Non: Week 11

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Course Work
Assignments Journal Pages Shared Class Journal Pages
BIOL368/S20:Week 1 BIOL368/S20:Class Journal Week 1
BIOL368/S20:Week 2 Non: Week 2 BIOL368/S20:Class Journal Week 2
BIOL368/S20:Week 3 Non: Week 3 BIOL368/S20:Class Journal Week 3
BIOL368/S20:Week 4 Non: Week 4 BIOL368/S20:Class Journal Week 4
BIOL368/S20:Week 5 Non: Week 5 BIOL368/S20:Class Journal Week 5
BIOL368/S20:Week 6 Non: Week 6 BIOL368/S20:Class Journal Week 6
BIOL368/S20:Week 8 Non: Week 8 BIOL368/S20:Class Journal Week 8
BIOL368/S20:Week 10 Non: Week 10 BIOL368/S20:Class Journal Week 10
BIOL368/S20:Week 11 Non: Week 11 BIOL368/S20:Class Journal Week 11
BIOL368/S20:Week 13 Non: Week 13 BIOL368/S20:Class Journal Week 13
BIOL368/S20:Week 14 Non: Week 14 BIOL368/S20:Class Journal Week 14


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)



  • 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
  • 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


  • ‘’’ 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


  • 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


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.



  • 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)