Physical Principles in the Self-Assembly of a Simple Spherical Virus

Abstract

Viruses are unique among living organisms insofar as they can be reconstituted "from scratch", that is, synthesized from purified components. In the simplest cases, their "parts list" numbers only two: a single molecule of nucleic acid and many (but a very special number, i.e., multiples of 60) copies of a single protein. Indeed, the smallest viral genomes include essentially only two genes, on the order of a thousand times fewer than the next-simplest organisms like bacteria and yeast. For these reasons, it is possible and even fruitful to take a reductionist approach to viruses and to understand how they work in terms of fundamental physical principles. In this Account, we discuss our recent physical chemistry approach to studying the self-assembly of a particular spherical virus (cowpea chlorotic mottle virus) whose reconstitution from RNA and capsid protein has long served as a model for virus assembly. While previous studies have clarified the roles of certain physical (electrostatic, hydrophobic, steric) interactions in the stability and structure of the final virus, it has been difficult to probe these interactions during assembly because of the inherently short lifetimes of the intermediate states. We feature the role of pH in tuning the magnitude of the interactions among capsid proteins during assembly: in particular, by making the interactions between proteins sufficiently weak, we are able to stall the assembly process and interrogate the structure and composition of particular on-pathway intermediates. Further, we find that the strength of the lateral attractions between RNA-bound proteins plays a key role in addressing several outstanding questions about assembly: What determines the pathway or pathways of assembly? What is the importance of kinetic traps and hysteresis? How do viruses copackage multiple short (compared with wild-type) RNAs or single long RNAs? What determines the relative packaging efficiencies of different RNAs when they are forced to compete for an insufficient supply of protein? And what is the limit on the length of RNA that can be packaged by CCMV capsid protein?

Single Particle Observation of SV40 VP1 Polyanion-Induced Assembly Shows That Substrate Size and Structure Modulate Capsid Geometry

“…One possible basis for the difference in substrate preference is rooted in the mechanism of assembly. CCMV assembly appears to depend on initial formation of a nonicosahedral nucleoprotein complex that eventually anneals into a capsid 5, 6, 26 . For SV40, in a SAXS study, assembly on RNA appeared to be a two-state reaction with no large diameter intermediates observed 42 .…”

“…For SV40, in a SAXS study, assembly on RNA appeared to be a two-state reaction with no large diameter intermediates observed 42 . The differences in these mechanisms are likely to arise from the strength of protein-protein interactions relative to protein-RNA interactions, with stronger protein-protein interactions favoring the two-state mechanism 6, 13, 26, 45 . Conversely, weak protein-protein interactions will favor initial formation of an amorphous nucleoprotein complex and are more compatible with a large unstructured substrate such as PSS.…”

“…On ssRNA substrates, VP1 forms uniform 22 nm diameter T=1 virus-like particles (VLPs) under mild solution conditions (50 mM MOPS, pH 7.2, 125 mM NaCl) in the absence of cellular or external factors 23 . Recent studies on Cowpea Chlorotic Mottle Virus (CCMV) demonstrated how different RNA sizes affect the capsid geometry and assembly efficiency 24–26 . However, CCMV is assembled from 90 dimers and may follow a complex assembly path to generate the mixture of pentameric and hexameric vertices found in a T=3 icosahedron 6, 27 .…”

Effects of RNA branching on the electrostatic stabilization of viruses

“…It is difficult to determine the topology of large singlestranded viral RNAs in solution, but recent experiments indicate that the secondary structure does play an important role in the efficient packaging of RNA [10,14]. The secondary structures can be predicted using a number of softwares, such as RNAsubopt (a program in the Vienna RNA package [48]), RNAfold (another program in the Vienna RNA package [48]) and mfold [49].…”

pH Reversible Encapsulation of Oppositely Charged Colloids Mediated by Polyelectrolytes

“…The reversibility is governed by a pH-responsive charge density of the individual capsid proteins due to the presence of acidic and basic moieties. 19−22 …”