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. 2002 Jan;76(2):633-43.
doi: 10.1128/jvi.76.2.633-643.2002.

GRP78, a coreceptor for coxsackievirus A9, interacts with major histocompatibility complex class I molecules which mediate virus internalization

Kathy Triantafilou  1 , Didier Fradelizi, Keith Wilson, Martha Triantafilou
Affiliations

GRP78, a coreceptor for coxsackievirus A9, interacts with major histocompatibility complex class I molecules which mediate virus internalization

Kathy Triantafilou et al. J Virol. 2002 Jan.

Abstract

It is becoming apparent that over the years cell infection by virus seems to have evolved into a multistep process in which many viruses employ distinct cell surface molecules for their attachment and cell entry. In this study the attachment and entry pathway of coxsackievirus A9 (CAV-9), a member of the Picornaviridae family, was investigated. It has been known that, although integrin alpha(v)beta3 is utilized as a receptor, its presence alone is insufficient for CAV-9 infection and that CAV-9 also requires a 70-kDa major histocompatibility complex class I (MHC-I)-associated protein (MAP-70) as a coreceptor molecule. We document by protein isolation and peptide sequencing that the 70-kDa protein is GRP78, a member of the heat shock protein 70 family of stress proteins. Furthermore we show by using fluorescence resonance energy transfer (FRET) that GRP78 is also expressed on the cell surface and associates with MHC-I molecules. In addition CAV-9 infection of permissive cells requires GRP78 and also MHC-I molecules, which are essential for virus internalization. The identification of GRP78 as a coreceptor for CAV-9 and the revelation of GRP78 and MHC-I associations have provided new insights into the life cycle of CAV-9, which utilizes integrin alpha(v)beta3 and GRP78 as receptor molecules whereas MHC-I molecules serve as the internalization pathway of this virus to mammalian cells.

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Figures

FIG. 1.
FIG. 1.
Identification of MAP-70. (A) Tryptic peptide mixture analysis via a matrix-assisted laser desorption ionization mass spectrometer. (B) Immunoblotting of protein with goat Igs followed by HRP-conjugated donkey anti-goat Ig (1), with only HRP-conjugated donkey anti-goat Ig (2), or with HRP-conjugated goat GRP78-specific serum (3). NEPHGE, nonequilibrium pH gradient gel electrophoresis.
FIG. 2.
FIG. 2.
Inhibition of CAV-9 infectivity by antibodies against its cellular receptors. Shown are percentages of inhibition of CAV-9 infectivity of GMK cells in the presence of MAbs (A) and combinations of MAbs (B) at concentrations of 2.5, 5, and 10 μg. Error bars, standard deviations over a number of independent experiments.
FIG. 3.
FIG. 3.
Inhibition of CAV-9 binding by antibodies against its cellular receptors. Shown are percentages of inhibition of CAV-9 binding to GMK cells. The inhibition of CAV-9 binding to GMK cells was measured in the presence of MAB1976 (integrin αvβ3 specific), MCA757G (integrin αvβ3 specific), and combinations of GRP78-specific serum with MAB1976, MCA757G, or MCA757G, and also GRP78-specific serum with irrelevant antibody M073401 (transferrin receptor specific). Inhibition in the presence of an irrelevant serum was also tested. Error bars, standard deviations over a number of independent experiments.
FIG. 4.
FIG. 4.
FRET measurements for CAV-9 and GRP78. Energy transfer between CAV-9 and GRP78 can be detected from the increase in donor fluorescence after acceptor photobleaching. (A) Donor (Cy3) image after acceptor photobleaching. (B) E image. (C) E as a function of the fluorescence at donor/acceptor antibody ratios of 1:1 (solid squares), 1:2 (open squares), and 1:4 (open circles). Scale bar, 10 μm.
FIG. 5.
FIG. 5.
FRET measurements for MHC-I and GRP78. Energy transfer between MHC-I and GRP78 can be detected from the increase in donor fluorescence after acceptor photobleaching. (A) Donor (Cy3) image after acceptor photobleaching. (B) E image. (C) E as a function of the fluorescence of donor/acceptor antibody ratios of 1:1 (open ellipse), 1:2 (filled circles), and 1:4 (filled squares). Scale bar, 10 μm.
FIG. 6.
FIG. 6.
MHC-I and GRP78 interactions in the absence and presence of virus. Measurement of energy transfer on GMK cells between MHC-I and GRP78 in the absence (A and B) and presence (C and D) of CAV-9 particles. The energy transfer can be detected from the increase in donor fluorescence after acceptor photobleaching. (A and B) Measurements in the absence of CAV-9, where the donor (Cy3) image after acceptor photobleaching (A) and the E image (B) are shown. (C and D) Measurements in the presence of CAV-9, where the donor (Cy3) image after acceptor photobleaching (C) and the E image (D) are shown. Scale bar, 10 μm.
FIG. 7.
FIG. 7.
Role of MHC-I in the CAV-9 infectious cycle. Experiments testing the inhibition of CAV-9 infection on GMK cells were performed. The following were used: MCA1115 (β2-microglobulin-specific MAb) (A), W6/32 (MHC-I-specific MAb) (B), an isotype control MAb (C), and infection with CAV-9 particles (D). These plates are representative of a number of independent experiments. CAV-9 binding to GMK cells in the presence of MCA1115, W6/32, an isotype control MAb, an irrelevant serum, and also a combination of W6/32 and MCA1115 was tested by flow-cytometric analysis. Binding was assayed by incubation with CAV-9 rabbit-specific serum and developed using an appropriate dilution of FITC-conjugated swine anti-rabbit Ig. (E) Percentage inhibition of CAV-9 binding to GMK cells. Error bars are calculated from standard deviations over a number of independent experiments.
FIG. 8.
FIG. 8.
Expression of GRP78 and αvβ3 on Daudi and GMK cells. Shown is flow-cytometric analysis of integrin αvβ3 and GRP78 expression on GMK, Daudi, and Daudi-MHC+ cells. Control GMK, Daudi, and Daudi-MHC+ cells were incubated with FITC-conjugated rabbit anti-mouse IgG. GMK cells were incubated with integrin αvβ3-specific MAb MAB1976 (A) and GRP78-specific serum (B). Daudi cells were incubated with integrin αvβ3-specific MAb MAB1976 (C) and GRP78-specific serum (D). Daudi-MHC+ cells were incubated with integrin αvβ3-specific MAb MAB1976 (E) and GRP78-specific serum (F). The histograms display relative cell numbers as a function of relative fluorescence intensities.
FIG. 9.
FIG. 9.
Infectivity assay. Shown are the results of plaque assay of GMK cells when Daudi cell lysate (A), Daudi-MHC+ (B), and GMK lysate (C) were added.
FIG. 10.
FIG. 10.
nCAV-9 binding on MHC+ and MHC− cells. Flow-cytometric analysis of CAV-9 binding to cells. GMK cells, Daudi cells, and Daudi-MHC+ cells were incubated without CAV-9 particles, as a control. CAV-9 binding on GMK cells (A), Daudi cells (B), and Daudi-MHC+ cells (C) was then assayed. Virus binding was assayed by incubation with CAV-9-specific serum and detected using an appropriate dilution of FITC-conjugated swine anti-rabbit Ig. The histograms display relative cell numbers as a function of relative fluorescence intensities.
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