In new study led by Xiaohui Ju, we define how mutations to Chikungunya virus envelope proteins affect entry in human vs mosquito cells. Sheds light on functional constraints & enables us to make loss-of-tropism mutants, which could be of use for vaccines. biorxiv.org
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As background, Chikungunya virus has transmission cycle that involves infecting both mosquitoes & humans or other primates. Infection can cause fever and severe joint pain in humans. Outbreaks are growing due to expanding mosquito range: nytimes.com
Chikungunya virus enters cell using its envelope proteins, which are also target of neutralizing antibodies and vaccine design. A receptor for these viral proteins in mammalian cells is the protein MXRA8, but receptor in mosquito cells is unknown.
We used pseudovirus deep mutational scanning to measure effects of all mutations to envelope proteins in context of single-cycle pseudotyped particles that provide a safe way to study viral protein mutations outside context of fully infectious virus.
We first measured how mutations affect entry in 293T cells expressing human receptor MXRA8. Below is constraint mapped on structure; see dms-vep.org for interactive heatmap of these data.
We then measured how mutations affect entry in two other cells: the mosquito cell-line C6/36, and 293T cells expressing TIM1 which enables envelope protein independent cell binding by virion associated phosphatidylserine. Most mutations similarly affect entry in all three cells, but some have cell-specific effects. For instance, mutations at E2 site 119 are generally tolerated in C6/36 and 293T-TIM1 cells, but deleterious in 293T-MXRA8 cells. (See dms-vep.org for interactive plot.)
Sites where mutations specifically impair entry in 293T-MXRA8 cells mostly at MXRA8 binding interface. We also find sites where mutations specifically impair entry in C6/36 cells. Although mosquito receptor unknown, we hypothesize these sites at its binding interface.
We next used non-replicative single-cycle alphavirus reporter particles (which provide another safe way to study mutations) to validate that mutations identified in deep mutational scanning indeed specifically impaired entry in human or mosquito cells only.
After using pseudoviruses & reporter particles to show mutations *loss* of function, we engineered into Chikungunya virus: mutants lost ability to infect human or mosquito cells. So we reduced natural tropism for both human & mosquito cells to just one type of cell.
Overall, these results shed light on how Chikungunya virus naturally infects cells from highly diverse species. Sequence-function information can aid in immunogen engineering, and loss-of-tropism mutants could be useful in vaccines as well. Data in interactive form at dms-vep.org Thanks to Xiaohui Ju for leading study Special thanks to @msdiamondlab.bsky.social for help Also Will Hannon, Caelan Radford, Brendan Larsen, Daved Fremont, Ofer Zimmerman, Tomasz Kaszuba, Chris Nelson, Israel Baltazar-Perez, Samantha Nelson