Anna Horvath Week 3

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Anna Horvath

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Journal Pages

Class Journal Pages

Purpose

The purpose of this assignment is to improve our ability to read and analyze journal articles, specifically focusing on evaluating how 2019-nCoV enters its host cells.

Biological Terms Defined

Orthologues: a gene, protein, or biopolymeric sequence that is evolutionarily related to another by descent from a common ancestor (Oxford Dictionary of Biochemistry and Microbiology, 2020)
Palm Civet: any of various small to medium-sized, chiefly arboreal cats of the civet family, of southeastern Asia (Dictionary.com, 2020)
Infectivity: the characteristic of a disease agent that embodies capability of entering, surviving in, and multiplying in a susceptible host (Biology Online, 2020)
Pathogenesis: the origin and development of disease (Biology Online, 2020)
Reiterative: to say or do again or repeatedly; repeat, often excessively. (Dictionary.com, 2020)
Radial: pertaining to a radius or ray; consisting of, or like, radii or rays; radiated; as, the radial artery (Biology Online, 2020)
Phylogram: a phylogenetic tree that indicates the relationships between taxa and also conveys a sense of time or rate of evolution (Oxford Dictionary of Biochemistry and Microbiology, 2020)
Iterative: repeating; making repetition; repetitious. (Dictionary.com, 2020)
Angiotensin: a family of oligopeptides associated with increased blood pressure, mainly by causing vasoconstriction (Biology Online, 2020)
Dipeptidyl peptidase: any member of a group of enzymes belonging to the sub‐subclass EC 3.4.14, dipeptidylpeptide and tripeptidylpepide hydrolases (Oxford Dictionary of Biochemistry and Microbiology, 2020)

Article Outline

Abstract and Introduction

2019-nCoV, colloquially referred to as COVID-19, originated in Wuhan, China and produces symptoms similar to those of SARS-CoV
Previous research has found interactions between SARS-CoV spike protein receptor-binding domain (RBD) and host receptor angitensisn-converting enzyme 2 (ACE2)
Bats and palm civets were found to be the natural reservoirs for the virus.
- Transmission from palm civet to human occurred in an animal market
SARS-CoV RBD has a core structure, of which a series of crystal structures can bind to ACE2
- ACE2 was found to have two virus-binding locations
- Amino acids at positions 442, 472, 479, 480, and 487 were found to be significant in the process of binding
  - When all five residues are present in a human ACE2, the binding of RBD is very efficient
This research aims to predict the host infectivity for the receptors in order to analyze strains similar to SARS
The significance of this work is to be able to create predictions for SARS-CoV viruses to better study their effects and predict the spread of the virus.
The previous study that lead them to this work was the analysis of SARS-CoV RBM and ACE2 binding from the 2002 and 2003 strains of SARS-CoV.
The main result presented in this paper is the 2019-nCoV was transmitted from bats and likely uses a similar ACE2 binding receptor as previous SARS-CoV strains.

Results

Sequence similarities between 2019-nCoV and SARS-CoV are
- Between 76-78% for the entire protein
- Between 73-76% for the RBD
- Between 50-53% for the RBM
These sequence similarities between 2019-nCoV and SARS-CoV mean they may share the same receptor ACE2
- These two were also found to not have any insertions or deletions
- Of the fourteen sequences found in the ACE2 residues of the RBD between the two, nine are conserved
  - These sequences are conserved across species - namely in humans, bats, and civets
Residue 493 in 2019-nCoV RBD, a glutamine, is located near ACE2’s reside of Lys31. These two residues form an ionic salt bridge
Residue 501 (asparagine, N) in 2019-nCoV RBD is near Lys353 on human ACE2, forming a salt bridge that is located in a hydrophobic environment
- A mutation from SARS-CoV (2002) has caused increased efficiency in the binding of these residues
Residues 455 (leucine, L), 486 (phenylalanine, F), and 494 (serine, S) play significant roles in binding.
- Their roles are not as significant as the aforementioned residues 479 and 487.
It is likely that 2019-nCoV uses civet ACE2 as a receptor.
- However, this is not optimally evolved at this time, as there are many unfavorable interactions between the residues at the binding site.

