Macie Duran Week 3

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Macie M. Duran

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Purpose

The purpose of this assignment is to critically analyze a primary scientific article and use the study as a basis for further research.

Defined Terms

  1. Palm civet: any of several black-spotted or black-striped yellowish gray or brownish gray civets of Paradoxurus or related genera that are widely distributed in Southeastern Asia and the East Indies (Merriam-Webster, n.d.)
  2. Receptor-binding domain (RBD): a short immunogenic fragment from a virus that binds to a specific endogenous receptor sequence to gain entry into host cells (Shabir, 2020)
  3. Phylogram: a phylogenetic tree that indicates the relationships between taxa and also conveys a sense of time or rate of evolution (Cammack, 2008)
  4. Salt bridge: any electrostatic bond, between positively and negatively charged groups of amino-acid residues of a protein, that contributes to the stability of the protein (Cammack, 2008)
  5. Orthologue: a gene, protein, or biopolymeric sequence that is evolutionarily related to another by descent from a common ancestor, having diverged as a result of a speciation event (Cammack, 2008)
  6. Residue: any of the monomers comprising a polymer, or any of the parts that integrate to make up a larger molecule (Biology Online Dictionary, n.d.)
  7. Pathogenesis: the origin and development of disease (Biology Online Dictionary, n.d.)
  8. Angiotensin: a family of oligopeptides associated with increased blood pressure, mainly by causing vasoconstriction (Biology Online Dictionary, n.d.)
  9. Dipeptidyl peptidase: any member of a group of enzymes belonging to the sub‐subclass EC 3.4.14, dipeptidylpeptide and tripeptidylpepide hydrolases (Cammack, 2008)
  10. Steric: relating to effects involving arrangements of atoms in space (Cammack, 2008)

Article Outline

Abstract & Introduction

  • 2019-nCoV, a novel coronavirus that recently originated in Wuhan, China, is showing similar symptoms to SARS-CoV
  • Previous research on SARS-CoV, which caused an outbreak in 2002, has shown interactions between the spike protein receptor-binding domain and the host cell receptor ACE2
  • 2019-nCoV's sequence is similar to that of SARS-CoV, indicating that it uses the same ACE2 receptor
  • Several of 2019-nCoV's receptor-binding motif residues interact favorably with the human ACE2 receptor, indicating the possibility for human cell infection, as well as human-to-human transmission
  • Phylogenetic analysis of 2019-nCoV indicates its origin in bats, but there is also a possibility of intermediate hosts in a variety of animals
    • It is believed that bats were the natural reservoir for SARS-CoV, and that palm civets were the intermediate reservoir
  • Similar to SARS-CoV, 2019-nCoV is thought to have passed from an animal reservoir to humans in an animal market
  • SARS-CoV and 2019-nCoV both belong to the family of coronaviruses, which are single-stranded enveloped RNA viruses
  • When entering a host cell, coronaviruses utilize a spike protein. They first bind to the host cell, and then fuse the viral and host membranes.
  • The SARS-CoV spike proteins have receptor-binding domains (RBD) that specifically recognize angiotensin-converting enzyme 2 (ACE2), the host cell receptor
    • Host susceptibility has shown to be dependent on the compatibility between the viral RBD and the host ACE2
  • Crystal structures of SARS-CoV RBDs from different host species showed that the RBD has a core structure and a receptor-binding motif (RBM) that binds to ACE2
    • Human ACE2 has two virus-binding hot spots
    • RBM mutations near these hot spots were shown to impact the host range of SARS-CoV
    • Amino acids at the 442, 472, 479, 480, and 487 positions were shown to enhance binding to human ACE2 and civet ACE2
      • When these residues were all combined into one RBD, it was able to bind to ACE2 with super affinity
  • These results can be used to predict infectivity and animal sources of future coronaviruses similar to SARS-CoV and 2019-nCoV

Materials & Methods

  • Softwares Coot and PYMOL were used to prepare structural models and introduce mutations.
  • Geneious Prime and the Jukes-Cantor genetic distance model were used to create consensus radial phylograms and show phylogenetic relationships between 2019-nCoV and other coronaviruses.
  • Clustal Omega was used to create protein sequence alignments.

