Ian R. Wright Week 3

Ian Wright's Bioinformatics Portfolio

Ian R. Wright's User Page

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Assignment Pages

BIOL368/F20:Week 1

BIOL368/F20:Week 2

BIOL368/F20:Week 3

BIOL368/F20:Week 4

BIOL368/F20:Week 5

BIOL368/F20:Week 6

BIOL368/F20:Week 8

BIOL368/F20:Week 9

BIOL368/F20:Week 10

BIOL368/F20:Week 11

BIOL368/F20:Week 12

BIOL368/F20:Week 14

Individual Journal Entries

1. Ian R. Wright Week 1
2. Ian R. Wright Week 2
3. Ian R. Wright Week 3
4. Ian R. Wright Week 4
5. Ian R. Wright Week 5
6. Ian R. Wright Week 6
7. Ian R. Wright Week 7
8. Therapeutic Target Database (TTD) Review
9. Ian R. Wright Week 9
10. Ian R. Wright Week 10
11. Ian R. Wright Week 11
12. The D614G Research Group Week 12
13. Ian R. Wright Week 14
14. The D614G Research Group Week 14

Class Journals

BIOL368/F20:Class_Journal_Week_1

BIOL368/F20:Class_Journal_Week_2

BIOL368/F20:Class_Journal_Week_3

BIOL368/F20:Class_Journal_Week_4

BIOL368/F20:Class_Journal_Week_5

BIOL368/F20:Class_Journal_Week_6

BIOL368/F20:Class_Journal_Week_7

BIOL368/F20:Class_Journal_Week_8

BIOL368/F20:Class_Journal_Week_11

BIOL368/F20:Class_Journal_Week_14

Glossary of Terms

• Motif- A recurring pattern of protein folding
• Palm Civet- An Asian and African tree cat
• Residues- Any of the monomers making up a polymer
• Salt bridge- A link between electrically charged acidic and basic groups, especially on different parts of a large molecule such as a protein
• Bifurcated- Divided into two branches or forks
• Intermediate Host- an organism that passes a virus from one species to another
• Pathogenesis- Development of a disease
• Iterative- Replicative of a procedure or process
• Consensus Radial Phylogram- Phylogenetic tree with estimates of the support for each branch of the tree
• Neighbor-joining build method- Bottom up clustering method for the joining of phylogenetic trees

Purpose

The purpose of this exercise is to discover the affinity of 2019-nCoV spike proteins to ACE-2 receptor proteins. The purpose is to pinpoint critical residue mutations that allow for the human-human transmission of 2019-nCoV.

Receptor Recognition by the Novel Coronavirus from Wuhan by Wan et.al (2020): An Outline

The following is an outline of Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus by Wan et. al (2020)

Abstract

• Background
  • The focus of the study is 2019-nCoV, a novel coronavirus that entered the human population in Wuhan, China.
  • 2019-nCoV is similar to SARS-CoV:
    • Outbreak 2002-2003
    • Acute respiratory syndrome symptoms
    • Bats are natural viral reservoir
    • Belong to same genus of coronavirus: the Beta-Genus
  • Structural studies of SARS-CoV have revealed protein interactions
    • Spike protein receptor-binding domain (RBD) interacts with host cell angiotensin-converting enzyme 2 (ACE2)
  • 2019-nCoV genome has been sequenced prior to study
  • This sequence can be compared to decades of research on SARS-CoV to study similarities
• What was done?
  • Using 2019-nCoV gene sequence and knowledge on SARS-CoV, 2019-nCoV receptor usage was analyzed
• Summary of Findings
  • 2019-nCoV RBD sequence is similar to RBD sequence of SARS-CoV
    • Suggests that 2019-nCoV also binds to host ACE-2
  • Identified critical residues of RBD that have high interaction with human ACE-2
  • 2019-nCoV recognizes ACE-2 from large variety of animals
    • Suggests there are more natural and immediate reservoirs to be discovered
• Relevance
  • Knowledge can assist epidemic surveillance
  • Useful in medical communities in developing methods to combat virus
  • Testing ground for SARS-CoV receptor analysis framework
    • Framework allows for predictions of host cell infectivity and receptor usage as well as identification of animal sources and models of inter-species disease spread

