key: cord-0875048-ri1g9z8x
authors: Barrantes, Francisco J.
title: The constellation of cholesterol-dependent processes associated with SARS-CoV-2 infection
date: 2022-05-02
journal: Prog Lipid Res
DOI: 10.1016/j.plipres.2022.101166
sha: 379be26de6f74039a79df25d0420eb226bc23e36
doc_id: 875048
cord_uid: ri1g9z8x

The role of cholesterol in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronavirus-host cell interactions is currently being discussed in the context of two main scenarios: i) the presence of the neutral lipid in cholesterol-rich lipid domains involved in different steps of the viral infection and ii) the alteration of metabolic pathways by the virus over the course of infection. Cholesterol-enriched lipid domains have been reported to occur in the lipid envelope membrane of the virus, in the host-cell plasma membrane, as well as in endosomal and other intracellular membrane cellular compartments. These membrane subdomains, whose chemical and physical properties distinguish them from the bulk lipid bilayer, have been purported to participate in diverse phenomena, from virus-host cell fusion to intracellular trafficking and exit of the virions from the infected cell. SARS-CoV-2 recruits many key proteins that participate under physiological conditions in cholesterol and lipid metabolism in general. This review analyses the status of cholesterol and lipidome proteins in SARS-CoV-2 infection and the new horizons they open for therapeutic intervention.

where it represents ~50% of the total membrane lipids [20, 21] . The non-homogeneous distribution in the plane of the membrane -observed in both native and synthetic membranes [22] -and its asymmetry along the axis perpendicular to the membrane normal, i.e., the transbilayer cholesterol asymmetry, are two additional manifestations of cholesterol´s remarkable distribution, which carry important functional implications. The former gave rise to the concept of "lipid rafts" [23] [24] [25] . A consensus definition was adopted at the 2006 Keystone Symposium [26] : rafts are lipid domains characterized by their relative enrichment in cholesterol intermixed with sphingolipids and glycerophospholipids with saturated acyl chains (relative to the rest of the bilayer lipid), and display the physico-chemical characteristics of liquid-ordered (Lo) gels [27] . It was subsequently found that raft lipid domains are very dynamic structures of nanometric lateral dimensions (10-200 nm) [28, 29] . The trans-bilayer asymmetry of cholesterol is less understood;

in model membranes made up cholesterol and phosphatidylcholine with fatty acids of different acyl chain length/degrees of saturation, cholesterol reorients rapidly in the plane perpendicular to the membrane bilayer, adopting a tilted orientation in regions with shorter acyl chains, as assessed by neutron scattering and solid-state 2H NMR, implying that the hydrophobic thickness is the primary driving force of cholesterol topography across the membrane [30, 31] . Finally, the ultimate asymmetry of cholesterol is inherent to its molecule proper: it possesses a smooth α face, with which it interacts with other lipids, e.g., with sphingomyelins, and a rough β face, suited to match the cavities, grooves and irregular contours of many transmembrane protein surfaces and of other irregularly shaped lipids like the gangliosides [32] .

The unique features of cholesterol distribution are also manifested at the organism level: the brain constitutes roughly 20% of the body´s weight, yet it is the organ with the highest J o u r n a l P r e -p r o o f concentration of the neutral lipid (approximately 23% of the body's cholesterol content), mainly due to the high membrane/volume ratio of neurons and the high proportion of myelin [33] . The source of brain´s cholesterol is not the neuronal cell, but the oligodendrocyte, which synthesizes the sterol to build up the myelin sheath of axons and the biomembranes of neuronal and glial cells alike. A particularly important function of glia-derived cholesterol complexed to apolipoprotein E (ApoE)-containing lipoproteins is brain synaptogenesis [34] [35] [36] [37] .

