Mking44 Week 11

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Purpose

The purpose of this paper is to study the structure, function, and mechanism of the SARS-CoV-2 S glycoprotein binding to host cells, and therefore possibly providing vaccine or therapeutic design for the newly emerging pandemic.

Preparation for Journal Club

The article my group is going to do journal club on is Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. DOI: 10.1016/j.cell.2020.02.058

Ten Biological Terms

1. furin (n): serine endopeptidase that processes various secretory proproteins (including some growth factors) by cleavage at paired basic amino acids. Will cleave the HIV envelope glycoprotein (gp160) into two portions, gp120 and gp41, making the virus fusion-competent. It is inhibited by serpin B8, (A Dictionary of Biomedicine, 2010).
2. biogenesis (n): the synthesis of a substance in a living organism; biosynthesis, (Oxford Dictionary of Biochemistry and Molecular Biology, 2006).
3. sialosides (n): Sialic acid-containing carbohydrates, play critical roles in many biological events and in diseases, including viral and bacterial infections, the immune response, the progression of tumor cell metastasis, (SialoGlyco Chemistry and Biology II. Topics in Current Chemistry, 2014).
4. angiotensin-converting enzyme (n): The dipeptidyl carboxypeptidase that converts angiotensin I into the biologically active form, angiotensin II. Angiotensin converting enzyme 2 is a carboxypeptidase which converts angiotensin I to angiotensin 1–9, a peptide of unknown function, and angiotensin II to angiotensin 1–7, a vasodilator. Polymorphisms in ACE1 are associated with susceptibility to ischaemic stroke and diabetic nephropathy and mutations can cause renal tubular dysgenesis, (A Dictionary of Biomedicine, 2010).
5. cryoelectron microscopy (n): a method for imaging frozen-hydrated specimens at cryogenic temperatures by electron microscopy. Specimens remain in their native state without the need for dyes or fixatives, allowing the study of fine cellular structures, viruses and protein complexes at molecular resolution, (Nature, 2020).
6. pneumocyte (n): a type of cell that lines the walls separating the air sacs in the lungs. Type I pneumocytes are flat and inconspicuous. Type II pneumocytes are cuboidal and secrete surfactant, (Concide Medical Dictionary, 2010).
7. Bio-Layer Interferometry (n): a label-free technology for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time, (ForteBio, 2020).
8. virus-neutralizing antibody (n): An antibody that binds to a virus and interferes with its ability to infect a cell, (NCI Dictionary of Cancer Terms).
9. Newcastle disease (n): An acute, infectious disease which affects birds, including domestic fowl. Although not necessarily fatal, the disease is economically important since affected birds are less productive. The disease affects mainly the respiratory tract, but the nervous system may become involved. Mortality rates vary. It is caused by a paramyxovirus, (A Dictionary of Zoology, 2009).
10. polyclonal antibody (n): An antibody produced by several clones of B lymphocytes and potentially directed at multiple epitopes. Antibody produced by immunization of an animal will be polyclonal, (A Dictionary of Biomedicine, 2010).

Outline

Introduction

Importance/Significance: Unlike other coronaviruses, SARS-CoV-2 is a now considered a worldwide pandemic, so it is important to understand the structure and function of the S glycoprotein in order to develop therapies or vaccines for this virus.

• Coronavirus enters the host cell by the S (spike) glycoprotein that forms a trimer with 3 identical units sticking out of the viral surface. S glycoprotein has two subunits: S1 which binds to the host cell receptor, and S2 which fuses the viral and cell membrane together.
• The S protein is cleaved between the S1 and S2 subunits for much coronaviruses. Then it is further cleaved by host proteases at the S2' site.
  • This cleavage has been studied to activate the glycoprotein to fuse the membranes together by irreversible protein conformation changes.
• Different CoVs use distinct domains to recognize receptors on the host membrane.
  • SARSr-COV (SARS related coronaviruses) interact with angiotensin-converting enzyme 2 (ACE2) to enter target cells.
• Since the S glycoprotein is protuding out of the membrane and permits entry into host cells, it is the main target for neutralizing antibodies (Abs) for therapies and vaccine design.

Limitations of previous study: SARS-CoV-2 is a different, new virus that emerged in December 2019, so there aren't really limitations, just that it is a new virus. A limitation could be that other researchers mostly studied the amino acid or DNA sequences between different SARS viruses.

• They overcame these limitations by studying SARS-CoV-2 and seeing how it binded with the host cell receptors and the mechanism of inhibition.

Main result: SARS-CoV-2 engages with ACE2 similarily to SARS-CoV-1. However, there was a furin cleavage site between S1 and S2 subunits, which is different than other SARS related viruses. They also found that the S glycoprotein trimer has multiple conformational changes that are similar to other SARS related viruses. Finally, they found that Abs of SARS-CoV-1 can inhibit SARS-CoV-2 pseudoviruses.

