Subcellular localisation of Theiler's Murine Encephalomyelitis Virus (TMEV) capsid subunit VP1 vis-á-vis host protein Hsp90

Abstract

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 5 hours 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.