Subcellular localisation of Theiler's murine encephalomyelitis virus (TMEV) capsid subunit VP1 vis-á-vis host protein Hsp90

Abstract

Highlights TMEV VP1 localises to the perinuclear region and cytoplasm of infected cells  TMEV VP1 and Hsp90 colocalise in the perinuclear region and cytoplasm  TMEV VP1 did not localise to the nucleus during infection  A typical NLS was absent from the TMEV VP1 sequence  TMEV VP1 localises in a similar manner to FMDV and EV71 VP1 AbstractThe 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.

Resilience in Long-Term Viral Infection: Genetic Determinants and Interactions

“…The “outlier” of the URs for the Resistant group was heat shock protein 990 (HSP-990), a synthetic HSP90 inhibitor with potential therapeutic use in cancer treatment [ 41 ]. Hsp90 has been identified as an important host factor in the life cycle of TMEV [ 42 ]: Hsp90 colocalizes with the VP1 subunit of TMEV during infection [ 43 ].…”

The In Silico Prediction of Hotspot Residues that Contribute to the Structural Stability of Subunit Interfaces of a Picornavirus Capsid

“…Flow diagram of the methodology used in the study. Theiler's murine encephalomyelitis virus (TMEV) subcomplexes representing the different subunit interfaces of TMEV were extracted from the homology model of the TMEV GDVII capsid generated previously [37,38]. The complexes were submitted to five tools for hotspot prediction.…”

“…Due to icosahedral symmetry, the protomer subunits and contacts between them are repeated 60 times across the picornavirus capsid. To reduce computing time, single complexes representing the intraprotomer, interprotomer and interpentamer interfaces were generated by extracting the relevant protomer subunits from a homology model of the complete biological assembly of TMEV GDVII [37,38]. Protomers in the biological assembly are numbered P1-P60 according to their position in the capsid, where P1-P5 constitutes the first pentamer and P6-P10 the second pentamer.…”

“…The second complex comprised the same protomer (P1), but the second protomer was the adjacent protomer (P23) in the adjacent pentamer located next to the three-fold axis of symmetry ( Figure 2D). GDVII [37,38]. Protomers in the biological assembly are numbered P1-P60 according to their position in the capsid, where P1-P5 constitutes the first pentamer and P6-P10 the second pentamer.…”