ACE2 Is an Entry Receptor for SARS-CoV-2

Methods: murine leukemia virus (MLV) pseudotyping system (synthetic viruses), examined both SARS-CoV-1 and SARS-CoV-2 entry into VeroE6 cells (known to express ACE2) as well as BHK cells.

• Figure 1A: SARS-CoV-1 S and SARS-COV-2 S and SARS-CoV-2 S fur/mut into VeroE6 cells measured in relative luminescence units. Data shows they enter equally well.
• Figure 1B: SARS-CoV-2 S or SARS-CoV-2 S fur/mut but into BHK cells and transfected with hACE2.
  • SARS fur/mut has a higher transduction efficieny in Figure 1A and the opposite trend for Figure 1B, which suggests that s1/s2 cleavage was not necessary for S entry in the conditions of the experiment
• This data shows that ACE2 is the receptor for SARS-CoV-2 and agrees with other evidence found.

Methods: sequence analysis of amino acid sequence and Western blot analysis allowed for a discovery of a furin cleavage system. Therefore, a S mutant was designed to not have the four A.A. residue and furin cleavage site (S fur/mut) in order for it to act like SARS-CoV related viruses.

• Figure 1C: Sequence alignment of SARS 2 and related viruses. There is a four amino acid insertion between the S1 and S2 subunits in comparison to other SARS related viruses, which introduces the furin cleavage site, which is conserved in 144 SARS-2 isolates sequenced to date.
• Figure 1D: SARS-2 is at 75 kDA, but the mutant and SARS-CoV are at 150 kda, which indicates only SARS-2 was cleaved.
  • The discovery of the cleavage site could expand its transmissibility compared with the other SARS viruses due to furin-like proteases and effects on other viruses

SARS-CoV-2 Recognizes hACE2 with Comparable Affinity to SARS-CoV

Methods: In order to study interaction of SARS-CoV and SARS-CoV-2, biolayer interferometry was used to study the kinectics and affinity of hACE2 to both SARS viruses with different dissocation constants. S^B mutations of SARS that caused severe disease in 2002 epidemic were used.

• Figure 2A: SARS-CoV-2 binding to hACE2. (1.2 nM=Kd)
• Figure 2B: SARS-CoV S binding to hACE2 (5 nM=Kd)
• Table 1: SARS-CoV-2 binds a little tighter to hACE2, and off rates were higher for SARS-CoV S^B which goes along with past studies.

Methods: Previous studies identified 14 positions that are key for binding of CoV-S^B to hACE2. Sequence analysis was used for SARS-Cov-2 to compare

• Figure 2C: 8/14 positions are conserved in CoV-2, while the other 6 are semi-conserved.
  • This can explain the similar binding affinities of CoV-2 and CoV S^B for hACE2.
  • SARS-Cov-2 is well adapted to hACE2 receptors as SARS-CoV strains of 2002, which explain rapid spread of the virus in humans.

Architecture of the SARS-CoV-2 S Glycoprotein Trimer

Methods: cryo-EM to study the 3D structure of the glycoprotein, the authors designed a trimer with a furin cleavage site.

• Figure 3: 3D analysis of cryo-EM data showed multiple conformational states of CoV-2 S which corresponds to the S^B domains of the S1 subunit. Half of them were open trimers, and the other half of them were closed.
  • This is similar to other SARS viruses, but Walls et al. did not detect more than one domain open
  • Overall, the SARS-CoV- S protein is 160A long trimer with triangular cross section which resembles SARS-CoV S structure.
  • SARS-CoV-S^B opening is expected to be necessary for interacting with ACE2 and initating the conformational changes which leads to cleavage.
  • Sequence and conserved conformational changes of the SARS and SARS-2 suggest that the antibodies may cross react and neutralize both viruses.
• Figure 4: SARS-CoV-2 is shown to have 22 N-linked glycans per protomer , which participate in S folding and modulate antibody recognition. Oligosaccharides are resolved in 16 of those sites.
  • Previously, SARS-CoV S possesses 23 N linked glycans per protomer, and 19 of them are glycosylated.
• Table 2: 20 out of 22 N linked SARS-2 glycans are conserved in SARS S. (All 9 conserved in S2 subunit)
  • This suggests that fusion to antibodies are comparable among these viruses

SARS-CoV S Elicits Neutralizing Abs Against SARS-CoV-2 S

Methods: Mapping of sequence conservation in different SARS related viruses shows high conservation

• Figure 5A and 5B: side (A) and top (B) view of sequence conservation show that S2 is more conserved than S1 subunit and highest divergence is within SA and SB.
  • Some of the SARS viruses use ACE2, and S1 subunit is more exposed which leads to more selection pressure and thus variation.

Methods: plasma from four mice immunized with SARS-CoV S to inhibit SARS-CoV-2 S and SARS-CoV S entry into target cells.

• Figure 5C: Entry of SARS-CoV and SARS-CoV-2 is greatly inhibited by the mouse polyclonal plasma (10%)
  • Immunity against one virus of SARS can potentially provide protection against realted viruses.

