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

J. Zachary Porterfield, Adam Zlotnick

Research output: Contribution to journalArticlepeer-review

141 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 languageEnglish
Pages (from-to)281-288
Number of pages8
JournalVirology
Volume407
Issue number2
DOIs
StatePublished - 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.

Keywords

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

ASJC Scopus subject areas

  • Virology

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