key: cord-345730-bxwsup70
authors: Kočar, Eva; Režen, Tadeja; Rozman, Damjana
title: Cholesterol, lipoproteins, and COVID-19: basic concepts and clinical applications
date: 2020-11-04
journal: Biochim Biophys Acta Mol Cell Biol Lipids
DOI: 10.1016/j.bbalip.2020.158849
sha: 
doc_id: 345730
cord_uid: bxwsup70

This review provides an overview of lipids and lipid metabolism in relation to COVID-19, with special attention on cholesterol. Cholesterol enriched lipid rafts represent a platform for viruses to enter the host cell by endocytosis. Generally, higher membrane cholesterol coincides with higher efficiency of COVID-19 entry. Inversely, patients with COVID-19 show lowered levels of blood cholesterol, high-density and low-density lipoproteins. The modulated efficiency of viral entry can be explained by availability of SR-B1 and LDL-receptors. Especially HDL seems to have a variety of roles, from being itself a scavenger for viruses, an immune modulator and mediator of viral entry. Due to inverse roles of membrane cholesterol and lipoprotein cholesterol in COVID-19 infected patients, treatment of these patients with cholesterol lowering statins remains controversial. In conclusion, cholesterol and lipoproteins are potential markers for monitoring the viral infection status where mechanistic inconsistencies warrant immediate further research.

: Characteristics comparison between SARS-CoV, MERS-CoV and SARS-CoV-2 at the time of writing this article (8 July, 2020) [1, 3, 5, 10, 11] 

Coronaviruses (CoVs) are widely distributed among humans and animals ( Table 2 ). In addition to the respiratory system, they can also infect the enteric, hepatic and central nervous systems of humans and other species [7, 12] . As RNA viruses, CoVs have a higher mutation rate and frequently undergo recombination. This leads to a greater genetic diversity, which hinders the development of vaccines [13] . In order to complete their replication cycle, they require a set of four major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) that are indispensable for the assembly of the virion and the infection of the host cell [12] . SARS-CoV-2 is a positive-sense, single-stranded RNA virus, surrounded by lipid envelope and is, with its ~30kb, one of the largest known viral RNAs [6] . Like SARS-CoV, it is of zoonotic origin and belongs to the family Coronaviridae, genus β-coronaviruses lineage B (β-B-CoVs), while MERS-CoV belongs to another lineage of the same genus (β-C-CoVs) [1, 5, 12, 13, 21] . The genome of the viral SARS-CoV-2 has been sequenced [14] , revealing 96.2% identity with a bat coronavirus (BatCoV RaTG13). In addition, SARS-CoV-2 shares 79.6% homology with SARS-CoV [14] , also derived from bats, and palm civet [1, 3] , and 51.8% identity with MERS-CoV [7, 22] . Although the data are consistent with bats representing the reservoir of the newly CoV, the natural secondary host through which the virus reached humans has not yet been identified [22] . SARS-CoV, MERS-CoV and SARS-CoV-2 share many common characteristics, from epidemiology, clinical features, molecular mechanism and underlying infection process. However, unlike other β-B-CoVs, the spike protein of SARS-CoV-2 harbors furin-detectable site between the S1 and S2 subunits [1] , indicating a potentially unique infectious property that could significantly increase the capacity of the viral spike protein [21] .

required to maintain membrane integrity and plays a pivotal role in modulating membrane fluidity and segregation, thereby affecting membrane heterogeneity [25, 26] . Within lipid rafts, cholesterol also affects membrane permeability, signaling and transport [27] . In addition, it serves as an essential precursor for the synthesis of oxysterols, steroid hormones and bile acids [28] , while cholesterol in the form of lipoproteins serves as a carrier of antioxidants, fat-soluble proteins, drugs and toxins [29] . Cholesterol is also a signaling molecule, regulates its own synthesis, the cell cycle and can modify proteins [28] . Due to its versatile roles and involvement in numerous physiological processes, the organism must maintain the cholesterol homeostasis, unless its excess could be potentially toxic. This is achieved by sophisticated regulation of de novo synthesis of cholesterol, its deposition in membranes, integration into lipoproteins or its storage in the form of cholesteryl esters and lipid droplets. The receptor-mediated uptake of cholesterol from the circulatory system and its metabolism into aforementioned downstream metabolites is also important for homeostasis [30] . The biosynthesis of cholesterol is an indispensable metabolic process in almost all mammalian cells,

where the major site of its production is the liver. Congenital errors in cholesterol synthesis lead to deaths before birth, but when they are compatible with life, they result in congenital defects and severe malformations [31, 32] . Cholesterol synthesis, its diurnal cycle and novel roles are discussed in more detail elsewhere [28, 30, 39, [31] [32] [33] [34] [35] [36] [37] [38] .

