Non: Week 11
From OpenWetWare
Jump to navigationJump to search
Purpose
The main purpose of this week's lab is to analyze a primary research article on SARS-CoV-2 that studies the structural and functional elements of the relatively new virus.
New Terms
- glycoprotein - a conjugated protein in which the non‐protein group is a carbohydrate; the sugar moiety occurs most commonly as oligosaccharide (Carmmack, et al. 2006)
- fusogenic - any agent, or set of conditions, that gives rise to fusion of membranes, including cell membranes (and hence of cells) (Carmmack, et al. 2006)
- furin (cleavage site) – 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. (Lackie, 2007)
- ectodomain – The extracellular domain of several membrane-anchored proteins (Hayashida, et al. 2010)
- pseudotyping – A process by which the host range of retroviral vectors including lentiviral vectors can be expanded or altered (Cronin, et al. 2005)
- biolayer interferometry – a label-free biosensor technology that allows for real-time analysis of the kinetics of molecular interactions by detecting changes in interference patterns of white light reflected from the surface of fiber-optic sensor tips.
- abrogated – to suppress or prevent (a biological function or process and especially an immune response) (Merriam Webster)
- sequons – a sequence of consecutive amino acids in a protein that can serve as the attachment site to a polysaccharide, frequently an N-linked-Glycan (Sperelakis, 2012)
- sarbecovirus – subgenus of the genus Betacoronavirus; includes the species SARS-CoV and SARS-CoV2 (Gorbalenya, et al. 2020)
- tropism – movement directed towards some positive stimulus, or away from some negative stimulus (Carmmack, et al. 2006)
Outline
Summary
- SARS-CoV-2 – 90,000 infections and 3,000 deaths
- spike glycoproteins responsible for entry; target of antibodies
- ACE2 receptor used by SARS-COV-2
- SARS-COV-2 has a furin cleavage site in the glycoprotein – differentiates itself from other SARS viruses
- SARS-COV-2 spike glycoprotein structure determined via cryo-EM
=== Introduction
- coronaviruses responsible for three pandemics in 21st century: SARS-CoV, MERS-CoV and now, SARS-CoV-2
- SARS-CoV in 2002, infected 8,098 people and killed 774
- MERS-CoV infected 2,494 and killed 858
- SARS-CoV-2 has infected over 90,000 and killed more than 3,000 though that is rapidly increasing
- coronaviruses likely originated in bats but have intermediate hosts for zoonotic transmission
- intermediate host currently unknown for SARS-CoV-2
- no vaccines have been approved for any human-affecting coronaviruses
- the transmembrane spike (S) glycoprotein forms homotrimers that protrude from viral surface
- has two functional subunits
- S1 – binding to host cell receptor, comprises receptor binding domains, stabilizes prefusion state of S2
- S2 – fusion of viral and cellular membranes
- has two functional subunits
- S is cleaved by host proteases at S2 site upstream of the fusion peptide in all coronaviruses
- entry into the cell is a process that involves receptor binding and proteolytic processing of the S protein to have virus and cell fusion
- coronaviruses have different distinct domains in the S1 to recognize different attachment receptors
- the S glycoprotein is the main target for antibodies as the region is surface exposed
- previously, the anti-SARS-CoV ab S230 mimicked receptor attachment and promoted conformational changes
- ACE2 could mediated SARS-CoV-2 S-mediated entry into cells
- similar affinity to ACE2 with SARS-CoV
- a furin cleavage site was identified at the S1-S2 boundary
- preventing the cleavage affected entry into cells but may also increase tropism
- cryo-EM found that SARS-COV-2 adopted multiple Sb conformations similar to SARS-CoV and MERS-CoV
Results
- ‘’’ ACE2 is an entry receptor for SARS-CoV-2’’’
- shares 76% sequence identity with SARS-CoV; 80% sequence identity with bat SARS viruses
- most similar to bat SARSr-CoV RaTG13: 97% sequence identity
- compared the transduction of SARS-CoV and SARS-CoV-2 into VeroE6 cells with a murine leukemia virus backbone to study ACE2
- later used baby hamster kidney cells (BHK) that were transfected with human ACE2 receptos to confirm that SARS-CoV-2 could utilize ACE2
- found a four amino acid residue sequence between S1 and S2 that introduces a furin cleavage site
- conserved in all SARS-CoV-2 isolates; not found the bat RaTG13 S
- tested using western blot; found that it was processed at the S1-S2 site
- studied the importance of this furin cleavage site by generating a mutant that lacked the site; found that cleavage was not necessary for S-mediated entry but increased tropism and transmissibility
