MALDI-TOF mass spectroscopy detects the capsid structural instabilities created by deleting the myxoma virus cupro-zinc SOD1 homolog M131R.
The myxoma virus M131R gene encodes a catalytically inactive homolog of cellular Cu-Zn superoxide dismutase (SOD1) and this 17,786 Da protein is a major virion component. We have used matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS) to study the effect(s) of deleting the gene on virion composition and structure. This approach confirmed that the M131R gene product is an abundant virion component. This conclusion was based upon the ready detection of a 1805.3 Da peptide released from the N-terminus of the myxoma SOD1 protein by mild trypsin treatment, as well as the detection of a 17,790 Da protein in HPLC fractionated virus extracts, which subsequently yielded M131R-encoded tryptic peptides. Neither peptide nor protein was detected in particles bearing a genome encoding an M131RDelta deletion mutation. Curiously, more proteins and tryptic peptides were detected when M131RDelta mutant virions were subjected to MALDI-TOF MS analysis compared with wild-type virus particles. This suggested that particles assembled in the absence of myxoma SOD protein are structurally unstable. Plaque analysis confirmed this conjecture by showing that SOD-deficient MYX particles are unusually heat labile and trypsin sensitive. Mutant Shope fibroma virus exhibited the same phenotype. Thus a previously unappreciated feature of MALDI-TOF MS is that the method can sometimes detect alterations in virion stability.
Department of Molecular Biology and Genetics, The University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Alicja
A
Zachertowska
Dyanne
D
Brewer
David H
DH
Evans
D036022:Capsid Proteins / Q000032:analysis / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D019286:Endopeptidase K / Q000378:metabolism; D017353:Gene Deletion; D005814:Genes, Viral; D006358:Hot Temperature; D009233:Myxoma virus / Q000235:genetics / Q000502:physiology*; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000032:analysis / Q000235:genetics*; D014357:Trypsin / Q000378:metabolism; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics; D019065:Virus Assembly
D036022:Capsid Proteins; D010455:Peptides; D014764:Viral Proteins; D015678:Viral Structural Proteins; D013482:Superoxide Dismutase; D014357:Trypsin; D019286:Endopeptidase K