Determinants of human versus mosquito cell entry by Chikungunya virus envelope
Jesse Bloom
Fred Hutch Cancer Center / HHMI
Chikungunya virus (CHIKV) is mosquito-borne virus that causes fever and joint pain
- CHIKV is an alphavirus
- Transmitted by Aedes aegypti and Aedes albopictus mosquitoes
- Causes severe joint pain and swelling
New outbreaks are growing due to climate-change induced spread of mosquito hosts
As part of its transmission cycle, CHIKV infects both mosquito and human hosts
CHIKV enters cells via its envelope proteins, which are synthesized as a polyprotein
Envelope proteins are E3-E2-6K-E1
E2 is primary receptor binding protein
E1 is type II fusion protein
E2 and E1 form trimers in mature protein
Receptor in human cells is Mxra8, the receptor in mosquito cells is unknown
Defining host-specific sequence determinants of cell entry
Pseudovirus deep mutational scanning
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.
Each lentiviral genome encodes a protein mutant followed by a barcode
Lentiviral genomes encode no viral proteins other than the CHIKV envelope proteins, and so cannot undergo multi-cycle replication
Barcoded pseudovirus library produced via two-step process
Two-step process needed to create genotype-phenotype link because transfected cells take up many plasmids and lentviruses pseudodiploid
Barcoded pseudovirus library produced via two-step process
Barcodes linked to protein variants after integration into cells due to lentiviral recombination
Measurement of how well each mutant can enter Mxra8-expressing cells
Protein mutants have a range of abilities to enter cells
A more negative score means worse cell entry
How nearly all mutations affect cell entry
How nearly all mutations affect cell entry
How do mutations affect entry in different cell lines?
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.
Most mutations similarly affect entry in all three cells, but some cell-specific effects
Some mutations are better tolerated in 293T-TIM1 cells (which enable envelope-proteins independent cell binding), especially in the E2 receptor binding protein.
Mutations worse for entry in 293T-Mxra8 vs 293T-TIM1 cells are near Mxra8 in structure
Mosquito receptor unknown, but can see where mutations impair C6/36 entry
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.
To validate the pseudovirus results, we first used alphavirus reporter particles
Transfecting cells with above plasmids produces alphavirus particles, but they do not encode structural proteins (capsid and envelope), so can only enter cells once.
Mutations impair entry in Mxra8-expressing human or mosquito cells
Mxra8-expressing human cells
mosquito cells
After confirming mutations were loss-of-tropism in pseudovirus and reporter particles, we engineered them into replicative CHIKV
Replicative loss-of-tropism CHIKV mutants in human or mosquito cells
Mxra8-expressing human cells
mosquito cells
Conclusions
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.
Thanks
Will Hannon
Caelan Radford
Brendan Larsen
Bloom lab
Washington University
Michael Diamond
Daved Fremont
Ofer Zimmerman
Tomasz Kaszuba
Chris Nelson
Israel Baltazar-Perez
Samantha Nelson
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