In mixed artificial membrane-mimetic systems, the presence of cholesterol generates the separation and coexistence of liquid-liquid phases, one the liquid-ordered phase (Lo), rich in the sterol and saturated lipids, and the other a liquid-disordered (Ld) phase, enriched in unsaturated acyl chain lipids. These physicochemical characteristics are at the root of the concept of "lipid rafts" [23] [24] [25] . According to this concept, lateral membrane heterogeneities like those observed in model systems also occur in biological membranes at sites enriched in cholesterol intermixed with sphingolipids and glycerophospholipids with saturated acyl chains. These specialized lipid domains would exhibit the physico-chemical properties of liquid-ordered (Lo) gels [27] . The functional relevance of these lipid domains is that they serve as signal transduction platforms for a wide palette of proteins involved in various cellular phenomena, and their participation at various stages of the viral infection process at the plasma membrane and at later stages of the virus cycle inside the cell are beginning to be delineated. The lipid raft concept has had an important influence on the field of viral infection, to the point that virus formation has been considered "unlikely to occur from a single raft (quoting the 10-50 nm raft size of ref. [38] ) and raft-dependent J o u r n a l P r e -p r o o f viruses have to organize their membrane by recruitment or coalescence of pre-existing small rafts into larger microdomains or by de novo assembly of a microdomain", with suggestions that this may occur at the plasma membrane of the host cell [39] .

The lipidomic profile of enveloped virus is acquired at late stages of the virus cycle and not by physical coalescence of membrane patches during the virus-host cell fusion process.

The Pestivirus bovine viral diarrhoea virus is among the few viruses whose lipidome has been characterized; its envelope is rich in sphingomyelin, cholesterol and hexosylceramide and although the virus buds from the ER, its lipid composition differs from that of a typical ER membrane, indicating that there is lipid sorting during virion formation [40] , further evidence against the coalescence hypothesis.

Another well characterized lipidomic profile is that of the human immunodeficiency virus type 1 (HIV-1), a retrovirus. Brügger and coworkers [41] resorted to mass spectrometry to quantitatively analyze the lipid composition of the viruses budding from the infected cells and of the host cell membranes. They found that inhibition of sphingolipid biosynthesis of the host cell resulted in diminished viral infectivity concomitant with enrichment in an unusual sphingolipid, dihydrosphingomyelin, thus disclosing a critical role of this lipid class in the HIV-1 morphogenesis and infectivity. Furthermore, the lipid composition of indirectly pointing to the influence of host-cell cholesterol on viral infection is that of Campbell and coworkers, who found that dietary-induced hypercholesterolemia made mice highly susceptible to coxsackievirus B infection [50] . Fusion-resistant L-2 fibroblast mutants regained the ability to fuse with the CoV murine hepatitis virus (MHV) upon supplementation with cholesterol [51] . Fusogenic activity was also related to the fatty acid metabolism of these cells.

These authors did not find MHV to be sensitive to hypercholesterolemia as reported by Campbell [52, 53] at the surface of e.g., epithelial cells in the mucosae that line the upper respiratory tract. Cholesterol co-clusters with sialic acidcontaining glycolipids in the host plasma membrane, augmenting IAV binding affinity; binding exhibits a cooperative dependence on the concentration of receptors. Using single-molecule superresolution microscopy, these authors also show that this entails the formation of nanoclusters of glycosphingolipid receptors [52] . However, as discussed in the section above on lipid domains in the virus envelope, other authors find that it is the cholesterol content in the membrane bilayer of the IAV proper that matters, and not the sterol in the host cell membrane [48] .

In the case of the CoVs, the role of cholesterol in plasma membranes has been centred around two related areas: i) the role of cholesterol-rich lipid domains at the plasmalemma as a platform for virus entry and ii) the concentration of the cell-surface metalloprotease, the angiotensin converting enzyme 2 (ACE2), considered to be the canonical receptor for SARS-CoV-2 and SARS-CoV viruses, at these cholesterol-enriched lipid domains. The two issues have been, and J o u r n a l P r e -p r o o f Journal Pre-proof remain, matters of controversy. Binding of SARS-CoV-2 to ACE2 is mediated by the receptorbinding domain (RBD) harboured in the S1 subunit of the S spike protein, close to the N-term region [54] . The RBD-ACE2 recognition process is seconded by auxiliary co-receptors like the transmembrane (TM) protease serine 2 precursor (TMPRSS2), furin, or cathepsin B or L, depending on the target host-cell type [55] . Antibodies directed against ACE2 provide only partial suppression of the binding process, whereas some monoclonal antibodies generated in convalescent COVID-19 patients against the N-term domain of the S1 protein neutralize this process but do not bind the RBD [56, 57] , raising the possibility that the virus could engage other host-cell receptors or co-receptors for cell entry and/or involve alternative mechanistic steps. For instance, acidic glycolipids or glycosphingolipids [52] co-clustered with ACE2 could act as coreceptors, thus explaining the partial suppression of binding by anti-ACE2 antibodies.

