ABSTRACT
SARS-CoV-2 is an RNA virus that has had immense ramifications around the globe over the past year. An analysis of the replication cycle of SARS-CoV-2 offers important clues about the mutation potential of the virus. Research suggests SARS-CoV-2 actually has a slightly lower mutation rate than most other RNA viruses due to a proofreading mechanism it employs during replication. Furthermore, most mutations that do occur to SARS-CoV-2 during replication are harmful to or have no effect on the structure and function of the pathogen. Despite this, unmitigated spread, high infection rates, and substandard countermeasures have allowed a select few variants with mutations that increase viral infectivity to propagate around the world. Understanding the mechanisms behind mutations in SARS-CoV-2 offers insight on the likelihood of mutations that increase the fitness of the pathogen and the efficacy of established and proposed control measures. These measures can ultimately be used to strengthen humanity’s response to the virus and limit further spread.
INTRODUCTION
Viruses are nonliving entities that require entry into host cells and access to a host cell’s cellular machinery for the replication and propagation of their genetic material. While often showcasing immense variation in shape and structure, all viruses consist of a protein capsid, and some have an envelope that serves to protect the genetic material found within the capsid (Mateu, 2013). This genetic material, either RNA or DNA, serves as a set of instructions for translation (and transcription, in the case of DNA) in the host cell (Mateu, 2013). During translation, mRNA is translated by host ribosomes into viral proteins. Upon completion of replication and reassembly, new viral particles are free to leave the host cells via exocytosis to continue propagation (Mateu, 2013). Coronaviruses are a specific family of viruses that use positive-sense single-strand RNA as genetic material (Mateu, 2013). Coronaviruses share similar protrusions from their envelopes that resemble solar coronas--the aura surrounding individual solar bodies--hence the name coronavirus. This spike protein mediates virus-host cell attachment and fusion of the envelope into the host cell membrane (Li, 2016). As a result, variation in structure or composition of the spike protein (such as those found in various types of coronaviruses) can alter infectivity and propagation via altered interactions between the protein and receptors of the host cell (Li, 2016). SARS-CoV-2 is a newfound coronavirus that infects respiratory cells and presents itself via respiratory tract symptoms, in a similar fashion to other known coronaviruses (such as SARS-CoV and MERS) with a lesser fatality rate but increased infectivity (Liu et al., 2020). When the viral load of SARS-CoV-2 was measured in infected patients with mild to no symptoms, the results indicated increased total viral load (up to 1000 times higher), decreased mean time to peak viral RNA concentration (fewer than 5 days compared to 7-10), and increased active viral load in the upper respiratory tract when compared to patients with SARS-CoV (Wolfel et al., 2020). This indicates that SARS-CoV-2 is more efficient at transmitting between hosts, especially in mild and asymptomatic cases. Enhanced transmission, minimal preparedness of society for the propagation of a newly discovered zoonotic virus, and inefficient intervention in preventing spread, have all allowed for SARS-CoV-2 to proliferate exponentially around the globe.
RNA viruses, such as SARS-CoV-2, have a high chance of potential mutation that can be attributed to the mechanism by which these viruses replicate their genetic material in host cells (Rosenberg et al., 2014). Mutations, or base pair changes in the genome of an entity, account for much of the novelty we see in viral evolution, and, in some cases, may enhance the pathogen’s genetic fitness as a result of natural selection (Duffy, 2018). Early and thorough research on mutations in known viruses can lead to earlier intervention/control measures and potentially increase the effectiveness of these measures on novel viruses (Abdullahi et al., 2020). Further, understanding the mechanisms behind the occurrence of and likelihood of mutations can deliver insight on what to expect from emerging or yet-to-emerge viral pathogens in the future (Abdullahi et al., 2020).
This piece reviews existing literature on the replication cycle of SARS-CoV-2. It also discusses how the replication cycle impacts mutation rate and viral variation in the context of the pandemic and how it has impacted and may impact humanity.
