**4. Cholesterol recognition/interaction amino acid consensus (CRAC) motifs in viral proteins. Possible uses of CRAC-containing peptides**

As was shown earlier [16, 17, 93], some proteins involved in cholesterol-dependent cell functions possess the so-called cholesterol recognition/interaction amino acid consensus (CRAC) motifs – small regions with a specific set of amino acid residues involving a branched apolar aminoacid residue (Val (V), Leu (L), or Ile (I)), aromatic residue (Tyr (Y)), and cationic aminoacid residue (Arg (R) or Lys (K)); these motif-forming amino acids are separated by short segments of any 1–5 amino acid residues. In subsequent discussions, more candidates of aromatic amino acid residues were proposed, and the general formula for the CRAC motifs presumably involved in the interaction of protein with cholesterol presently appears as follows: V/L/I–X(1–5)–W/Y/F–(X)(1–5)–R/K, where X stands for any amino acid residue [94–100]. Although the predictive value of this formula has been questioned [95–97], the presence of this motif in many proteins and its participation in the protein–cholesterol interactions has been confirmed by different methods [16, 17, 93–95]. The formula of the CRAC motif can be further developed [94]; important is the very concept of a motif mediating the interactions of cholesterol-dependent proteins with cholesterol.

**67**

envelope.

coronavirus [111, 112].

*Cholesterol Recognition Motifs (CRAC) in the S Protein of Coronavirus: A Possible Target…*

CRAC motifs are found in many viral proteins, and their role in cholesteroldependent virus–cell interactions have been demonstrated. For example, CRAC motifs are present in the HIV matrix protein р17, which was shown to participate in virus entry through the raft domains of the cell membranes [27, 101]. The α-helical domain of the hepatitis C virus nonstructural protein NS5A, which is anchored at the cytoplasmic leaflet of the endoplasmic reticulum and is involved in replication hepatitis C virus, also contains CRAC motif [102]. Importantly, peptides derived from this domain were shown to exhibit a broad-spectrum anti-viral activity. Cheng et al. 2008 [103] reported that peptide C5A containing amino acid residues 3–20 of the amphipathic α-helical N-terminal domain of hepatitis A virus protein NS5A suppressed the virus replication by more than 5 orders of magnitude. The authors did not mention the CRAC concept; however, the active peptide C5A (SWLRDIWDWICEVLSDFK) clearly contains the CRAC motif: RDIWDWICEV. Later, the antiviral activity of this peptide C5A against HIV was also demonstrated [104]. It is possible that peptide C5A, owing to the presence of the CRAC motif, binds cholesterol and competes for cholesterol binding with viral protein and thus

CRAC motifs are found in alpha-helices of matrix protein M1 of influenza A virus [105–107]. An important role of these CRAC motifs in the organization of the raft structure of the virion membrane was substantiated by using the method of directed point mutations in the CRAC-containing α-helices in the M1 protein [106, 107]. Further studies revealed that M1-derived peptides containing CRAC motifs LEVLMEWLKTR, NNMDKAVKLYRKLK, GLKNDLLENLQAYQKR, corresponding to α-helixes 3 (aa 39–49), 6 (aa 91–105) and 13 (aa 228–243), respectively, to a different extent modulate cholesterol-dependent interactions of cultured macrophages with 2-μm particles that mimic bacteria (phagocytic index). Of the three peptides, NNMDKAVKLYRKLK was most potent and stimulated the cell activity by 50–60% at 35 μΜ [108]. Peptide RTKLWEMLVELGNMDKAVKLWRKLKR obtained by combining two of these short peptides and containing two CRAC motifs produced much stronger and more complex effect: in a narrow range of low concentrations (1–5 μM) the peptide exerted a stimulatory effect and at 50 μM the peptide was cytotoxic [109]. Reducing the cholesterol content in the cells with methyl-βcyclodextrin (mβCD) abolished the stimulatory component and lowered the peptide concentration required for the toxic effect. Substitution of the motif-forming amino acids abolished these effects [110]. The cytotoxic effect of the M1-derived peptide RTKLWEMLVELGNMDKAVKLWRKLKR can be explained by the binding (sequestration) of membrane cholesterol by the peptide; this can imitate removal of cholesterol from cell membranes, which occurs during the formation of the viral

S proteins of coronaviruses SARS-CoV and SARS-CoV2 also contain CRAC motifs [51, 109, 110]. For example, in the case of the S protein of coronavirus SARS-CoV, the CRAC motif YIKWPWYVW is located in the "aromatic" region of the transmembrane domain of the S-protein; this highly conserved region of

If the assumption about the essential role of sequestration and removal of membrane cholesterol by viral CRAC-containing proteins in the COVID-19 parthenogenesis is correct, then in order to prevent this destructive action for the cell, it is necessary to maintain a safe level of cholesterol in the plasma membrane. A significant decrease in total cholesterol and low-density lipoprotein (LDL) cholesterol levels in COVID-19 patients [13, 88] may be indicative of the critical loss of cholesterol by cells, and an efficient cholesterol delivery to the cholesteroldepleted cells may be helpful. Cyclodextrins are possible candidates as non-toxic

the S-protein was shown to be necessary for the infection of cells with

*DOI: http://dx.doi.org/10.5772/intechopen.95977*

inhibits the formation of the viral particle.