Discussion

• SARS-2 uses hACE2 as a receptor and has a similar binding affinity to the SARS isolates from the 2002 epidemic in other studies.
• The presence of a cleavage site processed by furin like proteases is similar to the sites in highly pathogenic viruses like Newcastle virus and avian influenza viruses in other studies.
  • Furthermore, SARS isolates from 2002 do not have this furin cleavage site, which sets SARS isolates and SARS-2 apart in other studies.
• SARS-CoV-2 was found to have multiple conformational states from the S^B opening in the trimer, which agrees with previous SARS related viruses.
  • However, only closed S trimers have been detected in other coronaviruses such as the common cold.
  • Therefore based on the data, the authors hypothesize that highly pathogenic coronaviruses have S glycoproteins in the partially open and closed states.
• SARS-2 and SARS having high sequence conservation and similar structure could explain the fact why they both use hACE2 to enter target cells.
  • Also the fact that SARS Abs neutralize SARS-2, and it is proposed that probably targets the S^2 based on its conservation between both SARS viruses. **This agrees with other 2020 papers and previous studies showing that it targets this region as well in SARS 2002 isolates.
• These findings demonstrate also that since SARS-2 and previous SARS viruses are so similar that specific assays will need to be carried out.
• Implications: these findings allow for possible therapeutic and vaccination efforts using polyclonal antibodies from SARS-CoV immune plasma and a framework to identify conserved epitopes across S glycoproteins.
• Future Directions: Testing different antibodies and/or treatments that worked for SARS-CoV on SARS-CoV-2 virions in order to see if they will inhibit attachment to hACE2 or use them as a framework to construct new antibodies or treatments.

Critiques

• Methods and Results are combined together with not much explanation on methods
• A lot of supplemental data
• Some of the graphs are scaled differently

Scientific Conclusion

SARS-CoV-2 is a growing pandemic worldwide, and has surpassed its predecessors greatly. By understanding it's structure, mechanism of action, and antigenicity, it can provide a framework to work towards vaccinations or treatments for the disease. The authors of this paper found that, like SARS-CoV, the S glycoprotein uses the ACE2 receptor to enter host cells and binds with similar affinity. Then, by sequencing, the authors found that the SARS CoV-2 S glycoprotein has a furin-cleavage site between the S1 and S2 subunits, which is different than SARS-related viruses. This could explain why it has spread so quickly versus the SARS-1 outbreak in 2002. After, by using cryo-electon microscopy, they discovered the different conformational structures of the glycoprotein, which resembles closely SARS-1. Lastly, they saw that by using neutralizing Abs from SARS-1, it inhibits activity of SARS-2 greatly, which suggests possible therapeutic design.

Acknowledgements

• I copied and modified the protocol from Week 11 for this assignment.
• My homework partners for this week's presentation are Nick and Nathan.
• Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Mking44 (talk) 15:17, 5 April 2020 (PDT)

References

1. Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. DOI: 10.1016/j.cell.2020.02.058
2. OpenWetWare. (2020). BIOL368/S20:Week 11. Retrieved April 5, 2020, from https://openwetware.org/wiki/BIOL368/S20:Week_11
3. Furin (2010). In Lackie, J., A Dictionary of Biomedicine. : Oxford University Press. Retrieved 5 Apr. 2020, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803095838895.
4. Biogenesis (2006). 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 5 Apr. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-2238.
5. Sialosides (2014). In Liang CH., Hsu CH., Wu CY., Sialoside Arrays: New Synthetic Strategies and Applications. In: Gerardy-Schahn R., Delannoy P., von Itzstein M. (eds) SialoGlyco Chemistry and Biology II. Topics in Current Chemistry, vol 367. Springer, Cham. Retrieved 5 Apr. 2020, from https://link.springer.com/chapter/10.1007%2F128_2014_602 DOI: 10.1007/128_2014_602.
6. Angiotensin converting enzyme (2010). In Lackie, J., A Dictionary of Biomedicine. : Oxford University Press. Retrieved 5 Apr. 2020, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803095413310?rskey=oSpjlZ&result=1
7. Cryoelectron microscopy. Retrieved April 5, 2020, from https://www.nature.com/subjects/cryoelectron-microscopy
8. Pneumocyte (2010) In Concise Medical Dictionary. : Oxford University Press. Retrieved 5 Apr. 2020, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803100332744?rskey=TBdol7&result=1
9. Bio-Layer Interferometry (BLI) Technology. Retrieved April 5,2020, from https://www.fortebio.com/applications/bli-technology
10. virus-neutralizing antibody (n.d.) In NCI Dictionary of Cancer Terms. Retrieved 5 Apr. 2020, from https://www.cancer.gov/publications/dictionaries/cancer-terms/def/virus-neutralizing-antibody
11. Newcastle Disease (2009). In Allaby, M. A Dictionary of Zoology. : Oxford University Press. Retrieved 5 Apr. 2020, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803100231107
12. Polyclonal Antibody (2010). In Lackie, J., A Dictionary of Biomedicine. : Oxford University Press. Retrieved 5 Apr. 2020, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803100335453