HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

The HIV-1 nucleocapsid is formed during protease (PR)-directed viral maturation, and is transformed into pre-integration complexes following reverse transcription in the cytoplasm of the infected cell. Here, we report a detailed transmission electron microscopy analysis of the impact of HIV-1 PR and reverse transcriptase (RT) on nucleocapsid plasticity, using in vitro reconstitutions. After binding to nucleic acids, NCp15, a proteolytic intermediate of nucleocapsid protein (NC), was processed at its C-terminus by PR, yielding premature NC (NCp9) followed by mature NC (NCp7), through the consecutive removal of p6 and p1. This allowed NC co-aggregation with its single-stranded nucleic-acid substrate. Examination of these co-aggregates for the ability of RT to catalyse reverse transcription showed an effective synthesis of double-stranded DNA that, remarkably, escaped from the aggregates more efficiently with NCp7 than with NCp9. These data offer a compelling explanation for results from previous virological studies that focused on i) Gag processing leading to nucleocapsid condensation, and ii) the disappearance of NCp7 from the HIV-1 pre-integration complexes. We propose that HIV-1 PR and RT, by controlling the nucleocapsid architecture during the steps of condensation and dismantling, engage in a successive nucleoprotein-remodelling process that spatiotemporally coordinates the pre-integration steps of HIV-1. Finally we suggest that nucleoprotein remodelling mechanisms are common features developed by mobile genetic elements to ensure successful replication

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

International audienceThe Bacillus subtilis LrpC protein belongs to the Lrp/AsnC family of transcriptional regulators. It binds the upstream region of the lrpC gene and autoregulates its expression. In this study, we have dissected the mechanisms that govern the interaction of LrpC with DNA by electrophoretic mobility shift assay, electron microscopy, and atomic force microscopy. LrpC is a structure-specific DNA binding protein that forms stable complexes with curved sequences containing phased A tracts and wraps DNA to form spherical, nucleosome-like structures. Formation of such wraps, initiated by cooperative binding of LrpC to DNA, results from optimal protein/protein interactions specified by the DNA conformation. In addition, we have demonstrated that LrpC constrains positive supercoils by wrapping the DNA in a right-handed superhelix, as visualized by electron microscopy

Specificity of the interaction of RocR with the rocG–rocA intergenic region in Bacillus subtilis

International audienceIn Bacillus subtilis, expression of the rocG gene, encoding glutamate dehydrogenase, and the rocABC operon, involved in arginine catabolism, requires SigL (sigma(54))-containing RNA polymerase as well as RocR, a positive regulator of the NtrC/NifA family. The RocR protein was purified and shown to bind specifically to the intergenic region located between rocG and the rocABC operon. DNaseI footprinting experiments were used to define the RocR-binding site as an 8 bp inverted repeat, separated by one base pair, forming an imperfect palindrome which is present twice within the rocG-rocABC intergenic region, acting as both a downstream activating sequence (DAS) and an upstream activating sequence (UAS). Point mutations in either of these two sequences significantly lowered expression of both rocG and rocABC. This bidirectional enhancer element retained partial activity even when moved 9 kb downstream of the rocA promoter. Electron microscopy experiments indicated that an intrinsically curved region is located between the UAS/DAS region and the promoter of the rocABC operon. This curvature could facilitate interaction of RocR with sigma(54)-RNA polymerase at the rocABC promoter

Assessing intratumor distribution and uptake with MBBG versus MIBG imaging and targeting xenografted PC12-pheochromocytoma cell line

The heterogeneity of tumor uptake is likely to substantially limit the effectiveness of metaiodobenzylguanidine (MIBG) therapy. This study was done to establish whether metabromobenzyl-guanidine (MBBG) can target neuroendocrine tumors and to provide intratumor biodistribution and uptake information in comparison to MIBG. Methods: MBBG and MIBG tumor uptake and kinetic studies were performed in experimental PC-12 pheochromocytoma grown in nude mice. Intratumor distribution studies were performed using autoradiography and secondary ion mass spectrometry (SIMS) microscopy, because the latter technique can detect and potentially quantify both drugs concomitantly within the same tumor specimen. Results: MBBG uptake in PC12 tumors was early (2 hr) and intense (80% ID/g). Retention values were similar for both drugs 24 hr postinjection. At the cellular level, MBBG mostly accumulated in the cytosol. At the multicellular level, cells exhibited staining, but in many areas, SIMS images of both drugs were not spatially correlated. Conclusion: MBBG targeted experimental pheochromocytoma efficiently with high early uptake values. Bromine-76-MBBG is a promising means of imaging and quantifying tumor uptake with PET. Both drugs were localized in the cytosol, but the correlation between the two distributions, as assessed by the values of the standardized local concentrations, was weak although significant multicellularly

Reversible Binding of DNA on NiCl 2 -Treated Mica by Varying the Ionic Strength

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Adsorption of DNA to Mica Mediated by Divalent Counterions: A Theoretical and Experimental Study

The adsorption of DNA molecules onto a flat mica surface is a necessary step to perform atomic force microscopy studies of DNA conformation and observe DNA-protein interactions in physiological environment. However, the phenomenon that pulls DNA molecules onto the surface is still not understood. This is a crucial issue because the DNA/surface interactions could affect the DNA biological functions. In this paper we develop a model that can explain the mechanism of the DNA adsorption onto mica. This model suggests that DNA attraction is due to the sharing of the DNA and mica counterions. The correlations between divalent counterions on both the negatively charged DNA and the mica surface can generate a net attraction force whereas the correlations between monovalent counterions are ineffective in the DNA attraction. DNA binding is then dependent on the fractional surface densities of the divalent and monovalent cations, which can compete for the mica surface and DNA neutralizations. In addition, the attraction can be enhanced when the mica has been pretreated by transition metal cations (Ni(2+), Zn(2+)). Mica pretreatment simultaneously enhances the DNA attraction and reduces the repulsive contribution due to the electrical double-layer force. We also perform end-to-end distance measurement of DNA chains to study the binding strength. The DNA binding strength appears to be constant for a fixed fractional surface density of the divalent cations at low ionic strength (I < 0.1 M) as predicted by the model. However, at higher ionic strength, the binding is weakened by the screening effect of the ions. Then, some equations were derived to describe the binding of a polyelectrolyte onto a charged surface. The electrostatic attraction due to the sharing of counterions is particularly effective if the polyelectrolyte and the surface have nearly the same surface charge density. This characteristic of the attraction force can explain the success of mica for performing single DNA molecule observation by AFM. In addition, we explain how a reversible binding of the DNA molecules can be obtained with a pretreated mica surface

Transmission Electron Microscopy Reveals an Optimal HIV-1 Nucleocapsid Aggregation with Single-stranded Nucleic Acids and the Mature HIV-1 Nucleocapsid Protein

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