The successful entry of the virus to the host is a prerequisite of cross-species transmission. Therefore, understanding the exact mechanism of viral interaction with target cell provides a valuable information on viral pathogenesis and helps in vaccine and drug target design. The infectivity of certain viruses can be regulated by naturally-derived substances, thereby reducing their infectivity by interference with membrane lipid composition and consequently altering the viral lipid-dependent attachment [40] . Enveloped viruses which also include CoVs primarily engage plasma membrane fusion or endocytosis for entering the host cell [41] . Lipid raft microdomains with the unique protein composition are involved in the endocytosis-mediated process and serve as a platform and docking site for viruses to enter the host cell and release their genome [40] . By increasing the local concentration of entry receptors, lipid rafts mediate the entry process and influence other steps of the life viral cycle, such as assembly and budding [27] .

Membrane composition plays a crucial role in the behaviour of fusion proteins and also influences membrane fusion by modulating the organization and dynamics of both included membranes [42] .

However, the data are still contradictory and much more research in this area is needed. Rising cholesterol levels in human plasma membranes increased the infection rate of CoVs, by promoting membrane fusion [37] . Furthermore, Meher et al. [42] reported the effect of membrane cholesterol on the structure and oligomeric status of the fusion peptide of SARS-CoV whose binding affinity J o u r n a l P r e -p r o o f increased proportionally with increasing levels of membrane cholesterol. In contrast, cholesterol depletion physically disrupts the virion membrane [43] . Through the interferance with lipid-dependent attachment to human host cells, naturally derived sterols and cyclodextins can reduce the infectivity of CoVs ( Fig. 1c ) [40] . In vitro depletion of membrane-bound cholesterol from Angiotensin-Converting Enzyme 2 (ACE2)-expressing cells led to a reduced infectivity of CoVs, since the binding of the spike protein was reduced by half [44] . Also, interaction of phytosterols with lipid raft molecules can lead to a reduction of membrane cholesterol content or destabilization of its stucture, thereby affecting viral infectivity (Fig. 1c ) [40] . In addition, the viral infectivity is modulated by homeostatic control of cholesterol content and fatty acid metabolism [45] . 49] . Remarkably, the spike protein of SARS-CoV-2 interacts with cholesterol (EC50=187.6±120.5 nM), while both the spike and its S1 subunit interact with high-density lipoprotein (HDL) particles, with spike exhibiting a 5-fold higher binding affinity, [41] . This indicates a possible manipulation of cholesterol metabolism by interaction of SARS-CoV-2 spike protien with HDL particles.

Recent studies have shown that individuals with AA genotype of SLC10A1 (encoding Na/taurocholate cotransporter NTCP, the entry receptor of Hepatitis B Virus) exhibit a decreased level of cholesterol as a result of impaired bile acid uptake which may enable escape from the Hepatitis B virus (HBV) infection [50] . In another cohort, the infectivity of the human parainfluenza virus type 3 (HPIV3) was markedly reduced due to an abnormal internalization capacity in the absence of viral envelope cholesterol, as shown in internalization assay in Human Embryonic Kidney 293T cell line (HEK293T) [27] .

SARS-CoV-2, similarly to SARS-CoV, acquires human ACE2 as a functional receptor for host cell invasion (Fig. 1a ) [1, 13, 14] . ACE2 resides mainly within lipid rafts [49] . It is widely expressed in organs that regulate blood pressure, in the heart, vessels, kidneys, the small intestine of gastrointestinal tract [2, 51] , and is abundantly distributed in alveolar type II epithelial cells [52, 53] , suggesting that these organs should be considered as potentially at high risk of infection. However, the expression level of ACE2 in the lungs which is the major site of SARS-CoV-2 infection is rather low [51] , indicating that other viral entry mechanisms might be involved. ACE2 is also expressed in the mucosa of oral cavity and is highly enriched in epithelial cells of the tongue, suggesting that the oral cavity is also a potentially high-risk route of SARS-CoV-2 infection [52] .

The spike glycoprotein of SARS-CoV-2 envelope has two subunits, S1 and S2, by which it attaches to the plasma membrane and after fusion mediates the viral entry [13, 54] . After binding to the receptor, and prior to internalization, the spike protein is functionally cleaved by host membrane Transmembrane Protease Serine 2 (TMPRSS2) serine protease [54] . The cleavage site is at the R685/S686, releasing the fusion peptide of spike and facilitating internalization of the virus [13, 21] .

