Quantitation of adenovirus type 5 empty capsids.
Adenovirus empty capsids are immature intermediates that lack DNA and viral core proteins. Highly purified preparations of empty and full capsids were generated by subjecting purified adenovirus preparations to repeated cesium chloride gradient separations. PAGE results revealed that empty capsids contain at least five bands that correspond to proteins absent from the mature virus proteome. Peptide mapping by matrix-assisted laser desorption/ionization time-of-flight MS revealed that three of these bands correspond to varying forms of L1 52/55kDa, a protein involved in the encapsidation of the viral DNA. One band at around 31kDa was found to include precursors to proteins VI and VIII. These precursors correspond to proteins that have not been cleaved by the adenovirus-encoded protease and are not present in the mature full capsids. The precursor to protein VIII (pVIII), a capsid cement protein, is used in this study as a marker in reverse-phased HPLC (RP-HPLC) analyses of adenovirus for the quantitation of empty capsids. A novel calculation method applied to the integration of RP-HPLC chromatograms allowed for the generation of a percentage empty capsid value in a given adenovirus preparation. The percentage empty capsid values generated to date by this method show a high degree of precision and good agreement with a cesium chloride gradient/SDS-PAGE quantitation method of empty capsids. The advantage of this method lies in the accurate, precise, and rapid generation of the percentage of empty capsids in a given purified virus preparation without relying on tedious and time-consuming cesium chloride gradient separations and extractions.
Takahashi
Eddie
E
Bioprocess and Bioanalytical Research, Bioprocess R&D, Merck Research Laboratories, West Point, PA 19486, USA.
Cohen
Steven L
SL
Tsai
P K
PK
Sweeney
Joyce A
JA
D000256:Adenoviridae / Q000378:metabolism*; D002213:Capsid / Q000378:metabolism* / Q000648:ultrastructure; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D046529:Microscopy, Electron, Transmission; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization