A Comprehensive Update on SARS-CoV-2 and COVID-19

Part I

The Biological Properties of SARS-CoV-2

Although, for the sake of illustration, I will sometimes refer to viruses as “live” or “dead,” these are not technically correct descriptors. Viruses are not living beings. Basically, they can be infectious or non-infectious and they can be replicating (producing virus offspring) or dormant [we often refer to viruses that persist in the body, but are not actively replicating as “latent,” an important qualifier as there are times  when the body is stressed and/or the immune system is compromised (such as an overwhelming infection, the administration of chemotherapy, or the administration of steroids) that these viruses can be reactivated and cause ongoing infection and inflammation].

Another thing that I will write or say that is not correct, but is a useful concept to explain some of the biological characteristics of viruses, is something that implies that the viruses have intention. For example, I have said things like, “viruses just want to make more viruses” as if viruses have some degree of consciousness or goals. They do not. Their activity is all in accordance with their collection of genetic information as opposed to any degree of cognition. Nevertheless, I find that framing virus behavior in this way can be useful in describing and assisting those who are trying to understand the biological operations of the virus.

At the most basic level, viruses are little more than a collection of genetic material that provides the blueprint for reproducing copies of its genetic code and producing proteins that when assembled can create new viruses surrounded by a protective protein coat. However, because viruses do not have everything they need in order to use their genetic code to produce new proteins that are needed to form new viruses, they must infect cells and take over their cellular “machinery” and proteins to make their own (more on this below).

So, the first distinction we can make when categorizing viruses is whether that genetic material is DNA or RNA allowing us to put viruses in the categories of DNA viruses and RNA viruses. This distinction alone will give rise to many differences between these two groups of viruses. DNA viruses include such viruses as herpesviruses, poxviruses, and papillomaviruses. RNA viruses include the coronaviruses, the rhinovirus (one of the common cold viruses), and the hepatitis A and C viruses.

First, the size of the genomes (the set of genes or genetic material) is larger in DNA viruses compared to RNA viruses. Further, as we saw with SARS-CoV-2 (an RNA virus), the rate of mutations (these are changes to the individual nucleotide bases that make up nucleic acids (either DNA or RNA) and that in groups of three provide the code for an amino acid to be added to other amino acids to make a new protein during the transcription and translation process (see below), most often occurring from an error during the copying of the genetic material to make new viruses) is much faster in RNA viruses than in DNA viruses.

When a DNA virus is making new viruses, that process generally begins in the nucleus of the cell (think of the yolk when you crack an egg open to put in a skillet and the yolk remains intact) whereas the replication of RNA viruses most often occurs in the cytoplasm of the cell (think of the white part of that cracked egg as the egg is cooked or fried).

Our cells have defense mechanisms to try to keep viruses from infecting them, and then have additional defenses inside the cell that try to make it harder for the virus to replicate within the cytoplasm (where all the parts are located that are used normally by the cell to make its own proteins, but are hijacked by viruses to make them preferentially make viral proteins that can then be assembled to make new viruses) and even harder for a virus to get into the nucleus.

As I mentioned above, the virus’ genetic material (whether DNA or RNA) is surrounded by a protein coat, because the genetic material is very sensitive and vulnerable to temperature and chemicals. That protein coat is called the capsid.

As I mentioned above, the genetic material (nucleic acid that is either DNA or RNA) is a series of nucleotide bases that are aligned in groups of three with each group being referred to as a codon (pronounced “code on”). Codons, determined by the exact type and order of nucleotide bases) specify which amino acid the cell is to add to the protein being made. The types, order and number of amino acids then determines what the protein is and what its function will be.

The genetic material (nucleic acid) surrounded by the protein coating (capsid) together is referred to as the nucleocapsid. Some, but not all, viruses will have an additional surrounding layer comprised of fats and protein called lipoproteins, and this additional outside layer is referred to as the envelope. The SARS-CoV-2 virus is one that does have this additional layer, and therefore we refer to it as an enveloped virus.

Figure 1.

So, we haven’t covered all of the above structures yet, but let’s just get an image of the virus in our minds. Figure 1 is a depiction of the SARS-CoV-2 virus. So, remember, it is an RNA virus, so we see the reference to RNA in the center of the picture that removes a portion of the outer virus allowing us to peer inside. Recall that RNA is made up of nucleotide bases that in groups of three are codons that tell the infected cell what amino acids it is to add and in what order to make proteins that the virus can’t make by itself, but can make the cell produce for making new viruses. Then, the RNA is covered with a protective protein and that protein with the RNA inside is referred to as the capsid and the protective protein is called the nucleocapsid protein for which we simply refer to as N or the N-protein.

