Subcellular localisation of Theiler's murine encephalomyelitis virus (TMEV) capsid subunit VP1 vis-á-vis host protein Hsp90.
The VP1 subunit of the picornavirus capsid is the major antigenic determinant and mediates host cell attachment and virus entry. To investigate the localisation of Theiler's murine encephalomyelitis virus (TMEV) VP1 during infection, a bioinformatics approach was used to predict a surface-exposed, linear epitope region of the protein for subsequent expression and purification. This region, comprising the N-terminal 112 amino acids of the protein, was then used for rabbit immunisation, and the resultant polyclonal antibodies were able to recognise full length VP1 in infected cell lysates by Western blot. Following optimisation, the antibodies were used to investigate the localisation of VP1 in relation to Hsp90 in infected cells by indirect immunofluorescence and confocal microscopy. At 5h post infection, VP1 was distributed diffusely in the cytoplasm with strong perinuclear staining but was absent from the nucleus of all cells analysed. Dual-label immunofluorescence using anti-TMEV VP1 and anti-Hsp90 antibodies indicated that the distribution of both proteins colocalised in the cytoplasm and perinuclear region of infected cells. This is the first report describing the localisation of TMEV VP1 in infected cells, and the antibodies produced provide a valuable tool for investigating the poorly understood mechanisms underlying the early steps of picornavirus assembly.
Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa.
Caroline
C
Ross
Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa.
Nicole
N
Upfold
Centre for Biomolecular Sciences, School of Biology, Biomolecular Sciences Building, University of St. Andrews, North Haugh, St. Andrews, Scotland KY16 9ST, UK.
Garry A
GA
Luke
Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa.
Özlem Tastan
ÖT
Bishop
Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa. Electronic address: caroline.knox@ru.ac.za.
Caroline
C
Knox
D000595:Amino Acid Sequence; D000818:Animals; D036022:Capsid Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D018188:Cardiovirus Infections / Q000276:immunology / Q000378:metabolism* / Q000821:virology*; D002460:Cell Line; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D000072417:Protein Domains; D021381:Protein Transport; D017945:Theilovirus / Q000502:physiology*
D036022:Capsid Proteins; D000939:Epitopes; D018841:HSP90 Heat-Shock Proteins; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; C442551:VP1 protein, Theilovirus
Capsid; Hsp90; Polyclonal antibody; Theiler's murine encephalomyelitis virus; VP1