In Vitro-Assembled Alphavirus Core-Like Particles Maintain a Structure Similar to That of Nucleocapsid Cores in Mature Virus

Abstract

In vitro-assembled core-like particles produced from alphavirus capsid protein and nucleic acid were studied by cryoelectron microscopy. These particles were found to have a diameter of 420 Å with 240 copies of the capsid protein arranged in a T‫4؍‬ icosahedral surface lattice, similar to the nucleocapsid core in mature virions. However, when the particles were subjected to gentle purification procedures, they were damaged, preventing generation of reliable structural information. Similarly, purified nucleocapsid cores isolated from virus-infected cells or from mature virus particles were also of poor quality. This suggested that in the absence of membrane and glycoproteins, nucleocapsid core particles are fragile, lacking accurate icosahedral symmetry.

The Packaging of Different Cargo into Enveloped Viral Nanoparticles

“…Like cytoplasmic cores isolated from virus-infected cells 47 and in previous studies using CLPs with 48mer DNA, 24 CLPs with pure single-stranded DNA or RNA showed dissociation temperatures of approximately 52°C (Figure 3). In contrast, CLPs containing heparin and Au-nanoparticles showed a slightly higher dissociation temperature, approximately 54°C (Figure 3).…”

“…Structural studies show that CLPs containing nucleic acid cargo are similar in size, morphology, and symmetry to authentic nucleocapsid cores. 24 To test the possibility that assembly entails a constant ratio of negative to positive charges, we quantitated the amounts of different anionic cargo incorporated into CLPs. Cargo molecules included heparin and DNA-derivatized Au-nanoparticle as well as DNA and RNA, and the number of negative charges per cargo molecule varies over a ∼200-fold range.…”

“…14,37,39 The C-terminal domain (residues 101−270) is a chymotrypsin-like domain that comprises a majority of the ordered nucleocapsid core seen in the alphavirus structure. 14,24 There are minimal CP−CP contacts in the C-terminal domain 14,40 suggesting electrostatic interactions between the basic charged N-terminal domain of CP and the acidic residues of the viral RNA drive nucleocapsid core formation.…”

“…23,24 Additional work has shown that RRV CLPs will form with size-selected gold nanoparticles that are surface modified with polyethylene glycol and DNA oligomers. 22 In this work, we assembled RRV CLPs using a range of cargo types and with labeled CP.…”

Core-like Particles of an Enveloped Animal Virus Can Self-Assemble Efficiently on Artificial Templates

“…24 The ability to carry out in vitro assembly of corelike alphavirus particles [25][26][27][28] has made them a good candidate for bionanotechnological applications.…”