As I mentioned, some, but not all, viruses have an additional lining over the capsid called an envelope. The envelope is made up of lipoproteins (fats [lipids] plus protein = lipoproteins) and you can see on the diagram of the SARS-CoV-2 virus the indication of the envelope. The envelope protein is sometimes simply referred to as E or the E-protein.

You will recall that one of the differences between DNA viruses and RNA viruses is the size of their genomes (the genetic material). The larger the genetic material (RNA or DNA) the more types of different proteins that genetic material can code for. The simplest virus we know of (a plant virus) only codes for 1 protein. Many viruses capable of infecting humans and causing disease code for 5 – 10 proteins. However, some of the DNA viruses I mentioned (poxviruses and herpesviruses) can code for up to 200 proteins. The SARS-CoV-2 virus RNA codes for 29 proteins, which is a lot for an RNA virus.

These 29 proteins can be placed into 3 categories that are useful for explaining how the virus does what it does. Those three categories are: (1) structural proteins (these are the proteins that form the structure of the virus, some of which I already pointed out to you – e.g., the N and E- proteins), (2) non-structural proteins, and (3) accessory proteins.

We are now ready to update you about many of the things we have learned about how this SARS-CoV-2 virus is structured, how it infects cells, how it replicates (makes new viruses), how the virus evolves to evade some of our immune responses and how it causes disease. On the basis of some very beneficial (to the virus, not to humans) several SARS-CoV-2 variants of concern acquired increased fitness through improved human-to-human transmission, evasion of previously acquired immunity, or changes in disease virulence over the course of the pandemic.

Here we go:

As already pointed out, this coronavirus is an RNA virus and has an envelope. SARS-CoV-2 belongs to a family of coronaviruses, which are so named because of the distinctive surface structural feature that resembles a solar corona (or crown) due to the presence of spike proteins (S-proteins). Take a look at figure 1 again to see these proteins illustrated.

As an RNA virus (opposed to DNA viruses), SARS-CoV-2 already replicates (reproduces itself) faster, and is more error-prone in translating its genetic code resulting in a higher mutation rate than what we see in DNA viruses. However, we have seen that even among RNA viruses, SARS-CoV-2 has higher mutation rates, and very high rates of recombination.

Recombination occurs when part of the genetic material of one virus gets swapped with another virus, so that instead of a point mutation, where upon sequencing, we can pinpoint the place in the RNA where a nucleotide base got dropped or substituted with a different one when a new copy was being made, we can see a length of RNA that is identifiable as being from a specific variant of SARS-CoV-2 combined with another stretch of RNA that is clearly from a different variant. Re-combinations can occur when a person is infected with two different variants at the same time or when a person was first infected with one variant, but failed to clear that virus (more on this in a later blog post) and then becomes infected with a new variant and the exchange genetic material when replicating. We think that this same process can occur in some of the animals that humans have infected. As an aside, this current pandemic is notably unusual for how long this virus has been able to continuously transmit, in large part due to its high rate of mutations that have allowed it to develop a certain degree of immune escape and for the fact that this is the first time that the predominant circulating variants have been recombinants going on two years now.

Mutations can advantage or disadvantage the virus relative to what we refer to as fitness or viral fitness – the ability of a variant or strain of virus to infect and to grow in proportion to a reference virus such that the variant or strain can predominate in transmission over the reference virus. A mutation (or set of mutations) can weaken the transmissibility and the infectivity of the virus to the extent that the virus strain or variant essentially dies out. A mutation (or a set of mutations) can advantage a strain or variant such that it undergoes rapid growth in transmission overtaking other viruses. However, most often, a mutation (or set of mutations) involves a trade-off. For example, an advantage in transmissibility through stronger ACE-2 receptor binding may come at a loss of immune escape or vice versa.

