A simple and general method for determining the protein and nucleic acid content of viruses by UV absorbance

J. Zachary Porterfield; Adam Zlotnick

doi:10.1016/j.virol.2010.08.015

A simple and general method for determining the protein and nucleic acid content of viruses by UV absorbance

J. Zachary Porterfield, Adam Zlotnick

Microbiology, Immunology, and Molecular Genetics

Research output: Contribution to journal › Article › peer-review

147 Scopus citations

Abstract

UV spectra of viruses are complicated by overlapping protein and RNA absorbance and light scattering. We describe and validate methodology for estimating RNA and protein concentration from such spectra. Importantly, we found that encapsidation did not substantially affect RNA absorbance. Combining absorbance data with a known T number, we confirmed that brome mosaic virus packages about 3100 nucleotides/capsid, consistent with its genome. E. coli-expressed hepatitis B virus (HBV) packages host RNA based on capsid charge and volume. We examined HBV capsid protein (Cp183, +. 15 charge) and a less basic mutant (Cp183-EEE, +. 12 charge) that mimics a phosphorylated state. Cp183-EEE packaged ~. 3450 nucleotides per T. =4 capsid and Cp183 packaged ~. 4800 nucleotides, correlating to the size of HBV's RNA pre-genome and mature DNA genome, respectively. The RNA:protein charge ratio (about 1.4 phosphates per positive charge) was consistent with that of other ssRNA viruses. This spectroscopic method is generalizable to any virus-like particle.

Original language	English
Pages (from-to)	281-288
Number of pages	8
Journal	Virology
Volume	407
Issue number	2
DOIs	https://doi.org/10.1016/j.virol.2010.08.015
State	Published - Nov 25 2010

Bibliographical note

Funding Information:
We thank Dr. Bogdan Dragnea and Dr. Stella Aniagyei for providing purified BMV capsids and Dr. Lingling Chen for providing GroEL samples. We also thank Dr. Todd Stone of the Indiana University Physical Biochemistry Instrumentation Facility. This work was supported by National Institutes of Health grant R01-AI077688 to AZ.

Funding

We thank Dr. Bogdan Dragnea and Dr. Stella Aniagyei for providing purified BMV capsids and Dr. Lingling Chen for providing GroEL samples. We also thank Dr. Todd Stone of the Indiana University Physical Biochemistry Instrumentation Facility. This work was supported by National Institutes of Health grant R01-AI077688 to AZ.

Funders	Funder number
National Institutes of Health (NIH)
National Institute of Allergy and Infectious Diseases	R01AI077688

Keywords

Absorbance spectroscopy
Brome Mosaic Virus
Capsid assembly
DNA content
Electrostatics
Hepatitis B virus
RNA content
Virus assembly

ASJC Scopus subject areas

Virology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.virol.2010.08.015

Cite this

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title = "A simple and general method for determining the protein and nucleic acid content of viruses by UV absorbance",

abstract = "UV spectra of viruses are complicated by overlapping protein and RNA absorbance and light scattering. We describe and validate methodology for estimating RNA and protein concentration from such spectra. Importantly, we found that encapsidation did not substantially affect RNA absorbance. Combining absorbance data with a known T number, we confirmed that brome mosaic virus packages about 3100 nucleotides/capsid, consistent with its genome. E. coli-expressed hepatitis B virus (HBV) packages host RNA based on capsid charge and volume. We examined HBV capsid protein (Cp183, +. 15 charge) and a less basic mutant (Cp183-EEE, +. 12 charge) that mimics a phosphorylated state. Cp183-EEE packaged ~. 3450 nucleotides per T. =4 capsid and Cp183 packaged ~. 4800 nucleotides, correlating to the size of HBV's RNA pre-genome and mature DNA genome, respectively. The RNA:protein charge ratio (about 1.4 phosphates per positive charge) was consistent with that of other ssRNA viruses. This spectroscopic method is generalizable to any virus-like particle.",

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N2 - UV spectra of viruses are complicated by overlapping protein and RNA absorbance and light scattering. We describe and validate methodology for estimating RNA and protein concentration from such spectra. Importantly, we found that encapsidation did not substantially affect RNA absorbance. Combining absorbance data with a known T number, we confirmed that brome mosaic virus packages about 3100 nucleotides/capsid, consistent with its genome. E. coli-expressed hepatitis B virus (HBV) packages host RNA based on capsid charge and volume. We examined HBV capsid protein (Cp183, +. 15 charge) and a less basic mutant (Cp183-EEE, +. 12 charge) that mimics a phosphorylated state. Cp183-EEE packaged ~. 3450 nucleotides per T. =4 capsid and Cp183 packaged ~. 4800 nucleotides, correlating to the size of HBV's RNA pre-genome and mature DNA genome, respectively. The RNA:protein charge ratio (about 1.4 phosphates per positive charge) was consistent with that of other ssRNA viruses. This spectroscopic method is generalizable to any virus-like particle.

AB - UV spectra of viruses are complicated by overlapping protein and RNA absorbance and light scattering. We describe and validate methodology for estimating RNA and protein concentration from such spectra. Importantly, we found that encapsidation did not substantially affect RNA absorbance. Combining absorbance data with a known T number, we confirmed that brome mosaic virus packages about 3100 nucleotides/capsid, consistent with its genome. E. coli-expressed hepatitis B virus (HBV) packages host RNA based on capsid charge and volume. We examined HBV capsid protein (Cp183, +. 15 charge) and a less basic mutant (Cp183-EEE, +. 12 charge) that mimics a phosphorylated state. Cp183-EEE packaged ~. 3450 nucleotides per T. =4 capsid and Cp183 packaged ~. 4800 nucleotides, correlating to the size of HBV's RNA pre-genome and mature DNA genome, respectively. The RNA:protein charge ratio (about 1.4 phosphates per positive charge) was consistent with that of other ssRNA viruses. This spectroscopic method is generalizable to any virus-like particle.

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KW - Electrostatics

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KW - Virus assembly

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A simple and general method for determining the protein and nucleic acid content of viruses by UV absorbance

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

UN SDGs

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