Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells

Carlón-Andrés, Irene; Padilla‐Parra, Sergi

doi:10.3390/v12020206

Cited by 7 publications

(5 citation statements)

References 18 publications

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“…During the fusion, the BlaM-Vpr is released into the host cell cytoplasm, where it cleaves its fluorescent substrate CCF2, loaded into the cell. The fusion is detected by a spectral shift of CCF2 [52,53]. This approach provides information about the global fusion efficacy.…”

Section: Viral Entrymentioning

confidence: 99%

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

Mukherjee

Boutant²,

Réal³

et al. 2021

Viruses

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During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step.

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Section: Viral Entrymentioning

confidence: 99%

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

Mukherjee

Boutant²,

Réal³

et al. 2021

Viruses

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“…Frame acquisition averaging was 100 to accumulate enough photons at the cell periphery and at lamellipodia ruffling edges. As described in detail in ( 79 , 80 ), to remove artifacts caused by noise or photobleaching and insufficient signal-to-noise ratio, cells with negligible amounts of bleaching and at least 200 to 1000 photons per pixel were only allowed in the analysis. The acquired fluorescence decay of each pixel was deconvoluted with the instrument response function (IRF) and then postprocessed by being fitted with two-exponential theoretical models using Leica Application Suite X from Leica Microsystems.…”

Section: Methodsmentioning

confidence: 99%

Clustering of phosphatase RPTPα promotes Src signaling and the arthritogenic action of synovial fibroblasts

et al. 2023

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Receptor-type protein phosphatase α (RPTPα) promotes fibroblast-dependent arthritis and fibrosis, in part, by enhancing the activation of the kinase SRC. Synovial fibroblasts lining joint tissue mediate inflammation and tissue damage, and their infiltration into adjacent tissues promotes disease progression. RPTPα includes an ectodomain and two intracellular catalytic domains (D1 and D2) and, in cancer cells, undergoes inhibitory homodimerization, which is dependent on a D1 wedge motif. Through single-molecule localization and labeled molecule interaction microscopy of migrating synovial fibroblasts, we investigated the role of RPTPα dimerization in the activation of SRC, the migration of synovial fibroblasts, and joint damage in a mouse model of arthritis. RPTPα clustered with other RPTPα and with SRC molecules in the context of actin-rich structures. A known dimerization-impairing mutation in the wedge motif (P210L/P211L) and the deletion of the D2 domain reduced RPTPα-RPTPα clustering; however, it also unexpectedly reduced RPTPα-SRC association. The same mutations also reduced recruitment of RPTPα to actin-rich structures and inhibited SRC activation and cellular migration. An antibody against the RPTPα ectodomain that prevented the clustering of RPTPα also inhibited RPTPα-SRC association and SRC activation and attenuated fibroblast migration and joint damage in arthritic mice. A catalytically inactivating RPTPα-C469S mutation protected mice from arthritis and reduced SRC activation in synovial fibroblasts. We conclude that RPTPα clustering retains it to actin-rich structures to promote SRC-mediated fibroblast migration and can be modulated through the extracellular domain.

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“…Only BlaM + cells loaded with an appropriate substrate (CCF4-AM, Thermo Fisher Sci.) convert CCF4-AM to fluorescent CCF4, which is retained in the cytosol and can be quantified via flow cytometry on single cell level as described in the context of HIV-1 (Cavrois et al, 2002), and later by us (Albanese et al, 2021) and others (Carlon-Andres and Padilla-Parra, 2020;Cavrois et al, 2014;Desai et al, 2017;Feeley et al, 2011;Jones and Padilla-Parra, 2016).…”

Section: Virus-free Neutralization Testmentioning

confidence: 97%

Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test

Roessler

Pich

Albanese

et al. 2021

Preprint

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Neutralizing antibodies (NAbs), and their concentration in sera of convalescents and vaccinees are a solid correlate of protection from COVID-19. The antibody concentrations in clinical samples that neutralize SARS-CoV-2 are difficult and very cumbersome to assess with conventional virus neutralization tests (cVNTs), which require work with the infectious virus and biosafety level 3 containment precautions. Alternative virus neutralization tests currently in use are mostly surrogate tests based on direct or competitive ELISA formats or use viral vectors with the spike protein as the single structural component of SARS-CoV-2. To overcome these obstacles, we developed a virus-free, safe and very fast (4.5 h) in vitro diagnostic test based on engineered yet authentic SARS-CoV-2 virus-like-particles (VLPs). They share all features of the original SARS-CoV-2 but lack the viral RNA genome and thus are non-infectious. NAbs induced by infection or vaccination, but also potentially neutralizing monoclonal antibodies can be reliably quantified and assessed with ease and within hours with our test, because they interfere and block the ACE2-mediated uptake of VLPs by recipient cells. Results from the VLP neutralization test (VLPNT) show excellent correlation to a cVNT with fully infectious SARS-CoV-2 and allow to estimate the reduced neutralization capacity of COVID-19 vaccinee sera with variants of concern of SARS-CoV-2.Author summaryThe current pandemic caused by SARS-CoV-2 is a major challenge not only for COVID-19 patients, medical staff, healthcare systems and the general public, but also virologists and clinical laboratories. A particular challenge are safety issues which require biological safety level 3 to work with and study the pathogen. An alternative are virus-like particles (VLPs) of SARS-CoV-2, which are authentic in terms of viral structure and function but are harmless bioproducts in nature. We engineered VLPs which are close-to-perfect mimics of SARS-CoV-2 by all structural, biochemical, physical and functional criteria tested. SARS-CoV-2 VLPs were used in virus neutralization tests (VNTs). Because high concentrations of neutralizing antibodies correlate with protection from COVID-19 practical VNTs are urgently needed. We developed an authentic, virus-free, thus safe yet very fast in vitro diagnostic test with SARS-CoV-2 VLPs. Virus neutralizing antibodies induced by natural infection or vaccination but also certain monoclonal antibodies inhibit VLP fusion with recipient cells carrying ACE2. Quantitative results from a conventional neutralization test with fully infectious SARS-CoV-2 and results from the VLP-based neutralization test correlate perfectly. The setup of the test is very flexible and allows to analyze sera for their neutralizing capacity against different variants of concern and in a standardized assay format.

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Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells

Cited by 7 publications

References 18 publications

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

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

Clustering of phosphatase RPTPα promotes Src signaling and the arthritogenic action of synovial fibroblasts

Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test

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