Figures

Figure 1
- Figure 1a. Depicts the structure of human SARS-CoV, highlighting the interaction between the RBD and ACE2
  - Figure was created to specifically show that that RBM and ACE2 regions are interacting, allowing for a visualization of the relative lengths of the protein domains
- Figure 1b. Table shows the five amino acid residues previously shown to be important in ACE2 recognition
  - Differences between the sequences are presented, combined with the adaptations the amino acids have undergone through natural selection
  - Arrows and equal signs indicate the relative strengths of the adaptations
- Figure 1c. Amino acid residues of SARS CoV RBD and human ACE2 are shown, with the interactions between amino acids on the chain numbered
  - Notably, the five critical amino acid residues are shown. Residue 442 is phenylalanine (F), 472 is phenylalanine (F), 479 is asparagine (N), 480 is aspartic acid (D), and 487 is threonine (T).
  - The five critical amino acid residues are shown in proximity to lysine (K), methionine (M), aspartic acid (D), and glutamic acid (E).
- Figure 1d. Depicts human 2019-nCoV and RBD model where mutations are introduced to the RBM’s five critical amino acids.
Figure 2. Depicts the phylogenetic tree of spike phylogeny among different coronavirus strains
- Using GenBank accession numbers, 2019-nCoV MN908947 can be seen diverging from BtSCoV-VZ45 and BtSCoV-VZXC21, which are sister taxa. In this tree, the common ancestor is BtSCoV
Figure 3
- Figure 3a. Depicts sequence alignments of human SARS (2002), civet SARS (2002), bat SARS (2003), and 2019-nCoV.
  - The rows under these sequences indicate the level of conservation between the residues of the four different sequences.
  - This figure serves to highlight the five critical amino acid residues identified for optimal binding interactions and how these sequences have been altered through natural selection.
  - Human and civet SARS show mostly conserved sequences for these five amino acid resideues, wheras 2019-nCoV is different than the 2002 SARS sequence at each of these five residues.
  - Aside from these five residues, the sequence is relatiovely well conserved among the four strains.
- Figure 3b. Depicts the percentage similarity between human SARS (2002), civet SARS (2002), bat SARS (2003), and 2019-nCoV.
  - SARS-human and SARS-civet have around a 98 percentage similarity to one another, indicating large conservation of the sequence.
  - The three original SARS sequences and 2019-nCoV have only fifty percent similarity in the RBM binding sequences.
- Figure 3c. Depicts MERS-CoV and HKU4-bat virus spike protein similarities across sequences.
  - The RBM sequence seems to be the least conserved between the two viruses, at only 40 percent, wheras the spike protein sequence shows a 67 percent similarity between the two sequences (which is similar to the percentage identified between the SARS 2002 and 2019-nCoV sequences in Figure 3b).
Figure 4
- Figure 4a. Portrays animal ACE2 recognition sequences across different species that host the virus.
  - Most host organisms, such as monkeys, orangutangs, cats, ferrets, and humans only appear to have one change among the five listed residues that differs from other host species.
  - Notably, the changes in each species are different from that of other species. Mice have three amino acid residue changes when compared to the residues in other host species. These changes occur at positions 31, 82, and 353.
- Figure 4b. Depicts the optimal interaction that results from SARS-CoV RBD and ACE2, both for civets.
  - Notably, the five critical amino acid residues are shown. Amino acid 487 is threonine (T), 479 is arginine (R), 480 is glycine (G), 442 is tyrosine (Y), and 472 is proline (P).
  - These resides interact with glutamic acid (E), which is negatively charged, lysine (K), which is positively charged, and threonine (T), which is uncharged and polar.
- Figure 4c. Depicts civet SARS-CoV RBD and ACE2 interactions model where mutations are introduced to the RBM’s five critical amino acids.

