RNA Control of HIV-1 Particle Size Polydispersity

HIV-1, an enveloped RNA virus, produces viral particles that are known to be much more heterogeneous in size than is typical of non-enveloped viruses. We present here a novel strategy to study HIV-1 Viral Like Particles (VLP) assembly by measuring the size distribution of these purified VLPs and subsequent viral cores thanks to Atomic Force Microscopy imaging and statistical analysis. This strategy allowed us to identify whether the presence of viral RNA acts as a modulator for VLPs and cores size heterogeneity in a large population of particles. These results are analyzed in the light of a recently proposed statistical physics model for the self-assembly process. In particular, our results reveal that the modulation of size distribution by the presence of viral RNA is qualitatively reproduced, suggesting therefore an entropic origin for the modulation of RNA uptake by the nascent VLP

Etudes biochimiques et structurales de la réparation des lésions multiples de l'ADN

L'auteur n'a pas fourni de résumé en anglais.L'auteur n'a pas fourni de résumé en français

Biochemical and structural studies of the repair of DNA clusters lesions

L'auteur n'a pas fourni de résumé en français.L'auteur n'a pas fourni de résumé en anglais

Structure and Dynamics of DNA Duplexes Containing a Cluster of Mutagenic 8-Oxoguanine and Abasic Site Lesions

International audienceClustered DNA damage sites are caused by ionizing radiation. They are much more difficult to repair than are isolated single lesions, and their biological outcomes in terms of mutagenesis and repair inhibition are strongly dependent on the type, relative position and orientation of the lesions present in the cluster. To determine whether these effects on repair mechanism could be due to local structural properties within DNA, we used 1H NMR spectroscopy and restrained molecular dynamics simulation to elucidate the structures of three DNA duplexes containing bistranded clusters of lesions. Each DNA sequence contained an abasic site in the middle of one strand and differed by the relative position of the 8-oxoguanine, staggered on either the 3′ or the 5′ side of the complementary strand. Their repair by base excision repair protein Fpg was either complete or inhibited. All the studied damaged DNA duplexes adopt an overall B-form conformation and the damaged residues remain intrahelical. No striking deformations of the DNA chain have been observed as a result of close proximity of the lesions. These results rule out the possibility that differential recognition of clustered DNA lesions by the Fpg protein could be due to changes in the DNA's structural features induced by those lesions and provide new insight into the Fpg recognition process

Role of Phosphorylation in Moesin Interactions with PIP 2 -Containing Biomimetic Membranes

International audienceMoesin, a protein of the ezrin, radixin, and moesin family, which links the plasma membrane to the cytoskeleton, is involved in multiple physiological and pathological processes, including viral budding and infection. Its interaction with the plasma membrane occurs via a key phosphoinositide, the phosphatidyl(4,5)inositol-bisphosphate (PIP2), and phosphorylation of residue T558, which has been shown to contribute, in cellulo, to a conformationally open protein. We study the impact of a double phosphomimetic mutation of moesin (T235D, T558D), which mimics the phosphorylation state of the protein, on protein/PIP2/microtubule interactions. Analytical ultracentrifugation in the micromolar range showed moesin in the monomer and dimer forms, with wild-type (WT) moesin containing a slightly larger fraction (∼30%) of dimers than DD moesin (10-20%). Only DD moesin was responsive to PIP2 in its micellar form. Quantitative cosedimentation assays using large unilamellar vesicles and quartz crystal microbalance on supported lipid bilayers containing PIP2 reveal a specific cooperative interaction for DD moesin with an ability to bind two PIP2 molecules simultaneously, whereas WT moesin was able to bind only one. In addition, DD moesin could subsequently interact with microtubules, whereas WT moesin was unable to do so. Altogether, our results point to an important role of these two phosphorylation sites in the opening of moesin: since DD moesin is intrinsically in a more open conformation than WT moesin, this intermolecular interaction is reinforced by its binding to PIP2. We also highlight important differences between moesin and ezrin, which appear to be finely regulated and to exhibit distinct molecular behaviors

Model of entropic selection of viral genome at fixed particle size.

<p><i>(a)</i> Cartoon of the self-assembly. The model is specialized to bimodal products of self-assembly, in which the size of particle are equal and the RNA content are different. (b) Typical large RNA titration computed thanks to the model detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083874#pone.0083874.s001" target="_blank">File S1</a>. The value of parameters chosen for the calculation are found in the <i>material and methods</i>. Blue circles correspond to particles with large RNA, and green crosses correspond to particles lacking large RNA.(c) Phase diagram <i>small RNA/large RNA</i>. The boundary for which the concentration of both particles are equal is shown by blue filled circles. The line joining the circles is drawn to guide the eye.</p

Biochemical characterization of VLPs and viral cores, and AFM imaging.

<p><i>(a)</i> Immunoblot of HIV-1 for mature and immature VLPs and cores. <i>(b)</i> Reverse transcription test on mature VLPs and cores in the presence or the absence of ψRNA in the VLPs or cores. <i>(c)</i> Typical images of mature VLPs. <i>(d)</i> Typical images of viral cores.</p

Combined model of viral genome and particle size entropic selection.

<p>The model is specialized to bimodal products of self-assembly, with different particle size and different RNA content. <i>(a)</i> Typical large RNA titration computed thanks to the model detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083874#pone.0083874.s001" target="_blank">File S1</a>. The value of parameters chosen for the calculation are found in the <i>material and methods</i>. In this case, the number of proteins in the “large particles” is twice the number of proteins in the small one. Red circles correspond to small particles with large RNA, and pink crosses correspond to large particles lacking large RNA. <i>(b)</i> Phase diagram <i>small RNA/large RNA</i>. The boundary for which the concentration of both particles are equal is shown by red filled circles. The line joining the circles is drawn to guide the eye. For comparison, the boundary at fixed particle sizes found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083874#pone-0083874-g005" target="_blank">figure 5b</a> is depicted by a blue dotted line.</p

Statistical analysis of size distributions of VLPs and cores obtained through automated image analysis.

<p>The number of particles is indicated by the value <i>N</i>. <i>(a)</i> and <i>(c)</i> 2D histograms of short and long diameters for respectively mature VLPs and cores. <i>(b)</i> Short diameter histogram obtained by projecting the 2D histograms for VLPs and cores. <i>(d)</i> and <i>(f)</i>, long diameter histogram obtained projecting the 2D histograms for VLPs and cores. <i>(e)</i> Typical example of short and long diameters measured on a single particle.</p

Influence of the presence or absence of viral ψRNA on particle morphogenesis.

<p>The number of particles is indicated by the value <i>N</i>. <i>(a)</i> and <i>(c)</i> 2D histogram of short and long diameters for respectively VLPs in the absence and in the presence of ψRNA. The size distribution is shifted toward smaller value, and its dispersion is reduced. <i>(b)</i> and <i>(d)</i> 2D histogram of short and long diameters for respectively cores in the absence and in the presence of ψRNA. The same shift in the distribution is observed, although with w weaker amplitude. <i>(e)</i> Box plot of equivalent diameters summarizing the previous results. The equivalent diameter is obtained by converting the 2D projected area of the particle into the diameter of a disk that would give the same area.</p