Figure 1
Replication Cycle of SARS-CoV-2
Note: From “The Human Coronavirus Disease COVID-19: Its Origin, Characteristics, and Insights into Potential Drugs and Its Mechanisms” by Lo’ai Alanagreh, Foad Alzoughool, and Manar Atoum, 2020, Pathogens, 9(5), 331. doi: 10.3390/pathogens9050331. CC BY 4.0.
Single strand RNA viruses make up the bulk of emerging and discovered viruses in recent years (Rosenberg, 2014). SARS-CoV-2, similarly to other single-strand RNA viruses, has a rate of mutation roughly one to two orders of magnitude greater than that of DNA viruses and several orders of magnitude greater than that of DNA in eukaryotic organisms (Belshaw et al., 2008). Heightened rates of mutation in viruses have been linked to the ability of these viruses to jump between species and infect new hosts such as humans (Lau and Chan, 2015). Furthermore, the expanded genome of SARS-CoV-2 and other coronaviruses has led to the addition of genes that code for accessory proteins that enhance the ability of coronaviruses to jump between species (Artika et al., 2020). Such viruses that exhibit the ability to jump between species, known as zoonoses, also make up a bulk of the recently discovered and emerging viruses (Rosenberg, 2014). This suggests that the rapid emergence and discovery of SARS-CoV-2 as a zoonosis likely arose from its large genome and ability to mutate.
Figure 1 displays the steps in the replication cycle of SARS-CoV-2 in order. When SARS-CoV-2 binds to the ACE2 receptor of a cell in the respiratory tract, the viral particle fuses with the membrane, enters the host cell, and unwinds its RNA strand into the cytoplasm (Artika et al., 2020). The unwound positive-sense single-strand RNA immediately binds to host cell ribosomes in the cytoplasm to begin translation of open reading frames near the 5’ end of the RNA strand, thus producing proteins involved in the replication-transcriptase complex as shown (Artika et al., 2020). This complex includes non-structural proteins such as RNA-dependent RNA polymerase, RNA helicase, endoribonuclease, exonuclease, various proteases, and more. These proteins are produced from the action of proteases that cleave the two large polyproteins, pp1a and pp1ab, which were produced during the translation of the viral RNA (Artika et al., 2020). The replication-transcriptase complex mediates the replication of viral RNA template strands and subgenomic RNA strands that are used for the translation of proteins and assembly of new viral particles (Artika et al., 2020). Transcription of the positive-sense strand into a negative-sense template strand is accomplished by RNA-dependent RNA polymerase, and the newly formed strand serves as a template for the transcription of new positive-sense RNA strands which will serve as the genetic material for newly assembled viral particles (Artika et al., 2020). The negative-sense subgenomic RNA strands, produced via discontinuous transcription, are used as templates for the production of positive-sense subgenomic RNA strands which are used in the translation of structural proteins, such as the spike protein, membrane protein, envelope protein, and more (Artika et al., 2020). These, then, are used to assemble the viral envelope of SARS-CoV-2 (Artika et al., 2020). Assembly, budding, and release of viral particles completes in the endoplasmic reticulum-golgi intermediate compartment (Saraste and Prydz, 2021).
In general, RNA viruses tend to have increased rates of mutation (as compared to DNA viruses) resulting from the lack of proofreading capability of RNA-dependent RNA polymerase during replication (Peck and Lauring, 2018). However, evidence suggests that members of the Nidovirales family, such as SARS-CoV-2, have a proofreading mechanism that is independent of RNA-dependent RNA polymerase and should lead to lower than expected rates of mutation (Peck and Lauring, 2018). This proofreading mechanism, known as the exoribonuclease complex, is an exonuclease that is highly conserved across all coronaviruses and acts as a part of the replication-transcriptase complex to remove mismatched nucleotides from replicated RNA strands (Bouvet et al., 2012). Despite the heightened fidelity of replication achieved as a result of its proofreading mechanism, SARS-CoV-2 has a rate of mutation just slightly lower than those of other RNA viruses (Koyama et al., 2020). This, alongside being highly conserved, suggests that the exoribonuclease complex does not serve purely to limit the mutation rate of SARS-CoV-2 and that it likely plays another key role in viral replication.
