Owen R. Dailey Week 3

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Purpose

The purpose of this assignment is to gain a fundamental understanding of the method of entry of n-COV-2 into host cells. Additionally, this assignment will be used to practice reading/analyzing primary scientific literature.

Biological Terms and Definitions

  • Domain: “Used to describe a part of a molecule or structure that shares common physicochemical features” (Lackie, 130).
  • Orthologue: “Genes related by common phylogenetic descent and usually with a similar organization” (Lackie, 304).
  • Motif: “A small structural unit that is recognizable in several proteins, e.g. alpha helix” (Lackie, 274).
  • Putative: "Commonly regarded as such; reputed; supposed” (Dictionary.com, 2020).
  • Peptidase: "Any enzyme that catalyzes the splitting of proteins into smaller peptide fractions and amino acids by a process known as proteolysis" (Biology Online, 2020).
  • Iterative: "Repeating; making repetition; repetitious" (Dictionary.com, 2020).
  • Pathogenesis: "The origin and development of disease" (Biology Online, 2020).
  • Angiotensin: “A peptide hormone. Angiotensinogen (renin substrate) is a 60-kDa polypeptide released from the liver and cleaved in the circulation by renin to form the biologically inactive decapeptide angiotensin I. This is in turn cleaved to form active angiotensin II by angiotensin converting enzyme (ACE). * Angiotensin II causes contraction of vascular smooth muscle, and thus raises blood pressure and stimulates aldosterone release from the adrenal glands. Angiotensin is finally broken down by angiotensinases” (Lackie, 27).
  • Infectivity: "The characteristic of a disease agent that embodies capability of entering, surviving in, and multiplying in a susceptible host" (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" (Cammack, 2020).

Article Outline

Introduction

  • The current coronavirus (2019-nCoV) pandemic poses a serious health threat to the modern world
  • The symptoms and transmission of 2019-nCoV is not unlike another coronavirus that emerged in the early 2000’s (SARS-CoV)
  • SARS-CoV had a natural and intermediate reservoir, similar to what 2019-nCoV is predicted to have
    • SARS-CoV: Bats->Palm Civets->Humans
  • Previous research has shown that the Receptor Binding Domain (RBD) of the spike protein SARS-CoV recognizes Angiotensin Converting Enzyme 2 (ACE2), the host cell receptor, and mediates viral entry
  • Previous research has also shown the RBD of SARS-CoV contains a Receptor Binding Motif (RBM)
    • Using SARS-CoV from various host species and ACE2 from various receptor species, it was found that the RBM is what binds to ACE2
  • There are five residues in the RBD of SARS-CoV that confer a higher binding affinity to human ACE2
    • These results were confirmed by placing all of the “human-ACE2-favoring” residues on one RBD and observing the super-affinity and super-efficiency of the virus
  • Figure 1A: Shows the interaction between human ACE2 and the RBD of SARS-CoV spike protein
    • Illustrates how the RBM is the portion of the RBD that interacts with ACE2
  • Figure 1B: Shows the differences in the five critical residues for CE2 binding between various strains of coronaviruses
  • Figure 1C: Shows the interaction between optimized human SARS-CoV RBD and human ACE2
    • 442: F (Phenylalanine): Hydrophobic
    • 472: F (Phenylalanine): Hydrophobic
    • 479: N (Asparagine): Polar uncharged
    • 480: D (Aspartic Acid): Negatively charged
    • 487: T (Threonine): Polar uncharged
  • Figure 1D: Shows the interaction between human 2019-nCoV RBD and human ACE2
    • 455: L (Leucine): Hydrophobic
    • 486: F (Phenylalanine): Hydrophobic
    • 493: Q (Glutamine): Polar uncharged
    • 494: S (Serine): Polar uncharged
    • 501: N (Asparagine): Polar uncharged
  • Significance of this Research
    1. Determine if the 2019-nCoV uses the ACE2 receptor to enter and infect cells
    2. Determine possibilities for the original host(s) and the intermediate host(s) of 2019-nCoV
    3. Gain a better understanding of the conserved and mutated residues of the RBD and RBM of 2019-nCoV that affect the virus' entry into cells
    4. 2019-nCoV uses ACE2 as its host receptor
    5. 2019-nCoV original host was likely a bat (similar to SARS-CoV)
    6. There is not a sufficient amount of evidence to state whether civets or any other animals, were intermediate hosts of 2019-nCoV

