Nyeo2 Week 11

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Purpose

The purpose of this weeks assignment is to learn about the SARS-CoV-2 spike glycoprotein and how knowledge about its structure, function, and relativity to those of past coronaviruses can propel vaccine and treatment efforts for the current pandemic.

Definitions

  1. furin: Subtilisin-like eukaryotic endopeptidase (a kexin) with substrate specificity for consensus sequence Arg-X-Lys/Arg-Arg at the cleavage site. Furin is known to activate the haemagglutinin of fowl plague virus and will cleave the HIV envelope glycoprotein (gp160) into two portions, gp120 and gp41, a necessary step in making the virus fusion-competent.
  2. zoonotic: viruses that can undergo transformations which allow them to cross various species boundaries to infect humans.
  3. sialoside: Sialic acid‐containing carbohydrates, or sialosides, play significant biological roles in cellular recognition, cell communication, pathogen infections, tumor metastasis, and disease states...
  4. virus-neutralizing antibody: An antibody that binds to a virus and interferes with its ability to infect a cell.
  5. tropism: movement directed towards some positive stimulus, or away from some negative stimulus.
  6. pseudovirions: a synthetic virus consisting of the protein coat from one virus and the DNA from a foreign source.
  7. biolayer interferometry: Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI experiments are used to determine the kinetics and affinity of molecular interactions.
  8. glycosylation sequons: addition of one or more sugars to other molecules such as lipids and proteins(glycosylation). The sequence of consecutive amino acids in a protein that can serve as the attachment site is the sequon.
  9. cryoelectron microscopy: electron microscopy that is performed on cryogenically cooled samples which are embedded in an environment of vitreous water.
  10. Newcastle disease: a highly contagious viral disease of domestic poultry, cage and aviary birds and wild birds.

Article Outline

Introduction

  • Importance/significance
    • The novel coronavirus, SARS-CoV-2, started in Wuhan, China, and has rapidly spread across the globe. Causing deadly pneumonia, the virus is now considered a global pandemic. It is for the benefit of mankind that the virus's structure is understood from a biochemical level so that vaccines/therapies can be developed for those at risk or those who are ill.
  • The three coronaviruses that have infected humans in the past 20 years, SARS-CoV, MERS-CoV, and SARS-CoV-2, are all beta-coronaviruses, which are highly transmissible, and come from other animals.
  • They invade host cells by using a transmembrane spike glycoprotein (S) that sticks out from the surface of the virus.
    • This S glycoprotein is made up of two subunits. S1 binds to the host cell receptor and S2 fuses the viral and cellular membranes.
  • There is usually a cleavage between the S1 and S2 subunits, and always another cleavage by host proteases at the S2' location. Each cleavage and subunit is responsible for a unique step in the entry/fusion process.
  • Each coronavirus species has distinct domains on the S1 subunit to permit entry into host cells via receptors.
    • SARS-CoV and related viruses interact with angiotensin-converting enzyme 2 (ACE2) to enter host cells.
  • The S glycoprotein is exposed on the surface of the virus and is responsible for entry into target cells, so it can be targeted by neutralizing antibodies (Abs), which are the focus for therapies/vaccines.
  • Previous limitations
    • Since SARS-CoV-2 was a previously unknown virus, there were no limitations by previous studies because there were none. However, the study of previous coronaviruses gave scientists a good base of knowledge with which to base their research on.
  • Main results
    • Strong binding of SARS-CoV-2 to hACE2 explains the effective and rapid spread in humans. There is a furin cleavage site between S1 and S2 on the new virus, and its avoidance has a largely unknown effect on entry and tropism of the virus. The virus's S ectodomain trimer can be seen in similar B binding site conformations to previous coronavirus strains. Finally, the researchers found that the polyclonal sera from SARS-CoV S glycoprotein from mice inhibited viral entry into host cells.

Results

ACE2 Is an Entry Receptor for SARS-CoV-2

  • Methods: Used a murine leukemia virus (MLV) pseudotyping system to compare transduction of SARS-CoV-2 S-MLV and SARS-CoV S-MLV into VeroE6 cells and BHK cells, which are known to express ACE2.
  • Figure 1A: SARS-CoV-2 s-MLV and SARS-CoV S-MLV pseudoviruses entered VerE6 cells at similar effectiveness levels.
  • Figure 1B: SARS-CoV-2 S and SARS-CoV-2 S fur/mut entered into BHK cells and were transfected with hACE2. The figure shows that the transfection with hACE2 allowed for the entry of SARS-CoV-2 S-MLV, but the one with the fur/mut mutated virus did not transduce as well.
  • Methods: Sequence analysis used to reveal amino acid insertion between S1 and S2. This, along with western blot analysis, allowed the scientists to find the furin cleavage site and then create an S mutant to see if cleavage was necessary for processing before entering the cell.
  • Figure 1C: The sequence shows the four amino acid insertion in between the S1 and S2 subunits in the alignments of SARS-CoV-2 S, but not in other SARS viruses.
  • Figure 1D: Western blot analysis showed that the SARS-CoV-2 S was processed at this site between the subunits, so there was a furin cleavage in this virus, but not in SARS-CoV S.
  • The cleavage site was found to not be necessary for entry into target cells, but its presence could increase SARS-CoV-2's tropism and transmissibility.