Results

  • Phylogeny of 2019-nCoV indicates it is ancestral to SARS-CoV strains that enter host cells through the ACE2 receptor. Sequence similarities between 2019-nCoV and SARS-CoV are:
    • 76-78% for whole protein,
    • 73-76% for RBD, and
    • 50-53% for RBM
  • Similarities between MERS-CoV and bat MERS-like coronavirus are lower, yet they still utilize the same receptor. This indicates that 2019-nCoV is likely to enter host cells through the ACE2 receptor.
  • Five of the residues in 2019-nCoV RBM underwent natural selections in SARS-CoV and were critical in its cross-species transmission.
    • Residue 493 in 2019-nCoV RBD corresponds with residue 479 in SARS-CoV. 493 is a glutamine located near human ACE2 hot spot Lys31, a salt bridge between Lys31 and Glu35. Gln493 is compatible with the Lys31 hotspot, indicating that 2019-nCoV can infect host cells through the ACE2 receptor.
    • Residue 501 in 2019-nCoV RBD corresponds with residue 487 in SARS-CoV. 501 is an asparagine and is located near hot spot Lys353, a salt bridge between Lys353 and Asp38. Asn501 provides more support than the previous Ser487 seen in the civet SARS-CoV from 2002, but it is not as compatible as the Thr487 seen in the human SARS-CoV isolated in 2002. However, this still indicates that 2019-nCoV is capable of transmitting from human to human.
    • Residues 455, 486, and 494 in 2019-nCoV correspond with residues 442, 472, and 480 in SARS-CoV, respectively. In 2019-nCoV they are leucine, phenylalanine, and serine. These residues play significant roles in SARS-CoV's ACE2 binding.
      • Tyr442 in SARS-CoV has an unfavorable reaction with ACE2 hot spot 31, but Leu455 in 2019-nCoV has a more favorable interaction. This means binding is enhanced.
      • Leu472 in SARS-CoV provides a favorable reaction with the ACE2 hot spot 31, but residue Phe486 observed in 2019-nCoV provides an even more favorable interaction. Again, this enhances its binding to the host cell's ACE2 receptor.
      • Asp480 in SARS-CoV provides favorable support for ACE2 hot spot 353. Ser494 in 2019-CoV is not as favorable as Asp480, but it is still positive support.
  • 2019-nCoV shows some unfavorable interactions with civet ACE2, indicating that it likely did not evolve for civet ACE2 binding. It also does not show favorable interactions with mice or rats.
  • It is likely that 2019-nCoV recognizes ACE2 with similar efficiency in humans, pigs, ferrets, cats, orangutans, and monkeys. It likely also recognizes bat ACE2 receptors.

Figures

Figure 1: Shows a structural model of human ACE2 recognition by both SARS-CoV and 2019-nCoV.

  • 1A) Shows the structure of the 2002 SARS-CoV RBD core and RBM, as well as the human ACE2 receptor.This figure not only gives a visual representation of their structures, but also demonstrates how SARS-CoV binds to the outer surface of the ACE2 receptor.
  • 1B) This is a table outlining all of the changes in the five critical residues that were analyzed. It shows which residues are observed in SARS-CoV RBD versus those in 2019-nCoV RBD. There are also optimized viruses that were designed and analyzed in vitro. The table not only shows what changes were made, but it indicates which changes are favorable and which are unfavorable.
  • 1C) Shows the structure of the optimized SARS-CoV that was designed in vitro. This model also shows the optimized strain's interaction with human ACE2. The five key residues that were found to be most favorable for binding to the human ACE2 receptor are F442, F472, N479, D480, and T487.
  • 1D) Shows the modeled structure of the 2019-nCoV RBD, along with its interactions with the human ACE2 receptor. Mutations were introduced to the optimized RBD model from Figure 1C based on sequence differences observed in SARS-CoV and 2019-nCoV.

Figure 2: Shows a phylogenetic tree of the beta-genus lineage b coronaviruses. This tree represents differences in the spike protein sequences observed in coronaviruses. The tree shows that 2019-nCoV's sister taxa are BtSCoV-VZXC21 and BtSCoV-VZC45. It is ancestral to human SARS-CoV and bat SARS-CoV strains.

Figure 3: Shows sequence alignment comparisons between SARS-CoV and 2019-nCoV.

  • 3A) Shows sequence alignments of RBDs obtained from human SARS-CoV (2002), civet SARS-CoV (2002), bat SARS-CoV (2013), and 2019-nCoV. Sequences for RBM residues are highlighted in magenta, and the five critical residues analyzed in the study are highlighted in blue. The asterisks, colons, and periods below the sequences represent how well-conserved the sequences are.
  • 3B) The table shows the calculated percentages of similarities between sequences from human SARS-CoV, civet SARS-CoV, bat SARS-CoV, and 2019-nCoV. The percentages are representative of similarities in spike protein, RBD, and RBM sequences.
  • 3C) The table shows the percentage of sequence similarities between MERS-CoV and HKU4 in the spike protein, RBD, and RBM. This table is included to show that the sequence similarities in these two viruses are lower than the percentages in panel 3B. Despite having less sequence similarities, MERS-CoV and HKU4 bind to the same receptor in host cells. This demonstrates that it is likely that 2019-nCoV would bind to the same receptor as SARS-CoV.

Figure 4: Shows structural analysis of animal ACE2 receptor recognition by SARS-CoV and 2019-nCoV.

