Thanks and that was very helpful and a good level for me.
I do have two questions, one just more a curiosity than really important.
I had been thinking that the spike binding with the ACE2 protein on the cell wall was actually the entry path—perhaps based on a misconception. My (limited) understanding is that the ACE2 protein actually forms a tunnel through the cell bi layer, so serves as a mechanism to allowing both compounds made by the cell’s machinery to exit the cell for external functions and/or allow the cell to allow something in it needs.
However, your description seem to be describing a case where the two lipid bi-layers really merge. Is that what is really happening, the virus is not really using the spike to actually puncture the human cell. It really only hooks on to the ACE2 protein and then the two walls, the human cell and the virus wrapper, just merge into a common wall? Maybe a bit like what happens internally when an organelle buds off from the smooth ER and then links up with the Golgi apparatus?
That was the curiosity question.
I’m still not clear how how the infection is really working here. The virus binds with the cell and so now the nucleocapsid is now inside the cell but still in its protein wrap so the RNA is not really exposed. How is the RNA exposed?
To your first point: my intuition is that ACE2 is far too small for the genome to pass through itself. ACE2 is an enzyme that’s bound to the membrane—it actually just cleaves angiotensin 1 to angiotensin 2 (hence ‘angiotensin cleavage enzyme, ACE2). It does pass through the membrane, but it’s not really a ‘channel’—it is simply localised to the cell membrane, and acts on substances extracellularly.
Enveloped viruses can enter cells in many ways (principles of virology chapter 5 is really excellent for this, if you’re interested). It seems that SARS-CoV (the original outbreak) enters cells primarily how it is implied above—simple, direct membrane fusion mediated by the ACE2 receptor. There is some speculation that it may under some circumstances be endocytosed (taken into the cell in a separate sphere of membrane) and then break free of the endosome (the bubble) in a pH-dependent way. Obviously this is further complicated by the fact that this is SARS-CoV-2 that we’re really interested in, so I thought it would be best to leave it blank. You’re right in thinking this process is similar to SER budding, though.
To your second point: I wasn’t actually sure! I’ve done some research, but honestly I’m still not as confident about this as about the rest. As far as I can tell, for most viruses nucleocapsid shedding is either mediated by substances or organelles inside the cytoplasm—ribosomes in particular, apparently, bind to the capsids of some viruses and destabilise them—or is part of the process of receptor binding. Some viruses, for instance, seem to be able to leave their nucleocapsid behind with their envelope so it coats one side of the cell membrane.
Sorry I can’t give a better answer, hope it helps!
Brook’s response is pretty good. I can provide a little more detail.
The spike protein of the virus both mediates binding to the ACE2 protein (which allows it to attach to the cell in the first place) and the fusion of the membranes. ACE2 is not involved in the fusion event, that is completely mediated by the spike, all ACE2 does is allow binding that brings the two membranes close together for a long time. The spike has two functional domains, one that is highly variable across coronaviruses that mediates attachment and is the reason different viruses attack different species and cell types, and one that is more highly conserved that triggers the membrane fusion. In order for the fusion to occur, the spike protein has to be processed by a protease that actually cuts the fusion domain apart from the binding domain. This does not make them fall off each other, they remain bound, but they no longer have a continuous backbone. This then allows a re-folding of the protein to a lower-energy state, which drives the fusion of the closely opposed membranes.
It appears from the literature I have found that the re-folding requires an acidic pH, suggesting that fusion probably requires endocytosis of the virus into the lysosome as it goes along for the ride with recycled ACE2 protein. (This is one of several reasons that chloroquine and hydroxychloroquine are being studied for efficacy, they are known to reduce the acidification of this cellular compartment.) They are still arguing about if the current virus has the spike protein cleaved during synthesis, or cleaved by proteases that are present in the lysosome where the ACE2 is recycled. One of the distinguishing characteristics of this virus compared to other coronaviruses is extra cuttable sequences between the two domains allowing more proteases to more easily cut the two domains apart, causing faster and more reliable viral entry. This has been noted in virulent strains of multiple other viruses in the past.
