Fluorescence Anisotropy-Based Salt-Titration Approach to Characterize Protein–Nucleic Acid Interactions

Rye-McCurdy, Tiffiny; Rouzina, Ioulia; Musier‐Forsyth, Karin

doi:10.1007/978-1-4939-2214-7_23

Cited by 22 publications

(48 citation statements)

References 37 publications

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“…For the FA binding assays, RNAs were fluorescently labeled at the 3 ′ -end with fluorescein-5-thiosemicarbazide (Invitrogen) using previously described methods (Pagano et al 2007;Rye-McCurdy et al 2015). cm −1 .…”

Section: Preparation Of Proteins and Nucleic Acidsmentioning

confidence: 99%

Anticodon-like binding of the HIV-1 tRNA-like element to human lysyl-tRNA synthetase

Liu

Comandur

Jones

et al. 2016

RNA

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A critical step in the HIV-1 lifecycle involves reverse transcription of the viral genomic RNA (gRNA). Human tRNA Lys3 serves as a primer for transcription initiation and is selectively enriched in virus particles. Human lysyl-tRNA synthetase (hLysRS) is also packaged into virions. Recently, a tRNA-like element (TLE) within the HIV-1 gRNA was shown to mimic the global tRNA fold and bind competitively to hLysRS, suggesting a mechanism of tRNA targeting to the primer binding site (PBS) and release from the synthetase. Here, we use NMR to investigate hLysRS anticodon-binding domain (ACB) binding to six RNA oligonucleotides, including a hairpin derived from the HIV-1 gRNA TLE. We show that ACB interacts with submicromolar affinity to U-rich RNA oligonucleotides-the tRNA Lys3 anticodon stem-loop (ACSL), the WT TLE, and a nonanucleotide, U9. In contrast, the ACB bound only weakly to two TLE loop mutants and a C9 nonanucleotide. NMR chemical shift perturbations induced by each RNA indicate that the ACSL and the WT TLE both interact with the ACB in a strikingly similar manner. Taken together, these findings support the conclusion that tRNA mimicry by the HIV-1 genome leads to a highly specific protein-RNA interaction that facilitates efficient primer release from hLysRS prior to reverse transcription.

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Section: Preparation Of Proteins and Nucleic Acidsmentioning

confidence: 99%

Anticodon-like binding of the HIV-1 tRNA-like element to human lysyl-tRNA synthetase

Liu

Comandur

Jones

et al. 2016

RNA

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“…Fluorescence anisotropy (FA) salt-titration assays have previously been shown to be capable of distinguishing the relative contribution of specific vs. non-specific or electrostatic interactions for any given NC-RNA binding event [27, 75]. Briefly, the FA signal emitted by fluorescently-labeled RNA is measured at fixed protein and RNA concentrations, while the NaCl concentration is varied.…”

Section: Resultsmentioning

confidence: 99%

“…The salt-titration binding assays were performed essentially as previously described [27, 75]. Briefly, a fixed concentration of either HIV-1 or SIV NC (400 nM) was incubated with refolded RNA (10 nM) in increasing NaCl concentrations (30–750 mM) together with 20 mM HEPES (pH 7.5), 20 µM Tris-(2-carboxyethyl)-phosphine, 5 mM 2-mercaptoethanol, and 1 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

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Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition

et al. 2016

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BackgroundThe nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC. Non-human primates are frequently used as a platform to study retroviral infection in vivo; thus, it is important to understand underlying mechanistic differences between HIV-1 and SIV NC.ResultsHere, we characterize SIV NC chaperone activity for the first time. Only modest differences are observed in the ability of SIV NC to facilitate reactions that mimic the minus-strand annealing and transfer steps of reverse transcription relative to HIV-1 NC, with the latter displaying slightly higher strand transfer and annealing rates. Quantitative single molecule DNA stretching studies and dynamic light scattering experiments reveal that these differences are due to significantly increased DNA compaction energy and higher aggregation capability of HIV-1 NC relative to the SIV protein. Using salt-titration binding assays, we find that both proteins are strikingly similar in their ability to specifically interact with HIV-1 Psi RNA. In contrast, they do not demonstrate specific binding to an RNA derived from the putative SIV packaging signal.ConclusionsBased on these studies, we conclude that (1) HIV-1 NC is a slightly more efficient NA chaperone protein than SIV NC, (2) mechanistic differences between the NA interactions of highly similar retroviral NC proteins are revealed by quantitative single molecule DNA stretching, and (3) SIV NC demonstrates cross-species recognition of the HIV-1 Psi RNA packaging signal.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0322-5) contains supplementary material, which is available to authorized users.

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“…Similar high-throughput assays have been previously developed for oncogenic mRNA binding protein IMP-1, 5 Rift Valley fever virus nucleocapsid protein, 6 and other protein nucleic acid interactions. 7 …”

Section: Methodsmentioning

confidence: 99%

Development of High-Throughput Screening Assay for Antihantaviral Therapeutics

Roy

Mir

2017

SLAS Discovery

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Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. There is no treatment for hantavirus diseases at present. Therapeutic intervention during early stages of viral infection can improve the outcome of this zoonotic viral illness. The interaction between an evolutionary conserved sequence at the 5′ terminus of hantaviral genomic RNA and hantavirus nucleocapsid protein plays a critical role in the hantavirus replication cycle. This unique interaction is a novel target for therapeutic intervention of hantavirus disease. We developed a very sensitive, tractable, and cost-effective fluorescence-based assay to monitor the interaction between the nucleocapsid protein and the target RNA sequence. The assay was optimized for high-throughput screening of chemical libraries to identify molecules that interrupt this RNA–protein interaction. The assay was validated using a library of 6880 chemical compounds. This validation screen demonstrated the reproducibility and validity of required statistical criteria for high-throughput screening. The assay is ready to use for high-throughput screening of large chemical libraries to identify antihantaviral therapeutic molecules and can be amenable to similar targets in other viruses.

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Fluorescence Anisotropy-Based Salt-Titration Approach to Characterize Protein–Nucleic Acid Interactions

Cited by 22 publications

References 37 publications

Anticodon-like binding of the HIV-1 tRNA-like element to human lysyl-tRNA synthetase

Anticodon-like binding of the HIV-1 tRNA-like element to human lysyl-tRNA synthetase

Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition

Development of High-Throughput Screening Assay for Antihantaviral Therapeutics

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