Final Report Summary - NUCARCHIV (Novel approaches based on bionanoscience to manipulate, study, visualize and dissociate the nucleocapsid complex of HIV)
The goal of our project was to use the recent developments and strategies of nano-biotechnology to form, manipulate and visualise, at the level of single molecule, the nucleocapsid (NC) complex of the human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS). HIV NC is a macromolecular architecture composed of about 2000 NC proteins (NCp7) that bind and condense the viral genome at the confine of the viral particle. A clear understanding of how this complex is formed, its structure and its behavior during the life cycle of HIV are essential for both explaining HIV biology and for unmasking new pharmaceutical targets. However, because of its size and complexity, the NC complex remains currently largely inaccessible with the state-of-the-art experimental methods. We have thus explored the principles of bottom-up self-assembly and atomic force microscopy (AFM) imaging to form biomimetic HIV NCs from their purified components (proteins and nucleic acids) and next to 'see' their remodeling with a resolution never explored before.
We have succeeded in these new approaches. We have setup a complete methodology to assemble biomimetic-NC complexes and to follow their transformations/modulations by other HIV proteins such as the protease (PR) and the reverse transcriptase (RT). AFM images acquisition was realised and showed for the first time single complexes of adequate shapes. Two consecutive reactions including PR and RT were successfully done together in an all-in-one assay and imaged with AFM, as proposed initially in the project. This reconstitution is the most complex functional biological system ever assembled by bottom-up assembly, with about 700 NC proteins, 50 RT and 50 PR 'working' in the same tube to assemble into a single condensed complex where the single stranded DNA scaffold is convertible into double stranded DNA. It paves the way to a future possible reconstitution of fully synthetic HIV-like particles assembled from bottom to top, with an important impact in the field of biotechnology and AIDS vaccine development. Apart of the technical challenge, our data highlight with a novel and original point of view some essential mechanisms of HIV biology and publications in top-ranked revues are expected from this research. We failed however in imaging these complexes in liquid environment due to their weak interaction with the surface. Another strategy using DNA nanotechnology was developed, which leaded to unexpected results with a direct demonstration that HIV NCp7 presents original properties potentially usable for the conception of nanodevices. Registration for a patent has been engaged for this particular part of the project.
A second project aimed at developing of a new class of anti-HIV molecule. The central role of NC protein in HIV replication and its very high conservation have soon inspired researches aiming at targeting it with small molecules. Some of them are actively studied for both the generation of therapeutic vaccine and the generation of a new class of microbicidal preparation. Our project was to develop a 'killer' substrate targeting NCp7, starting from proof-of-concept experiments. This molecule is a small four-stranded DNA structure capable of disrupting the biomimetic-NC complexes described above. As expected, we found that this molecule presents a satisfying antiviral activity. We next modified it with chemical functionalisation to enhance penetration in the virus. We discovered a very promising candidate able to strongly inhibit viral replication (IC50 close to 10 nM) without detectable toxicity. A patent on this molecule is under preparation as a potent low cost virucidal agent for AIDS prevention.
We have succeeded in these new approaches. We have setup a complete methodology to assemble biomimetic-NC complexes and to follow their transformations/modulations by other HIV proteins such as the protease (PR) and the reverse transcriptase (RT). AFM images acquisition was realised and showed for the first time single complexes of adequate shapes. Two consecutive reactions including PR and RT were successfully done together in an all-in-one assay and imaged with AFM, as proposed initially in the project. This reconstitution is the most complex functional biological system ever assembled by bottom-up assembly, with about 700 NC proteins, 50 RT and 50 PR 'working' in the same tube to assemble into a single condensed complex where the single stranded DNA scaffold is convertible into double stranded DNA. It paves the way to a future possible reconstitution of fully synthetic HIV-like particles assembled from bottom to top, with an important impact in the field of biotechnology and AIDS vaccine development. Apart of the technical challenge, our data highlight with a novel and original point of view some essential mechanisms of HIV biology and publications in top-ranked revues are expected from this research. We failed however in imaging these complexes in liquid environment due to their weak interaction with the surface. Another strategy using DNA nanotechnology was developed, which leaded to unexpected results with a direct demonstration that HIV NCp7 presents original properties potentially usable for the conception of nanodevices. Registration for a patent has been engaged for this particular part of the project.
A second project aimed at developing of a new class of anti-HIV molecule. The central role of NC protein in HIV replication and its very high conservation have soon inspired researches aiming at targeting it with small molecules. Some of them are actively studied for both the generation of therapeutic vaccine and the generation of a new class of microbicidal preparation. Our project was to develop a 'killer' substrate targeting NCp7, starting from proof-of-concept experiments. This molecule is a small four-stranded DNA structure capable of disrupting the biomimetic-NC complexes described above. As expected, we found that this molecule presents a satisfying antiviral activity. We next modified it with chemical functionalisation to enhance penetration in the virus. We discovered a very promising candidate able to strongly inhibit viral replication (IC50 close to 10 nM) without detectable toxicity. A patent on this molecule is under preparation as a potent low cost virucidal agent for AIDS prevention.