Introduction

• Coronavirus (both SARS-CoV and 2019-nCoV) background
  • Animal source of 2019-nCoV unknown
  • 2019-nCoV is capable of human-human transmission
  • Coronaviruses are RNA viruses
  • Host-cell entry is dependent on affinity between viral spike protein and host receptor
  • Subsequent to spike protein/host receptor interaction, viral and host membranes are fused
  • The last 10 years of SARS research have revealed a core structure to the spike protein along with highly variable Receptor-Binding Motif (RBM)
    • The RBM is the area of the spike protein that binds to ACE-2
    • RBM determines SARS host range

Methods

• Structural analysis of spike and ACE-2 proteins was done using Coot and PyMOL softwares. Coot was used to introduce mutations. PyMOL was used to create protein structure figures
• Phylogenetic tree was created using Geneious Prime software
• Sequence alignment was achieved using Clustal Omega

Results

• Figure 1
  • Figure 1 shows the structure of the Human ACE-2 receptor protein alongside the SARS-in-vitro-optimized RBD (C) and the Human 2019-nCoV RBD model
  • Figure part B is a table showing the pinpoint RBM residue mutations between various SARS viruses and 2019-nCoV
  • Residues 442, 472, 479, 480, and 487 were mutated between SARS-human, SARS-civet, SARS-bat, and the 2003 SARS-human viruses
    • These mutations were crucial to the transmission between species.
  • Each residue mutation is evaluated for its affinity to ACE-2
  • 2019-nCoV contains less favorable interactions to ACE-2 than SARS-human from 2002 but more favorable interactions to ACE-2 than SARS-human from 2003.
    • This suggests that 2019-nCoV uses ACE-2 as a receptor and is transmittable between humans

• Figure 2
  • Figure 2 shows phylogeny of spike proteins of the coronavirus beta-genus
  • It is stated that 2019-nCoV likely originated from bats due to a close relationship with bat beta-genus coronaviruses

• Figure 3
  • Figure 3A shows the annotated protein sequence of:
    • Human-SARS-2002
    • Civet-SARS-2002
    • Bat-SARS-2013
    • 2019-nCoV
  • All 5 critical residues are mutated between Human-SARS-2002 and 2019-nCoV
    • These mutations were analyzed for their compatibility with ACE-2
      1. Residue 493 is still compatible with ACE-2
      2. Residue 501 is less compatible than the analog for 2002 SARS but more compatible than 2003 SARS
      3. Residue 455 is still compatible
      4. Residue 486 is more compatible
      5. Residue 494 is less favorable but still compatible
    • This compatibility supports the claim that 2019-nCoV uses ACE-2 as its receptor

• • Overall sequence similarities (Figure 3B and C)
    • SARS-human to SARS-Civet
      • ~98% similarity between full spike sequence, RBD, and RBM
    • SARS-human to SARS-bat
      • 92-94% similarity between full spike, RBD, and RBM
    • SARS-human to 2019-nCoV
      • ~75% similarity between full spikes and RBDs
      • 50% similarity between RBMs
    • SARS-civet to SARS-bat
      • 92-94% similarity between spikes, RBDs, and RBM
    • SARS-civet to 2019-nCoV
      • ~75% similarity between full spikes and RBDs
      • 50% similarity between RBMs
    • SARS-bat to 2019-nCoV
      • ~75% similarity between full spikes and RBDs
      • 50% similarity between RBMs
    • Although RBM similarity is low between SARS and 2019-nCoV, note:
    • MERS-human compared to HKU4-bat
      • 40% similarity between RBMs
      • Both viruses bind to the same receptor (DPP4)
    • Therefore, it was concluded that 2019-nCoV could use same receptor as SARS