The high mutation rates of RNA viruses often teeter between proper genetic variation and instability in the viral genome. Mutations arise from the unreliability of RNA-dependent RNA polymerase and the exoribonuclease complex in catching mismatched nucleotides during the transcription of viral RNA (Fleischmann Jr., 1996). The fast pace at which viral RNA replication occurs also contributes to higher rates of mutation (Belshaw et al., 2008). High rates of infection around the globe have further stimulated the likelihood of mutation, and most are overwhelmingly neutral or unfavorable to the virus (Wang et al., 2021). However, in certain cases, mutations can be beneficial to the virus, and persistent and propagating strains likely remain as a result of the increased fitness derived from these mutations (Duffy, 2018).
Many mutations may go unnoticed in a viral genome because they often result in little to no change in structural and nonstructural proteins, thereby having little effect on the overall fitness of the virus (Fleischmann Jr., 1996). Some mutations can cause structural and functional changes within the virus and can affect the rate and outcome of infection. Such mutations that interfere with viral function do not allow for propagation and fizzle out quickly (Fleischmann Jr., 1996). On the other hand, other mutations are likely responsible for the zoonotic nature of such viruses and high rates of mutation may increase a virus' ability to become zoonotic, or transmissible from animal to human (Lau and Chan, 2015). While extremely rare in practice, SARS-CoV-2 strains with increased fitness often propagate, heighten infectivity for the virus across humans in general, and tend to go on to make up large proportions of circulating viral strains because of their potential to evade established control measures.
Finally, as of now, there is no way to accurately determine the rate at which viral variants may appear. However, work is being done to develop algorithms that can predict the rate and gauge the impact of potential variants of RNA viruses.
CONCLUSION
SARS-CoV-2 has developed variants with significant enough mutations to interfere with global countermeasures. Many variants have been discovered that have the potential to evade vaccination, transmit more easily, and/or thwart known treatments. As the total number of viral particles replicating increases, the number of mutations occurring during replication increases. Thus, despite most mutations and variants fizzling out, the unmitigated spread of SARS-CoV-2 worldwide has enabled the few more effective variants of the virus to develop and proliferate. Since any variant that begins to circulate has the potential to be of high consequence by completely counteracting countermeasures, it is of utmost importance to limit spread as much as possible to reduce the likelihood of mutations occurring overall. Since limiting spread is not always possible, it is important for health authorities to be proactive in keeping track of variants and in educating the general public. To do this, enough sequencing must be carried out and information about variants of importance must be heavily circulated. This information should include locations of circulation, location of origin, transmissibility, potential to evade current countermeasures, and new recommendations to limit spread if any are available. By keeping up with such information, countermeasures can evolve in different locations to mitigate circulating variants and infection rates.
SARS-CoV-2 has had a profound impact on the way humans live their lives and on the world around them. As of May 2021, over three million lives have been lost and many millions have been struck with long-term health issues. Still, the virus continues to infect and affect hundreds of thousands across the globe daily. Even as the vaccination campaign continues and infection rates decline, it is imperative for humanity to remain on top of the current situation to hinder more effective variants from taking hold.
The most effective way of limiting the potential development and subsequent spread of variants, while also decreasing stress on healthcare systems, is increasing accessibility to vaccination worldwide. Vaccine inequity has allowed for pockets around the world with low levels of vaccine-induced immunity. These pockets are ripe for mutation as the virus replicates more and for longer in unvaccinated individuals than in vaccinated individuals. Many of the variants of concern and interest (as designated by the WHO) were found and have propagated early in nations with lagging vaccine coverage/rollout. While vaccination rates continue to hit high levels in countries that can afford mass vaccination, the potential for the development of other variants of concern/interest will remain at a high level until wealthier countries facilitate mass vaccination in areas that are lagging behind.
