ABSTRACT
The SARS-CoV-2 virus is a newly emerged coronavirus that was first detected in the human population in December 2019 and became ubiquitous within months, infecting over 200 million people to date. The virus has undergone many mutations during the RNA replication process, some of which have led to advantageous characteristics such as higher transmissibility, thus increasing infection rates. Scientists and researchers have thoroughly studied the most prevalent variants in order to track, manage and predict virus behavior. The majority of mutations in the most widespread variants lie in small substitutions of nucleotides that code for the spike proteins on the surface of the virus. These spike proteins are the first point of host cell contact and entry and are therefore essential for successful viral replication and transmission. In more recent months, the newly emerging Delta variant with increased ability to attach to and fuse with host cells, demonstrates the continual evolutionary developments of this virus. These findings provide necessary information to develop possible interventions, such as vaccines, as well as a better understanding overall of how the virus evolves and develops new characteristics.
INTRODUCTION
Viruses are microscopic particles that contain DNA or RNA as their genetic material but cannot replicate without a host organism. They exist in a huge variety in all organisms on earth, and some can cause serious illness or death of the host. Viruses are made of a protective protein coat (capsid), in some cases surrounded by a lipid membrane, that holds their genetic material. When a virus comes into contact with a host cell, it uses proteins on its outer surface to bind to receptors on the host cells, where it enters the cell and utilizes that cells’ synthesis machinery to replicate its own genetic material (Isabel et al, 2021). This utilization and cooption of the host's cells organelles results in cell death.
Coronaviruses are a group of viruses named for their crown-like surface proteins and cause respiratory-related illness in humans and mammals. Other well-known coronaviruses include the Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The SARS-CoV-2 is similar to other coronaviruses but is unique in its novelty and extremely high rates of transmissibility. Although the rates of hospitalizations and fatalities are slightly lower for SARS-CoV-2 as compared to SARS and MERS, the high transmissibility leads to greater number of infected patients overall and therefore more total hospitalizations and deaths (Petersen et al., 2020). The coronavirus spreads through direct contact from an infected individual who sneezes, coughs or otherwise spreads infected particles to another individual. Unlike retroviruses such as HIV, the coronavirus does not need to gain entry to the host cell’s nucleus but can completely replicate itself once it enters the cytoplasm (Scudellari, 2021).
Since the winter of 2019-20, the SARS-CoV-2 coronavirus emerged into the human population and has led to a global pandemic of unprecedented impact, infecting some 200 million people and leading to 4,4 million deaths as of August 21, 2021 (World Health Organization [WHO], 2021). As the number of infections increases, so do the chances for the virus to mutate and acquire advantageous characteristics. SARS-CoV-2 is a positive-sense single-stranded RNA virus with low stability therefore acquires more mutations (Khateeb, 2021). Researchers have identified several variants of concern including the D614G variant, the “Cluster 5” variant, VOC 202012/01, 501Y.V2 variant and now the Delta variant (WHO, 2020). The focus of this paper is on the key molecular and structural characteristics of the D614G variant, which, before the emergence of the Delta and Omicron variants, had been the most common and widespread version of the SARS-CoV-2 globally (WHO, 2020).
As a newly evolving virus, the spread of SARS-CoV-2 has been difficult to control among humans who have little or no natural immunity. The virus binds to one of the most common cell receptor, angiotensin converting enzyme 2 (ACE2), and has a strong binding affinity that has increased in effectiveness as advantageous mutations arise (Ozono et al, 2021). During viral replication errors occur, some of which lead to phenotypic and molecular variations that improve functionality. Those random viral mutations that lead to selective advantage will be further replicated since the future virus generations will retain these changes, such as improved cell receptor binding (Gobeil et al, 2021). In the case of SARS-CoV-2, several variants have been identified as variants of concern, closely monitored by researchers due to their increased transmissibility and infectivity. Note that higher infectivity and viral load, not viral virulence (the severity of illness caused by the virus) is characteristic of the dominant variants (Martin et al, 2021).
Scientists and researchers across the globe have been mapping genetic sequences and gathering geographic data on these variants. This information allows scientists to understand and track how the virus evolves in order to develop treatments, observe vaccine effectiveness and predict future viral behavior. Genetic sequencing has revealed that most prevalent variants, including the D614G variant, contain very minor mutations in the genes that code for the spike (S) protein. The S protein is the structure that enables the virus to bind to the host cell receptor and enter the cell by membrane fusion (WHO, 2020). Other parts of the SARS-CoV-2 genome code for nucleocapsid, membrane, and envelope proteins to build new viruses, however fewer mutations are observed in these genes (Singh et al, 2021).