I remember well when we noted the first major change in the SARS-CoV-2 virus in the summer of 2020 that involved just a single substitution mutation (one nucleotide substituted for another) that we referred to as D614G, an indication of the nucleotides swapped for each other and the point along the genetic code of the RNA at which the substitution occurred. Just this one substitution resulted in a 20% growth advantage (increase in fitness) relative to the original (wild-type) virus. Then at the end of 2020, we began seeing variants emerging with much larger numbers of mutations. Additionally, each subsequent variant of concern (alpha, beta, gamma, delta, and omicron) from 2021 on gained transmission (fitness) advantages over the previous one and would become dominant regionally or globally eventually displacing the prior variant. Alpha (beginning of 2021) and Delta (fall/winter 2021) acquired mutations that promoted cleavage of the spike protein (see below) and as a consequence increased receptor binding and transmissibility conferring a 65% growth advantage over D614G for alpha and a 55% growth advantage for delta over beta and gamma. With each successive variant of concern, fitness was optimized by increasing transmissibility through improved cleavage, ACE-2 binding and infectivity. However, starting with omicron at the beginning of 2022, its fitness advantage was not tied to optimized cleavage of the spike protein, but rather significant immune evasion where neutralizing antibodies were far less effective in preventing infection.

Of the 29 proteins coded for by the SARS-CoV-2 virus, 4 are the core structural proteins that we illustrated in figure 1 – the nucleocapsid (N) protein, the spike (S) protein, the envelope (E) protein and the membrane (M) protein. These proteins are responsible for the assembly of proteins into new viruses and for the suppression of the host (whatever organism the virus is infecting) immune response.

The RNA also codes for 16 non-structural proteins, which play a role in viral replication and transcription (the process of making a copy of the RNA into what we call messenger RNA that can then be delivered to the protein making factory of the cell in the cytoplasm called ribosomes, where the mRNA is translated by reading the three-nucleotide sequences [codons] to provide the instructions for the amino acids that are to be assembled in order to produce viral proteins, such as the N, S, E and M proteins). They also are involved in virus-host interactions within the host cell.

The function of accessory proteins has not yet been fully characterized; however, these proteins can be involved in regulating viral infection, and many of these proteins are linked to immune evasion.

The S-protein (spike) is the only component of the virus given in the three US-approved or authorized vaccines. Of course, if you get infected, your body will be exposed to all of the proteins. The S-protein was selected because it is target-rich for antibodies, and because binding of the virus to a cell receptor in order to allow entry of the virus into the cell is through the receptor binding domain (RBD) within the spike protein, some of these antibodies attach to the virus in the area of the RBD thus preventing the virus from attaching to the receptor and from entering (infecting) the cell, and thus, we refer to these antibodies as neutralizing antibodies.

It is important to note that the virus configuration is not always the same. When enough mutations occur, and especially, when they appear in certain locations, they actually alter the shape of the virus. When the shape changes, targets for antibodies to attach on the virus may be no longer accessible.

Proteins normally fold upon themselves for reasons that we don’t need to get into for you to understand the main points. Think about origami. So, imagine that you take the piece of paper before it is folded and place six red colored dots with a marker at various places on the paper. Those dots will be very visible to all while the paper is lying flat on the table. Once the paper is folded into the shape of an animal or bird, perhaps only one or two of those dots will be visible. Now, let’s change the situation slightly such that instead of six red dots, there are three red dots within a black circle and three green dots outside of the black circle. The paper represents the spike protein and the origami animal or bird represents the protein as it is folded in its natural state. The black circle represents the receptor binding domain and the red dots represent sites where antibodies can attach and block the S-protein from binding to the receptors on the host cell (thus, these are neutralizing antibodies). The green dots also represent sites where antibodies can attach, but when they bind to the green dots they do not interfere with the S-protein binding to the host cell receptor and thus do not prevent the entry of the virus into the cell (thus, these are “binding antibodies,” but not “neutralizing antibodies.”)

What has happened as the SARS-CoV-2 virus has mutated or undergone recombination, or both, is that when there are enough mutations or mutations just in the right place, it has caused the origami bird to change form into a bear, or similar analogy. As you look at the origami animal, you might see one or two of the red dots with the first animal, but perhaps not see any of the red dots as the paper is morphed to make a different animal shape. Of course, when these protein shape changes occur in the actual SARS-CoV-2 virus, they can be either helpful to the virus (hiding the red dots) or they can make the virus more vulnerable (make the red dots more visible and accessible to the neutralizing antibodies). Those that become more vulnerable will have less ability to reinfect a population of people who have developed neutralizing antibodies through vaccination, infection or both, while those that develop confirmational changes that make existing antibodies less effective will gain a competitive advantage, referred to as increased fitness. This is one of the reasons that some variants take off and become the dominant circulating variant for a period of time, while other variants may appear alarming based on the large number of mutations acquired, yet the mutations may result in less favorable configurations with neutralizing antibody sites more exposed. There are other factors that determine a virus’ fitness, and we will discuss those below.

