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

Affiliation

School of Biological Sciences, University of Auckland, Auckland, New Zealand. Electronic address: [Email]

Abstract

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.

Keywords

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,