Recent data using genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-based studies have found that several cell lines that are susceptible to SARS-CoV-2 infection have very low ACE2 expression levels, suggesting the possibility of alternative ACE2independent entry routes [58] . Experimental evidence of an ACE2-independent pathway has recently been reported to operate in H522 human lung cells, which can be infected by the SARS-CoV-2 variant E484 (a spike S protein mutant) even in the absence of ACE2 expression [59] .

Infection requires heparan sulphates at the cell surface and involves clathrin-mediated endocytosis. Single-cell transcriptomics has shown that SARS-CoV-2 viral RNA is found in various types of immune cells, including myeloid cells with phagocytic activity (neutrophils and macrophages) and lymphocytes without such phagocytic activity (T, B, and NK cells).

Remarkably, SARS-CoV-2 RNA-positive immune cells do not co-express the canonical ACE2

and TMPRSS2 receptor/co-receptor, or other hypothetical entry co-factors [60] . T lymphocytes J o u r n a l P r e -p r o o f from COVID-19 patients have been found to contain SARS-CoV-2 RNA and viral antigen, and SARS-CoV-2 induces marked lymphopenia, suggesting that these cells are targets of the coronavirus. Using ACE2 knockdown or receptor blocking experiments, Shen and coworkers recently found that SARS-CoV-2 infection of T lymphocytes is spike-ACE2/TMPRSS2independent [61] . Another ACE2-independent mechanism of SARS-CoV-2 internalisation has recently been reported by Liu and coworkers, who showed that upon initial contact with ACE2 in cells of the respiratory tract, cell invasion by SARS-CoV-2 proceed via the ubiquitous cellsurface integrin α5β2 [62] .

The main argument in support of the participation of cholesterol-rich lipid domains in the initial binding/recognition step is based on the hypothesis that ACE2 or similar cell-surface receptors reside in raft lipid domains that purportedly serve as platforms for viral recognition and entry.

Evidence in support of this notion is available for several viruses. One of the human commoncold etiological agents, the coronavirus HCoV-229E, sediments in Triton X-100-resistant membrane fractions isolated from infected human fibroblasts, where the virus is recognized by its receptor, alanine aminopeptidase N, also termed CD13 [9] . Similar experimental approaches were used to arrive to analogous conclusions in the case of SARS-CoV [13] . Another experimental tool frequently used in attempting to demonstrate the lipid domain-dependence of viral infection is acute CDx-mediated cholesterol depletion from the plasma membrane. This procedure reduces porcine rotavirus infection by 99% [63] and infection by canine CoV by up to 68% [16] . Likewise, acute CDx-mediated cholesterol reduction decreases by ca. 50% the binding of the SARS-CoV S protein to ACE2-expressing hamster kidney cells and the entry of the virus into these cells [64] . A reduction of ~90% in the infectivity of Vero E6 cells by pseudo-typed SARS-CoV has also been reported upon CDx acute treatment [13] . However, several authors J o u r n a l P r e -p r o o f have specifically investigated whether ACE2 is localized in cholesterol-rich lipid domains and found no support for this claim using CHO cells transiently expressing ACE2 [65] or Vero E6 cells endogenously expressing ACE2 [12] . Interestingly, in the study of Li and coworkers, cholesterol depletion inhibited the release of SARS-CoV-2 particles from the infected cells, and cholesterol supplementation restored virion production, suggesting that cholesterol was implicated in stages of the virus cycle other than initial binding to ACE2 or subsequent viral entry.

Invasion of the brain parenchyma by SARS-CoV-2 virions via the general circulation involves the crossing of the virions through the blood-brain barrier, i.e., traversing the plasma membranes of endothelial cells, pericytes and smooth muscle cells [5] . A recent work employed fluorescently labeled spike protein to follow in vitro the possible pathways employed by the virus to engage with these cell types. Uptake was mediated by ACE2, requiring interaction with ganglioside GM1 in lipid rafts [66] , as suggested by the inhibition of S protein uptake by anti-ACE2 or anti-GM1 antibodies.

Cholesterol is ubiquitously present in cellular membranes, and although most of the neutral lipid occurs at the plasma membrane, the intracellular membrane pool also affects other steps of the viral life cycle, as exemplified by HIV. One of the natural target cells of HIV is the macrophage.

