Statin may increase COVID-19 infection – Researchers have identified critical molecular processes

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When a coronavirus – including SARS-CoV-2, which causes COVID-19 – infects someone, it hijacks the person’s cells, co-opting their molecular machinery for its own survival and spread. Researchers at Gladstone Institutes and the Chan Zuckerberg Biohub, in collaboration with scientists at UC San Francisco (UCSF) and Synthego Corporation, have identified critical molecular processes in human cells that coronaviruses use to survive.

They report, in a study published in the journal Cell, that targeting these processes with drugs may treat not only COVID-19 infections, but other existing and future coronaviruses.

“What is unique about our study is that we didn’t just look at SARS-CoV-2, but other coronaviruses at the same time,” says one of the leaders of the study, Melanie Ott, MD, Ph.D., director of the Gladstone Institute of Virology.

“This gives us a good idea of drug targets that could broadly suppress many coronaviruses.”

A large family of viruses, coronaviruses include common cold viruses as well as more severe viruses. The SARS-CoV virus that caused a deadly SARS epidemic in 2002 was a coronavirus, as is the MERS virus, which has caused outbreaks in the Middle East.

“There have now been multiple coronavirus outbreaks, so it’s clear this virus family has high pandemic potential,” says Andreas Puschnik, Ph.D., a principal investigator at the Chan Zuckerberg Biohub and the other leader of the study.

“COVID-19 is not the last coronavirus infection we’ll be dealing with.”

Comparing and Contrasting Coronaviruses

Like all viruses, coronaviruses can only grow inside host cells; they rely on the host cell’s molecules to multiply. Because of this, the team of researchers want to target human molecules that the viruses use to survive, rather than components of viruses themselves.

In the new study, they infected human cells with either SARS-CoV-2 or two other coronaviruses that cause common colds – and all three viruses killed the cells. Next, the team of researchers mutated the cells using CRISPR-Cas9 gene-editing technology and studied which mutations made the cells less vulnerable to the coronaviruses.

“We reasoned that the few cells that could survive these infections presumably had mutations in host molecules that the viruses use to infect them or to multiply,” explains Puschnik.

Some results were not surprising. For instance, the human ACE2 receptor is known to be required by SARS-CoV-2 to enter human cells. So, cells with a mutation in the ACE2 gene were no longer infected or killed by SARS-CoV2.

But other findings were less expected. The researchers found that certain genetic mutations prevented all three coronaviruses from successfully infecting and killing the cells.

These were mutations in genes known to control the balance of two types of lipid molecules in human cells, namely cholesterol and phosphatidylinositol phosphate (PIP).

Cholesterol is needed for some viruses to enter cells, but it hadn’t been studied in the context of coronaviruses when this study started. Similarly, PIP is known to play a role in forming the small vesicles that viruses often use to travel into and around cells, but it had not been directly linked to SARS-CoV-2 before.

A Pathway toward Therapeutics

To verify the importance of the cholesterol and PIP genes for coronavirus infection, the researchers engineered human cells that lack these genes completely and infected them with the virus. Cells lacking the genes were protected from infection by all three coronaviruses.

Similarly, when the team used existing compounds to disrupt the balance of PIP or cholesterol, the cells were less susceptible to infection by any of the viruses.

These results suggest that targeting cholesterol or PIP could be a promising strategy to combat multiple coronaviruses.

“For viruses, the traditional view has been that we design drugs against unique viral targets, and that means it takes time to develop a drug each time there’s a new virus,” says Ott, who is also a professor in the Department of Medicine at UCSF. “If we could develop a few broader antiviral drugs that target host cells’ molecules, that would go a long way toward making us better prepared for future pandemic viruses.”

Not all results were the same between the three studied viruses, however. Some human molecules required for SARS-CoV-2 infection weren’t needed by the two common cold coronaviruses, and vice versa. These findings could help explain what makes SARS-CoV-2 more deadly than the other two viruses.

More work is needed to test the effectiveness of drugs targeting PIP and cholesterol, and whether they can effectively stop viral growth without causing dangerous side effects.

The team would also like to repeat the screens using other coronaviruses – including the first SARS-CoV and MERS viruses – to determine just how universal the new targets they pinpointed are.

