Fred Hutch Cancer Research Center / HHMI
Slides at http://slides.com/jbloom/salzman-2020
virion image from https://phil.cdc.gov/Details.aspx?pid=23312
-Asn-Ile-Thr-Asn-Leu-
RBD phenotypes
- protein folding
- affinity for ACE2
- binding by antibodies
Structure still matters implicitly, as it largely determines mapping from mutation to phenotype.
-Asn-Ile-Thr-Asn-Leu-
-Asn-Ile-Thr-Glu-Leu-
-Asn-Lys-Thr-Asn-Leu-
(201 sites) X (19 amino-acid mutations per site) = 3,819 mutations
RBD
fluorescent ACE2
yeast
fluorescent tag on RBD
Click here for details on how library is made.
RBD
fluorescent ACE2
yeast
(1) label yeast at different ACE2 concentrations
RBD
fluorescent ACE2
yeast
fluorescent signal
(1) label yeast at different ACE2 concentrations
(2) measure FACS signal at each concentration
[ACE2]
RBD
fluorescent ACE2
yeast
fluorescent signal
(1) label yeast at different ACE2 concentrations
(2) measure FACS signal at each concentration
(3) binding curve of FACS signal versus [ACE2]
[ACE2]
fluorescent signal
[ACE2]
(1) label yeast at different ACE2 concentrations
(2) sort into FACS bins
Deep sequence to assign RBD mutants to bins and infer dissociation curves
(3)
This strategy is TiteSeq, an elaboration of deep mutational scanning.
yeast
fluorescent tag on RBD
In maps, tall letters indicate strong escape mutations
core RBD
receptor-binding motif
ACE2-contact residue
core RBD
receptor-binding motif
ACE2-contact residue
Subtle differences between COV2-2165 & COV2-2832 (e.g., sites 486, 487) validate in neutralization assays (see here).
core RBD
receptor-binding motif
ACE2-contact residue
Spike-expressing VSV (Case*, Rothlauf*, ..., Whelan. Cell Host & Microbe, 2020) in a real-time cell analysis assay (Gilchuk, ..., Crowe. Immunity, 2020).
See here for more details.
mutation
G446D
Q498R
count
3
2
mutation
G446D
Q498R
count
3
2
mutation
G446D
Q498R
count
3
2
mutation
G446D
Q498R
count
3
2
ACE2 binding
weak strong
effect on escape
ACE2 affinity
effect on escape
ACE2 affinity
0 escape mutants in 56 attempts
RBD expression
ACE2 binding
RBD expression /
ACE2 binding
weak strong
Red indicates escape mutations identified by Regeneron in Baum, ..., Kyratsous. Science (2020)
Red indicates escape mutations identified by Regeneron in Baum, ..., Kyratsous. Science (2020)
Tyler Starr
Allie Greaney
Sarah Hilton
Bloom lab (Fred Hutch)
James Crowe
Seth Zost
Pavlo Gilchuk
Crowe lab (Vanderbilt)
Tyler Starr
Allie Greaney
Sarah Hilton
Bloom lab (Fred Hutch)
James Crowe
Seth Zost
Pavlo Gilchuk
Crowe lab (Vanderbilt)
Tyler Starr
Allie Greaney
Sarah Hilton
Bloom lab (Fred Hutch)
Bloom lab (Fred Hutch)
Crowe lab (Vanderbilt)
Whelan lab (Wash U)
King lab (Univ Wash)
Veesler lab (Univ Wash)
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