<scp>FRET</scp> analysis of HIV‐1 Gag and GagPol interactions

Takagi, Shimon; Momose, Fumitaka; Morikawa, Yuko

doi:10.1002/2211-5463.12328

Cited by 10 publications

(9 citation statements)

References 40 publications

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“…We first assessed the efficiency of homodimerization among Gag-to-Gag (G-G), GagPol(WT)-to-GagPol(WT) and GagPol(IN:M50I)-to-GagPol(IN:M50I). Consistent with another report(17), FRET signals from G-G (Fig. 4a) were stronger than the signals from other pairs (Fig.…”

Section: Resultssupporting

confidence: 93%

“…The autoprocessing of GagPol polyprotein is dependent upon the formation of GagPol polyprotein homodimers (5, 16), thus we hypothesized that an IN:M50I mutation might lead to a structural hindrance in GagPol interfering with homodimerization. To determine whether or not the M50I mutation suppresses homodimer formation, we employed a Förster resonance energy transfer (FRET) assay using the plasmids pGag(MA/EGFP/CA), pGagPol(MA/mSB/CA), pGag(MA/mSB/CA), pGagPol(MA/EGFP/CA) (17). Since the pGag(MA/EGFP/CA) and pGagPol(MA/mSB/CA) contained RH:N79S, they were back-mutated to RH:S79N, and IN:M50I was induced in both plasmids.…”

Section: Resultsmentioning

confidence: 99%

“…These masks were then applied to both unfiltered FRET index, Donor and Acceptor images, hence allowing for unbiased analysis of FRET efficiency for each field of view as a whole, and not singled-out cells. Apparent FRET efficiencies were calculated as previously described (17,73), as a function of acceptor (EA). were identified as the templates of CCD with identity of 99.4 %; and finally two structures (PDB IDs: 5HOT and 1EX4) were chosen as the CTD templates with identity of 100 % and 94.7 %, respectively.…”

Section: Fret Index Image = Ifret -(Adidonor) -(Aaiacceptor)mentioning

confidence: 99%

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Natural Occurring Non-Synonymous Single Nucleotide Polymorphisms in Integrase and RNase H Regulate Assembly and Autoprocessing of HIV-1

Imamichi¹,

Bernbaum

Laverdure³

et al. 2021

Preprint

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Recently, a genome-wide association study using plasma HIV RNA reported that 14 naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) in HIV derived from anti-retrovirus naïve patients were associated with virus load (VL). However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP using site-directed mutagenesis and examined their replicative abilities and biological properties. An HIV variant containing Met-to-Ile change at codon 50 in integrase (HIV(IN:M50I)) was found an impaired virus. Despite the mutation being in integrase, a quantification assay demonstrated that the virus release was significantly suppressed (P<0.001). Transmission electron microscopy analyses revealed that the accumulation of abnormal shapes of buds on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins in the HIV(IN:M50I) particles. Förster Resonance Energy Transfer (FRET) assay displayed that GagPol containing IN:M50I (GagPol(IN:M50I)) significantly increased the efficiency of homodimerization (P<0.05) and heterodimerization with Gag (P<0.001), compared to GagPol(WT). HIV replication assay using a series of variants of HIV(IN:M50I) elucidated that the C-terminus residues, Asn at codon 288, plays a key role in the defect and the impaired maturation and replication capability was rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser change at codon 79 in RNase H. These data demonstrate that Gag and GagPol assembly, virus release and autoprocessing are not only regulated by integrase but also RNase H.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Fret Index Image = Ifret -(Adidonor) -(Aaiacceptor)mentioning

confidence: 99%

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Natural Occurring Non-Synonymous Single Nucleotide Polymorphisms in Integrase and RNase H Regulate Assembly and Autoprocessing of HIV-1

Imamichi¹,

Bernbaum

Laverdure³

et al. 2021

Preprint

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“…One solution to this problem was Förster resonance energy transfer (FRET) which can be used as a molecular ruler across small distances (1–10 nm). Despite its technical challenges, FRET has been applied to investigate virus-host interactions (Emmott et al 2015 ; Koh et al 2011 ; Takagi et al 2017 ). A fundamental solution that overcomes the limitations of Abbe’s law is super-resolution (SR) microscopy, which has been revolutionizing life sciences and is paving its way into single-particle virology.…”

Section: Viral Infection Tracking By Super-resolution Single-moleculementioning

confidence: 99%

Single-particle virology

et al. 2020

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The development of advanced experimental methodologies, such as optical tweezers, scanning-probe and super-resolved optical microscopies, has led to the evolution of single-molecule biophysics, a field of science that allows direct access to the mechanistic detail of biomolecular structure and function. The extension of single-molecule methods to the investigation of particles such as viruses permits unprecedented insights into the behavior of supramolecular assemblies. Here we address the scope of viral exploration at the level of individual particles. In an era of increased awareness towards virology, single-particle approaches are expected to facilitate the in-depth understanding, and hence combating, of viral diseases.

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“…In this case, the efficiency of FRET is measured as the lifetime of the donor fluorescence, which depends on the proximity to the acceptor. In sum, FRET is widely applicable for exploring host–virus interactions [ 198 , 199 , 200 ].…”

Section: Diffraction Limited Microscopymentioning

confidence: 99%

Concepts in Light Microscopy of Viruses

Witte

Andriasyan

Georgi

et al. 2018

Viruses

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Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research.

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FRET analysis of HIV‐1 Gag and GagPol interactions

Cited by 10 publications

References 40 publications

Natural Occurring Non-Synonymous Single Nucleotide Polymorphisms in Integrase and RNase H Regulate Assembly and Autoprocessing of HIV-1

Natural Occurring Non-Synonymous Single Nucleotide Polymorphisms in Integrase and RNase H Regulate Assembly and Autoprocessing of HIV-1

Single-particle virology

Concepts in Light Microscopy of Viruses

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