Mutations and re-combinations are not the only reasons for the spike protein to change shape. There is a pre-fusion conformation (pre-fusion being a reference to the virus prior to attaching to the cell receptor – the primary host cell receptor being the ACE-2 protein). Just prior to the SARS-CoV-2 virus binding with the receptor, a certain enzyme (a special type of protein that can promote chemical reactions) on the cell surface can cleave the spike protein in two parts, yielding S1 and S2 subunits of the S protein that when cleaved (split) open up to make the receptor binding site more accessible to bind with the receptor. Even though the receptor binding domain is on S1, when S2 is cleaved from S1, it undergoes conformational change that promotes viral-host cell membrane fusion and the subsequent release of the viral RNA into the cytoplasm.

Once the virus binds to the receptor, it prompts the cell to engulf the virus and carry it from outside the cell to the inside of the cell (cytoplasm). Once inside the cell, the virus commandeers the cell’s protein making factory. The RNA of the virus is copied into messenger RNA (mRNA). The mRNA then travels to a cell organelle called the ribosome. Think of the ribosome like a sewing machine. The mRNA carries the instructions for which amino acids and in what order are to be strung together to form a viral protein. As these new proteins are sewn together, they then move to other organelles in the cytoplasm referred to as endoplasmic reticulum and Golgi bodies. These are the assembly plants that take the various N-, S-, E-, M- and other proteins from all the different sewing machines (ribosomes) and assembles them together to form new virus offspring.

Once the new viruses are made, they are expelled to outside the cell by the reverse process of how the virus got into the cell, this reverse process being called exocytosis. These new viruses now may infect other cells.

Figure 2.

Figure 2 is an illustration of this process. It starts with the spike (S) protein of the virus approaching the ACE-2 receptor on a host cell. The enzyme that can cleave the S protein into two parts (S1 and S2) is on the cell surface of lung cells and vascular cells. It is called TMPRSS2 (transmembrane serine protease cell surface protein 2).  Other surface protein enzymes may accomplish cleavage on other types of cells, including cathepsin B and cathepsin L. However, TMPRSS2 cleavage allows for the cell surface entry process depicted above, which is simpler and faster, whereas the cathepsin activation requires the more involved process of cell membrane fusion followed by endosomal entry, which is somewhat like the membrane engulfing the virus and carrying it inside the cell.

The receptor binding domain of the virus is on S1. This promotes binding of the virus with the receptor, which in turn triggers the process of virus entry into the cell. All of this is represented by number 1 in the figure at the top.

Once inside the cell, the RNA of the virus is transcribed into mRNA, the mRNA is used to give the instructions for how to make all the component proteins of the virus (N, S, E, and M as well as other non-structural and accessory proteins) (steps 2 – 6 in the figure above) and the new proteins are packaged and assembled by the endoplasmic reticulum and Golgi complex (steps 7 and 8) and the new virus progeny are then exited out of the cell through exocytosis (steps 9 and 10) where they can now infect other cells and create more virus (the amount of virus in an infected host is referred to as the viral load).

However, this process is a bit risky for the new virus progeny as antibodies cannot get inside cells, but if the person has preexisting antibodies through vaccination or prior infection and these antibodies have neutralizing capabilities, they can bind the new viruses as they are expelled from the cells and prevent them from entering and infecting new cells. This will lower the viral load, which has been correlated with disease severity and is likely why vaccination, while not preventing initial infection, prevented many people from developing severe disease by reducing the ability of the virus to invade cells and make more virus.

In the absence of prior vaccination or infection, it can take as long as 7 – 10 days for the body to make antibodies that are of high quality that can neutralize virus. This gives the virus a big head start, and thus, many more unvaccinated individuals developed severe disease compared to vaccinated individuals. One therapy that could be used early on in the pandemic before the virus developed strong immune evasion to them was monoclonal antibodies (antibodies that were produced by pharmaceutical companies that were designed to specifically target these receptor binding domain sites on S1 that we knew would be neutralizing in their effect).

We saw that with certain variants, most notably Delta back in the fall and winter of 2021, that the virus acquired increased fusogenicity – the ability to cause infected cells to fuse together with their neighboring cells. This greatly thwarted the defense of neutralizing antibodies (remember, antibodies cannot enter cells, they merely circulate in the extracellular space) because instead of the virus progeny undergoing exocytosis into the extracellular space where the antibodies could bind to them, they could merely pass from cell to cell directly once they were fused. This caused very serious disease in the lungs.