Availability of the host proteases largely determines whether CoVs can enter the target cell through plasma membrane or endocytosis [1] . A lack of the host protease or incompatibility between the latter and the viral spike protein can inhibit virus entry [13] . After successful internalization, the virus uses the molecular machinery of the host in order to replicate, modifying the host metabolism and leading to major changes in the cellular lipid profile of the host [49, 55] .

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The role of lipoproteins as a first line of defense against microbes is well established [55] , with most of them being able to bind and neutralize Gram-negative and Gram-positive bacterial membrane components, such as lipopolysaccharides and lipoteichoic acid, respectively [56, 57] . Lipoprotein levels are altered during viral infections [56] . Hypolipidemia has been reported in critically ill patients, especially in septic conditions [56] . According to recent meta-analysis, the severity of a Dengue infection, a mosquito-borne tropical disease caused by the dengue virus, inversely correlates with total cholesterol (TC) and LDL-cholesterol [56] . HDL consists mainly of free cholesterol, phospholipids, triglycerides, cholesteryl esters and apolipoproteins (A1, A2), and has protective effects against oxidized lipids [49] . HDL particles are constantly modified in response to physiological, pathological and acute inflammatory conditions, which is reflected in their lipid and protein content [56, 58] . Gangliosides in reconstituted HDL particles protect the cells from the polymeric Cholera toxin, suggesting the possibility that HDL containing lipids exhibit anti-infectious activity [56] . Apolipoprotein A1 (ApoA1), a major protein component of the HDL particles, binds the Dengue virus and enlarges its infectivity by facilitating its access to the cell via the Scavenger

Receptor, class B type 1 (SR-B1) the functional HDL receptor enriched in lipid rafts [25, 56] . SR-B1

mediates the selective uptake of lipoprotein-derived cholesteryl esters into the cells [25] . It is also involved in reverse transport of cholesterol, the selective uptake of other HDL-bound lipid components, facilitates cholesterol secretion through bile acids and the outflow of cellular cholesterol to HDL particles [25] . Pretreatment of HEK293T cells with a potent SR-B1 antagonist, ITX5061 strongly inhibited the entry of SARS-CoV-2-S pseudovirus to the host cells, where the treatment had no cytotoxic effect on cell survival [41] . The remaining question is whether SARS-CoV-2 could, in addition to ACE2, engage another route of entering the host cell, possibly via lipoprotein receptors (Fig. 1b) .

HDL particles can have an antiviral eff ect on RNA and DNA viruses by neutralizing them, regardless of whether they are enveloped or not. However, the correlation between the latter and viral infections is not as clear as for bacteria [56] . The antiviral activity of HDL particles could be a consequence of ApoA1 interference with viral entry or with the target cell membrane during fusion, but HDL particles themselves could induce direct virus inactivation. Paraoxonase 1 (PON1) which is mainly transported by HDL displays antibacterial and antiviral properties [56] . By participating in cholesterol outflow from the cell membrane to HDL particles, PON1 contributes to lowering the cholesterol levels within lipid rafts, thus modulating viral infection (Fig. 1c) . Pleiotropic nature of HDL particles that play an important role in cholesterol transport (reverse or not), act antiinflammatory, and have antiviral and antioxidant properties, makes them a likely pathogen scavenger that could potentially be involved in the removal of infectious material [56, 58] . The day of infection and then began to recover [49] . Similar changes in the lipid profile were also reported by other groups [41, 59] . A significant decrease in the level of HDL-cholesterol was observed only in critical cases of COVID-19, while significant decrease of TC and LDL-cholesterol was observed in all patient groups (mild, severe, critical). Accordingly, it seems that hypolipidemia occurs in patients with mild symptoms and escalates with the progression and severity of the disease [59] . Taking into account increased serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST), the decrease of LDL-cholesterol may be explained by liver damage as a consequence of the SARS-CoV-2 infection. On the other hand, LDL-cholesterol levels may also decrease due to increase in Interleukin-6 (IL-6) [59] . In contrast, another study observed significantly increased level of serum LDL compared to the reference population where levels of HDL-cholesterol and TC were inversely correlated with the severity of COVID-19 [41] .

Meta-analysis showed an inverse association between serum cholesterol and non-cardiovascular mortality in respiratory and digestive diseases, some cancers, and other residual causes of infectious origin [29] . A weak but statistically significant inverse association was found between level of TC and the incidence of some infectious diseases diagnosed in hospital setting [61] .