Ott and Puschnik agree that the current study was made possible by researchers from many labs coming together without hesitation.

Puschnik has expertise in studying viral host factors, but didn’t have access to a Biosafety Level 3 (BSL-3) lab required to work with SARS-CoV-2.

Ott was spearheading Gladstone’s effort to open such a lab earlier this year and offered to collaborate. Scientists at Synthego provided the engineered cells needed to study the viruses, and Gladstone Senior Investigator Nevan Krogan, Ph.D., helped analyze the results of the CRISPR-Cas9 screen.

“Everybody was completely willing to roll up their sleeves, pool resources, and work together to help contribute to better understanding COVID-19,” says Puschnik.

The paper “Genetic screens identify host factors for SARS-CoV-2 and common cold coronaviruses,” was published online by the journal Cell on December 8, 2020.


Enveloped viruses like the Coronavirus acquire their envelope from the host cell membrane which is a bilayer of phospholipid interspersed with cholesterol molecules and proteins. Viruses enter their host cell by coming in contact with their specific receptors. These specific receptors then undergo conformational changes and induce fusion of the viral envelope with the host cell membrane leading to receptor-mediated endocytosis of the virus.

For Coronaviruses, their specific receptors are Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM-1).1Recent studies have shown that the receptors for the COVID-19 may be Angiotensin- Converting Enzyme (ACE)on the respiratory cells.

Experiments have shown that when cell membranes are depleted of cholesterol in vitro by Methyl beta-cyclodextrin (MβCD) these Coronaviruses are not able to enter the host cell membrane by the process of receptor-mediated endocytosis.1,2It is a well-known fact that cholesterols are vital for the stability and integrity of bilayer phospholipid membrane structure of eukaryotic cells and it may be equally vital for the stabilization of the Corona envelope.

Receptor-mediated  endocytosis  of  enveloped viruses requires a fusion of the envelope (which is also a bilayer of phospholipid) with the host cell membrane, and studies have shown that this process occurs in areas of lipid rafts.1-3

There are multiple areas in the eukaryotic cell membrane with a very high concentration of cholesterols and sphingolipids. These microdomains are known as lipid rafts (because they float like rafts on the water surface). There is strong evidence that enveloped animal viruses select lipid rafts for their assembly and budding.4

What statin does is, it inhibits the Mevalonate pathway that produces cholesterol along with many other very important substances in the body. The mechanism of the Mevalonate pathway exists in almost all the cells of our body because cholesterol is vital for the stability and integrity of the cell membrane as well as intracellular organelles.

The cell membranes and the intracellular organelles derive their cholesterol either from the endogenous source through the Mevalonate pathway or the exogenous source.5The exogenous source are either the cholesterol synthesized by hepatic cells and released in the bloodstream or the cholesterol that comes from the foodstuffs that we ingest.

Because the exogenous cholesterol is insoluble in the water of blood plasma it has to be transported in combination with protein which is soluble in water, so low-density lipoprotein (LDL)transportsthis vital cholesterol to all the cell membranes of our body.

Statin inhibits 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG Co-A reductase), a key enzyme in the Mevalonate pathway resulting in either very low or no production of endogenous cholesterol by the human cells. Homeostasis of cholesterol inside and outside of a cell is tightly regulated by a complex network of intracellular organelles, proteins, and enzymesthat involve in synthesis, import, export, esterification, and metabolism of cholesterol.

In the membrane of Endoplasmic Reticulum (ER), sterol regulatory element-binding proteins (SRE- BP), function as critical regulators of the genes involved in cholesterol uptake and biosynthesis, such as LDL receptors (LDL-R). Cholesterol level in ER acts as a sensor of intracellular cholesterol.

Statins, often prescribed lifelong, creates a chronic deficiency of endogenous cholesterol. The decrease in ER cholesterol induces the translocation of SRE-BP from the ER to the Golgi and then to the nucleus for the transcriptional activation of the target genes, including those involved in cholesterol uptake and biosynthesis6, resulting intoupregulation of LDL-R in the cell membrane.

This constant upregulation of LDL-R in the cell membrane may lead to more cholesterol getting incorporated into the cell membrane through LDL-C from the plasma creating a greater number of lipid raftssuitable for entry of enveloped viruses by receptor-mediated endocytosis.

