Single-molecule stretching studies of RNA chaperones

Wu, Hao; Rouzina, Ioulia; Williams, Mark C.

doi:10.4161/rna.7.6.13776

Cited by 15 publications

(19 citation statements)

References 104 publications

(140 reference statements)

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“…Comparison of the relative lengths of protein domains in the schematic shown in Figure 1a and the experimental data shown in Figure 3 suggest another possibility. The NC domain of Gag has been shown to have non-specific nucleic acid chaperone activity [52] and it is evident from cryo-EM of P22-∆MA-Gag VLPs that upon ∆MA-Gag binding, ssDNA must collapse to a distribution located close to the P22 procapsid surface, without distortion of the relative arrangement of the principal domains of Gag. As suggested by chromatography, the inward DNA strand is closer to the final state of DNA in an assembled VLP, than the outward DNA strand.…”

Section: Modification Of Templates To Establish Factors Affecting Gagmentioning

confidence: 99%

Virus Matryoshka: A Bacteriophage Particle—Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus

Saxena

Malyutin

et al. 2016

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Immature human immunodeficiency virus type 1 (HIV-1) is approximately spherical, but is constructed from a hexagonal lattice of the Gag protein. As a hexagonal lattice is necessarily flat, the local symmetry cannot be maintained throughout the structure. This geometrical frustration presumably results in bending stress. In natural particles, the stress is relieved by incorporation of packing defects, but the magnitude of this stress and its significance for the particles is not known. In order to control this stress, we have now assembled the Gag protein on a quasi-spherical template derived from bacteriophage P22. This template is monodisperse in size and electron-transparent, enabling the use of cryo-electron microscopy in structural studies. These templated assemblies are far less polydisperse than any previously described virus-like particles (and, while constructed according to the same lattice as natural particles, contain almost no packing defects). This system gives us the ability to study the relationship between packing defects, curvature and elastic energy, and thermodynamic stability. As Gag is bound to the P22 template by single-stranded DNA, treatment of the particles with DNase enabled us to determine the intrinsic radius of curvature of a Gag lattice, unconstrained by DNA or a template. We found that this intrinsic radius is far larger than that of a virion or P22-templated particle. We conclude that Gag is under elastic strain in a particle; this has important implications for the kinetics of shell growth, the stability of the shell, and the type of defects it will assume as it grows.

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Section: Modification Of Templates To Establish Factors Affecting Gagmentioning

confidence: 99%

Virus Matryoshka: A Bacteriophage Particle—Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus

Saxena

Malyutin

et al. 2016

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“…In addition, our DNA stretching assay also demonstrates the strong capability of FIV NC to aggregate dsDNA, as shown by the additional stretching force needed to unwind dsDNA (at low extensions) from its FIV NC-aggregated state (Fig. 4B, (Wu et al, 2010)). Also, in contrast to HIV-1 NC, which destabilizes the DNA duplex by lowering the overstretching transition midpoint, FIV NC appears to stabilize the duplex, as evidenced by the increased overstretching transition midpoint force relative to that observed in the absence of protein (Fig.…”

Section: Discussionmentioning

confidence: 55%

Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

Wu¹,

Wang²,

Naiyer³

et al. 2014

Virus Research

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Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic residue switches the binding mode of FIV NC from primarily electrostatic binding to more non-electrostatic binding, conferring upon it NA interaction properties comparable to that of HIV-1 NC.

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“…This additional force at low extensions is referred to as the DNA compaction force (F c ). The magnitude of the F c reflects the ability of the protein to attract dsDNA, which normally results in DNA aggregation in the absence of applied force [67, 68]. To quantify this compaction force, we used the method described in the legend to Fig.…”

Section: Resultsmentioning

confidence: 99%

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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Single-molecule stretching studies of RNA chaperones

Cited by 15 publications

References 104 publications

Virus Matryoshka: A Bacteriophage Particle—Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus

Virus Matryoshka: A Bacteriophage Particle—Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus

Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

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

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