Thanks and that was very helpful and a good level for me.
I do have two questions, one just more a curiosity than really important.
I had been thinking that the spike binding with the ACE2 protein on the cell wall was actually the entry path—perhaps based on a misconception. My (limited) understanding is that the ACE2 protein actually forms a tunnel through the cell bi layer, so serves as a mechanism to allowing both compounds made by the cell’s machinery to exit the cell for external functions and/or allow the cell to allow something in it needs.
However, your description seem to be describing a case where the two lipid bi-layers really merge. Is that what is really happening, the virus is not really using the spike to actually puncture the human cell. It really only hooks on to the ACE2 protein and then the two walls, the human cell and the virus wrapper, just merge into a common wall? Maybe a bit like what happens internally when an organelle buds off from the smooth ER and then links up with the Golgi apparatus?
That was the curiosity question.
I’m still not clear how how the infection is really working here. The virus binds with the cell and so now the nucleocapsid is now inside the cell but still in its protein wrap so the RNA is not really exposed. How is the RNA exposed?
To your first point: my intuition is that ACE2 is far too small for the genome to pass through itself. ACE2 is an enzyme that’s bound to the membrane—it actually just cleaves angiotensin 1 to angiotensin 2 (hence ‘angiotensin cleavage enzyme, ACE2). It does pass through the membrane, but it’s not really a ‘channel’—it is simply localised to the cell membrane, and acts on substances extracellularly.
Enveloped viruses can enter cells in many ways (principles of virology chapter 5 is really excellent for this, if you’re interested). It seems that SARS-CoV (the original outbreak) enters cells primarily how it is implied above—simple, direct membrane fusion mediated by the ACE2 receptor. There is some speculation that it may under some circumstances be endocytosed (taken into the cell in a separate sphere of membrane) and then break free of the endosome (the bubble) in a pH-dependent way. Obviously this is further complicated by the fact that this is SARS-CoV-2 that we’re really interested in, so I thought it would be best to leave it blank. You’re right in thinking this process is similar to SER budding, though.
To your second point: I wasn’t actually sure! I’ve done some research, but honestly I’m still not as confident about this as about the rest. As far as I can tell, for most viruses nucleocapsid shedding is either mediated by substances or organelles inside the cytoplasm—ribosomes in particular, apparently, bind to the capsids of some viruses and destabilise them—or is part of the process of receptor binding. Some viruses, for instance, seem to be able to leave their nucleocapsid behind with their envelope so it coats one side of the cell membrane.
Sorry I can’t give a better answer, hope it helps!
Brook’s response is pretty good. I can provide a little more detail.
The spike protein of the virus both mediates binding to the ACE2 protein (which allows it to attach to the cell in the first place) and the fusion of the membranes. ACE2 is not involved in the fusion event, that is completely mediated by the spike, all ACE2 does is allow binding that brings the two membranes close together for a long time. The spike has two functional domains, one that is highly variable across coronaviruses that mediates attachment and is the reason different viruses attack different species and cell types, and one that is more highly conserved that triggers the membrane fusion. In order for the fusion to occur, the spike protein has to be processed by a protease that actually cuts the fusion domain apart from the binding domain. This does not make them fall off each other, they remain bound, but they no longer have a continuous backbone. This then allows a re-folding of the protein to a lower-energy state, which drives the fusion of the closely opposed membranes.
It appears from the literature I have found that the re-folding requires an acidic pH, suggesting that fusion probably requires endocytosis of the virus into the lysosome as it goes along for the ride with recycled ACE2 protein. (This is one of several reasons that chloroquine and hydroxychloroquine are being studied for efficacy, they are known to reduce the acidification of this cellular compartment.) They are still arguing about if the current virus has the spike protein cleaved during synthesis, or cleaved by proteases that are present in the lysosome where the ACE2 is recycled. One of the distinguishing characteristics of this virus compared to other coronaviruses is extra cuttable sequences between the two domains allowing more proteases to more easily cut the two domains apart, causing faster and more reliable viral entry. This has been noted in virulent strains of multiple other viruses in the past.