• Figure 4
  • Part A
    • Amino acids of the five critical residues are compared between the ACE-2 proteins of human, civet, bat, mouse, rat, pig, ferret, cat, orangutan, and monkey
    • It is concluded that 2019-nCoV likely recognizes the ACE-2 receptor from pig, ferret, cat, orangutan, and monkey
  • Part B and C
    • Civet ACE-2 structure is shown with the RBD structure from CivetSARS and Human 2019-nCoV
    • There is little compatibility between Civet ACE-2 and 2019-nCoV RBD

Discussion

• 2019-nCoV most likely originated from bats due to close phylogenetic relationship with other bat SARS strains
• Implications of the study
  • Testing of the atomic-level iterative framework for virus-receptor interactions
  • Receptor recognition is a very important factor of determining inter- and intra-species spread of coronaviruses
  • High mutability of RBM implies inter-species adaptation can be common

very important

• • Affinity of 2019-nCoV RBD to human ACE-2 will be significantly enhanced upon an N501T mutation
• The authors should next analyze the affinity of 2019-nCoV to ACE-2 proteins of other various animals
• Critical analysis of conclusions
  • Earlier, it was stated that the authors predicted 2019-nCoV binds to ACE-2 based on the fact that MERS and HKU4 spikes have a low percentage of similarity. MERS and HKU4 bind to a completely different receptor protein (DPP4). The affinity of MERS/HKU4 spike proteins to DPP4 could be very simple in comparison to the ACE-2 connections. The 40% similarity between MERS/HKU4 could be accounting for 100% of the critical binding residues. 100% of the critical residues of 2019-nCoV were mutated from SARS-2002 so why should the authors make the conclusion that 2019-nCoV spike binds to ACE-2?
  • The assumption of 2019-nCoV spike affinity to ACE-2 was strongly validated by the atomic-level analysis of the spike protein/ACE-2 interactions.
  • Although I would not personally be able to understand the methods, there should have been an explanation of the atomic-level interactions that led them to the conclusions of increased or decreased affinity of each critical residue mutation.

Scientific Conclusion

The purpose of this exercise was to discover the affinity of 2019-nCoV spike proteins to ACE-2 receptor proteins. This purpose was achieved and critical residues were identified to describe spike protein compatibility with ACE-2.

Acknowledgements

• BIOL368/F20:Week_3 for providing instructions on how to complete exercise
• Collaboration with User:Jcorrey
  • Discussion on the meaning of Figure 3
  • Discussion on content requirements of outline
• User:Kam_D._Dahlquist for instruction on background information of 2019-nCoV spike protein
• Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

References

• Motif. (2010). Retrieved September 24, 2020, from https://www.genscript.com/molecular-biology-glossary/1943/motif
• Palm civet. (2006). Retrieved September 24, 2020, from https://www.britannica.com/animal/palm-civet
• Residue. (2004). Retrieved September 24, 2020, from https://www.biologyonline.com/dictionary/residue
• Salt Bridge. (2012). Retrieved September 24, 2020, https://www.lexico.com/definition/salt_bridge
• Bifurcate. (2012). Retrieved September 24, 2020, https://www.lexico.com/definition/bifurcate
• Intermediate Host. (2012). Retrieved September 24, 2020, https://www.lexico.com/definition/intermediate_host
• Pathogenesis. (2012). Retrieved September 24, 2020, https://www.lexico.com/definition/pathogenesis
• Iterative. (2012). Retrieved September 24, 2020, https://www.lexico.com/definition/iterative
• 12.4.1 Consensus Trees. (2005). Retrieved September 24, 2020, http://assets.geneious.com/manual/8.1/GeneiousManualsu91.html
• The Neighbor Joining Method. (2010). Retrieved September 24, 2020, http://www.deduveinstitute.be/~opperd/private/neighbor.html
• Wan, Y., Shang, J., Graham, R., Baric, R. S., & 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).