A new study published on the preprint server bioRxiv* in May 2020 suggests that the strikingly increased mortality in COVID-19 patients who are either old or also have high blood pressure, diabetes, cardiovascular disease is due to high tissue cholesterol levels. It also indicates the role of a healthy diet, by showing that polyunsaturated fatty acids (PUFA) in blood oppose the effects of cholesterol and thus the risk for severe COVID-19 symptoms.
Study: The role of high cholesterol in age-related COVID19 lethality. Image Credit: Naeblys / Shutterstock
Variation in Infectivity Between Adults and Children
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19 disease, is known to infect and enter host cells employing the ACE2 receptor molecule present on the outside of the host cell membrane. It was initially thought that low ACE2 levels in children caused the decreased risk for this condition, but recent studies show that this is not necessarily always true. Some children show high ACE2 levels in early childhood.
The current study aims to answer the question as to what controls the entry of SARS-CoV-2 into the host cell.
The Role of Cholesterol in Viral Entry
Earlier SARS-CoV viral research might help provide the answer. This virus enters the cell only in the presence of cholesterol and monosialotetrahexosylganglioside1 (GM1) in the form of lipid rafts, in culture. Cholesterol is required for the formation of these GM-1 lipid rafts, and viruses that attach here enter the cell by endocytosis.
These GM-1 rafts migrate from areas of disordered lipids or polyunsaturated fats.
Cholesterol is a fat found in cell membranes all over the body and deposited in macrophage-rich tissues with chronic inflammation. In lung disease, lung cells, as well as macrophages, are observed to take cholesterol in and out. The study, therefore, focused on finding the relationship between the infective potential of the virus and the concentration of cholesterol.
Cholesterol Promotes Viral Entry
First, using a pseudovirus expressing the spike (S) protein of SARS-CoV-2, they loaded cholesterol into cultured cells along with the carrier protein apoE, and blood serum. The S protein is responsible for viral entry. In the cholesterol-bound form, the apoE protein binds to the LDL receptor of the cell to load cholesterol into the cell. If free, its binding promotes the unloading of cholesterol from the cell.
In the cholesterol loading condition, the viral entry increased markedly by 50%, while it was significantly reduced in the unloading state. The addition of a cholesterol-removing chemical called methyl-beta-cyclodextrin (MβCD) to the cell culture duplicated the viral entry-inhibiting effect of cholesterol unloading. The researchers concluded that the lack of virus entry with the unloading of cholesterol is not due to a lack of ACE2 receptor, because the latter was expressed at normal high levels in the presence of both apoE and MβCD.
Having indicated the cholesterol-dependent entry of the virus, they examined the underlying molecular mechanisms. Cholesterol is essential to the number and size of GM1 rafts as well as the in and out movement of proteins into the rafts in the cell membrane. With an increase in these, the viral entry also goes up.
Cholesterol Facilitates Greater ACE2 Availability
The movement of ACE2 receptors to the viral entry site also increases with the cholesterol load. To find out how much of the receptor is localized in these raft sites, and how much this varies with the cholesterol concentration, they used fluorescent-labeled antibodies to ACE2 to cells treated with apoE and serum and used dSTORM (direct stochastical optical reconstruction microscopy) to acquire images of the lipid domain structure at the nanoscale.
The researchers found that ACE2 receptors were correlated with GM1 lipid rafts more than 3-fold in the loaded condition of the cell. The opposite effect (an over 70% reduction in localization to the rafts) was seen with the addition of MβCD. Thus apoE-mediated cholesterol loading causes localization of ACE2 at the point of viral entry.
Why is COVID-19 So Much More Dangerous in the Old and Sick?
This cholesterol-dependent mechanism of ACE2 localization, as well as virus entry, could be why COVID-19 is so much more lethal in the elderly. Tissue cholesterol levels increase with age as a result of chronic disease conditions like atherosclerosis and other acute or chronic inflammations. These are the same conditions that are found to increase the severity and likelihood of symptomatic COVID-19 disease.
