Experiments carried out in kidney cells seem to show that the D614G mutation helps stabilize the spike protein, and might be responsible for its increase throughout the globe. These experiments were not carried out in settings similar to an actual COVID infection, and some results contradict previous work on the subject. Therefore, the results should be viewed with a healthy dose of skepticism.
- The D614G mutation does not seem to directly affect the binding of the spike protein to human cells (the strength of binding between the original form and the D614G version seems to be similar).
- The spike protein consists of 2 segments. The first is responsible for attaching the virus to the human lung cells, and the second segment is responsible for taking the virus inside the human cells. The two parts are connected together by a thread that can be cut by a human protein. The D614G mutation stabilizes the interaction between the two parts, and helps the spike protein not fall apart even after the two parts’ connecting threads are cut.
- This stabilization seems to help the virus stick to human cells and infect them. However, this experiment was carried out under conditions that are very different than would happen under a normal COVID infection, with some essential pieces missing. These results need to be replicated under closer conditions to COVID infection (using real viral particles, and maybe human lung cells), before the results can be accepted.
- While it seems like this mutation under lab settings can increase infection rate, the mechanism of this increase remains very much clouded with multiple studies proposing different or opposing mechanisms.
Why is this important?
Mutations in viruses are one way for viruses to survive, evolve and continue infecting hosts. This is why we need a new flu vaccine every year. Keeping up to date with the mutations that are appearing in COVID is very important both in terms of preparing for changes in the pandemic, as well as making sure our therapeutics and preventatives are as effective against the virus with these new mutations as they were against the original form of the virus. The D614G mutant was identified at the end of April. This mutation seems to spread much more quickly than the original version of the virus, and by April had become the dominant form of the virus. The original study was not able to show any clinical differences between patients with the different forms of the virus in terms of length of hospital stay or mortality. Nevertheless, such a rapid rate of growth for the mutant form was very unusual and required further investigation.
The current work tries to see if they can show, under lab settings, if there is a difference between the two forms of the virus. As working with the virus carries a lot of risk and requires more sophisticated labs, most researchers work on human viruses using what are known as Virus Like Particles (VLPs) or pseudoviruses (PVs). In the case of COVID, the researchers take an unrelated virus (that is often not harmful to humans) and add the protein of interest from COVID, or they create a virus particle that lacks essential proteins, which means it is not able to replicate in human cells. The danger of this approach is that if the new conditions are too far away from the normal conditions of COVID infection, the results from these experiments might not be the same as the results if the experiments were carried out with the real virus on the correct cells.
What did the study do?
Chose 2 forms of pseudoviruses to study the D614G mutant version of spike protein
The researchers tested their hypothesis that the D614G mutant is more transmittable by using a cellular model. As the virus is dangerous and you need special training and equipment to handle it, researchers often use parts of the virus that they want to study and place them in an unrelated virus (usually harmless to humans), which allows them to study the virus while reducing the risk of working with it.
Used two different cellular models
The first model that the researchers used added the COVID spike protein to a mouse virus that cannot normally infect human cells. They used this modified virus to infect human kidney cells. The protein on lung cells that the spike protein recognizes is not present on those human kidney cells, so those cells also had to be modified to include this protein on their surface. Now, the researchers had a model that they could use. The second cellular model that they used was made up of the COVID virus with important parts missing, which made it no longer risky to humans.
Models might be too simplistic
Certain proteases (such as furin and TMPRSS2), which are enzymes that breakdown proteins, have shown to be very important for virus entry and infection may be missing in these models. For example, multiple peer-reviewed articles have stated that blocking these proteases leads to much lower virus infectivity. However, one of the controls in this paper is removal of the part of the virus that protease furin can cut, and the researchers showed that not only did the virus not have lower infectivity, but it had a higher level of infectivity. This likely points to the fact that the virus and the infection can behave differently in this simplified lab model as compared to the infection out in the world.
Measured the outcome of mutation using their simplified model
The researchers then used their model to track the effects of the D614G mutation using a variety of lab-based experiments. As long as we accept the context of these experiments (carried out in simplified model systems), we can judge the validity of the experiments themselves.
Mutation increased rate of infection in lab setting by ~9-fold
The experiments that were designed and carried out seem to show that the D614G mutation increased how efficiently the virus particles are able to infect human cells expressing the needed protein for the virus to recognize.
D614G mutation seems to reduce “S1-shedding”
S1-shedding refers to the first part of the spike protein (the part responsible for binding to the human cell) can fall away. This process seems to be essential for virus entry into human cells, however, it is possible that if this is done at the wrong time, the virus can lose its ability to target the human cells. Think of it like making pasta. You have to drain the water after the pasta is boiled to have a good meal. However, if you drain the water before you put the pasta in it, you will not have a good dish waiting for you. This is similar. While the shedding of the S1 part seems very important for the virus to enter the human cells, this has to happen after the S1 part has performed its duty in attaching the virus to the human cells. It looks like the D614G mutation strengthens the interaction between the 2 parts of the spike protein, and allows it to hold onto this part longer. This could be good or bad (avoids throwing the pasta water before you add the pasta, but might make it harder to drain the pasta after it is cooked).
Virus neutralized by antibodies from recovered patients
This was an area of concern specifically with regards to immunity and vaccine development. There was, at least initially, a concern that the patients who had gotten the original form of the virus may not be protected against the new mutant version (D614G). This paper, along with another more recent one, provide evidence that patients who have gotten any form of COVID seem to have neutralizing antibodies that do not distinguish between the two forms of the virus (in the second source, 38/41 patients showed comparable levels of neutralization of infection for either form). The caveat here is that the built up immunity in these cases was due to the patients getting and subsequently recovering from COVID, whereas this can still be an area of concern when developing vaccines against the virus.
D614G mutation can lead to an increase in infectivity
This paper, alongside a slightly more recent pre-print, both point towards an evolutionary advantage provided to the COVID virus by the D614G mutation, when tested on a simplified model. Both sets of researchers used a similar model and got very similar results. While the results they got seem valid and scientifically sound, the main question with regards to this work is how much will these results in a simplified model match the course of the infection in real humans. And while both of these works add to our knowledge about this mutation and its possible effects, more broad generalized conclusions should be viewed rather skeptically. It is absolutely possible that this virus can lead to an increased efficiency of infections in the real world, however we cannot base that conclusion solely on these two papers.
Mutation can help two parts of the spike protein interact more strongly
The original study that identified the D614G mutation proposed that this mutation might weaken the interaction between the two parts of the spike protein, and that might lead to an increase in infection efficiency. It is clear from the results from these two new pre-prints that the original proposal is wrong, and that the interaction between the two parts of the spike protein are actually strengthened by this mutation. Based on the strength of the evidence, this conclusion is likely to hold even in the case of the virus during a real human infection.
Patients who recovered seem to have neutralizing antibodies regardless of this mutation
Both studies show that antibodies from the blood of patients who recovered from COVID are able to stop the infection in the simplified lab setting for either form of the virus.
Not enough evidence to conclude that the D614G mutation can lead to an increase in infectivity
While the researchers provide ample evidence that the D614G mutation can increase the efficiency of infection in a simplified model under lab settings, it is unclear if these results can be translated directly to the case of the virus in the real world.