Nanoparticle-directed assembly of enveloped virus components and applications

Gold nanoparticles have been used as templates to promote self-assembly of enveloped virus shells. This is the first time templated assembly of virus-like particles (VLPs) has been demonstrated for mammalian viruses. This doctoral thesis introduces two new model VLPs derived from HIV-1 and alphavirus, both important viral systems.;The first project is focused on the design of VLPs formed from alphavirus coating proteins. Capsid-like particles have been assembled with gold nanoparticles and used as simplified models for studying assembly mechanisms. Optimal gold core dimensions that fit within the internal capsid space of the virus were determined by measuring nanoparticle encapsulation efficiency as a function of core size. Moreover, a minimum charge of 8000 e- on the core is required for assembly to occur. Complete reconstitution of alphaviruses requires the acquisition of two protein cages, first the capsid interacting directly with the core, and second the envelope membrane that provides specificity towards the host cell. Confocal fluorescence spectroscopy of fluorescently-labeled VLPs confirmed the preservation of their functional character as shown by the successful internalization of virus particles into epithelial cells in vivo.;The second part of this work is dedicated to the study of immature HIV-1 model VLPs. The size polydispersity of immature HIV-1 represents a challenge for traditional methods of biological ultra-structural analysis. An in vitro model for the immature HIV-1 was constructed from recombinant HIV-1 Gag proteins lacking residues 16-99 and gold nanoparticles functionalized with DNA. VLPs containing a 60 nm gold core were successful in decreasing the wide polydispersity of immature-like HIV viruses from 40% to 5% relative standard deviation. Moreover, VLPs obtained from HIV-1 have been shown to form homogeneous monodisperse particles suitable for structural analysis by single-particle EM reconstruction. Our HIV-like particles were used to construct a 3D model for immature non infectious HIV-1. The presence of an extended defect in the protein shell corresponds to a seam that separates the particle into two approximately equal parts. This result introduced a new paradigm in the way how a virus that is made up of rod-like protein subunits grows into a spherical shape.