Quantitative monitoring of the cytoplasmic release of NCp7 proteins from individual HIV-1 viral cores during the early steps of infection

Abstract

Fluorescence microscopy imaging of individual HIV-1 viruses necessitates a specific labeling of viral structures that minimally perturbs the infection process. Herein, we used HIV-1 pseudoviruses containing NCp7 fused to a tetracystein (TC) tag, labeled by a biarsenical fluorescein derivative (FlAsH) to quantitatively monitor the NCp7 protein concentration in the viral cores during the early stages of infection. Single particle imaging of individual pseudoviruses with defined ratios of TC-tagged to non tagged NCp7 proteins, together with theoretical modeling of energy transfer between FlAsH dyes, showed that the high packaging of TC-tagged proteins in the viral cores causes a strong fluorescence quenching of FlAsH and that the fluorescence intensity of individual viral complexes is an appropriate parameter to monitor changes in the amount of NCp7 molecules within the viral particles during infection. Interestingly, we observed a dramatic fluorescence increase of individual FlAsH-labeled pseudoviruses containing 100% TC-tagged NCp7 proteins in infected cells at 8 and 16 h post-infection. This effect was significantly lower for pseudoviruses expressing TC-tagged integrase. Therefore, this fluorescence increase is likely related to the cytoplasmic viral transformation and the release of NCp7 molecules from the viral complexes. This loss of quenching effect is largely reduced when reverse transcriptase is inhibited, showing that NCp7 release is connected to viral DNA synthesis. A spatial analysis further revealed that NCp7-TC release is more pronounced in the perinuclear space, where capsid disassembly is thought to be completed. Quantification of NCp7-TC content based on fluorescence quenching presented in this study evidences for the first time the cytoplasmic release of NCp7 during the remodeling of HIV-1 viral particles on their journey toward the nucleus. The developed approach can be applied to quantify dye concentrations in a wide range of nano-objects by fluorescence microscopy techniques.

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

“…Likewise, a cytoplasmic release of NCp7 during the early stages of infection has been shown in a fluorescence quenching-based microscopy study ( Figure 3 A–C) [ 22 ]. The fluorescent signal of TC/FlAsH labeled NCp7 in VSV-G pseudotyped HIV-1 was monitored during the first 8 h and 16 h of infection in HeLa cells.…”

“…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 ].…”

“…Time-lapse microscopy in living cells has provided critical information on the kinetics of infection, the intracellular transport mechanism [ 3 , 4 , 5 , 10 , 11 ] and has unveiled unexpected steps such that the prolonged viral docking at the nuclear envelope which is crucial for its nuclear entry [ 12 , 13 ]. Smart labeling strategies have been developed to visualize membrane fusion [ 14 ], loss of viral core integrity [ 15 , 16 ], uncoating [ 13 , 17 , 18 , 19 ], release of viral proteins [ 20 , 21 , 22 ], and remodeling of the viral complex. Original tools have been developed to visualize the viral genome and to follow the nuclear trafficking of the PIC leading to integration.…”

Elucidating the microscopic and computational techniques to study the structure and pathology of SARS‐CoVs

“…Apart from the structural analysis of the virus, AFM was used to study the adsorption of virus particles in (Lichtman & Conchello, 2005). Fluorescence microscopy has been used for investigating viruses and their interactions in cellular systems (Beilstein et al, 2019;Dáder et al, 2019;Kumar et al, 2017;Mazumder et al, 2013;Zgheib et al, 2019). SARS-CoV is a highly contagious virus and various pathological facet of the virus yet unknown.…”

“…With the rapid development of the fluorescence technique, various modern microscopies such as laser scanning confocal microscope and superresolution microscopes are used mainly for biomedical applications due to less destructive process as compared with an electron microscope (Lichtman & Conchello, 2005). Fluorescence microscopy has been used for investigating viruses and their interactions in cellular systems (Beilstein et al, 2019; Dáder et al, 2019; Kumar et al, 2017; Mazumder et al, 2013; Zgheib et al, 2019). SARS‐CoV is a highly contagious virus and various pathological facet of the virus yet unknown.…”