ReAsH/tetracystein-based correlative light-electron microscopy for HIV-1 imaging during the early stages of infection

Abstract

Visualization of viruses in the host cell during the course of infection by correlative light-electron microscopy (CLEM) requires a specific labelling of the viral structures in order to recognize the nanometric viral cores in the intracellular environment. For Human immunodeficiency virus type 1 (HIV-1), the labelling approaches developed for fluorescence microscopy are generally not suited for transmission electron microscopy (TEM), so that imaging of HIV-1 particles in infected cells by CLEM is not straightforward. Herein, we adapt the labeling approach with a tetracystein tag (TC) and a biarsenical resorufin-based label (ReAsH) for monitoring the HIV-1 particles during the early stages of HIV-1 infection by CLEM. In this approach, the ReAsH fluorophore triggers the photo-conversion of 3,3-diaminobenzidine tetrahydrochloride (DAB), generating a precipitate sensitive to osmium tetroxide staining that can be visualized by transmission electron microscopy. The TC tag is fused to the nucleocapsid protein NCp7, a nucleic acid chaperone that binds to the viral genome. HeLa cells, infected by ReAsH-labeled pseudoviruses containg NCp7-TC proteins exhibit strong fluorescent cytoplasmic spots that overlap with dark precipitates in the TEM sections. The DAB precipitates corresponding to single viral cores are observed all over the cytoplasm, and notably near microtubules and nuclear pores. This work describes for the first time a specific contrast given by HIV-1 viral proteins in TEM images and opens new perspectives for the use of CLEM to monitor the intracellular traffic of viral complexes.

Imaging Viral Infection by Fluorescence Microscopy: Focus on HIV-1 Early Stage

“…In parallel, labeling systems with smaller genetic tags and fluorescent markers have been implemented. For example, the tetracystein tag (TC-tag; molecular weight = 585 Da), that binds FlAsH or ReAsH fluorophores, has been successfully fused to CA, IN, and Nucleocapsid protein 7 (NCp7) without drastically compromising the viral infectivity [10,22,35,36]. NCp7-TC/FlAsH, ReAsH [22,36] vRNA APOBEC3F-FP (A3F-FP) (cytidine deaminase that is incorporated into virions during production) [37] MICDDRP (multiplex immunofluorescent-cell-based detection of DNA, RNA and proteins; bDNA FISH based vDNA and vRNA labeling combined with immunostaining) [39] [Ru(phen) 2 (dppz)] 2+…”

Indirect visualization of endogenous nuclear actin by correlative light and electron microscopy (CLEM) using an actin-directed chromobody

“…First of all, we established a correlative light and electron microscopy staining protocol. We adopted a CLEM approach based on several published protocols to best suit our aim to morphologically characterize nuclear actin (Fabig et al 2012; Kukulski et al 2011, 2012; Lysova et al 2018; Schwarz and Humbel 2014). First, the sample preparation through high-pressure freezing, freeze substitution and low-temperature embedding in LR gold guaranteed a high level of ultrastructure preservation while maintaining antigenicity.…”

“…For this, a correlative light and electron microscopy (CLEM) protocol was established allowing for simultaneous localization of fluorescently-labeled actin molecules (in the light microscope) and immunogold-labeled actin molecules [in the electron microscope; (Roth et al 1978)] on one section of the same cell. CLEM is a useful versatile approach to localize different macromolecules of interest as shown before (Fabig et al 2012; Kukulski et al 2011, 2012; Lysova et al 2018; Schwarz and Humbel 2014).…”

Single virus tracking

“…成像分辨率的提 高可以实现对病毒各组分动态行为更加深入地解析, 有利于推进侵染机制的探究和相关抗病毒药物的开 发 [47] . 高性能荧光材料的开发和人工智能技术的迭代 也将进一步推进超高分辨率显微镜在单病毒示踪领域 中的应用 [48] . 融合进入宿主细胞的, 该过程发生在酸性内体中 [52] .…”