Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis - PubMed (original) (raw)

Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

Matthias Nitschke et al. Virology. 2008.

Abstract

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kidney cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments.

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Figures

Fig. 1

Fig. 1

Role of clathrin-dependent endocytosis in infection of BHK cells by EAV. BHKasc cells were preincubated in the presence (tc+) and absence (tc−) of tetracycline. In the latter case antisense RNA against clathrin heavy chain is expressed suppressing clathrin-dependent endocytosis. A. Alexa Fluor 633 transferrin uptake. CHC expressing cells (tc+) display fluorescent transferrin (Tf) with high intensities in intracellular vesicles, whereas cells (tc−) with suppressed CHC expression show no or less intense intracellular internalisation of transferrin. Uptake was measured one min after addition of transferrin (37 °C). B.Infection of BHKasc cells by EAV. At 2h p.i. and 24h p.i. cells were fixed and examined, stained with a primary antibody against nucleocapsid protein and TRITC labelled secondary antibodies, and examined by confocal microscopy. Fluorescence images were recorded at identical gain settings. PC — phase contrast microscopy. DIC — differential interference microscopy.

Fig. 2

Fig. 2

Influence of chlorpromazine on EAV infection of BHK-21 cells. BHK-21 cells were incubated with chlorpromazine and infection by EAV (filled circles) or by SV5 (open circles) was assessed by the plaque assay. Data present mean ± standard error of estimate of three independent experiments.

Fig. 3

Fig. 3

Influence of the lysosomotropic agents on EAV infection of BHK-21 cells. BHK-21 cells were incubated with ammonium chloride (squares) (A), with the vacuolar type H+-ATPase inhibitors (B) bafilomycin A1 (circles) and (C) concanamycin A (triangles up), and (D) with monensin (triangles down), and infection by EAV (filled symbols) or by SV5 (open symbols) was assessed by the plaque assay. Data present mean ± standard error of estimate of twelve (A) and at least three (B–D) and independent experiments.

Fig. 4

Fig. 4

Influence of preincubation at acidic pH. EAV (filled circles) or SV5 (open circles) were incubated at the indicated pH for 1h at 37 °C. After pH neutralization of virus suspension, viruses were adsorbed to BHK cells and the plaque assay was performed (see Materials and methods). Data present mean ± standard error of estimate of three independent experiments.

Fig. 5

Fig. 5

Low pH-bypass of inhibition of virus infection. BHK cells were pretreated as described above either with chlorpromazine (60 µM), concanamycin A (4nM), monensin (400nM), or MβCD. After the first 60 min of virus adsorption at 37 °C, the pH was lowered to 5.0 for 15 min. After neutralization, virus–cell complexes were incubated for further 45 min and the plaque assay was performed as described in Materials and methods. Control — mock pretreatment, no low pH exposure. w/o — without low pH exposure. w — with low pH exposure. Data present mean ± standard error of estimate of three independent experiments.

Fig. 6

Fig. 6

Influence of cholesterol depletion on EAV infection of BHK-21 cells. (A) After pretreatment of BHK cells with MßCD or (B) incubation with filipin III infection of cells by EAV (filled circles) or by VSV (open circles) was assessed by the plaque assay. For treatment of the cells with substances see Materials and methods. Data present mean ± standard error of estimate of three independent experiments.

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References

    1. Brindley M.A., Maury W. Endocytosis and a low-pH step are required for productive entry of equine infectious anemia virus. J. Virol. 2005;79(23):14482–14488. - PMC - PubMed
    1. Brindley M.A., Hughes L., Ruiz A., McCray P.B., Jr., Sanchez A., Sanders D.A., Maury W. Ebola virus glycoprotein 1: identification of residues important for binding and postbinding events. J. Virol. 2007;81(14):7702–7709. - PMC - PubMed
    1. Chandran K., Sullivan N.J., Felbor U., Whelan S.P., Cunningham J.M. Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science. 2005;308(5728):1643–1645. - PMC - PubMed
    1. Choi K.S., Aizaki H., Lai M.M. Murine coronavirus requires lipid rafts for virus entry and cell–cell fusion but not for virus release. J. Virol. 2005;79(15):9862–9871. - PMC - PubMed
    1. de Vries A.A., Chirnside E.D., Horzinek M.C., Rottier P.J. Structural proteins of equine arteritis virus. J. Virol. 1992;66(11)):6294–6303. - PMC - PubMed

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