Using acute cyclodextrin-mediated depletion, statin-mediated chronic metabolic inhibition [69, 70] . Working on the nicotinic acetylcholine receptor protein we identified the inverse or "mirror" image of CRAC, which we coined CARC ( [71] . In subsequent work we found that these two cholesterol consensus motifs are present in a great variety of membrane proteins [72] , including the superfamily of ligand-gated ion-channel (LGIC) proteins [73] and other channels like the transient receptor potential (TRP) channel [74] . Other groups searched for cholesterol motifs in cell-surface receptors, including the large superfamily of G/protein coupled receptor (GPCR) proteins [75] [76] [77] . CRAC/CARC sequences share a central aromatic residue, like tyrosine for CRAC, and tyrosine, phenylalanine, J o u r n a l P r e -p r o o f or tryptophan for CARC, flanked on both sides by one to five amino acid residues ending in a basic (arginine or lysine) and an apolar terminus (valine or leucine). Both CARC and CRAC are vectorial motifs, with the CARC motif preferentially, albeit not exclusively, located in the exofacial membrane leaflet, whereas CRAC most often occurs in the cytoplasmic-facing leaflet of the plasma membrane [72] , a condition met by single-passage membrane proteins with exofacial N-term and cytoplasmic-facing C-term.

The presence of cholesterol-recognition motifs has recently been reported in the primary sequence of SARS-CoV-2 spike S homotrimeric glycoprotein [78] that interacts with the host cell-surface canonical receptor ACE2 via its receptor binding domain (RBD) [79] . The ACE2

metalloprotease is bound to the host-cell membrane and protrudes into the extracellular space, and the RBD-ACE2 binding reaction occurs in this compartment, outside the bilayer. In contrast, the S2 subunit crosses the viral envelope lipid bilayer, laying its three-footed base inside the host cell. The S2 transmembrane domain consists of three portions: a juxtamembrane aromatic section, a central hydrophobic section, and a cysteine-rich section [80] . The transmembrane domain is highly conserved in the three highly pathogenic members of the heptad human CoVs, SARS-CoV, MERS-CoV, and SARS-CoV-2 [55] , all sharing the same cholesterol-recognition motif topography, with CARC deeper in the aromatic amino acid-rich part of the transmembrane domain and CRAC laying immediately adjacent to it, partly embedded in the juxtamembrane portion, in the so-called tail-to-tail disposition [81] .

As its name indicates, another portion of the S2 subunit, the so-called fusion peptide, is involved In the case of enveloped viruses, release of their genome into the host-cell requires the fusion of their membrane bilayer to either the plasma membrane or the endocytic membrane of the target cell. Despite the high resolution information made available in recent months on the structure of the SARS-CoV-2 spike glycoprotein (see review in [79] ), we still lack atomic resolution data on the section of the viral machinery involved in the fusion process, the so-called fusion peptide, a polypeptide region contained in its S2 subunit [82] . Molecular dynamics simulations have attempted to fill this gap [82] [83] [84] [85] [86] [87] [88] [89] .

One of the clinical features and indication of severity of COVID-19 is the so-called hyperreactive cytokine release syndrome or cytokine storm (see reviews in [5, 90, 91] ). The pathognomonic cytokine and interferon profile elicited by SARS-CoV-2 virosis involves the recruitment of interferon-stimulated genes [92] . One such gene, CH25H, codes for cholesterol 25-hydroxylase, the enzyme that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol [93] , a metabolite that represses cholesterol biosynthesis, is an intermediate in the pathway to the GPRC GPR183/EB12 (a chemotactic receptor for lymphoid cells), and also participates in another cholesterol metabolic pathway, acting as a potent suppressor of the sterol regulatory element-binding protein (SREBP) pathway [94] (see Figure 1 in section below). The oxysterol 25-hydroxycholesterol has been found to exert a broad anti-viral [92] and anti-CoV action [95] .

The latter authors disclosed a mechanism involved in SARS-CoV-2 inhibition of fusion at the plasma membrane to the ER had been previously found to be dissociable from the ACAT inhibition by 25-hydroxycholesterol [94] . Experimentally, 25-hydroxycholesterol was found to inhibit SARS-CoV-2 pseudovirus infection of lung and colorectal epithelial cell lines and human lung organoids [95] . 7α-hydroxycholesterol, structurally similar to 25-hydroxycholesterol, did not exert anti-SARS-CoV-2 activity via such mechanism [95] .

