Encapsidation of DNA, a protein and a fluorophore into virus-like particles by the capsid protein of cucumber mosaic virus

J Gen Virol. 2012 May;93(Pt 5):1120-1126. doi: 10.1099/vir.0.040170-0. Epub 2012 Jan 25.

Abstract

An important property of some spherical plant viruses is their ability to reassemble in vitro from native capsid protein (CP) and RNA into infectious virus-like particles (VLPs). Virions of cucumber mosaic virus (CMV) are stabilized by protein-RNA interactions and the nucleic acid is essential for assembly. This study demonstrated that VLPs will form in the presence of both ssDNA and dsDNA oligonucleotides, and with a lower size limit of 20 nt. Based on urea disruption assays, assembled VLPs from CMV CP and RNA (termed ReCMV) exhibited a level of stability similar to that of virions purified from plants, whilst VLPs from CMV CP and a 20mer exhibited comparable or greater stability. Fluorescent labelling of VLPs was achieved by the encapsidation of an Alexa Fluor 488-labelled 45mer oligonucleotide (ReCMV-Alexa488-45) and confirmed by transmission electron and confocal microscopy. Using ssDNA as a nucleating factor, encapsidation of fluorescently labelled streptavidin (53 kDa) conjugated to a biotinylated oligonucleotide was observed. The biological activity and stability of ReCMV and ReCMV-Alexa488-45 was confirmed in infectivity assays and insect vector feeding assays. This work demonstrates the utility of CMV CP as a protein cage for use in the growing repertoire of nanotechnological applications.

MeSH terms

  • Animals
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism*
  • Cucumovirus / genetics*
  • DNA / metabolism*
  • DNA, Single-Stranded / metabolism*
  • Fluorescent Dyes / metabolism*
  • Insecta
  • Microscopy, Confocal
  • Microscopy, Electron, Transmission
  • Protein Multimerization
  • Virosomes / genetics
  • Virosomes / metabolism*

Substances

  • Capsid Proteins
  • DNA, Single-Stranded
  • Fluorescent Dyes
  • Virosomes
  • DNA