Image credit: “Mouse Intestine Tissue Autophagy” by Dimitra Kalamida is licensed under a CC BY 4.0 license.
This article was reviewed by Dr. Phil Lange, one of our subject matter experts.
The paper we’ll be demystifying can be found here, if you’d like to follow along!
TL;DR: Researchers from the Chinese Academy of Sciences found a SARS-CoV-2 protein named ORF3a that impairs the process of autophagy, a form of programmed cell death used keep the rest of the body safe.
Nobody likes clutter. We clean our homes, organize our closets, and delete spam, all in the pursuit of removing clutter and maximizing efficiency.
As it turns out, this practice goes down to the cellular level. Our cells have their own process, named autophagy, that breaks down and recycles any useless or broken cellular components. With “auto” referring to self and “phagy” meaning eating, autophagy is essentially your cells chewing up its unwanted parts to cook up new ones using vesicles, membrane-bound capsules of protein. It also protects the cell, allowing it to destroy any invaders, such as viruses and bacteria.
Ever the thorn in our side, SARS-CoV-2 seems to be able to avoid this fate, persisting against chemical attack like a cellular cockroach. A team of researchers from the Chinese Academy of Sciences endeavored to figure out why, and perhaps how we could circumvent it.
The team began by using HEK293T and HeLa cells, which are human in origin. Genes encoding SARS-CoV-2 proteins were placed in a plasmid vector, a hula-hoop-shaped DNA loop that can be incorporated and used by cells. In this case, the plasmids were given to the HEK293T and HeLa cells, allowing the researchers to examine the effect of individual SARS-CoV-2 proteins on autophagy.
The researchers looked for puncta, clusters of proteins seen as bright, starry points when viewed with specialized microscopy techniques. The puncta were made of two autophagic proteins: LC3 and p62. During autophagy, LC3 binds to autophagosomes, vesicles full of autophagic proteins, and otherwise float diffusely around the cell. p62 also aggregates in early autophagy phases but is later broken down by enzymes. There were significantly more puncta of both proteins in cells that were given plasmids to express the viral genes ORF3a, ORF7a, M and NSP6. ORF3a had the greatest impact on puncta, so the team focused their efforts on ORF3a from then onwards.
To figure out when in the clean-up process ORF3a interferes, the team used an RFP-GFP-LC3 assay, which dyes cellular structures in different colours. They tracked how autophagosomes and amphisomes, a later iteration of autophagosomes, merge with lysosomes, bubbles of acids and digestive enzymes, to form autolysosomes during autophagy. The test dyed the unmerged components yellow, and merged vesicles red. When autophagy was induced, cells with ORF3a-expression had yellow puncta, while those in control cells were red, showing that ORF3a impairs autophagy before these merged vesicles form. Essentially, ORF3a messes with the cell’s ability to destroy invaders by preventing vesicles from becoming an effective weapon.
Image credit: “Stressed Fibroblast” by Glyn Nelson is licensed under a CC BY-NC-SA 2.0 License.
They zeroed in on this phase by examining the levels of certain markers during these stages, finding a dramatic rise in lysosomal fusion markers, ATG14 and STX17, in ORF3a-expressing cells. Levels of WIPI2 also increased, associated with a rise in unfused autophagosomes and their predecessors, isolation membranes (IMs). This indicated that ORF3a acts either at the creation or fusion of autophagosomes.
They found that ORF3a acts at the point of fusion by finding high colocalization between LC3 puncta and late endosomes and low colocalization between the puncta and lysosomes. While endosomes, autophagosomes, and lysosomes normally merge during autophagy, the viral ORF3a protein seems to stop this event in its tracks.
Examining the autophagosome, researchers performed a series of tests for closure, essentially the autophagosome going from an open sac to sealing itself up. Both tests found that LC3 puncta in ORF3a-expressing cells were at their highest on the inner membranes of closed, mature autophagosomes. Researchers then zoomed into the workings of ORF3a-expressing cells using transmission electron microscopy (TEM). The cells were seen to have accumulated autophagosome and amphisomes, but not autolysosomes. These results mean that ORF3a causes autophagosomes and amphisomes to mature and accumulate instead of fusing with lysosomes to continue the process of autophagy, preventing the cell from melting down virus particles.
Next, the researchers tested their ORF3a theory by infecting cells with the entire SARS-CoV-2 virus. They found the same increase in LC3 and p62 puncta in the infected cells. It also seemed to follow the same timeline, with an RFP-GFP-LC3 assay showing the same yellow puncta from unmerged autolysosome components, and colocalization was low between puncta and lysosomes. Using TEM again, the team also found the same accumulation of autophagosome and amphisomes, but a closer look showed that these vesicles were different! They were full of viral RNA and whole SARS-CoV-2 virions. It seems that SARS-CoV-2 prevents lysozyme fusion to provide a cozy place to replicate itself.
To find the exact mechanism, the team checked for co-immunoprecipitation, essentially looking for any proteins stuck to ORF3a. VPS39, VPS41 and VPS33A were strongly co-immunoprecipitated by ORF3a. All three are part of the HOPS complex, a seahorse-shaped assembly of proteins vital in autophagy. A similar, more selective technique called in vitro pulldown found that only VPS39 directly interacted with ORF3a.
The HOPS complex promotes lysosomal fusion, so it would make sense that ORF3a interacts with it. It does so by promoting the formation of another complex: the STX17-SNAP29-VAMP8 SNARE complex. SNAREs, or “SNAp Receptors”, control membrane fusion between vesicles, in this case between lysosomes and autophagosomes. When testing co-immunoprecipitation with the three SNARE components, STX17, SNAP29 and VAMP8, binding of both SNAP29 and VAMP8 to the tagged STX17 was drastically lower in ORF3a-expressing cells, demonstrating that assembly of the SNARE is disrupted by ORF3a.
Lysosomes themselves were also affected by ORF3a. As expected, the number of lysosomes were greater in ORF3a-expressing cells, as they couldn’t fuse with autophagosomes. Additionally, a test of function revealed that lysosomes were not as active in ORF3a-expressing cells. Researchers determined so with DQ-BSA, a part-protein, part-fluorescent molecule that glows when it is broken down by lysosomes, like a glow stick crunched by a partygoer. There were lower levels of fluorescence in ORF3a-expressing cells than in control. This may be due to galectin 3, a protein that damages lysosomes, which was present is higher levels in ORF3a-expressing cells.
Oddly enough, SARS-CoV (note the lack of a “-2”) has none of these evasive abilities. When cells were engineered to express SARS-CoV ORF3a, the researchers couldn’t find any significant changes in LC3 and p62 puncta, GFP-RFP-LC3 assays displayed red puncta, SNAREs were intact, and DQ-BSA showed no change in lysosomal function.
While finding out that SARS-CoV-2 can throw yet another wrench in our cellular gears is disheartening, researchers also discovered a method to reverse the process! O-linked β-N-acetylglucosamine (O-GlcNAc) transferase, as known as OGT, is known to mediate the formation of SNAREs by interacting with one of its three pieces: SNAP29. By removing OGT from ORF3a-expressing cells, the team noted red puncta, meaning successful merging with lysosomes, and greater SNARE complex formation. They basically took the brakes out to compensate for ORF3a slowing autophagy down.
It’ll take a lot more work to adapt the OGT deletion into something medically workable. Knocking out a gene safely in humans carries a significant risk and is yet to be accomplished in a clinical setting. Trying to find a drug that inhibits the OGT protein is a good plan but also a considerable undertaking. Regardless, the team from Chinese Academy of Sciences has given us a clear new target to aim at.
In the meantime, stay safe and wear a mask.