The work of Wang and coworkers also has wider implications on the entry mechanism of SARS- [82] proposed that rather than simply More recently, Sanders et al. [45] showed that treatment of SARS-CoV-2 with methyl-βcyclodextrin prior to exposure of cells to SARS-CoV-2 blocked virus infection, pointing out that the cholesterol content of SARS-CoV-2 viral particles is critical to infectivity. Lai and Freed [84] described that although SARS-CoV-2 fusion peptide and SARS-CoV fusion peptide have ~93% identity, the former induces greater membrane ordering than SARS-CoV fusion peptide, possibly due to its greater hydrophobicity. These authors concluded that this effect could explain, at least in part, why SARS-CoV-2 is better able to infect host cells [84] .

Interconnected metabolic pathways intervene in cholesterol homeostasis and its regulation. Figure 1 and reviews in [107, 110] ) that activate the full set of genes involved in cholesterol synthesis and uptake from LDL. In the case of cholesterol biosynthesis, this implies activating transcription of the key reductase HMGCR, the rate-limiting enzyme in the mevalonatecholesterol biosynthetic pathway.

As mentioned above, cleavage of the SCAP-SRBP-1 complex is precluded by high cholesterol levels. Viral infection by human CMV overrides this inhibition and increased cleavage of the complex, the translocation of SREBPs to the nucleus, and the activation of genes involved in lipogenesis [111] (Figure 1 ), a requisite for CMV growth in cells [112] .

Pathogens, including viruses, can activate SREBP-2. Epstein-Barr virus induces its expression (but not that of SRBP-1), but, somewhat unexpectedly, instead of synthesizing cholesterol, the mevalonate-cholesterol pathway is geared towards the production of geranylgeranyl pyrophosphate that contributes to the outgrowth of newly infected B-cells and downstream activation of the small Ras superfamily G-protein Rab13 [113] . In the mevalonate-cholesterol pathway, acetyl-CoA can be reduced by HMG-CoA-reductase to produce mevalonate and converted into farnesyl pyrophosphate, which can either be used to synthesize squalene for cholesterol biosynthesis or be diverted toward GGPP synthesis, the route induced by the Epstein-Barr virus.

In the context of COVID-19, SREBPs have been recently identified as targets of AM580 using a lipidomic screening, a retinoid derivative and retinoic acid receptor-α agonist having antiinflammatory and neuroprotective effect [114] (see Figure 3 below). In addition to these effects, AM580 displays a potent and broad-spectrum antiviral activity against MERS-CoV, as well as IAV, in vitro and in vivo [115] . SREBP-2 regulates the production of interleukin-1β and tumour necrosis factor α and through these effector molecules is directly related to one of the complications of COVID-19, the hyperreactive cytokine syndrome, involving the disruption of cholesterol biosynthesis [116] . Different CoVs produce different hyperreactive cytokine responses [5] . Transcription of the sestrin-1 gene (SESN1), also known as p-53 regulated protein PA26, which mediates p53 inhibition of cell growth, is regulated by SRBP-2; SESN1 is a causal gene associated with plasma cholesterol levels [117] . Conversely, sestrin-1 inhibits SRBP-2- 

The Aster/GRAMD1 proteins constitute a family of evolutionarily conserved ER-resident sterol transporter proteins that mediate non-vesicular cholesterol transport from the plasma membrane to the ER. Sandhu and coworkers described the Aster A, B and C cholesterol-binding proteins [118] . The central domain of Aster-A is similar to the sterol-binding fold of mammalian StARD proteins, whereas the Aster N-term GRAM domain, exposed to the cytosol, binds phosphatidylserine and is responsible for Aster recruitment to plasma membrane-ER contact sites when cholesterol accumulates in the plasma membrane. GRAMD1 proteins are required for instance for delivery of HDL-cholesterol to the adrenal cortex, where the biosynthesis of steroid hormones proceeds from their precursor, cholesterol [119] . Under lipid-poor conditions GRAMD1 localizes at the ER membrane; in response to excess cholesterol in the plasma membrane, GRAMD1 is recruited to the ER-plasma membrane contact sites (EPCS) via its GRAM domain (Figure 1 ). At the EPCS, the sterol-binding VASt/ASTER domain of GRAM binds to cholesterol at the plasma membrane ( Figure 1 ) and catalyses its transfer from the cell surface to the ER. GRAMD1 proteins were shown to possess synergistic but distinct sites than can sense free cholesterol and anionic lipids in close vicinity in the plasma membrane, a topographical relationship that is also regulated by sphingomyelins [120] . The crystal structure of AsterA (GramD1a) with bound cholesterol molecules is shown in Figure 2 . (PDB accession number 6GQF). The crystal structure of the protein (blue ribbon) and sterol molecules (green ball-and-stick rendering, oxygen in red) was obtained by X-ray crystallography at 2.9 Å resolution [118] . Molecular coordinates were downloaded from the PDB data bank and structures produced using the CCP4MG Molecular Graphics Program of the University of York, U.K. [121] .