Experiments have shown that increasing cholesterol concentration in the cell membrane enhances Coronavirus fusion with the cell membrane leading to enhanced infection and depletion of cholesterol from the cell membrane decreases the entry of these viruses into their host cells.1

It is a paradox that statins increase cholesterol in the cell membranes which may enhance Coronavirus infection.

Lipid rafts also appear to be involved in the uptake of the malaria parasite Plasmodium falciparum by erythrocytes, a cell type that is normally incapable of endocytosis or phagocytosis.7There may be a common mechanism by which the anti-malarial drug, chloroquine, which has been proposed for COVID-19 infection works.

Recent epidemiological data show that hypertensives, diabetics, coronary artery disease, and cerebrovascular disease patients are the ones that are developing fulminant COVID-19 disease8 and these are the patients who are most frequently prescribed statins for either primary or secondary prevention of cardiovascular diseases.

According to an article published in the British Medical Journal in 2004 France, Germany, Italy, Spain and the UK of the European continent are the countries with the highest rate of statin intake.9 The USA is another country where statins are extensively prescribed.

These are the parts of the globe with the highest number of Corona infections. Whereas a study published in 2016 in BMJ Open Diabetes Research and Care10 analyzed prescription data in India and found only 50% of patients with diabetes were prescribed with statins much lower compared to western countries. This could partly explain the relatively lower frequency of Corona infection in the Indian subcontinent.

I would thus like to conclude by putting forward my hypothesis that statins create a constant deficiencyof endogenous cholesterol content of cells leading to constant upregulation of LDL-R, in turn leading to excessive incorporation of exogenous cholesterol into the cells and their cell membrane. This process leads to multiple lipid rafts on the cell membrane and enhances accessibility for Coronaviruses. This will need to be tested in Virology labs by looking for whether there is a significant difference in Coronavirus infectivity of cells of patients taking statin and cells of individuals not taking a statin.

REFERENCES

  1. Edward B, Thorp and Thomas M, Gallagher. Requirements for CEACAMs and Cholesterol during Murine Coronavirus Cell Entry. Journal Of Virology 2004; 2682–92. DOI: 10.1128/JVI.78.6.2682-2692.2004
  2. Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. J Clin Invest 2002; 110(5):597-603. DOI: 10.1172/JCI16390
  3. Rawat SS, Viard M, Gallo SA, et al. Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review). Molecular Membrane Biology 2003; 20: 243-54. DOI: 10.1080/0968768031000104944
  4. Bavari S1, Bosio CM, Wiegand E, et al. Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J Exp Med 2002; 195(5):593-602 https://doi.org/10.1084/jem.20011500
  5. Junfang Lyu, Eun Ju Yang and Joong Sup Shim. Cholesterol Trafficking: An Emerging Therapeutic Target for Angiogenesis and Cancer. Cells 2019; 8: 389; doi:10.3390/cells8050389.
  6. Ikonen, E. Cellular cholesterol trafficking and compartmentalization. Nat Rev Mol Cell Biol 2008; 9: 125–38. doi: 10.1038/nrm2336.
  7. Lauer S, VanWye J, Harrison T, et al. Vacuolar uptake of host components, and a role for cholesterol and sphingomyelin in malarial infection. EMBO Journal 2000; 19: 3556-64. https://doi.org/10.1093/emboj/19.14.3556
  8. Fang L, Karakiulakis G, Roth M. Are patients with hypertension and diabetes mellitus at increased risk for COVID-19 infection? Lancet Respir Med 2020 Published Online March 11, 2020. https://doi.org/10.1016/PII
  9. Walley T, Folino-Gallo P, Schwabe U, et al. Variations and increase in use of statins across Europe: data from administrative databases. BMJ 2004;328:386–7.doi:“https://dx.doi. org/10.1136%2Fbmj.328.7436.385”10.1136/bmj.328.7436.385
  10. Gupta R, Lodha S, Sharma KK, et al. Evaluation of statin prescriptions in type 2 diabetes: India Heart Watch-2. BMJ Open Diabetes Research and Care 2016;4: e000275. doi:10.1136/bmjdrc-2016-000275

More information: Cell (2020). DOI: 10.1016/j.cell.2020.12.004 , www.cell.com/cell/fulltext/S0092-8674(20)31626-3

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