and economic impact of the COVID-19 pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been met with an equally unprecedented scientific response. Much of this response has focused, appropriately, on the mechanisms of SARS-CoV-2 entry into host cells, and in particular the binding of the spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. This Review provides the structural and cellular foundations for understanding the multistep SARS-CoV-2 entry process, including S protein synthesis, S protein structure, conformational transitions necessary for association of the S protein with ACE2, engagement of the receptor-binding domain of the S protein with ACE2, proteolytic activation of the S protein, endocytosis and membrane fusion. We define the roles of furin-like proteases, transmembrane protease, serine 2 (TMPRSS2) and cathepsin L in these processes, and delineate the features of ACE2 orthologues in reservoir animal species and S protein adaptations that facilitate efficient human transmission. We also examine the utility of vaccines, antibodies and other potential therapeutics targeting SARS-CoV-2 entry mechanisms. Finally, we present key outstanding questions associated with this critical process.

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Mechanisms of SARS-CoV-2 entry into cells.

The SARS-CoV-2 infection is likely from a zoonotic source. Transmissibility between people happens principally
 through beads and surface contacts. People with the infection may shed it while suggestive or asymptomatic. Despite
 the fact that brooding period is as long as 12 days, middle term of viral shedding can be as long as 20 days. Clinical
 manifestations change generally and might be mellow (80%), moderate (15%), or serious (5%).

https://www.longdom.org/open-access/coronavirus-and-covid19.pdf

SARS-CoV-2 Infection

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2014.11.036

Roles and regulation of phospholipid scramblases

Cell entry by SARS-CoV-2.

Transcription and replication of the influenza A virus (IAV) genome occur in the nucleus of infected cells and are carried out by the viral ribonucleoprotein complex (vRNP). As a major component of the vRNP complex, the viral nucleoprotein (NP) mediates the nuclear import of the vRNP complex via its nuclear localization signals (NLSs). Clearly, an effective way for the host to antagonize IAV infection would be by targeting vRNP nuclear import. Here, we identified phospholipid scramblase 1 (PLSCR1) as a binding partner of NP by using a yeast two-hybrid (Y2H) screen. The interaction between NP and PLSCR1 in mammalian cells was demonstrated by using co-immunoprecipitation and pull-down assays. We found that the stable overexpression of PLSCR1 suppressed the nuclear import of NP, hindered the virus life cycle, and significantly inhibited the replication of various influenza subtypes. In contrast, siRNA knockdown or CRISPR/Cas9 knockout of PLSCR1 increased virus propagation. Further analysis indicated that the inhibitory effect of PLSCR1 on the nuclear import of NP was not caused by affecting the phosphorylation status of NP or by stimulating the interferon (IFN) pathways. Instead, PLSCR1 was found to form a trimeric complex with NP and members of the importin α family, which inhibited the incorporation of importin β, a key mediator of the classical nuclear import pathway, into the complex, thus impairing the nuclear import of NP and suppressing virus replication. Our results demonstrate that PLSCR1 negatively regulates virus replication by interacting with NP in the cytoplasm and preventing its nuclear import.

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Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication.

Human phospholipid scramblase 4 (hPLSCR4), an isoform of the scramblase family, is a type II single-pass transmembrane protein whose function remains unknown. To understand its role, recombinant hPLSCR4 was obtained by cloning the ORF into a pET28 a(+) vector and overexpressed in Escherichia coli. Functional assay showed that Ca2+, Mg2+, and Zn2+ activate hPLSCR4 and mediate scrambling activity independent of the phospholipid head group. Far-UV-CD and fluorescence spectroscopy revealed that Ca2+ and Mg2+ binding induces conformation change in hPLSCR4, exposing hydrophobic patches of the protein, and Ca2+ has more affinity than Mg2+ and Zn2+. Stains-all studies further confirm that hPLSCR4 is a Ca2+-binding protein. Point mutation (Asp290→Ala) in hPLSCR4 decreased the Ca2+-binding affinity as well as Tb3+ luminescence, suggesting residues of the predicted Ca2+-binding motif are involved in Ca2+ binding. Functional reconstitution with (Asp290→Ala) mutant led to ~50% and ~40% decrease in scramblase activity in the presence of Ca2+ and Mg2+, respectively.

Biochemical and functional characterization of human phospholipid scramblase 4 (hPLSCR4).

Snail interacts with hPLSCR1 promoter and down regulates its expression in IMR-32

http://www.jbc.org/article/S0021925822007621/pdf

The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamics

Human phospholipid scramblase 1 (hPLSCR1) is a Ca2+-dependent protein known to scramble phospholipids in the plasma membrane resulting in loss of membrane asymmetry It has been reported that hPLSCR1 exhibits Ca2+- independent activity at low pH However, the conformational changes induced at low pH leading to functional activation are not known Our results showed that recombinant hPLSCR1 was functionally activated at low pH, which is similar to the behavior of natively extracted hPLSCR1 Tryptophan fluorescence measurements showed a decrease in Ca2+-binding affinity at low pH, although not at pH 55 Far and near UV-CD revealed that low pH induced structural changes, with a significant increase in the β-sheet content of the protein At the physiological level, decreased hPLSCR1 expression was observed after a period of exposure to low pH The effect occurred at the promoter level The expression levels of hPLSCR1 directly correlated with the sensitivity of HEK293 to apoptosis Based on these results, we conclude that the mechanisms of Ca2+- and pH-induced functional activation of hPLSCR1 are different and that hPLSCR1 expression regulated by low pH could provide insights into the role of hPLSCR1 in cancer progression

Biochemical evidence for Ca2+-independent functional activation of hPLSCR1 at low pH.

During the maturation process spermatozoa undergo a series of changes in their lateral and horizontal lipid profiles. However, lipid metabolism in spermatozoa is not clearly understood for two reasons: i) the mature spermatozoa are devoid of endoplasmic reticulum, which is the major site of phospholipid (PL) synthesis in somatic cells, and ii) studies have been superficial due to the difficulty in culturing spermatozoa. We hypothesize that spermatozoa contain biogenic membrane flippases since immense changes in lipids occur during spermatogenic differentiation. To test this, we isolated spermatozoa from bovine epididymides and reconstituted the detergent extract of sperm membranes into proteoliposomes. In vitro assays showed that proteoliposomes reconstituted with sperm membrane proteins exhibit ATP-independent flip-flop movement of phosphatidylcholine (PC), phosphatidylserine, and phosphatidylglycerol. Half-life time of PC flipping was found to be ∼3.2±1 min for whole sperm membrane, which otherwise would have taken ∼11-12 h in the absence of protein. Further biochemical studies confirm the flip-flop movement to be protein-mediated, based on its sensitivity to protease and protein-modifying reagents. To further determine the cellular localization of flippases, we isolated mitochondria of spermatozoa and checked for ATP-independent flippase activity. Interestingly, mitochondrial membranes showed flip-flop movement but were specific for PC with half-life time of ∼5±2 min. Our results also suggest that spermatozoa have different populations of flippases and that their localization within the cellular compartments depends on the type of PL synthesis.

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Vincent Gerard Francis

Papers

Biochemical and functional characterization of human phospholipid scramblase 4 (hPLSCR4).

Snail interacts with hPLSCR1 promoter and down regulates its expression in IMR-32

The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamics

Biochemical evidence for Ca2+-independent functional activation of hPLSCR1 at low pH.

Biochemical evidence for energy-independent flippase activity in bovine epididymal sperm membranes: an insight into membrane biogenesis