RESULTS
Like all coronaviruses, the genetic material is protected by a capsid and a lipid envelope that has spike (S) proteins embedded. The S proteins recognize and bind to specific receptors on the host cell. The SARS-CoV-2 receptor binding process is mediated by the entry receptor angiotensin-converting enzyme 2 (ACE2) and is cleaved with the transmembrane protease serine 2 (TMPRSS2) (Kwarteng et al, 2021 & Ozono et al, 2021). Both enzymes are commonly found in cells in the airways, lungs, and nasal/oral mucosa, allowing for easy entry to the host cells if the virus comes into contact with them (Ozono et al, 2021). The spread of SARS-CoV-2 is limited when the tissues where these cells are found are covered (by PPE) or through social distancing.
However, if a virus comes into contact with a host cell with the matching receptor, it must first bind to the cell and then enter the cell via membrane fusion (endocytosis). Membrane fusion is how the virus gains entry to the host cell where it releases its genome. Once inside the cell's cytoplasm, the viral RNA is released and directly translated by the host cell's ribosomes into viral capsid and matrix proteins, then new viruses are assembled in the Golgi body (Figure 1).
Figure 1
SARS-CoV-2 Viral Life Cycle
Note: Reprinted from SARS-CoV-2 Mechanism of Cell Entry and Replication, In Tocris Bioscience, Retrieved May 26, 2021,. https://www.tocris.com/research-area/covid-19-research.
The S proteins are large structures whose molecular arrangement determines the function of the protein. The S protein is composed of two subunits, the S1 subunit which is the extracellular receptor binding area, and the S2 subunit which facilitates membrane fusion and undergoes conformational change (Kwarteng et al, 2021 & Gobeil et al, 2021).
Changes to the molecular and 3-D structure of the S protein can significantly alter the effectiveness of viral attachment to the receptor which is necessary for cell entry, thereby affecting overall viral transmissibility. Proteins are made of long, polypeptide chains of amino acids that form a specific structure. The amino acids that form the polypeptides are coded by specific nucleotide sequences in the viral RNA. Mutations occur randomly during the viral replication process and a change of even one nucleotide in the RNA can code for a different amino acid in the polypeptide chain. This molecular change can result in a structural change, depending on which amino acid is coded for, such as in the D614G substitution in the S protein receptor binding domain (RBD). This amino acid change results in improved binding function and conformational changes that allow host enzymes to aid in membrane fusion (Zhou et al, 2021).
While many mutations have been identified since the emergence of this virus, most mutations in SARS-CoV-2 are located in the genes that code for the S protein (Isabel et al, 2021). The D614G variant is a single nucleotide substitution change from a negatively charged amino acid aspartate (D) to glycine (G) in the S1 subunit (Mohammed et al, 2020, p. 611). Due to this minor molecular change, the result is the loss of a hydrogen bond which allows for two major changes: 1) increased flexibility of the S2 subunit when binding the cell, allowing for greater receptor binding ability, and 2) increased flexibility that allows more accessibility to the cleave or entry site (Mohammed et al, 2020, p. 611). Unlike the rigid spike proteins of influenza virus, the SARS-CoV-2 has 3 hinge sites that allow for greater orientational movement when binding and fusing and for multiple viruses to bind to one cell (Turoňová et. al., 2020). This improved binding capability directly increases membrane cleavage and the capacity of the virus to replicate and release these copies into the host for further infection.
CONCLUSION
The findings of this study reveal that the majority of advantageously selected-for mutations of the SARS-CoV-2 are in genes that code for the spike protein. This shows us that viral success relies primarily on its ability to bind to and enter the host cell. Knowing that viruses cannot survive or replicate without a host, we can use this information to develop strategies to prevent the virus by controlling how it binds to and enters cells. In fact, the vaccines developed by Moderna and Pfizer utilize this knowledge by giving our immune cells the genetic code (mRNA) for the spike protein. Once this protein is recognized by the immune system, our bodies can develop a more effective immune response without the risk of developing the disease (CDC, 2021). Further research into which specific genes code for the spike protein can allow us to target the expression of these genes as a means of viral control. For example, if gene A codes for the spike protein amino acids that improve binding affinity, we may be able to disable or modify this gene or genetic sequence resulting in a virus unable to effectively bind to the host cell receptor, and therefore unable to release its RNA and replicate.
This study supports epidemiologists’ emphasis on vaccination, the importance of social distancing, wearing PPE, and getting vaccinated as extremely effective tools to prevent the spread, and, more importantly, limit the frequency of mutations of the virus. Every time an individual contracts SARS-CoV-2, the virus can replicate, making thousands of copies of itself, and every replication is a chance for errors leading to possible advantageous mutations. As we saw with the D614G variant, one single nucleotide substitution can result in higher infectivity and viral spread. Now the Delta variant is showing us that new mutations continue to arise and result in nucleotide substitutions that improve the virus' transmissibility and ability to evade the immune system. Though these mutations are random, the molecular and structural changes that improve survival will always be selected for. By limiting the number of times the virus can replicate, we are directly limiting its ability to evolve and possibly develop further mechanisms to increase transmissibility or infectivity.
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