Lipid-specific labeling of enveloped viruses with quantum dots for single-virus tracking

Abstract

24Quantum dots (QDs) possess optical properties of superbright fluorescence, excellent photostability, narrow 25 emission spectra, and optional colors. Labeled with QDs, single molecules/viruses can be rapidly and 26 continuously imaged for a long time, providing more detailed information than labeled with other 27 fluorophores. While they are widely used to label proteins in single-molecule tracking studies, QDs have 28 rarely been used to study virus infection, mainly due to lack of accepted labeling strategies. Here, we report 29 a general method to mildly and readily label enveloped viruses with QDs. Lipid-biotin conjugates were used 30 to recognize and mark viral lipid membranes, and streptavidin (SA)-QD conjugates were used to light them 31 up. Such a method allowed enveloped viruses to be labeled in 2 hours with specificity and efficiency up to 32 99% and 98%. The intact morphology and the native infectivity of viruses could be furthest preserved. With 33 the aid of this QD labeling method, we lit wild-type (WT) and mutant Japanese encephalitis virus (JEV) up, 34 tracked their infection in living Vero cells, and found that H144A and Q258A substitutions in the envelope 35 (E) protein didn't affect the virus intracellular trafficking. The lipid-specific QD labeling method described 36 in this study provides a handy and practical tool to readily "see" the viruses and follow their infection, 37 facilitating the widespread use of single-virus tracking and the uncovering of complex infection 38 mechanisms. 39Author summary 40 Virus infection in host cells is a complex process comprising a large number of dynamic molecular events. 41Single-virus tracking is a versatile technique to study these events. To perform this technique, viruses must 42 be fluorescently labeled to be visible to fluorescence microscopes. Quantum dot is a kind of fluorescent tags 43 that has many unique optical properties. It has been widely used to label proteins in single-molecule tracking 44 studies, but rarely used to study virus infection, mainly due to lack of accepted labeling method. In this 45 study, we developed a lipid-specific method to readily, mildly, specifically, and efficiently label enveloped 46 viruses with quantum dots by recognizing viral envelope lipids with lipid-biotin conjugates and recognizing 47 these lipid-biotin conjugates with streptavidin-quantum dot conjugates. Such a method is superior to the 48 commonly used DiD/DiO labeling and the other QD labeling methods. It is not only applicable to normal 3 49 viruses, but also competent to label the key protein-mutated viruses and the inactivated high virulent viruses, 50 providing a powerful tool for single-virus tracking. 51 Introduction 52 Single-particle tracking is a powerful tool to study the dynamic molecular events in living cells. An essential 53 prerequisite to perform this technique is fluorescently labeling the targets. In the past decade, various 54 fluorescent tags such as organic dyes [1], fluorescent proteins [2], metal complex of dppz [3], and QDs ...

Applications of nanotechnology in virus detection, tracking, and infection mechanisms

“…Hence, a handy and sturdy tool to facilitate the SVT by following the virus infection path for better understanding of the complex infection mechanisms (L.‐J. Zhang et al, 2020).…”

“…Furthermore, precise tracking of pathogens within the host cell is need of the hour to determine the pathogenic mechanism and to pave the way for drug and vaccine development (Nikaeen et al, 2020; L.‐J. Zhang et al, 2020).…”

“…A variety of nanomaterials for virus detection and tracking have been invented, contributing to the elucidation of virus infection mechanisms, such as magnetic and gold (Au) NPs, ZnO/Pt‐Pd, graphene and quantum dots (QDs) (Chan & Nie, 1998; Wen et al, 2014; L.‐J. Zhang et al, 2020).…”

Leveraging 3D Model Systems to Understand Viral Interactions with the Respiratory Mucosa

“…Individual virions can be tracked in real time by directly labelling viral particles with reactive, lipophilic, or intercalating dyes [ 287 ]. Quantum dots, a type of semiconductor nanoparticles, can also be used to label virions [ 288 ] without significantly impacting infectivity [ 289 ]. Once labeled, particles can be imaged directly [ 290 ] in mucus or engineered surrogates [ 273 ].…”

Lipid-mediated biosynthetic labeling strategy for in vivo dynamic tracing of avian influenza virus infection

“…For example, many glycolipids and lipoproteins incorporated into cell surface may be modified with chemical functional groups during the process of lipid metabolism, which might disturb cell membrane activities. 31,32 Recently, Biotin-Cap-PE lipid as a conventional phospholipid-modified agent, could be more optimal for binding streptavidin moieties to the surface of bilayers through the high efficient biotin-avidin conjugation system. 33,34 Thus, it is quite feasible that the metabolic modification of virions through the Biotin-Cap-PE lipid biosynthesis will offer another artificial labeling for enveloped influenza viruses.…”