REFERENCES
Abdullahi, I. N., Emeribe, A. U., Ajayi, O. A., Oderinde, B. S., Amadu, D. O., & Osuji, A. I. (2020). Implications of SARS-CoV-2 genetic diversity and mutations on pathogenicity of the COVID-19 and biomedical interventions. Journal of Taibah University Medical Sciences, 15(4), 258-264. doi:10.1016/j.jtumed.2020.06.005
Alanagreh, L., Alzoughool, F., & Atoum, M. (2020). The human coronavirus disease COVID-19: Its origin, characteristics, and insights into potential drugs and its mechanisms. Pathogens, 9(5), 331. doi:10.3390/pathogens9050331
Artika, I. M., Dewantari, A. K., & Wiyatno, A. (2020). Molecular biology of coronaviruses: Current knowledge. Heliyon, 6(8). doi:10.1016/j.heliyon.2020.e04743
Belshaw, R., Gardner, A., Rambaut, A., & Pybus, O. G. (2008). Pacing a small cage: Mutation and RNA viruses. Trends in Ecology & Evolution, 23(4), 188-193. doi:10.1016/j.tree.2007.11.010
Bouvet, M., Imbert, I., Subissi, L., Gluais, L., Canard, B., & Decroly, E. (2012). RNA 3-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex. Proceedings of the National Academy of Sciences, 109(24), 9372-9377. doi:10.1073/pnas.1201130109
Duffy, S. (2018). Why are RNA virus mutation rates so damn high? PLOS Biology, 16(8). doi:10.1371/journal.pbio.3000003
Fleischmann Jr., R. W. (1996). Medical Microbiology. 4th edition.
Koyama, T., Platt, D., & Parida, L. (2020). Variant analysis of SARS-CoV-2 genomes. Bulletin of the World Health Organization, 98(7), 495–504. doi:10.2471/BLT.20.253591
Lau, S. K., & Chan, J. F. (2015). Coronaviruses: Emerging and re-emerging pathogens in humans and animals. Virology Journal, 12(1). doi:10.1186/s12985-015-0432-z
Li, F. (2016). Structure, function, and evolution of coronavirus spike proteins. Annual Review of Virology, 3(1), 237-261. doi:10.1146/annurev-virology-110615-042301
Liu, J., Xie, W., Wang, Y., Xiong, Y., Chen, S., Han, J., & Wu, Q. (2020). A comparative overview of COVID-19, MERS and SARS: Review article. International Journal of Surgery, 81, 1-8. doi:10.1016/j.ijsu.2020.07.032
Mateu, M. G. (2013). Structure and physics of viruses: An integrated textbook.
Peck, K. M., & Lauring, A. S. (2018). Complexities of viral mutation rates. Journal of Virology, 92(14). doi:10.1128/jvi.01031-17
Rosenberg, R. (2014). Detecting the emergence of novel, zoonotic viruses pathogenic to humans. Cellular and Molecular Life Sciences, 72(6), 1115-1125. doi:10.1007/s00018-014-1785-y
Saraste, J., & Prydz, K. (2021). Assembly and cellular exit of coronaviruses: Hijacking an unconventional secretory pathway from the pre-golgi intermediate compartment via the golgi ribbon to the extracellular space. Cells, 10(3), 503. doi:10.3390/cells10030503
Wang, R., Chen, J., Gao, K., Hozumi, Y., Yin, C., & Wei, G. (2021). Analysis of SARS-CoV-2 mutations in the United States suggests presence of four substrains and novel variants. Communications Biology, 4(1). doi:10.1038/s42003-021-01754-6
Wölfel, R., Corman, V. M., Guggemos, W., Seilmaier, M., Zange, S., Müller, M. A., . . . Wendtner, C. (2020). Virological assessment of hospitalized patients with COVID-2019. Nature, 581(7809), 465-469. doi:10.1038/s41586-020-2196-x
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