Metabolic labeling of enterovirus 71 with quantum dots for the study of virus receptor usage

Xianliang Ke,Hanzhong Wang,Mingyuan Du,Zhike He,Zhenhua Zheng,Yan Liu,Dan Luo,Ting Wang,Zhongyuan Tan,Yuan Zhang,Chunjie Li

doi:10.1186/s12951-021-01046-5

Abstract

Fluorescent labeling and dynamic tracking is a powerful tool for exploring virus infection mechanisms. However, for small-sized viruses, virus tracking studies are usually hindered by a lack of appropriate labeling methods that do not dampen virus yield or infectivity. Here, we report a universal strategy for labeling viruses with chemical dyes and Quantum dots (QDs). Enterovirus 71 (EV71) was produced in a cell line that stably expresses a mutant methionyl-tRNA synthetase (MetRS), which can charge azidonorleucine (ANL) to the methionine sites of viral proteins during translation. Then, the ANL-containing virus was easily labeled with DBCO-AF647 and DBCO-QDs. The labeled virus shows sufficient yield and no obvious decrease in infectivity and can be used for imaging the virus entry process. Using the labeled EV71, different functions of scavenger receptor class B, member 2 (SCARB2), and heparan sulfate (HS) in EV71 infection were comparatively studied. The cell entry process of a strong HS-binding EV71 strain was investigated by real-time dynamic visualization of EV71-QDs in living cells. Taken together, our study described a universal biocompatible virus labeling method, visualized the dynamic viral entry process, and reported details of the receptor usage of EV71.Graphic

Highlights

Human enterovirus 71 (EV71) is the major causative pathogen of the human hand, foot, and mouth disease (HFMD) [1]
We took advantage of the discovery that a mammalian methionyl-tRNA synthetase (MetRS) mutant could charge methionine tRNA with ANL [25]
An expression cassette of the human MetRS mutant, termed MetRS* in this report, was inserted into the genome of RD cells to generate a cell line that could translate the ATG codon to ANL. When this cell line was used for virus production, ANL was incorporated into the viral structural protein, preparing the progeny virus for selective reaction with fluorescent molecules harboring a DBCO group through copper-free click chemistry

Summary

Introduction

Human enterovirus 71 (EV71) is the major causative pathogen of the human hand, foot, and mouth disease (HFMD) [1]. EV71 infects millions of children in the Asian-Pacific area and causes thousands of deaths every year. The cell entry process is a key step for viruses to establish successful infection in cells and usually provides promising targets for antiviral therapeutic intervention [2]. Many cellular molecules, such as vimentin, SCARB2, PSGL-1, nucleolin, fibronectin, and heparan sulfate proteoglycans, have been proven to serve as cellular receptors for EV71 infection [3,4,5,6,7]. The mechanism of cell entry mediated by the receptors, as well as viral preferences among them, are not fully understood

Methods

Results

Conclusion