Phosphatidylserine has been shown to modulate the transport of LDL-derived cholesterol from lysosomes to the ER [122] and play a role in the localization of cholesterol in the cell [120, 123] .

When the plasma membrane cholesterol exceeds a certain threshold, excess cholesterol moves to the ER in a phosphatidylserine-dependent manner [122] . In a homeostatic feedback mechanism, the surplus cholesterol in the ER reduces external cholesterol uptake, preventing further cholesterol accumulation. It becomes apparent that the complex regulation of cholesterol metabolism is intimately related to the fate of other lipid components in the cell, with a close functional and topographical relationship between cholesterol and anionic phosphoglycerides, particularly phosphatidylserine.

The transmembrane protein 41B (TMEM41B) is best known for its participation at early stages of autophagosome biogenesis from ER membranes, in which process TMEM41B mobilizes neutral lipids from lipid droplets. In the context of viral infection, TMEM41B

plays a key additional role: it is an absolute requirement to infect the host cell for all J o u r n a l P r e -p r o o f members of the Flaviridae family of viruses, which includes the mosquito-and tick-borne human pathogenic yellow fever virus, the Zika virus and the West Nile virus [124] . The mechanism does not involve autophagosome formation; TMEM41B apparently lowers the free-energy required for induction of membrane curvature [124] at the virus-induced membrane complex known as replication-transcription complexes. These complexes are employed by Flaviviruses like the Zika virus [125] , and CoVs like MHV [126] , SARS-CoV [127] , and SARS-CoV-2 [128, 129] . Replication complexes can be envisaged as a virus-induced organelle derived from the ER-Golgi complex (ERGIC) characteristically enriched in 200-300 nm double-membrane vesicles (DMVs) [79] . The liquid-ordered domain lipid sphingomyelin is essential for the formation of the DMVs required for the replication of hepatitis C virus and other +-stranded RNA viruses [130] . A recently conducted search using genome-wide CRISPR genetic knockout screens showed that SARS-CoV-2 and three other seasonal Coronaviridae viruses (HCoV-OC43, HCoV-NL63, and HCoV-229E) require TMEM41B for infection and replication [131] .

TMEMB1 and VMP1 have recently been found to play a role in the distribution of free cholesterol and phosphatidylserine [132] . In the absence of these two proteins, more free cholesterol becomes available on the cytoplasmic-facing leaflet of the plasma membrane, and an abnormal increase in cell-surface cholesterol content may ensue. Moreover, TMEM41B and VMP1 have phospholipid scramblase enzymatic activity, thus contributing to the maintenance of phospholipid distribution in the cell [132] . Scramblases possess linear cholesterol-recognition motifs [133] .

Ubiquitously located at the surface of a great variety of cells, scavenger receptor class B type 1 (SR-B1) is a multifunctional membrane-bound protein mainly expressed in liver and one of the heavy-density lipoprotein (HDL) receptors (see review in [19] ). SR-B1 is considered to be a major player in inflammation and atherosclerosis, and as such to be an important modifiable risk factor in hypercholesterolemia, coronary heart disease, hypertension, obesity, and stroke [134] .

SR-B1 participates in the selective transport and regulation of cholesteryl esters and other lipids, but it possesses the capacity to bind many other ligands, including proteins, proteoglycans, phospholipids, and carbohydrates, and intervenes in immune surveillance, pathogen infection, and cancer [135] .

