A Simple RNA-DNA Scaffold Templates the Assembly of Monofunctional Virus-Like Particles

Abstract

Using the components of a particularly well-studied plant virus, cowpea chlorotic mottle virus (CCMV), we demonstrate the synthesis of virus-like particles (VLPs) with one end of the packaged RNA extending out of the capsid and into the surrounding solution. This construct breaks the otherwise perfect symmetry of the capsid and provides a straightforward route for monofunctionalizing VLPs using the principles of DNA nanotechnology. It also allows physical manipulation of the packaged RNA, a previously inaccessible part of the viral architecture. Our synthesis does not involve covalent chemistry of any kind; rather, we trigger capsid assembly on a scaffold of viral RNA that is hybridized at one end to a complementary DNA strand. Interaction of CCMV capsid protein with this RNA-DNA template leads to selective packaging of the RNA portion into a well-formed capsid, but leaves the hybridized portion poking out of the capsid through a small hole. We show that the RNA-DNA protruding from the capsid is capable of binding DNA and DNA-functionalized colloidal particles. Additionally, we show that the RNA-DNA scaffold can be used to nucleate virus formation on a DNA-functionalized surface. We believe this self-assembly strategy can be adapted to viruses other than CCMV.

Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome

“…To perform such measurements, we use interferometric scattering microscopy (15). We tether the RNA genome of bacteriophage MS2—a T=3, positive-sense single-stranded RNA virus that infects Escherichia coli bacteria—to the surface of a functionalized coverslip using DNA linkages (16) (Fig. 1 B ).…”

Nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging genome or polyelectrolyte

“…The cowpea chlorotic mottle virus (CCMV) is an icosahedral, single-stranded (ss)RNA plant virus widely used in physical 6 , 8 , 28 and nanotechnological 9 , 10 , 19 , 40 studies. Its T = 3 capsid is made up of 90 chemically identical protein dimers—hereafter called subunits—enclosing the genome shared between four ssRNA segments.…”

A modelling paradigm for RNA virus assembly

“…Moreover, molecular dynamics simulations of capsid assembly, both in the absence and presence of different types of cargoes, have made important contributions to our understanding of virus assembly [ 23 •• , 24 ]. Indeed, viral capsids can be assembled in vitro around different types of cargoes, including anions [ 25 , 26 , 27 ]. The models presented here go one step further.…”