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An improved labeling strategy enables automated detection of single-virus fusion and assessment of HIV-1 protease activity in single virions

Published on Dec 8, 2017in Journal of Biological Chemistry
· DOI :10.1074/jbc.M117.818088
Chetan Sood4
Estimated H-index: 4
(Emory University),
Ashwanth C. Francis5
Estimated H-index: 5
(Emory University)
+ 1 AuthorsGregory B. Melikyan23
Estimated H-index: 23
(Emory University)
Sources
Abstract
Abstract Enveloped viruses transfer their genomes into host cells by fusing their membrane to that of the cell. To visualize single-virus fusion in living cells, researchers take advantage of HIV-1's proteolytic maturation, which can generate free fluorescent proteins within the viral particle. Co-labeling viruses with a content marker and a fluorescently tagged Vpr (a viral core protein) enables detection of single-virus fusions, but a major limitation of this approach is that not all viral particles incorporate both markers. Here, we designed a labeling strategy based on the bifunctional mCherry-2xCL-YFP-Vpr construct, in which 2xCL denotes a tandem cleavage site for the viral protease. This bi-functional marker was efficiently cleaved during virus maturation, producing free mCherry and the core-associated YFP-Vpr. A nearly perfect colocalization of these two markers in virions and their fixed 1:1 ratio enabled automated detection of singleparticle fusion in both fixed and live cells based upon loss of the mCherry signal. Furthermore, a drop in FRET efficiency between YFP and mCherry due to cleavage of the bi-functional marker, which manifested as a marked shift in the normalized YFP/mCherry fluorescence ratio, reliably predicted viral protease activity in single virions. This feature could discriminate between the particles containing free mCherry, and therefore likely representing mature viruses, and immature particles whose fusion cannot be detected. In summary, our new labeling strategy offers several advantages compared with previous approaches, including increased reliability and throughput of detection of viral fusion. We anticipate that our method will have significant utility for studying viral fusion and maturation.
  • References (44)
  • Citations (7)
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References44
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#1Joao Filipe Inacio Mamede (NU: Northwestern University)H-Index: 5
#2Gianguido C. Cianci (NU: Northwestern University)H-Index: 13
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After fusion, HIV delivers its conical capsid into the cytoplasm. To release the contained reverse-transcribing viral genome, the capsid must disassemble in a process termed uncoating. Defining the kinetics, dynamics, and cellular location of uncoating of virions leading to infection has been confounded by defective, noninfectious particles and the stochastic minefield blocking access to host DNA. We used live-cell fluorescent imaging of intravirion fluid phase markers to monitor HIV-1 uncoating...
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#2Kevin TartourH-Index: 5
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To better characterize the behavior of HIV-1 capsids we developed EURT, for Entry/Uncoating assay based on core-packaged RNA availability and Translation. EURT is an alternative to Blam-Vpr, but as reporter RNA translation relies on core opening, it can be used to study viral capsids behavior. Our study reveals the existence of two major capsid species, a dead end one in which the viral genome is readily exposed to the cytoplasm and a functional one in which such exposure requires artificial cor...
13 CitationsSource
#1Yoonhyeun Oum (Emory University)H-Index: 5
#2Tanay M. Desai (Emory University)H-Index: 8
Last. Gregory B. Melikyan (Emory University)H-Index: 23
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Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion. We have previously demonstrated that incorporation of fluorescent labels int...
8 CitationsSource
#1Ashwanth C. Francis (Emory University)H-Index: 5
#2Mariana Marin (Emory University)H-Index: 18
Last. Gregory B. Melikyan (Emory University)H-Index: 23
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Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that C...
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ABSTRACT Ebola virus (EBOV) makes extensive and intricate use of host factors in the cellular endosomal/lysosomal pathway to release its genome into the cytoplasm and initiate infection. Following viral internalization into endosomes, host cysteine proteases cleave the EBOV fusion glycoprotein (GP) to unmask the binding site for its intracellular receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). GP-NPC1 interaction is required for viral entry. Despite these and other recent discoveri...
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#1Chetan Sood (Emory University)H-Index: 4
#2Mariana Marin (Emory University)H-Index: 18
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HIV-1 fusion leading to productive entry has long been thought to occur at the plasma membrane. However, our previous single virus imaging data imply that, after Env engagement of CD4 and coreceptors at the cell surface, the virus enters into and fuses with intracellular compartments. We were unable to reliably detect viral fusion at the plasma membrane. Here, we implement a novel virus labeling strategy that biases towards detection of virus fusion that occurs in a pH-neutral environment—at the...
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Ebolavirus, a deadly hemorrhagic fever virus, was thought to enter cells through endolysosomes harboring its glycoprotein receptor, Niemann-Pick C1. However, an alternate model was recently proposed in which ebolavirus enters through a later NPC1-negative endosome that contains two-pore Ca2+ channel 2 (TPC2), a newly identified ebolavirus entry factor. Here, using live cell imaging, we obtained evidence that in contrast to the new model, ebolavirus enters cells through endolysosomes that contain...
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Background HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm.
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We have produced a novel, simple and rapid method utilising genetically encodable FRET-based biosensors to permit the detection of HIV-1 virion fusion in living cells. These biosensors show high sensitivity both spatially and temporally, and allow the real-time recovery of HIV-1 fusion kinetics in both single cells and cell populations simultaneously.
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