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.
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
authors.forename: J Zachary
authors.initials: JZ
authors.lastname: Porterfield
authors.forename: Adam
authors.initials: A
authors.lastname: Zlotnick
mesh_terms: D017795:Bromovirus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002213:Capsid / Q000737:chemistry / Q000378:metabolism; D036022:Capsid Proteins / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D006515:Hepatitis B virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D012367:RNA, Viral / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation / Q000379:methods; D014764:Viral Proteins / Q000032:analysis*; D014773:Virology / Q000379:methods*; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; D012367:RNA, Viral; D014764:Viral Proteins