A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the...

Full description

Saved in:
Bibliographic Details
Published in:Virology (New York, N.Y.) N.Y.), 2019-01, Vol.527, p.159-168
Main Authors: Webby, Melissa N., Sullivan, Matthew P., Yegambaram, Kavestri M., Radjainia, Mazdak, Keown, Jeremy R., Kingston, Richard L.
Format: Article
Language:English
Subjects:
Analytical Ultracentrifugation
Biuret assay
Circular dichroism spectroscopy
Electron Microscopy
Inductively coupled plasma mass spectrometry
Negative-sense ssRNA viruses
Nucleocapsid
Paramyxovirus
Protein and Nucleic acid quantification
UV absorbance spectroscopy
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable. •Methods are needed to analyze the composition of recombinant viral particles.•Phosphorus often provides a unique marker of bound nucleic acids.•Phosphorus can be quantified using Inductively Coupled Plasma-Mass Spectrometry.•Protein can be quantified using the biuret assay.•Reliable estimates for the protein-to-nucleic-acid ratio result.
ISSN:0042-6822
1096-0341
DOI:10.1016/j.virol.2018.11.013