*Cholesterol Recognition Motifs (CRAC) in the S Protein of Coronavirus: A Possible Target… DOI: http://dx.doi.org/10.5772/intechopen.95977*

CRAC motifs are found in many viral proteins, and their role in cholesteroldependent virus–cell interactions have been demonstrated. For example, CRAC motifs are present in the HIV matrix protein р17, which was shown to participate in virus entry through the raft domains of the cell membranes [27, 101]. The α-helical domain of the hepatitis C virus nonstructural protein NS5A, which is anchored at the cytoplasmic leaflet of the endoplasmic reticulum and is involved in replication hepatitis C virus, also contains CRAC motif [102]. Importantly, peptides derived from this domain were shown to exhibit a broad-spectrum anti-viral activity. Cheng et al. 2008 [103] reported that peptide C5A containing amino acid residues 3–20 of the amphipathic α-helical N-terminal domain of hepatitis A virus protein NS5A suppressed the virus replication by more than 5 orders of magnitude. The authors did not mention the CRAC concept; however, the active peptide C5A (SWLRDIWDWICEVLSDFK) clearly contains the CRAC motif: RDIWDWICEV. Later, the antiviral activity of this peptide C5A against HIV was also demonstrated [104]. It is possible that peptide C5A, owing to the presence of the CRAC motif, binds cholesterol and competes for cholesterol binding with viral protein and thus inhibits the formation of the viral particle.

CRAC motifs are found in alpha-helices of matrix protein M1 of influenza A virus [105–107]. An important role of these CRAC motifs in the organization of the raft structure of the virion membrane was substantiated by using the method of directed point mutations in the CRAC-containing α-helices in the M1 protein [106, 107]. Further studies revealed that M1-derived peptides containing CRAC motifs LEVLMEWLKTR, NNMDKAVKLYRKLK, GLKNDLLENLQAYQKR, corresponding to α-helixes 3 (aa 39–49), 6 (aa 91–105) and 13 (aa 228–243), respectively, to a different extent modulate cholesterol-dependent interactions of cultured macrophages with 2-μm particles that mimic bacteria (phagocytic index). Of the three peptides, NNMDKAVKLYRKLK was most potent and stimulated the cell activity by 50–60% at 35 μΜ [108]. Peptide RTKLWEMLVELGNMDKAVKLWRKLKR obtained by combining two of these short peptides and containing two CRAC motifs produced much stronger and more complex effect: in a narrow range of low concentrations (1–5 μM) the peptide exerted a stimulatory effect and at 50 μM the peptide was cytotoxic [109]. Reducing the cholesterol content in the cells with methyl-βcyclodextrin (mβCD) abolished the stimulatory component and lowered the peptide concentration required for the toxic effect. Substitution of the motif-forming amino acids abolished these effects [110]. The cytotoxic effect of the M1-derived peptide RTKLWEMLVELGNMDKAVKLWRKLKR can be explained by the binding (sequestration) of membrane cholesterol by the peptide; this can imitate removal of cholesterol from cell membranes, which occurs during the formation of the viral envelope.

S proteins of coronaviruses SARS-CoV and SARS-CoV2 also contain CRAC motifs [51, 109, 110]. For example, in the case of the S protein of coronavirus SARS-CoV, the CRAC motif YIKWPWYVW is located in the "aromatic" region of the transmembrane domain of the S-protein; this highly conserved region of the S-protein was shown to be necessary for the infection of cells with coronavirus [111, 112].

