Abstract
The outbreak of Zika virus (ZIKV) in 2016 created worldwide health emergency which demand urgent research efforts on understanding the virus biology and developing therapeutic strategies. Here, we present a time-resolved chemical proteomic strategy to track the early-stage entry of ZIKV into host cells. ZIKV was labeled on its surface with a chemical probe, which carries a photocrosslinker to covalently link virus-interacting proteins in living cells on UV exposure at different time points, and a biotin tag for subsequent enrichment and mass spectrometric identification of the receptor or other host proteins critical for virus internalization. We identified Neural Cell Adhesion Molecule (NCAM1) as a potential ZIKV receptor and further validated it through overexpression, knockout, and inhibition of NCAM1 in Vero cells and human glioblastoma cells U-251 MG. Collectively, the strategy can serve as a universal tool to map virus entry pathways and uncover key interacting proteins.
Highlights
The outbreak of Zika virus (ZIKV) in 2016 created worldwide health emergency which demand urgent research efforts on understanding the virus biology and developing therapeutic strategies
We devised and synthesized a multifunctional chemical probe (Fig. 1a; Supplementary Figs. 1–3) bearing a labeling group that conjugates the probe to the ZIKV surface, a photo-reactive group that allows for covalent crosslinking of ZIKV proteins to interacting host cell proteins upon UV exposure, and an isolation tag of biotin for purifying the interacting proteins for quantitative mass spectrometry (MS) analysis, facilitating the investigation of host–pathogen interactomes in a time-resolved manner (Fig. 1b)
According to the structure of mature ZIKV determined by cryoelectron microscopy by our group[2], there are 13 cysteines in ZIKV E (12 in the ectodomain, 1 in the transmembrane domain) and no cysteine in M protein (Supplementary Fig. 4a; cysteine residues are highlighted as gray spheres in the structures)
Summary
We did not identify any peptide from virus membrane (M) protein, capsid (C), or any of the nonstructural proteins of ZIKV, further confirming the exclusive tagging of virus surface with the reagent. This result is primarily owing to the membrane impermeable attribute of the chemical proteomic probe imparted by the PEGylated linkers. We used the labeled ZIKV to infect Vero cells and interacting proteins were crosslinked at fixed time points to identify the virus–host factors and elucidate the virus entry mechanism (Fig. 1c). We reasoned that the chemical proteomic probe on the virus surface only crosslinks with proteins in direct contact with ZIKV, which can subsequently withstand vigorous washing conditions to remove a Diazirine c (cross-linking group)
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