Results

  • Percent similarities between SARS-CoV and 2019-nCoV
    • Spike Protein: 76%-78%
    • Spike Protein RBD: 73%-76%
    • Spike Protein RBM: 50%-53%
  • Although the percent similarity may seem low human MERS-CoV and bat MERS-CoV have a lower percent similarity and still bind to the same host receptor
    • This suggests that it is not unlikely that 2019-nCoV binds to ACE2 like SARS-CoV
  • More evidence:
    • There is only one insertion/deletion in the RBM of 2019-nCoV when compared to the RBM of SARS-CoV
    • 9/14 ACE2 interacting amino acids are conserved and 4/14 are partially conserved between 2019-nCoV and SARS-CoV from human, civet, and bat
  • Figure 2: Shows spike protein of 2019-nCoV phylogenetically compare to other coronavirus spike proteins
  • Figure 3A: Sequence alignment of the RBD of SARS-CoV and 2019-nCOV
  • Figure 3B: Percent similarities between the RBD, RBM, and whole spike protein of SARS-CoV and 2019-nCOV
  • Figure 3C: Percent similarities between the RBD, RBM, and whole spike protein of human MERS-CoV and bat HKU4
  • Residue 493 (glutamine) in 2019-nCoV corresponding to residue 479 (asparagine) in human SARS-CoV
    • Near virus binding hotspot, residue 31 (lysine), on human ACE2
      • Hotspot: Salt bridge between residue 31 and residue 35 in a hydrophobic environment
    • Residue 479 (lysine) in civet-SARS-CoV cause steric hindrance with the hotspot
      • Thus, human SARS-CoV mutation allows for better viral binding to human ACE2
    • Residue 493 (glutamine) in 2019-nCoV is compatible with hotspot 31
      • Suggests 2019-nCoV can recognize human ACE2
  • Residue 501 (asparagine) in 2019-nCoV corresponding to residue 487 (threonine) in human SARS-CoV (2002) and residue 487 (serine) in human SARS-CoV (2003)
    • Near virus binding hotspot, residue 353 (lysine), on human ACE2
      • Hotspot: Salt bridge between residue 353 and residue 38 in a hydrophobic environment
    • Residue 487 (lysine) in civet-SARS-CoV cause steric hindrance with the hotspot
      • Human SARS-CoV (2002) mutation allows for better viral binding to human ACE2 because threonine can interact favorably with residue 353 of human ACE2 (human-human transmission)
      • Human SARS-CoV (2003) mutation allows for worse viral binding to human ACE2 because serine can’t interact favorably with residue 353 of human ACE2 (no human-human transmission)
    • Residue 501(asparagine) in 2019nCoV provides more support to hot spot 353 than Ser487 but less than Thr487
      • Recognizes human ACE2 better than SARS-CoV (2003) but worse than SARS-CoV (2002)
  • Residues 455, 486, and 494 in 2019-nCov
    • Residues are leucine, phenylalanine, and serine
    • Leu455, Phe486, and Ser494 of 2019-nCoV RBD support the idea that 2019-nCoV recognizes human ACE2
    • These residues do not play as big of a role as residue 493 and residue 501
  • Interaction of 2019-nCoV of ACE2 receptors from other species
    • “2019-nCoV likely still uses civet ACE2 as its receptor, although it appears that 2019-nCoV RBD has not evolved adaptively for civet ACE2 binding”
    • 2019-nCoV can’t recognize mice or rat ACE2 receptors because of a histidine at position 353
    • 2019-nCoV RBD can recognize ACE2 from pigs, ferrets, cats, orangutans, monkeys, and humans because the ACE2 receptors are identical/similar
  • Figure 4A: The five hotspot residues of ACE2 orthologues
  • Figure 4B: Civet SARS-CoV optimized’s interaction with civet ACE2
  • Figure 4C: Human 2019-nCoV’s interaction with civet ACE2

Discussion

  • Confident that human 2019-nCoV use ACE2 as its host receptor
    • Uses ACE2 more efficiently than SARS-CoV (2003) but less efficiently than SARS-CoV (2002)
    • Possibly more easily transmitted in humans if 2019-nCoV’s residue 501 mutates from an asparagine to a threonine such as the one that occurred in SARS-CoV (2003)
  • 2019-nCov most likely originated in bats and can recognize a large amount of ACE2 orthologues
    • No mutations for binding to civet ACE2
      • Civets weren’t intermediate host
      • Civets transmitted 2019-nCoV to humans before it could mutate and adapt to civet ACE2

Materials and Methods

  • Structural analysis (PyMOL: Protein structure analysis)
  • Phylogenetic analysis (Geneious Prime)
  • Sequence analysis (Clustal Omega)

Implications

  • 2019-nCoV uses the ACE2 receptor for host cells; thus, mutations that occur in the RBD/RBM of 2019-nCoV that confer an increased interaction with ACE2 could significantly increase the infectivity of the virus
    • Specifically a mutation in residue 501
  • Possible drug research could target human ACE2 to prevent the entry of the virus into the host cell

Future Directions

  • Experiment with drugs that would change the structure of 2019n-CoV RBD or human ACE2 in order to prevent viral entry into the host cell

Critical Evaluations

  • My only critique would be that this experiment would be hard to replicate because of the limited materials and methods section, so they did not obey the principles of open science
  • Overall, I think the researchers did a good job of comparing the new sequence of 2019-nCoV to the sequences of past SARS-CoV and explaining the effects of the conserved/mutated residues

Figure Analysis

  • Figure 1A: Structural analysis proving that the RBM is the portion of 2019-nCoV that interacts with ACE2 receptor
  • Figure 1B: The five residues in the RBD that interact with the ACE2 receptor can and have been mutated and adapted
  • Figure 1C: The five residues in the RBD of SARS-CoV interact closely with ACE2 and allow the virus to enter the host cell
  • Figure 1D: Although mutations have occurred, 5 residues are still interacting with ACE2
  • Figure 2: 2019-nCoV is closely related to SARS-CoV
  • Figure 3: There are 5 critical residues that need to interact with ACE2, they can be mutated
  • Figure 4A: ACE2 residues are fairly conserved
  • Figure 4B: There is optimal binding for Civet ACE2 that 2019-nCoV does not have
  • Figure 4C: 2019-nCoV could interact with Civet ACE2

Conclusion

The 2019-nCoV is a virus that most likely came from bats and it uses ACE2 as its host cell receptor. The amino acid sequence of the virus should be monitored closely as a mutation could confer more optimal binding with human ACE2 and leader to higher rates of transmission. Given the current 2019-nCoV pandemic, reading, and understanding scientific literature is imperative.

Aknowledgements

  • I contacted my homework partner, Nathan Beshai, via text to discuss the figure in the article that was assigned to us.
  • I copied and modified the procedures shown on the Week 3 Page.
  • Referenced and copied questions from the BIOL368/F20 week 3 page.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Owen R. Dailey (talk) 19:35, 23 September 2020 (PDT)

References