Discussion

Through their modeling and knowledge of previous SARS-CoV strains, researchers believe 2019-nCoV uses ACE2 as a receptor.
- It likely binds with better efficiency than the 2003 SARS-CoV virus did
Mutations at amino acid 501 may enhacne the binding capability of 2019-nCoV to ACE2
2019-nCoV likely originated from bats
- Shows a close phylogenetic relationship to beta-genus bat lineages.
- Mice and rats were rules out as intermediary hosts.
- Palm civets were likely not intermediary hosts, as the palm civet ACE2 receptor is not similar to that of the human and bat ACE2 receptor.

Materials and Methods

Used the software PYMOL in order to build structural models that detail interactions between ACE2 and SARS-CoV
Using Geneious Prime, phylograms were created to show phylogenetic relationships between SARS-CoV strains
Clustal Omega was employed in order to align protein sequences

Implications

This work shows that 2019-nCoV strains utilize an ACE2 receptor, similar to the one used by SARS-CoV. This indicates that future strains of SARS-CoV will likely employ a similar binding process. If this can be disrupted, SARS-CoV strains would likely not be able to cause such large problems throughout the population.

Future Directions

Some future directions the study can take would be looking at how ACE2 and SARS-CoV RBD domain interactions can be inhibited in humans. In this way, this study can be used as a preventative measure.

Critical Evaluations

As this study was published in March of 2020, when 2019-nCoV was still largely unknown, I believe it did a good job analyzing the binding interactions of the virus. I would have liked to see more of their methods, as this section was very brief and did not give me insight into how they chose the SARS-CoV strains to specifically study. Overall, I believe this research was well-done, as they had limited information to work with, yet were able to come to a logical conclusion.

Conclusion

This paper aimed to show the binding methodology of 2019-nCoV, using information taken from previous SARS-CoV strains in 2002 and 2003. It presented an important analysis, determining that 2019-nCoV uses an RBM region with five critical amino acid residues in order to bind to human ACE2 receptor. They also determined that the virus originated from bats. These discoveries are important in the understanding of 2019-nCoV's interactions. If these interactions can be disrupted in some way, the virus would cease to cause such high rates of infection.

Acknowledgements

I spoke with my homework partner, Taylor Makela, over Zoom to discuss our shared figure for journal club.
I contacted my TA, Annika Dinulos, to ask about the proper way to format the figure analysis.
I copied and modified the procedures shown on the Week 3 page.
I used the Wan et. al - Receptor Recognition by the Novel Coronavirus from Wuhan paper for the summary.
I used definitions from the following sources: Biology Online Dictionary, Dictionary.com, and Oxford Dictionary of Biochemistry and Molecular Biology
Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Anna Horvath (talk) 11:36, 22 September 2020 (PDT)

References

Biology Online. (2020). Angiotensin. Retrieved 22 September 2020, from https://www.biologyonline.com/dictionary/angiotensin

Biology Online. (2020). Infectivity. Retrieved 22 September 2020, from https://www.biologyonline.com/dictionary/infectivity

Biology Online. (2020). Pathogenesis. Retrieved 22 September 2020, from https://www.biologyonline.com/dictionary/pathogenesis

Biology Online. (2020). Radial. Retrieved 22 September 2020, from https://www.biologyonline.com/dictionary/radial

Dictionary.com. (2020). Iterative. Retrieved 22 September 2020, from https://www.dictionary.com/browse/iterative

Dictionary.com. (2020). Palm civet. Retrieved 22 September 2020, from https://www.dictionary.com/browse/palm-civet

Dictionary.com. (2020). Reiterative. Retrieved 22 September 2020, from https://www.dictionary.com/browse/reiterative

OpenWetWare. (2020). BIOL368/F20:Week 3. Retrieved https://openwetware.org/wiki/BIOL368/F20:Week_3

Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Dipeptidyl-peptidase. In Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. : Oxford University Press. Retrieved 22 Sep. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-5318.

Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Orthologue. In Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. : Oxford University Press. Retrieved 22 Sep. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-14381?rskey=HuHbPl&result=14281

Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Phylogram. In Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. : Oxford University Press. Retrieved 22 Sep. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-15568?rskey=qsnCBm&result=15487

Wan, Y., Shang, J., Graham, R., Baric, R., & Li, F. (2020). Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. Journal Of Virology, 94(7). doi: 10.1128/jvi.00127-20