SARS-CoV-2 News: Phosphatidylinositol Phosphate, Cholesterol And Genes TMEM106B, VAC14, SCAP Used By SARS-CoV-2 To Infect Human Cells
: Scientist from at Gladstone Institutes and the Chan Zuckerberg Biohub, in collaboration with scientists at University of California- San Francisco (UCSF) and Synthego Corporation, have identified critical molecular processes in human cells that coronaviruses including the SARS-CoV-2 virus uses to survive.
According to the study team’s abstract, the Coronaviridae
are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics.
The study team conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E) and glycosaminoglycans (for OC43).
Importantly, the study team identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses.
By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.
The study findings were published in the peer-reviewed journal Cell. https://www.cell.com/cell/fulltext/S0092-8674(20)31626-3
Typically when a coronaviruses 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.
The study team report that targeting these processes with drugs may treat not only COVID-19 infections, but other existing and future coronaviruses.
Co-researcher Dr Melanie Ott, MD, Ph.D., director of the Gladstone Institute of Virology told Thailand Medical News, "What is unique about our study is that we didn't just look at SARS-CoV-2, but other coronaviruses at the same time. This gives us a good idea of drug targets that could broadly suppress many coronaviruses."
Coronaviruses are a large family of viruses that 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.
Dr Andreas Puschnik, Ph.D., a principal investigator at the Chan Zuckerberg Biohub and co-researcher said,"There have now been multiple coronavirus outbreaks, so it's clear this virus family has high pandemic potential. COVID-19 is not the last coronavirus infection we'll be dealing with."
Similar to all viruses, coronaviruses can only grow inside host cells; they rely on the host cell's molecules to multiply. Becau
se of this, the team of researchers wants to target human molecules that the viruses use to survive, rather than components of viruses themselves.
For the study, the researchers 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.
Dr Puschnik explained, "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."
Certain study findings 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.
However other findings were less expected. The study team 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).
Although 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. https://www.thailandmedical.news/news/covid-19-research-study-shows-that-high-density-lipoproteins-hdl-or-good-cholesterol-facilitates-sars-cov-2-cell-entry
In order 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.
Interesting, it was found that 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 study findings suggest that targeting cholesterol or PIP could be a promising strategy to combat multiple coronaviruses.
The screen for SARS-CoV-2 host factors using Huh7.5.1-ACE2- 427 IRES-TMPRSS2 cells identified the known SARS-CoV-2 entry factors, such as ACE2 and heparan sulfate, supporting its validity. Additional notable candidate host factors are TMEM106B, VAC14, cholesterol regulators and subunits of the exocyst. Remarkedly, the majority of these genes were independently identified in a CRISPR screen using Huh7.5 cells, the parental line of the Huh7.5.1 cells used in the study, underscoring the reproducibility and importance of these host factors for SARS-CoV-2 infection. https://www.biorxiv.org/content/10.1101/2020.10.07.326462v1
TMEM106B was additionally found in a third study. https://www.biorxiv.org/content/10.1101/2020.09.28.316281v1
While the exact molecular function of TMEM106B for SARS-CoV-2 infection remains to be determined, its importance was confirmed in several in cell lines (including lung cells).
Dr Ott, who is also a Professor in the Department of Medicine at UCSF said, "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. 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."
However, not all results were the same between the three studied viruses,
Certain human molecules required for SARS-CoV-2 infection weren't needed by the two common cold coronaviruses, and vice versa.
The study findings could help explain what makes SARS-CoV-2 more deadly than the other two viruses.
Additional research and 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.
Dr Ott and Dr Puschnik agree that the current study was made possible by researchers from many labs coming together without hesitation. Dr 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 Dr Nevan Krogan, Ph.D., helped analyze the results of the CRISPR-Cas9 screen.
Dr Puschnik said, "Everybody was completely willing to roll up their sleeves, pool resources, and work together to help contribute to better understanding COVID-19."
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