COVID-19 Latest: Yet Another Way SARS-CoV-2 Messes Up Host Immune System By Targeting ULK1 Kinase Enzymes To Disrupt Cellular Autophagy!

COVID-19 Latest: Researchers from a Canada in a new study have found that the SARS-CoV-2 has yet another manner of messing up the human host immune system by using its viral papain proteases to target the ULK1 kinase enzymes which leads to disruption of cellular autophagy, a critical component of the immune function.


 
The study findings were published on a preprint server but are currently being peer-reviewed. https://www.biorxiv.org/content/10.1101/2020.10.23.353219v1
 
The current COVID-19 pandemic sweeping through the world has led to enormous efforts to understand the SARS-CoV-2 coronavirus that causes the COVID-19 disease and its pathogenesis.
 
In the immune system, autophagy is a cellular mechanism that removes damaged or unnecessary cell components, allowing the body to get rid of these cells and, in addition, acting against infecting pathogens. The process is highly regulated and is affected by different factors like oxidative stress, deficiency or excess of nutrients, and viral infection.
 
A protein kinase is a kinase enzyme that modifies other proteins by chemically adding phosphate groups to them (phosphorylation). Phosphorylation usually results in a functional change of the target protein (substrate) by changing enzyme activity, cellular location, or association with other proteins. The human genome contains about 500 protein kinase genes and they constitute about 2% of all human genes. Protein kinases are also found in bacteria and plants. Up to 30% of all human proteins may be modified by kinase activity, and kinases are known to regulate the majority of cellular pathways, especially those involved in signal transduction.
 
It is known that the serine/threonine unc-51-like kinase (ULK1) plays a critical role in autophagy and is believed to be involved in many diseases like cancer, neurodegeneration, and inflammatory disorders. ULK1 has an N-terminal kinase domain and C-terminal early autophagy targeting domain that helps ULK1 interact with its various substrates.https://pubmed.ncbi.nlm.nih.gov/23685627/

It is known that positive-sense RNA viruses like betacoronovirus use autophagy to utilize cellular double-membrane vesicles as surfaces for RNA synthesis, but how they subvert autophagy components for their use is not well understood till now.
 
The study team from the University of British Columbia and St. Paul’s Hospital-Vancouver, discovered in this study that the betacoronavirus-encoded papain-like protease disrupts autophagy, partly by targeting ULK1.
 
In order to understand the regulation and function of ULK1 during betacoronavirus infection, the researchers used mouse hepatitis virus A59 as a model and an infected murine fibroblast cell line 17C11.
 
The study team observed loss of ULK1 starting about 12 hours after infection using anti-ULK1 antibodies. A previous study also found reduced ULK1 levels during porcine betacoronavirus infection. Using polyclonal antibodies, they observed a potential cleavage fragment that was not detected using monoclonal antibo dies.
 
The phenomena prompted the scientists to look into whether betacoronavirus proteases might be responsible. Of the two proteases found in the viruses, the team made a construct expressing the papain-like cysteine protease of SARS-CoV-2, which has 63% similarity to that of the mouse virus.
 
In order to determine if the papain protease targets ULK1, they used HEK293T cells. Again, the studyt eam found reduced ULK1 levels and a smaller molecular weight cleaved fragment.
 
The Canadian researchers also tested to see if the other protease in betacoronaviruses, a chymotrypsin-like cysteine protease, was responsible for the reduction in ULK1 levels or cleavage using an in vitro assay. They found this protease was not involved in ULK1 cleavage, suggesting the effect of only the papain protease.
 
Subsequently, the study team determined the location on ULK1, where the papain protease acts. Using previous studies, they identified two potential cleavage sites. The team mutated the potential cleavage sites, and transfected wild-type or mutant ULK1 along with control or the papain protease construct into HEK293T cells.
 
The study team found cleavage in the wild-type and G531 mutant ULK1, but not in the G499 mutant. This suggests the protease cleaves ULK1 after G499, separating the N-terminal kinase domain from the C-terminal domain that binds to substrates.
 
In addition, the scientists found that the cleavage results in reduced co-immunoprecipitation of ULK1 with ATG13, a molecule that binds to ULK1 and a protein in the autophagy-initiating ULK1 complex, suggesting cleavage of ULK1 results in reduced binding. The ULK1 complex is required for starvation-induced autophagy.
 
Significantly, the cleavage resulted in reduced autophagic degradation, and they found that cells expressing the papain protease did not initiate autophagy.
 
Importantly, when a ULK1 inhibitor was added to cells infected with the mouse virus, the team found that viral RNA replication was reduced significantly. Furthermore, a non-cleavable ULK1 mutant suppressed viral replication compared to the wild-type ULK1. This indicates betacoronaviruses need ULK1 before cleavage for replication.
 
Past studies on SARS-CoV reported deubiquitinase activity by recognizing the LXGG motif. The cleavage site of the ULK1 in SARS-CoV-2 after glycine is also similar. Targeting ULK1 and other host proteins with LXGG could be another new mechanism that betacoronaviruses use to disrupt cellular autophagy.
 
It was found that ULK1 levels started decreasing later in the mouse virus infection, coinciding with the observation of cleavage fragments. This suggests ULK1 function, although necessary for viral replication in the initial stages of infection, might be dispensable in later stages of viral infection, or cleavage may have new functions that serve the virus. Non-cleavage of ULK1 showing low virus replication suggests continued ULK1 activity throughout the infection may be bad for the virus.
 
Hence the study provides new mechanisms by which the SARS-CoV-2 virus uses papain protease to disrupt cellular autophagy.

It should be noted that one effort toward treatment has been the repurposing of autophagy-modulating drugs like chloroquine and hydroxychloroquine. This failed as at the time as no researchers knew about the viral papain proteases targeting the ULK1 kinases.
 
Thailand Medical News is researching the usage of plant based phenolic phytochemicals from seeds of  the Psoralea corylifolia plant,  lycorine from the Amaryllidaceae species and cinnamic amides from Tribulus terrestris as potential viral papain proteases inhibitors for usage in our various therapeutic tea blends. https://www.thailandmedical.news/news/new-therapeutic-teas-
 
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