In SR-B1 serves as cell-surface receptor for the Flavivirus HCV, together with tetraspanin CD81

and claudin-1 and occludin (two tight junction proteins) [136, 137] . Upon recognition by the extracellular domain of SR-B1, the ensuing internalization of the virus proceeds via clathrinmediated endocytosis and pH-dependent fusion with endosomal membranes [137] . SR-B1 can also interact with virus-associated lipoproteins [19] . 

Another genome-wide CRISPR genetic screening of SARS-CoV-2 and HCoV-229E disclosed further associations between viral infection of these two CoVs and virus-specific and/or shared host-cell factors, including the genes coding for the autophagy regulator TMEM41B and the enzyme phosphatidyl-inositol-kinase type 3 (PI3K) [58] . The kinase, which catalyses the biosynthesis of the important inositol signalling molecule phosphatidyl-inositol-3-phosphate, serves as a host-factor for the common-cold CoVs HCoV-229E and HCoV-OC43, and to a lesser extent for SARS-CoV-2. In addition, these authors found that SARS-CoV-2 requires the lysosomal protein TMEM106B to infect human cell lines and primary pulmonary cells in culture.

TMEM106B is an endosome/lysosome-resident protein that has gained attention because of its role in frontotemporal dementia, a form of pre-senile neurodegenerative neuropsychiatric disease [138] . TMEM106B was found to be the strongest dependency host factor for SARS-CoV-2 infection, whereas HCoV-229E infection requires lysosomal acidification, but is TMEM106B- the detergent-extraction criterion [8] .

Clinical studies disclose prognostic value of SREBP2 in COVID-19

A recent retrospective analysis of 861 patients with mild, moderate, severe, or critical (the latter with respiratory failure, shock or organ failure) clinical presentations of COVID-19 evaluated the possible association of plasma cholesterol and HDL levels on morbidity and prognosis of the disease [141] . The levels of total cholesterol and HDL were found to be inversely correlated with severity, and severely ill patients with high HDL had a better prognosis. Survivors also had higher cholesterol and HDL levels. Furthermore, pre-treatment with ITX 5061, a potent antagonist of SR-B1 that increases HDL levels, competitively inhibited in vitro the SARS-CoV-2 infection of HEK-293T cells [141] . ITX-5061 and TX 7650 are two small organic molecules that inhibit HCV entry into cells. They do so by competing for HDL-mediated lipid transfer acting as a pharmacological antagonist of SR-B1 [142] . Recent studies confirm the lower-than-normal levels of plasma cholesterol, HDL and LDL in COVID-19 patients [143] . interleukins are characteristic noxa of the injured endothelium [5] .

Therapeutic targeting of the lipidome in COVID-19

One can envisage two approaches to anti-viral therapy: interfering with the virus proper or addressing the host-cell, in the latter case either inhibiting the pathways employed by the virus or reinforcing the endogenous defense mechanisms. The use of statins in COVID-19 has been pondered in three contexts: i) in an attempt to inhibit SARS-CoV-2 entry and endocytic trafficking; ii) to mitigate the vascular compromise observed in the disease [5] and iii) to increase the therapeutic levels in COVID-19 patients with hyperlipidemia, history of congestive heart disease or myocardial infarction, frequent comorbidities and risk factors of worse outcomes of COVID-19.

In the case of both SARS-CoV-2 and SARS-CoV, S1 glycoprotein-ACE2 recognition/ binding is the first therapeutically addressable mechanistic step. Based on the purported location of ACE2 in cholesterol-enriched lipid domains, statin-mediated reduction of cholesterol levels in the plasma membrane has been attempted in several viral diseases, and, indeed, found to lower viral titres and, in some cases, arrest the internalization of viruses [7] . By the same token, cholesterol analogues have been proposed as small drugs to interfere with SARS-CoV-2 entry [144] .

