Characterization of the major capsid proteins of myxoma virus particles using MALDI-TOF mass spectrometry.
The protein composition of poxvirus particles remains uncertain because of their large size and structural complexity. This has complicated the characterization of even well-studied Orthopoxviruses, like vaccinia virus, and little or nothing is known about the capsid composition of viruses belonging to other poxvirus genera. This paper describes methods that address this problem and have been used to identify 17 different Leporipoxvirus capsid proteins. Myxoma virus particles were purified using sucrose and Nicodenz gradient centrifugation and subfractionated into membrane and core fractions by thiol and detergent treatment. These materials were further fractionated using reverse-phase chromatography and SDS-PAGE and the resulting proteins identified by mass spectroscopy. Most of the myxoma proteins identified in this manner were homologs of either vaccinia virus structural proteins (F17R, L4R, J1R, H3L, A3L, A10L, A27L, and A45R) or virion-associated enzymes (I7L, H4L, D11L, A7L, and A22R). However, the myxoma homolog of the vaccinia P4a/A10L protein (M099L) differs from P4a protein in being proteolytically cleaved only once. M095L and M151R were also detected in core fractions. M095L and M151R are homologs of vaccinia A6L and B13R proteins, respectively, and poxvirus proteins not previously known to be capsid components. M093L, a protein of unknown function and having no certain Orthopoxvirus homolog associates with membrane fractions. These studies illustrate the conservation of Chordopoxvirion architecture and the methods that can be used to elucidate the proteins comprising these structures.
Zachertowska
Alicja
A
Department of Molecular Biology and Genetics, The University of Guelph, Guelph, Ont., Canada N1G 2W1.
Brewer
Dyanne
D
Evans
David H
DH
D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D009233:Myxoma virus / Q000737:chemistry* / Q000302:isolation & purification; D011205:Povidone; D017386:Sequence Homology, Amino Acid; D012822:Silicon Dioxide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013395:Sucrose; D014616:Vaccinia virus / Q000235:genetics
D036022:Capsid Proteins; C104449:Nicodenz; D013395:Sucrose; D012822:Silicon Dioxide; D011205:Povidone