If the assumption about the essential role of sequestration and removal of membrane cholesterol by viral CRAC-containing proteins in the COVID-19 parthenogenesis is correct, then in order to prevent this destructive action for the cell, it is necessary to maintain a safe level of cholesterol in the plasma membrane. A significant decrease in total cholesterol and low-density lipoprotein (LDL) cholesterol levels in COVID-19 patients [13, 88] may be indicative of the critical loss of cholesterol by cells, and an efficient cholesterol delivery to the cholesteroldepleted cells may be helpful. Cyclodextrins are possible candidates as non-toxic

*Management of Dyslipidemia*

oxidative stress and JNK-dependent apoptosis [85].

virus acts like a lysosomotropic drug. Cholesterol supplementation was also shown to reverse a strong cytotoxic effect on colon cancer cells caused by a low molecular weight compound TASIN-1 producing cholesterol-dependent ER stress triggering

Thus, virus–cell interactions lead to significant modulations in the lipid composition of cell membranes. A decrease in cholesterol in cell membranes owing to the formation of viral envelopes can be one of the most dangerous consequences of the virus particle assembly, as the amount of cholesterol removed from the cell membranes by newly formed viruses can exceed the compensatory resources of the cell. If the delivery of cholesterol to the cells is insufficient, deregulation of cholesteroldependent processes can lead to massive cell death, which manifests itself in the clinical course of the disease and a poor prognosis. In this connection, it should be noted that in patients infected with COVID-19, a significant decrease (several fold) in total cholesterol and low-density lipoprotein (LDL) cholesterol levels was recorded [13, 88], and cholesterol-lowering treatments (such as statins) may not be advisable for patients with life-threatening COVID-19 infection, at least until they recover from the infection [88]. Such a drop of the LDL cholesterol level in COVID-19 patients may reflect an enhanced recruitment of circulating cholesterol by the cells to compensate for its loss associated with virus reproduction. Perhaps the clinical prognosis depends on the timely and successful delivery of cholesterol required for cell membrane repair. Another direction in the development of drugs for the treatment of the disease is the search for agents that interfere with the interactions of viral proteins with cholesterol, and this search should be based on an understanding of the mechanisms of these interactions. So far, the only drugs for which clinically significant results have been demonstrated against COVID-19, are dexamethasone and some other corticosteroids [89–91]; secosteroids (vitamin D) are shown to help, too [92]. The use of dexamethasone led to a reduction in mortality to one third of hospitalized patients with severe respiratory complications from COVID-19. It seems possible that the action of steroids may be associated with the repair of a cell membrane damaged by virus-induced depletion of cholesterol. How does the virus manage to bind and remove cholesterol from cell

**4. Cholesterol recognition/interaction amino acid consensus (CRAC) motifs in viral proteins. Possible uses of CRAC-containing peptides**

As was shown earlier [16, 17, 93], some proteins involved in cholesterol-dependent cell functions possess the so-called cholesterol recognition/interaction amino acid consensus (CRAC) motifs – small regions with a specific set of amino acid residues involving a branched apolar aminoacid residue (Val (V), Leu (L), or Ile (I)), aromatic residue (Tyr (Y)), and cationic aminoacid residue (Arg (R) or Lys (K)); these motif-forming amino acids are separated by short segments of any 1–5 amino acid residues. In subsequent discussions, more candidates of aromatic amino acid residues were proposed, and the general formula for the CRAC motifs presumably involved in the interaction of protein with cholesterol presently appears as follows: V/L/I–X(1–5)–W/Y/F–(X)(1–5)–R/K, where X stands for any amino acid residue [94–100]. Although the predictive value of this formula has been questioned [95–97], the presence of this motif in many proteins and its participation in the protein–cholesterol interactions has been confirmed by different methods [16, 17, 93–95]. The formula of the CRAC motif can be further developed [94]; important is the very concept of a motif mediating the interactions of cholesterol-dependent

**66**

proteins with cholesterol.

membranes?

cholesterol transporters [113–116], which can redistribute cholesterol from endogenous and/or exogenous sources. The use of cyclodextrins increased the lifespan of NPC1−/− experimental mice [117] and improved the condition of patients with Nieman–Pick disease [118]. Another alternative is to prevent viral proteins from interacting with membrane cholesterol. At least some of the drugs that are currently tested – for example, polyphenolic substances like quercetin and saponin glycyrrhizin [119, 120] – can act at the protein/cholesterol interface and hinder cholesterol binding by the CRAC motif of the viral S protein and thus inhibit the assembly of new viral particles. Glycyrrhizin, an active component of liquorice roots, was shown to inhibit SARS-CV replication in Vero cells and replication of SARS-associated coronavirus [121, 122]. However, such agents are not very selective and can affect other cholesterol-dependent proteins and therefore cause side effects. Perhaps specially designed CRAC-peptides specifically blocking the interactions of S-protein with cholesterol will prevent the cellular cholesterol loss leading to permeabilization of membranes, oxidative stress, and cell death. The ability of CRAC-containing peptides to regulate cholesterol-dependent cell functions has been demonstrated in a number of works [17, 109, 122, 123], and studies of the antiviral activity of these peptides may be useful and promising.