Based on these findings, the researchers propose that cholesterol is essential for SARS-CoV-2 infection. Low cholesterol levels provide few entry points, and reduce their size, as in children. However, with aging, higher cholesterol levels cause the number and size of entry points, accounting for the much higher likelihood of infection.
The researchers next attempted to find out whether the reduced viral entry was due to less receptor binding. They tested the binding of the virus to the receptor-binding domain (RBD) of the ACE2 receptor on the culture cells, both with and without the addition of MβCD to the cells. The slight reduction of the RBD binding due to the presence of the chemical was insignificant.
The next step was to find out how viral entry was affected by ACE2 expression and low cholesterol levels. They found that with higher ACE2 concentrations, the receptors moved away from the vicinity of the GM1 lipid rafts, possibly because the excess ACE2 migrated to the disordered lipid region. Though the virus attaches to the ACE2 in this region, it cannot enter the cell, and thus this state protects against infection, as seen in children.
Cholesterol Increases Furin Availability
The virus requires protease-mediated priming to enter the host cell. In SARS-CoV-2, this is performed by furin, which has a cleavage site, unique to this virus, between S1 and S2 subunits of the S protein.
In the pseudovirus, this site is cleaved in the cells that manufacture the virus. On the other hand, another site cleaves a fusion peptide and allows the virus to enter the host cell membrane. This is supposed to be cleaved by the proteases TMPRSS2 or cathepsin.
To test this, they labeled both furin and TMPRSS2 along with the GM1 rafts. They found only furin was associated with the rafts. In low cholesterol conditions, this association disappeared.
Finally, the study also reveals the attachment of palmitoyl residues during post-translation modification. These 16-carbon lipids attach to many proteins to facilitate their entry and exit from GM1 lipid rafts. The SARS-CoV endodomain must be palmitoylated to allow viral fusion and entry via the S protein. All the palmitoylation sites considered to be present in this virus are also found in SARS-CoV-2, with one more, which may indicate that this alteration favors virus survival.
Multiple Mechanisms of Cholesterol-Dependent Infectivity
The model shows that in high cholesterol regions, more and larger GM1 lipid rafts are formed in the cell membrane due to cholesterol loading. ACE2 moves into the GM1 lipid raft and enters the cell via endocytosis. The spike protein covering the virus surface binds to ACE2, possibly pulling the GM1 lipids over the virus and so facilitating endosomal entry.
Overall, therefore, the study suggests 3 distinct mechanisms that could explain the differences in infectivity between population subgroups with comorbidities and chronic inflammation.
The first is the cholesterol-dependent increase in the number and size of GM1 lipid rafts, and hence entry points for the virus. Secondly, the receptor can dock into the sites that offer the most significant benefit for viral entry, again in a cholesterol-dependent manner. And thirdly, the proteases required for virus activation prefusion are also absent in low cholesterol conditions.
In children, who typically have surface viral entry only rather than lipid-raft-mediated endocytosis, the limited viral entry leads to mild infection. Low cholesterol reduces the infective potential of the virus still more.
With higher cholesterol, RBD binding improves, which could indicate the preferential use of endocytosis by the virus for cell entry. The increase in TMPRSS2 is linked to higher infectivity. However, if this protease is preferred in children, it might be ineffective in older and sicker patients.
How is This Important To COVID-19 Treatment?
The fact that most cell cultures for SARS-CoV-2 are carried out without a cholesterol source like serum is vital in developing therapies for COVID-19, in the light of these findings. It is vital to note that a high tissue level of cholesterol and not a high blood level is the driver of infectivity with this virus. This means that current tests for blood cholesterol do not always indicate the actual risk of severe or critical disease.
One key example of this seeming paradox is the presence of chronic inflammation, where tissue cholesterol levels are high though blood cholesterol is low. The reason is that cholesterol unloading in the tissue is inhibited, while macrophages load it into the cells.
The study could thus help forward research into the role of cholesterol in viral infection in COVID-19. More research is needed to find the role of other proteins and lipids in the complex membrane environment, which could significantly influence the virus entry and infection of the host cell.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.