- ’’’SARS-CoV-2 recognizes hACE2 with comparable affinity to SARS-CoV’’’
- binding affinity of SARS-CoV for hACE2 corresponds with rate of replication, transmissibility and disease severity
- used biolayer interferometry to study binding kinetics and affinity of ACE2 to SARS-CoV and SARS-CoV-2
- comparison of 14 previously identified positions that were important for binding in SARS-CoV found that 8 of those positions are conserved, 6 were semi conserved in SARS-CoV-2
- ‘’’Architecture of the SARS-CoV-2 S glycoprotein trimer’’’
- designed a prefusion stabilized ectodomain trimer construct
- found presence of multiple conformational states that corresponded to distinct organization of a single Sb domain with S1
- some had a single open Sb domain; some had all domains closed
- conformational variability similar to SARS-CoV and MERS-CoV
- similar to other betacoronavirus S glycoproteins, S1 has a v-shaped architecture
- SARS-CoV-2 Sb opening is expected to be important for ACE2 interaction and initiating conformation changes that lead to cleavage, membrane fusion and entry
- coronavirus S glycoproteins densely covered by heterogeneous N-linked glycans that are involved in S folding, affecting priming by host proteasesm and might modulate antibody recognition
- SARS-CoV has 23 N-linked glycosylated sequons per protomer, SARS-CoV-2 has 22
- all S2 glycans are conserved; 9/13 are conserved in S1
- ‘’’SARS-CoV S elicits neutralizing Abs against SARS-CoV-2’’’
- the higher divergence in S1 can be explained by the fact that they face more evolutionary selective pressure from the immune system
- plasma from mice with stabilized SARS-CoV S resulted in a 90% reduction in transduction
Discussion
- receptor recognition is a key factor in host cell and tissue tropism
- ability to work with ACE2 from different species reflects the zoonotic transmission from animals to humans
- priming of the S glycoprotein by host proteases through cleavage at the S1-S2 site is another important factor in tropism and pathogenicity
- presence of polybasic cleavage site is a signature of avian influenza diseases
- furin cleavage site sets SARS-CoV-2 apart from SARS-CoV
- coronaviruses use conformation masking and glycan shielding to limit recognition by immune response
- removal of S1 crown is likely necessary to allow for S2 conformational changes that lead to fusion of membranes
- structural and sequence similarity between SARS-CoV and SARS-CoV-2 shows close relationship
Conclusion
The close relationship between SARS-CoV and SARS-CoV-2 is seen both structurally and functionally. SARS-CoV-2 has a furin cleavage site that SARS-CoV does not have and may explain the increased tropism.
Files
Acknowledgements
- My homework partners for this week were Madeleine and Nick, where we worked together in creating a presentation
- I copied and used the Week 11 Protocol].
- Except for what is noted above, this individual journal entry was completed by me and not copied from another source.
Non (talk) 23:37, 15 April 2020 (PDT)
References
- BLI • Biolayer Interferometry • Kinetics Alternative to SPR and Biacore. (n.d.). Retrieved April 15, 2020, from https://2bind.com/bli/
- (2006). glycoprotein. 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 15 Apr. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-8228.
- (2006). fusogen. 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 15 Apr. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-7527.
- (2006). tropism. 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 15 Apr. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-20058.
- Cronin, J., Zhang, X. Y., & Reiser, J. (2005). Altering the tropism of lentiviral vectors through pseudotyping. Current gene therapy, 5(4), 387-398.
- Gorbalenya, A.E., Baker, S.C., Baric, R.S. et al. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5, 536–544 (2020). https://doi.org/10.1038/s41564-020-0695-z
- Hayashida, K., Bartlett, A. H., Chen, Y., & Park, P. W. (2010). Molecular and cellular mechanisms of ectodomain shedding. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology, 293(6), 925-937.
- Lackie, J. M., & Lackie, J. M. (Eds.). (2007). The dictionary of cell and molecular biology. Retrieved from https://electra.lmu.edu:2110
- Merriam-Webster. (n.d.). Abrogate. In Merriam-Webster.com dictionary. Retrieved April 15, 2020, from https://www.merriam-webster.com/dictionary/abrogate
- Sperelakis, N. (2012). Cell Physiology Source Book Essentials of Membrane Biophysics. London: Academic Press. doi: https://doi.org/10.1016/B978-0-12-387738-3.00015-9
- 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