Lovastatin is reported to decrease viral load and increase CD4+ cell counts in models of acute infection and in chronically HIV-1 patients [145] .The polyomavirus BK virus infection is abrogated by pravastatin [146] . Simvastatin (or CDx) inhibits poliovirus [147] or Epstein-Barr J o u r n a l P r e -p r o o f virus [113] infection; however, simvastatin-mediated abrogation of poliovirus infection appears not to be mediated by inhibition of cholesterol biosynthesis but rather is due to the production of vesicles that cannot sustain viral RNA transcription at a later stage of the virus cycle [147] . This and other studies reinforce the view that besides inhibiting HMG-CoA-reductase, statins have pleiotropic effects, upregulating LDL receptors, increasing the clearance of LDL-cholesterol, and acting as anti-inflammatory, anti-thrombotic and immunomodulatory drugs (see recent review in [148] ). Even when they operate on the mevalonate pathway statins may exert effects other than inhibition of cholesterol synthesis, as exemplified by the effect of Lovastatin on HIV-1 infection: the mechanism impeding HIV-1 entry and budding involves prenylation of small Gproteins and downregulation of Rho GTPase activity [145] . Attempting to establish cause-effect links based exclusively on the main effect of statins on the rate-limiting enzymatic step of the mevalonate-cholesterol biosynthetic pathway is an oversimplification.

The notion that cholesterol is involved in virus trafficking and exit from the host cell in association with cholesterol-rich platforms has prompted several authors to suggest the use of drugs that interfere with these pathways as possible therapeutic agents against SARS-CoV-2 [7, 149, 150] , sometimes in combination with a variety of drugs of still unproven efficacy such as chloroquine or hydroxychloroquine, with the argument that such drugs destabilize the order of liquid-ordered lipid domains [149] . Targeting lipid "rafts" has also been the argument behind suggesting the use of cholesterol-sequestering cyclodextrins as the "best candidate to improve complex therapies" in COVID-19 [149] . Cyclodextrins have a dose-dependent toxicity on the human organism, particularly on the renal parenchyma, and require chemical modifications to be used as anti-viral agents. Mercaptoundecane sulfonic acids have been recently employed to produce biocompatible broad-spectrum antivirals, tested on herpes simplex virus, dengue virus, J o u r n a l P r e -p r o o f respiratory syncytial virus, and Zika virus [151] . Modified cyclodextrins are also finding applications as carriers, adjuvants, cryo-preservation agents, and stabilizers of anti-viral drugs [152] . [155] .

Thrombotic and thromboembolic complications have increasingly been documented as the COVID-19 pandemic evolved [159] [2] . Sepsis with affectation of the microcirculatory endothelium has also been reported [160, 161] . Post-mortem studies of COVID-19 patients showed (micro)thrombotic/thromboembolic lesions in the CNS and olfactory mucosae [162] .

Pulmonary arterial thrombosis of mid-size and small vessels has been described as a cause of death [163] . In another study 35% of hospitalized critically ill patients were found to present thrombotic complications [164] . As analyzed in more detail in a recent review [5] , endothelial dysregulation appears to be at the root of the thrombogenic endothelial dysregulation in COVID-19 [165] . It is in this context that statins have been considered as pharmacological agents for the prevention of thrombosis and the hyper-inflammatory complications and severe forms of the disease, and to potentially minimize tissue injury through production of angiotensin [1] [2] [3] [4] [5] [6] [7] propensity-scores matching retrospective studies, which match COVID-19 patients who were on statin therapy with those who were not, suggest that statin medication is mainly associated with a reduction of mortality, other evidence being largely anecdotal (reviewed in [168] ). This outcome is observed, however, with high doses of statins and not with low dosage; no link has been found between antecedent statin therapy and admission to intensive care units [169] . We must therefore wait for randomized controlled trials of statin use in COVID-19 [170] to assess its therapeutic value, appropriate dosage, and mechanism of action.

The identification of SR-B1 as a SARS-CoV-2 entry cofactor (see Figure 3 ) has opened new therapeutic horizons for COVID-19 treatment. As recently proposed ( [78, 141] , drugs that target SR-B, such as AM580 (Figure 3 ) could be employed as SARS-CoV-2 antivirals. As a proof of concept, these authors showed that ITX 5601, a clinically approved inhibitor of HCV infection, J o u r n a l P r e -p r o o f inhibits SARS-CoV-2 infection in vitro. Other metabolic steps offer prophylactic or therapeutic possibilities for inhibiting SARS-CoV-2, such as 25-hydroxycholesterol [95] .

As sterol molecules (green ball-and-stick rendering, oxygen in red) was obtained by X-ray crystallography at 2.9 Å resolution [118] . Molecular coordinates were downloaded from the PDB data bank and structures produced using the CCP4MG Molecular Graphics Program of the University of York, U.K. [121] . 

The author declares no conflicts of interest.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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