Cellular IP₆ Levels Limit HIV Production while Viruses that Cannot Efficiently Package IP₆ Are Attenuated for Infection and Replication

Summary: HIV-1 hijacks host proteins to promote infection. Here we show that HIV is also dependent upon the host metabolite inositol hexakisphosphate (IP6) for viral production and primary cell replication. HIV-1 recruits IP6 into virions using two lysine rings in its immature hexamers. Mutation of either ring inhibits IP6 packaging and reduces viral production. Loss of IP6 also results in virions with highly unstable capsids, leading to a profound loss of reverse transcription and cell infection. Replacement of one ring with a hydrophobic isoleucine core restores viral production, but IP6 incorporation and infection remain impaired, consistent with an independent role for IP6 in stable capsid assembly. Genetic knockout of biosynthetic kinases IPMK and IPPK reveals that cellular IP6 availability limits the production of diverse lentiviruses, but in the absence of IP6, HIV-1 packages IP5 without loss of infectivity. Together, these data suggest that IP6 is a critical cofactor for HIV-1 replication

IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis.

The HIV capsid is semipermeable and covered in electropositive pores that are essential for viral DNA synthesis and infection. Here, we show that these pores bind the abundant cellular polyanion IP6, transforming viral stability from minutes to hours and allowing newly synthesised DNA to accumulate inside the capsid. An arginine ring within the pore coordinates IP6, which strengthens capsid hexamers by almost 10°C. Single molecule measurements demonstrate that this renders native HIV capsids highly stable and protected from spontaneous collapse. Moreover, encapsidated reverse transcription assays reveal that, once stabilised by IP6, the accumulation of new viral DNA inside the capsid increases >100 fold. Remarkably, isotopic labelling of inositol in virus-producing cells reveals that HIV selectively packages over 300 IP6 molecules per infectious virion. We propose that HIV recruits IP6 to regulate capsid stability and uncoating, analogous to picornavirus pocket factors. HIV-1/IP6/capsid/co-factor/reverse transcription

A lysine ring in HIV capsid pores coordinates IP6 to drive mature capsid assembly.

The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection

Binding of Transcription Factor GabR to DNA Requires Recognition of DNA Shape at a Location Distinct from its Cognate Binding Site

Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small-angle X-ray scattering and multi-angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR–DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR–DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location from the cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites

Direct Determination of Absolute Molecular Stereochemistry in Gas Phase by Coulomb Explosion Imaging

Bijvoet’s method, which makes use of anomalous x-ray diffraction or dispersion, is the standard means of directly determining the absolute (stereochemical) configuration of molecules, but it requires crystalline samples and often proves challenging in structures exclusively comprising light atoms. Herein, we demonstrate a mass spectrometry approach that directly images the absolute configuration of individual molecules in the gas phase by cold target recoil ion momentum spectroscopy after laser ionization–induced Coulomb explosion. This technique is applied to the prototypical chiral molecule bromochlorofluoromethane and the isotopically chiral methane derivative bromodichloromethane

The Relative Importance of Topography and RGD Ligand Density for Endothelial Cell Adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×102–6×1011 RGD/mm2. We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×105 RGD/mm2 on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×108 RGD/mm2 irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry

Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly

The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 Å resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly

Interactions of tropomyosin Tpm1.1 on a single actin filament: A method for extraction and processing of high resolution TIRF microscopy data.

Skeletal muscle tropomyosin (Tpm1.1) is an elongated, rod-shaped, alpha-helical coiled-coil protein that forms continuous head-to-tail polymers along both sides of the actin filament. In this study we use single molecule fluorescence TIRF microscopy combined with a microfluidic device and fluorescently labelled proteins to measure Tpm1.1 association to and dissociation from single actin filaments. Our experimental setup allows us to clearly resolve Tpm1.1 interactions on both sides of the filaments. Here we provide a semi-automated method for the extraction and quantification of kymograph data for individual actin filaments bound at different Tpm1.1 concentrations. We determine boundaries on the kymograph on each side of the actin filament, based on intensity thresholding, performing fine manual editing of the boundaries (if needed) and extracting user defined kinetic properties of the system. Using our analytical tools we can determine (i) nucleation point(s) and rates, (ii) elongation rates of Tpm1.1, (iii) identify meeting points after the saturation of filament, and when dissociation occurs, (iv) initiation point(s), (v) the final dissociation point(s), as well as (vi) dissociation rates. All of these measurements can be extracted from both sides of the filament, allowing for the determination of possible differences in behaviour on the two sides of the filament, and across concentrations. The robust and repeatable nature of the method allows quantitative, semi-automated analyses to be made of large studies of acto-tropomyosin interactions, as well as for other actin binding proteins or filamentous structures, opening the way for dissection of the dynamics underlying these interactions

Timing performance of NicoLase controller.

<p>A 10 Hz square wave signal from a function generator (blue) provides a trigger signal to the NicoLase Arduino controller (‘IRF + Arduino’, purple) or to the diode laser directly (‘IRF’, green). Mean rise times to half amplitude of normalized signal (dashed lines with standard deviations in gray) are as indicated. Occasional (1 of 57 here) triggers are caught on a second clock cycle by the controller, with a maximum observed delay between incoming trigger and laser firing of 12.38 <i>μ</i>s.</p