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

  • Methods: Compared infectivity of SARS-CoV-2 and SARS-CoV by using biolayer inferometry to study the affinity of these to viruses for hACE2.
  • Figure 2A: It was found that hACE2 bound to SARS-CoV-2 B binding site with an equilibrium dissociation constant of 1.2 nM
  • Figure 2B: hACE2 bound to SARS-CoV B binding site with an equilibrium dissociation constant of 5.0 nM
  • Table 1: The off-rate was higher was higher for SARS-CoV S^B, and SARS-CoV-2 binds tighter to hACE2.
  • Figure 2C: 8 out of the 14 positions necessary for binding to hACE2 are conserved between SARS-CoV-2 S^B and SARS-CoV S^B, which explains similar binding affinities.

Architecture of the SARS-CoV-2 S Glycoprotein Trimer

  • Methods: To study the SARS-CoV-2 S glycoprotein with cryo-EM, the scientists created a prefusion stabilized ectodomain trimer without the furin cleavage site.
  • Figure 3: Through cryo-EM, multiple 3D conformational states of the SARS-CoV-2 S S^B domains in the S1 subunit were found. The images show a variety of open, closed, and partially open states. This open and closed variety is similar to that of SARS-CoV S and MERS-CoV S trimers. The SARS-CoV-2 S S^B domain is a 160-A-long trimer with a triangular cross-section, which is also similar to that of SARS-CoV. The open conformation is said to be necessary to interact with hACE2, which is how the virus enters the cell. This area can be grounds for antibody neutralizations.
  • Figure 4: This figure is a diagram of the SARS-CoV-2 S glycoprotein structures with N-linked glycans. The glycoprotein contains 22 N-linked glycosylation sequons per protomer, which help in S folding and priming by host proteases. SARS-CoV S had 23 of these sequons.
  • Table 2: 20 of 22 SARS-CoV-2 S glycosylation sequons are conserved and 9 out of 13 glycans in the S1 subunit and all 9 glycans in the S2 subunit are conserved.

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

  • Methods: Mapped the sequence conservation in S sequences in sarbecoviruses.
  • Figure 5A/5B: The S2 subunit is more conserved than S1(S1 more exposed on surface) and S^A and S^B have the highest divergence.
  • Methods: Tested plasma from four mice immunized with SARS-CoV S to see if it would inhibit SARS-CoV-2 S and SARS-CoV S.
  • Figure 5C: Polyclonal immune plasma from four mice with SARS-CoV S inhibits entry of SARS-CoV S-MLV and SARS-CoV-2 S-MLV into VeroE6 cells.

Discussion

  • How do the results of this study compare to the results of previous studies (See Discussion).
    • It was found that SARS-CoV-2 had similar affinity to hACE2 as SARS-CoV from 2002-2003.
    • SARS-CoV-2 S has a furin cleavage site that SARS-CoV S does not have, which can lead to increased cell and tissue tropism, as well as transmissibility. This furin cleavage site, which is polybasic, can be seen in highly transmissible avian influenza and Newcastle disease virus.
    • SARS-CoV-2 S trimers exist in many conformational states, which differ at the B domain opening. This is similar to works that state that these states are necessary in the fusion process of the other two deadly coronaviruses: SARS-CoV S and MERS-CoV S.
      • In human coronaviruses that cause colds, the S trimers exist is only closed states, while in more deadly coronaviruses, the trimer is partially open. This leads the scientists to think that the more pathogenic coronaviruses have these trimers in both open and closed states.
    • The study showed that the S2 subunit of the S glycoprotein was highly conserved, which explains the shared hACE2 entry site in SARS-CoV-2 S and SARS-CoV S. Furthermore, SARS-CoV S can evoke polyclonal antibodies against SARS-CoV-2 by targeting the S2 subunit.
    • The amino acid sequences between the new coronavirus and the 2002-2003 virus very similar, so specific assays will need to be designed to definitively test exposure to SARS-CoV-2.
  • What are the important implications of this work?
    • This work will allow for vaccine efforts to move forward, as scientists will now be able to identify potential epitopes on the S glycoprotein to target.
  • What future directions should the authors take?
    • Authors should start to test these vaccine findings on subjects to see if they inhibit SARS-CoV-2 S-mediated entry in to target cells. An incredible amount of people are dying and being infected every day, so the sooner a vaccine can be approved to administer to humans, the better.

Critical Evaluation

  • I was not fond of how the methods were not their own separate section from the results, so it was hard to pinpoint which results were for which experiment.
  • The graph scales vary, but I think that the data shows what they intended to prove relatively well. They cite quite a bit of information from previous papers on old coronaviruses, and those papers help show that the new virus is similar, but different enough from the old one that vaccine efforts need to be helped.

Scientific Conclusion

The novel coronavirus, SARS-CoV-2, has quickly spread across the globe and is still a growing pandemic. The paper at hand tries to lay out groundwork for vaccines and therapies that would undoubtedly save many lives. What the researchers found was that SARS-CoV-2 is very similar to the two other deadly coronaviruses of the 21st century, most notably SARS-CoV from 2002-2003. SARS-CoV-2 uses a spike glycoprotein (S) to bind to host receptors and enter into target cells. The glycoprotein in SARS-CoV-2 uses hACE2 to enter into cells, and it has a similar binding affinity to that of SARS-CoV. Sequence comparison revealed that only the new virus contains a furin cleavage site between the S1 and S2 subunits on the S glycoprotein, and later, the S^B domain on S2 was shown to exist in multiple open and closed conformational states, each with differing roles in virus entry and pathogenicity. All of this knowledge was used to test immune plasma from mice exposed to SARS-CoV. It is promising results that the antibodies from this plasma inhibits the entry of SARS-CoV-2 into cells.

Acknowledgements

  • I used the protocol and the questions found on Week 11.
  • My homework partners this week were Nathan, Lizzy, and Madeleine.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Nyeo2 (talk) 16:51, 15 April 2020 (PDT)

References

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