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

Producción científica: Article › revisión exhaustiva

170 Citas (Scopus)

Resumen

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.

Idioma original	English
Páginas (desde-hasta)	281-288
Número de páginas	8
Publicación	Virology
Volumen	407
N.º	2
DOI	https://doi.org/10.1016/j.virol.2010.08.015
Estado	Published - nov 25 2010

Nota bibliográfica

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.

Financiación

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.

Financiadores	Número del financiador
National Institutes of Health (NIH)
National Institute of Allergy and Infectious Diseases	R01AI077688

ODS de las Naciones Unidas

Este resultado contribuye a los siguientes Objetivos de Desarrollo Sostenible

Good health and well being

ASJC Scopus subject areas

Virology

Acceder al documento

10.1016/j.virol.2010.08.015

Otros archivos y enlaces

Citar esto

@article{6c53dfe666c543f8825c54b39bfdbff8,

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 \textasciitilde{}. 3450 nucleotides per T. =4 capsid and Cp183 packaged \textasciitilde{}. 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.",

keywords = "Absorbance spectroscopy, Brome Mosaic Virus, Capsid assembly, DNA content, Electrostatics, Hepatitis B virus, RNA content, Virus assembly",

author = "Porterfield, \{J. Zachary\} and Adam Zlotnick",

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.",

year = "2010",

month = nov,

day = "25",

doi = "10.1016/j.virol.2010.08.015",

language = "English",

volume = "407",

pages = "281--288",

number = "2",

}

TY - JOUR

T1 - A simple and general method for determining the protein and nucleic acid content of viruses by UV absorbance

AU - Porterfield, J. Zachary

AU - Zlotnick, Adam

N1 - 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.

PY - 2010/11/25

Y1 - 2010/11/25

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.

KW - Absorbance spectroscopy

KW - Brome Mosaic Virus

KW - Capsid assembly

KW - DNA content

KW - Electrostatics

KW - Hepatitis B virus

KW - RNA content

KW - Virus assembly

UR - https://www.scopus.com/pages/publications/77957689491

UR - https://www.scopus.com/pages/publications/77957689491#tab=citedBy

U2 - 10.1016/j.virol.2010.08.015

DO - 10.1016/j.virol.2010.08.015

M3 - Article

C2 - 20850162

AN - SCOPUS:77957689491

SN - 0042-6822

VL - 407

SP - 281

EP - 288

JO - Virology

JF - Virology

IS - 2

ER -

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

Resumen

Nota bibliográfica

Financiación

ODS de las Naciones Unidas

ASJC Scopus subject areas

Acceder al documento

Otros archivos y enlaces

Huella

Citar esto