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Abstract

Chikungunya virus (CHIKV), a positive stranded RNA alphavirus, recently reemerged causing multiple outbreaks around the world. Generally, alphaviruses enter the cell via clathrin-mediated endocytosis. Entry is supported by the structural envelope proteins E2 and E1. Different experimental approaches have been applied previously to identify receptor(s) molecules responsible for CHIKV binding and entry in human and mosquito cells. However they cannot account for all CHIKV entry events in all susceptible cell types.

We performed affinity purification coupled to mass spectrometry to identify entry factors of CHIKV in both human and mosquito cells. We transiently expressed the N-terminally Strep-tagged, full-length envelope gene in 293T and C6/36 cells. Affinity purifications were digested on-bead and analyzed by mass spectrometry. MiST analysis allowed the identification of 39 human proteins with a confidence score above 0.8. Twelve proteins were selected for validation with CRISPR/Cas9 knock-out cells. Three separate AP-MS experiments in C6/36 cells led to the identification of 31, 58 and 11 proteins with a MiST score higher than 0.7, of which 19 proteins were chosen for further analysis.

Using knock-out experiments in 293T cells and a reporter CHIKV (ECSA strain) resulted in the identification of the CD147-CD98 protein complex on human cells as possible entry factor. Repetition of this knock-out experiment using an Asian CHIKV strain combined with E2 staining, confirmed these results. CD147 contains 2 immunoglobulin domains which is similar to MXRA8, a previously identified alphavirus entry factor. The interaction of CD147 with E2 was validated on Western Blot after affinity purification.

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/content/journal/acmi/10.1099/acmi.imav2019.po0032
2019-12-01
2020-01-24
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http://instance.metastore.ingenta.com/content/journal/acmi/10.1099/acmi.imav2019.po0032
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