Jesse Bloom PRO
Scientist studying evolution of proteins and viruses.
Fred Hutch Cancer Center / HHMI
Envelope proteins are E3-E2-6K-E1
E2 is primary receptor binding protein
E1 is type II fusion protein
E2 and E1 form trimers on mature virions
Create library of lentiviral particles (pseudoviruses) each expressing different envelope proteins mutant on its surface.
These pseudoviruses encode a nucleotide barcode in their genomes identifying its envelope proteins mutant.
The pseudoviruses can only undergo a single round of cell entry, and so are not human pathogens and can be safely studied at biosafety-level-2.
Lentiviral genomes encode no viral proteins other than the CHIKV envelope proteins, and so cannot undergo multi-cycle replication
Two-step process needed to create genotype-phenotype link because transfected cells take up many plasmids and lentviruses pseudodiploid
Barcodes linked to protein variants after integration into cells due to lentiviral recombination
A more negative score means worse cell entry
293T-Mxra8 cells: Human cells expressing the known human receptor.
293T-TIM1 cells: Human cells expressing TIM1, which enables envelope-protein independent cell binding via virion associated phosphatidylserine.
C6/36 cells: Aedes albopictus mosquito cell line, does not express either Mxra8 (not present in mosquitoes) or TIM1.
Some mutations are better tolerated in 293T-TIM1 cells (which enable envelope-proteins independent cell binding), especially in the E2 receptor binding protein.
Two views of E2 and E1 with sites where mutations worse for entry in C6/36 vs 293T-TIM1 cells in red. The human receptor (Mxra8) is shown in two binding modes in light cartoon.
Transfecting cells with above plasmids produces alphavirus particles, but they do not encode structural proteins (capsid and envelope), so can only enter cells once.
Mxra8-expressing human cells
mosquito cells
Mxra8-expressing human cells
mosquito cells
We measured how all mutations to the CHIKV envelope proteins affected entry in three different cells.
Although we still do not know the mosquito receptor, we identified which mutations specifically impair human versus mosquito cell entry.
We used this information to rationally design CHIKV mutants that could only infect human or mosquito cells.
These loss-of-tropism mutants are potential vaccine candidates, and our data can also be used to engineer the envelope proteins as immunogens.
Will Hannon
Caelan Radford
Brendan Larsen
Bloom lab
Washington University
Michael Diamond
Daved Fremont
Ofer Zimmerman
Tomasz Kaszuba
By Jesse Bloom
Pseudovirus deep mutational scanning of the CHIKV envelope proteins in different cell lines