  • 4A) The table shows critical changes of ACE2 residues in a variety of host animals. The table demonstrates strong similarities in the ACE2 receptors of humans, bats, pigs, ferrets, cats, orangutans, and monkeys. However, mice, rats, and civets have less similar residues and likely have more unfavorable interactions with 2019-nCoV RBD residues.
  • 4B) Shows an optimized model of civet SARS-CoV RBD and its interaction with the civet ACE2 receptor. The five critical residues on the civet SARS-CoV RBD are T487, R479, G480, Y442, and P472.
  • 4C) Shows a model of human 2019-nCoV RBD and its interaction with the civet ACE2 receptor. Mutations were introduced to the structure from Figure 4B based on sequence differences observed in SARS-CoV and 2019-nCoV. This model shows a much less favorable interaction between the RBD and the ACE2 receptor, as compared to the interaction shown in the optimized model in Figure 4B. The five residues on the human 2019-nCoV RBD are N501, S494, Q493, L455, and F486. The phenylalanine in position 486 on 2019-nCoV shows an unfavorable interaction with the civet ACE2 receptor's threonine in position 82. Furthermore, leucine in position 455 and glutamine in position 493 of 2019-nCoV have unfavorable interactions with civet ACE2. However, they would still be compatible. Based on this model, it is likely that the 2019-nCoV RBD did not evolve for civet ACE2 binding, but it is still able to use civet ACE2 as its receptor.

Discussion

  • Analyzing and understanding virus-receptor interactions in previous virus strains allows us to better predict the interactions in new strains, such as 2019-nCoV. Because of previous studies on SARS-CoV and its interaction with ACE2 in a variety of hosts, researchers had a starting point for analysis of 2019-nCoV.
  • The structural analyses outlined in the article indicate that 2019-nCoV uses hosts' ACE2 receptors for entry.
    • When comparing 2019-nCoV to previous strains, it is likely that the strain uses ACE2 less efficiently than SARS-CoV isolated in 2002, but more efficiently than SARS-CoV isolated in 2003.
    • ACE2 binding affinity plays a key role in 2019-nCoV's ability to infect human cells and to be transmitted from human to human.
    • The models indicate that a mutation in residue 501 (from asparagine to thymine) could significantly enhance the strain's binding affinity.
  • Because a single mutation could significantly enhance the virus's ability to infect host cells, it is important to closely monitor mutations.
  • Based on phylogenetic analysis, 2019-nCoV likely originated in bats, which is consistent with the SARS-CoV origin.
  • 2019-nCoV likely recognizes ACE2 receptors in a variety of host animals.
    • 2019-nCoV RBD shows no adaptations in its critical residues that would indicate palm civets as an intermediate reservoir. Because of this, it is likely that palm civets were either not an intermediate reservoir, or the virus quickly passed to humans and did not evolve to civet ACE2 receptors.
    • Rats and mice are unlikely intermediate hosts for 2019-nCoV.
    • ACE2 receptors in pigs, ferrets, cats, orangutans, and monkeys show more favorable interactions with 2019-nCoV RBD and would serve as better model animals or intermediate reservoirs.
  • Additional factors play a role in 2019-nCoV's infectivity and pathogenesis. These factors should also be studied and considered.

Importance & Implications

This study demonstrates the importance of analyzing virus-receptor interactions in order to be able to use previous knowledge as a basis for studying and understanding emerging viruses. The structural modeling indicates that 2019-nCoV likely uses ACE2 as a receptor in host cells. Knowing how a virus enters and infects host cells is important in determining how to prevent infection and transmission.

Future Directions

Because the study gives us a better understanding of how 2019-nCoV enters host cells, we have a starting point for determining prevention of infection.

Critical Evaluation

The authors did a good job of backing up all claims with evidence. As far as limitations, this study was conducted and published fairly early on in the pandemic. Very little was understood about COVID-19 at the time, so being able to compare protein sequencing to that of SARS-CoV was a good basis for analysis. However, very little was explained in the materials and methods. Because of this, it is difficult to verify how the sequences were obtained in order to model and compare SARS-CoV and 2019-nCoV. I imagine that replicating the study would also be difficult due to the lack of information.

Conclusion

Reading this article gave me a better understanding of the similarities between SARS-CoV and SARS-CoV-2. The study provided strong evidence backing up that SARS-CoV-2 utilizes the ACE2 receptor for entry, and that the virus originated in bats. Having a basic understanding of the virus's origin and host cell entry provides a great basis for future studies.

Acknowledgements

  1. I contacted my homework partner, Aiden Burnett, once over Zoom and once over text in regard to analyzing Figure 2 in the assigned article.
  2. I copied and modified the protocol outlined on the Week 3 assignment page.
  3. I used the Wan et al. article to create my outline.
  4. I used the following sources to define terms: Biology Online Dictionary, Oxford Dictionary of Biochemistry and Molecular Biology, Merriam-Webster, and News Medical.
  5. Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

(Macie Duran (talk) 22:55, 23 September 2020 (PDT))

References

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