Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent, such as RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with a combinatorial number of intermediates. Hence, the mechanism of the reaction is poorly understood. In this paper, we combined solution small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (TEM), and computational modeling to determine the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by SAXS and cryo-TEM. To analyze the data, we performed Langevin dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures consistent with the experimental data. We found that close to physiological ionic strength, $$T=1$$VLPs coexisted with VP1 pentamers. At lower or higher ionic strengths, incomplete particles coexisted with pentamers and $$T=1$$particles. Including the simulated structures was essential to explain the SAXS data in a manner that is consistent with the cryo-TEM images.

The current challenges of structural biophysics include determining the structure of large self-assembled complexes, resolving the structure of ensembles of complex structures and their mass fraction, and unraveling the dynamic pathways and mechanisms leading to the formation of complex structures from their subunits. Modern synchrotron solution X-ray scattering data enable simultaneous high-spatial and high-temporal structural data required to address the current challenges of structural biophysics. These data are complementary to crystallography, NMR, and cryo-TEM data. However, the analysis of solution scattering data is challenging; hence many different analysis tools, listed in the SAS Portal (http://smallangle.org/), were developed. In this review, we start by briefly summarizing classical X-ray scattering analyses providing insight into fundamental structural and interaction parameters. We then describe recent developments, integrating simulations, theory, and advanced X-ray scattering modeling, providing unique insights into the structure, energetics, and dynamics of self-assembled complexes. The structural information is essential for understanding the underlying physical chemistry principles leading to self-assembled supramolecular architectures and computational structural refinement.

{Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent like RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with combinatorial intermediates, and therefore the mechanism of the reaction is poorly understood. In this paper, we determined the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by solution small-angle X-ray scattering (SAXS) and cryo-TEM. To analyze the data, we performed Brownian dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures that were consistent with the experimental data. We found that close to physiological ionic strength

Virus are remarkable self-assembled nanobiomaterial-based machines, exposed to a wide range of pH values. Extreme pH values can induce dramatic structural changes, critical for the function of the virus nanoparticles including assembly and genome uncoating. Tuning cargo - capsid interactions is essential for designing viral-based delivery systems. Here we show how pH controls the structure and activity of wild-type simian virus 40 (wtSV40) and the interplay between its cargo and capsid. Using cryo-TEM and solution X-ray scattering, we found that wtSV40 was stable between pH 5.5 and 9, and only slightly swelled with increasing pH. At pH 3, the particles aggregated, while capsid protein pentamers continued to coat the virus cargo but lost their positional correlations. Infectivity was only partly lost after the particles had been returned to pH 7. At pH 10 or higher, the particles were unstable, lost their infectivity, and disassembled. Using time-resolved experiments we discovered that disassembly began by swelling of the particles, poking a hole in the capsid through which the genetic cargo escaped, and followed by a slight shrinking of the capsids and complete disassembly. These findings provide insight into the fundamental intermolecular forces, essential for SV40 function, and for designing virus-based nanobiomaterials, including delivery systems and antiviral drugs.

Viruses have been of interest to mankind since their discovery as small infectious agents in the nineteenth century. Because many viruses cause diseases to humans and agriculture, they were rigorously studied for biological and medical purposes. Viruses have remarkable properties such as the symmetry and self-assembly of their protein envelope, maturation into infectious virions, structural stability, and disassembly. Solution X-ray scattering can probe structures and reactions in solutions, down to subnanometer spatial resolution and millisecond temporal resolution. It probes the bulk solution and reveals the average shape and average mass of particles in solution and can be used to study kinetics and thermodynamics of viruses at different stages of their life cycle. Here we review recent work that demonstrates the capabilities of solution X-ray scattering to study in vitro the viral life cycle.