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Theiler’s murine encephalomyelitis virus infection induces a redistribution of heat shock proteins 70 and 90 in BHK-21 cells, and is inhibited by novobiocin and geldanamycin

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Cell Stress and Chaperones Aims and scope

Abstract

Theiler’s murine encephalomyelitis virus (TMEV) is a positive-sense RNA virus belonging to the Cardiovirus genus in the family Picornaviridae. In addition to other host cellular factors and pathways, picornaviruses utilise heat shock proteins (Hsps) to facilitate their propagation in cells. This study investigated the localisation of Hsps 70 and 90 in TMEV-infected BHK-21 cells by indirect immunofluorescence and confocal microscopy. The effect of Hsp90 inhibitors novobiocin (Nov) and geldanamycin (GA) on the development of cytopathic effect (CPE) induced by infection was also examined. Hsp90 staining was uniformly distributed in the cytoplasm of uninfected cells but was found concentrated in the perinuclear region during late infection where it overlapped with the signal for non-structural protein 2C within the viral replication complex. Hsp70 redistributed into the vicinity of the viral replication complex during late infection, but its distribution did not overlap with that of 2C. Inhibition of Hsp90 by GA and Nov had a negative effect on virus growth over a 48-h period as indicated by no observable CPE in treated compared to untreated cells. 2C was detected by Western analysis of GA-treated infected cell lysates at doses between 0.01 and 0.125 μM, suggesting that processing of viral precursors was not affected in the presence of this drug. In contrast, 2C was absent in cell lysates of Nov-treated cells at doses above 10 μM, although CPE was evident 48 hpi. This is the first study describing the dynamic behaviour of Hsps 70 and 90 in TMEV-infected cells and to identify Hsp90 as an important host factor in the life cycle of this virus.

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Abbreviations

ADP:

Adenosine diphosphate

ATP:

Adenosine triphosphate

CPE:

Cytopathic effect

DAPI:

4’,6-Diamino-2-phenylindole dihydrochloride

DMEM:

Dulbecco’s modified Eagle Medium

DMSO:

Dimethyl sulphoxide

DTT:

Dithiothreitol

EDTA:

Ethylene diamine tetra-acetic acid

EMCV:

Encephalomyelitis virus

FMDV:

Foot-and-mouth disease virus

GA:

Geldanamycin

Hop:

Hsp70/Hsp90 organising protein

hpi:

Hours post-infection

Hsp:

Heat shock protein

MOI:

Multiplicity of infection

Nov:

Novobiocin

PMSF:

Phenyl methyl sulphonyl fluoride

PV:

Poliovirus

TMEV:

Theiler’s murine encephalomyelitis virus

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Acknowledgements

We gratefully acknowledge T. Michiels for providing TMEV GDVII cDNA used for preparation of virus. This work was supported by a National Research Foundation grant (NRF, South Africa) and Joint Research Council funding (Rhodes University, South Africa).

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Authors and Affiliations

  1. Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, 6140, Grahamstown, South Africa

    Lorraine Z. Mutsvunguma, Boitumelo Moetlhoa & Caroline Knox

  2. Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, 6140, Grahamstown, South Africa

    Adrienne L. Edkins & Gregory L. Blatch

  3. Centre for Biomolecular Sciences, School of Biology, Biomolecular Sciences Building, University of St Andrews, North Haugh, St Andrews, Scotland, KY16 9ST, UK

    Garry A. Luke

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  1. Lorraine Z. Mutsvunguma
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  2. Boitumelo Moetlhoa
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  6. Caroline Knox
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Correspondence to Caroline Knox.

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Mutsvunguma, L.Z., Moetlhoa, B., Edkins, A.L. et al. Theiler’s murine encephalomyelitis virus infection induces a redistribution of heat shock proteins 70 and 90 in BHK-21 cells, and is inhibited by novobiocin and geldanamycin. Cell Stress and Chaperones 16, 505–515 (2011). https://doi.org/10.1007/s12192-011-0262-x

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  • DOI: https://doi.org/10.1007/s12192-011-0262-x

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