If antibodies cannot neutralize the virus, then we have to rely on so-called T-cells, a type of white blood cell that can recognize markers on the cell that indicate that the cell is infected, triggering the T-cells to release chemicals that perforate the lining of the cells and destroy the cell, while also destroying the virus. This is great in that it kills the virus, but it can also be bad in that it causes destruction to cells and tissues, which if significant enough, can leave the patient with impaired functioning of that tissue (in this case, the lungs); a process that is referred to as immunopathology. As another element of immune evasion, we saw that some SARS-CoV-2 variants also developed the ability to impair the ability of cells to put up the marker on their cell surface that signals the T-cells to destroy the cell because it is infected.

The complex immune response will be a common theme as we explore the health consequences of COVID-19. Without our immune systems, we would be vulnerable to every pathogen out there and likely die at a very early age. However, the immune system works in a very careful balanced way. Think of it as a scale. If we tilt the scale by weakening the immune response (age, medications, disease), then infections that most people can tolerate well become serious infections because our immune system does not control the infection. But, if we tilt the scale the other way with an overly exuberant immune response, the excessive release of chemical messengers from immune cells can be like dropping a bomb on a building where there are many people merely to kill a single shooter. This is often what happened when we saw severe disease in older individuals and even younger adults with underlying medical conditions in roughly the second week of their illness. We also see this in children and in some adults who develop multisystem inflammatory syndrome (MIS) weeks to months after their infection (possibly triggered by viral persistence that results in ongoing inflammation and overstimulation of the immune system).

While not certain what many of the other proteins’ roles were at the onset of the pandemic, we have learned a lot. The E (envelope) protein plays an important role in the release of the viral genome once the virus enters the cell, as well as a role in the viral assembly of the various proteins to form new viruses.

Not surprising is the role of the N (nucleocapsid) protein to protect the viral genome. But, it appears that the N protein may also be able to antagonize a number of the host immune responses.

Unfortunately, because we have done little globally to prevent or diminish the transmission of this virus, it has had millions of hosts to serve as living laboratories to mutate and recombine and then for those mutations that enhanced the fitness of the virus to take over and cause new waves of infection. These evolutions of the virus have helped the virus to achieve improved host recognition and cellular entry, genome replication, assembly and release of progeny viruses, and host immune surveillance evasion.

SARS-CoV-2 infection damages mitochondria, organelles within cells that generate energy for the cell. This damage has been implicated in Long COVID, which will be discussed in later blog posts. Mitochondria are unique among organelles of cells in that they contain maternal DNA. When the mitochondria are damaged, they leak this DNA into the cytoplasm, which triggers an immune response within the cell that is aimed at both DNA and RNA viruses, with the presumption that the leak must be caused by a viral infection. The SARS-CoV-2 has evolved proteins that help fight this attack on it.

SARS-CoV-2 is also able to downregulate (turn off or at least turn down) the cellular gene that makes the signal that I mentioned above that tells cytotoxic T-cells that the cell is infected and they should destroy the cell to kill the virus.

Interferon is one of the body’s early responses to a viral infection that stimulates certain cells of the innate immune system (the first responders that can respond to an invader without prior exposure to the invader that is needed for antibodies and T-cells), including natural killer cells that can kill cells that are infected by the virus. Interferons also serve to warn cells to increase their defenses, which makes it harder for viruses to enter cells. Part of the immune evasion defense SARS-CoV-2 has evolved is the ability of certain of its proteins to inhibit this interferon defense.

We have learned a lot about this virus, but there remains much to learn. In subsequent parts of this blog series, we will review what we have learned about its transmission, its pathogenesis (how it causes disease), and I particularly want to elaborate on comments that I have been making for years now as to “signals” that I see about the potential for long-term health consequences in some persons from COVID-19, and particularly from repeated reinfections, and why I think the public’s, and especially the health professions’ complacency about COVID-19 may be foolish. We have much more to cover in future blog posts.

References:

1. Basic Virology, 4^th ed. Hewett, M.J., Camerini D., and Bloom, D.C.
2. Fenner and White’s Medical Virology, 5^th ed. Burrell, C.J., Howard, C.R., and Murphy, F.A.
3. Virology, Transmission and Pathogenesis of SARS-CoV-2 https://www.bmj.com/content/371/bmj.m3862.
4. Structural biology of SARS-CoV-2 and implications for therapeutic development https://doi.org/10.1038/s41579-021-00630-8.
5.  SARS-CoV-2 biology and host interactions SARS-CoV-2 biology and host interactions | Nature Reviews Microbiology.
6. SARS-CoV-2 variant biology: immune escape, transmission and fitness. https://doi.org/10.1038/s41579-022-00841-7