Patients with familial hypercholesterolemia (FH) have a lifelong increase in plasma LDL-cholesterol [8, 62] . They are at high risk of cardiovascular disease, and therefore have an increased risk of suffering a severe course of COVID-19 [8, 62] . Since statins, the first-choice treatment against FH may have a protective role against endothelial dysfunction and an acute coronary event, a

consequence of viral infection, should not be withheld [8] . inhibitors could interfere with infectivity of SARS-CoV-2 via numerous lipid-dependent mechanisms [65] .

Non-alcoholic Fatty Liver Disease (NAFLD), a hepatic manifestation of the metabolic syndrome [32, 39, 66] , is the most common liver disease of developed world [67] as a result of obesity and diabetes epidemic [68] . NAFLD is a multifactorial condition that defines a spectrum of hepatic changes, ranging from simple steatosis, steatohepatitis (NASH), further progressing to fibrosis and cirrhosis, and eventually leading to hepatocellular carcinoma (HCC) [39, [66] [67] [68] [69] . The disease is characterized by intrahepatic deposition of excess triglycerides, which continue to cause lipotoxicity, aggravate liver damage, and may lead to hepatocyte death [66, 67, 69] . Metabolomic analysis have shown increased cholesterol synthesis in NAFLD patients, while absorption of cholesterol was decreased.

Plasma levels of LDL were also elevated, with abnormalities in lipoprotein incidence reflected in altered homeostasis of the major lipid components; cholesterol, lipoproteins, cholesterol esters and triglycerides [39] . Apart from limitations regarding the early prediction of NAFLD, there are no drugs for the direct treatment of the disease. However, patients are often treated with statins alone or in combination with antioxidants (e.g. vitamin E) [39, 67] . Yet, the most effective treatment strategy for NAFLD is lifestyle intervention through a combination of diet, exercise and weight loss [67, 70] .

Therefore, it is intriguing to contemplate whether NAFLD patients without treatment are more J o u r n a l P r e -p r o o f susceptible for SARS-CoV-2 infection, or whether statin application may directly affect the entry of SARS-CoV-2 into the host cell by regulating cholesterol cell levels.

Statins have pleiotropic properties but are best known as cholesterol-lowering agents (Fig. 1c) that inhibit a rate-limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) [28, 30, 55, 70, 71] . Transcriptome analyses performed by our group revealed a modifying effect of rosuvastatin and atorvastatin on the expression of many metabolic and signaling pathways in the liver, including drug metabolism [72] . Statins are also ligands of Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR), both members of the nuclear receptor protein family [71] . Despite having adverse effects on the liver, statins are considered beneficial for the treatment of NASH [70] .

Along with beneficial effects on cardiovascular and pulmonary function, statins can also strengthen the host defense. They have substantial anti-inflammatory and immunomodulatory effects, which is why they may be used as a host-targeted treatment against pathogen infections. Lipid-lowering treatment and lipid raft disruption have already been shown for other CoVs [73] . As lipid lowering drugs, statins might thus significantly reduce the attachment and internalization of SARS-CoV-2 by lowering membrane cholesterol levels (Fig. 1c ) [37] . However, the results of clinical studies on the effect of statin therapy in viral infections are contradictory [37] . An in-silico study showed that fluvastatin, lovastatin, pitavastatin and rosuvastatin may inhibit the main protease of SARS-CoV-2 [62] . Lowering cellular cholesterol might also trigger a greater uptake of cholesterol from the bloodstream, thereby lowering serum HDL-and LDL-cholesterol levels. Consequently, this would plausibly lead to an upregulation of the LDL-and SR-B1 receptors, and to incorporation of cholesterol into the plasma membranes, resulting in higher SARS-CoV-2 infection rate.

The recent COVID-19 outbreak caused by SARS-CoV-2 poses a threat to the human population with an urgent need for rapid development of effective antiviral therapeutic agents. Understanding the exact molecular mechanism of viral pathogenesis is a fundamental step towards infection prevention.

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Funding: The work was funded by Slovenian Research Agency (ARRS) programme grant P1-0390. project J1-9176 and the PhD grant for young researchers (EK)

Conflict of interest/Competing interests: The authors declare no conflict of interest

Authors contribution: EK performed the literature search and wrote the manuscript, TR critically revised the lipid and lipoprotein part of the review; DR designed the manuscript and its contents. All authors actively contributed to writing and revising the text.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.J o u r n a l P r e -p r o o f