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. 2008 Feb;82(3):1581-90.
doi: 10.1128/JVI.01375-07. Epub 2007 Nov 21.

Calpain 1 and 2 are required for RNA replication of echovirus 1

Paula Upla  1 , Varpu Marjomäki, Liisa Nissinen, Camilla Nylund, Matti Waris, Timo Hyypiä, Jyrki Heino
Affiliations

Calpain 1 and 2 are required for RNA replication of echovirus 1

Paula Upla et al. J Virol. 2008 Feb.

Abstract

Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.

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Figures

FIG. 1.
FIG. 1.
Calpain inhibitors block EV1 infection. (A) Immunofluorescent labeling of SAOS-α2β1 cells infected with EV1, using virus-specific antiserum with or without calpeptin (calp.; 140 μM) treatment after viral attachment (0 h) or 2, 4, 6, and 8 h p.i. Images are of sections through the center area of the cells. Bars, 10 μm. (B) Western blot analysis of the time course of EV1 3D protein expression in SAOS-α2β1 cells. C, uninfected control. Anti-tubulin antibody was used as a loading control. (C) Western blot analysis of EV1 3D protein in SAOS-α2β1 cells 6 h p.i., with or without calpeptin (calp.; 140 μM) treatment. Anti-caveolin-1 antibody was used as a loading control. (D) Plaque titration assay showing the production of infectious EV1 in SAOS-α2β1 cells in the presence or absence of 140 μM calpeptin. (E) The number of EV1-infected cells 6 h p.i. among siRNA-positive cells was determined after siRNA transfection of calpain 1 (1-1 and 1-2) and 2 (2-1 and 2-2) siRNAs for 72 h. A scrambled (S) siRNA was used as a control. (F) Blocking effects of calpain inhibitor 1 and calpain inhibitor 2 on EV1 infection, determined 6 h p.i. in SAOS-α2β1 cells. Inh., inhibitor. (G) The effect of several protease inhibitors with increasing concentrations on EV1 infection in SAOS-α2β1 cells calculated 6 h p.i. The inhibitors used were antipain (Ant; 125, 250, 500, and 1,000 μM), leupeptin (Leu; 125, 250, 500, and 1,000 μM), aprotinin (Apr; 1.25, 2.5, 5, and 10 U/ml), trypsin inhibitor (Try; 0.5, 1, 2, and 4 mM), and elastatinal (Ela; 62.5, 125, 250, and 500 μM).
FIG. 2.
FIG. 2.
Effect of calpain activity on HPEV1 and CVB3 infections. (A) The proportion of HPEV1-infected A549 cells in the absence or presence of calpeptin (140 μM), determined 6 h p.i. Error bars show the standard errors. C, untreated control. (B) The number of HPEV1-infected A549 cells 6 h p.i. among siRNA-positive cells was determined after transfection of calpain 1 (1-1 and 1-2) and 2 (2-1 and 2-2) siRNAs for 72 h prior to infection. A scrambled (S) siRNA was used as a control. (C) The proportion of CVB3-infected SAOS-α2β1 and GMK cells as well as EV1-infected HeLa MZ and 293 cells in the presence or absence of 140 μM calpeptin (Calp.). Error bars indicate the standard errors.
FIG. 3.
FIG. 3.
Calpain activity assay. (A) Calpain activity was measured from detergent-soluble and -insoluble fractions of serum-starved SAOS-α2β1 cells after different periods of TPA (100 ng/ml) treatment. C, untreated control. (B) Calpain activity in the detergent-soluble fraction of uninfected and EV1-infected SAOS-α2β1 cells at the indicated time points p.i. (C) Calpain activity measured from EV1-infected SAOS-α2β1 cells 3 h p.i. The values are averages from four identical and separately performed experiments with detergent-soluble fractions. The error bar shows the standard error. (D) Protein levels of calpain 1 and calpain 2, detected by Western blot analysis 3 h p.i. using specific antibodies.
FIG. 4.
FIG. 4.
Importance of calpains at late stages of EV1 infection. (A) The effect of calpeptin (calp.) on the internalization of EV1 or antibody-clustered α2β1 integrin in SAOS-α2β1 cells. (B) The blocking effect of calpeptin, added at the indicated time points p.i., on EV1 infection in SAOS-α2β1 cells. C, untreated control. (C) The inhibitory effects of calpain inhibitor 1 and calpain inhibitor 2 on EV1 infection in SAOS-α2β1 cells 6 h p.i. The inhibitors were added at the indicated time points p.i. Error bars show the standard errors.
FIG. 5.
FIG. 5.
Localization of calpain 1 and calpain 2 in intracellular EV1-containing vesicles. (A) Immunofluorescent labeling of calpain 1 and 2 (green) together with GPI-APs (aerolysin [ASSP]; red) in uninfected SAOS-α2β1 cells. White rectangles indicate the magnified portions (blow-ups). Bars, 10 μm. (B) Calpain 1 and 2 expression levels detected by Western blot analysis from detergent-soluble and -insoluble fractions of uninfected SAOS-α2β1 cells. (C) Immunofluorescent labeling of infected SAOS-α2β1 cells with EV1 antiserum together with antibodies against calpain 1 and 2 and caveolin-1. Confocal images are three-dimensional projections of scanned cells. White rectangles indicate the locations of the magnified portions. Bars, 10 μm. (D) An immunoelectron microscopy micrograph visualizing an intracellular vesicle containing protein A-gold-labeled (10 nm) antibody-induced α2β1 integrin clusters from SAOS-α2β1 cell lysates. The integrin clustering was done simultaneously with EV1 attaching to the cells, followed by incubation at 37°C for 30 min. The membranous location of calpain 2 (arrows) is indicated by immunostaining the cell lysates without permeabilization with anti-calpain 2 antibody linked to protein A-gold (5 nm). Bar, 100 nm.
FIG. 6.
FIG. 6.
Effect of calpain inhibition on EV1 RNA replication. (A) The proportion of infected cells, following EV1 RNA microinjection, in the presence or absence of calpeptin (140 μM) in SAOS-α2β1 cells 5 h p.i. The numbers of negative-strand (B) and genomic-strand (C) EV1 RNA copies analyzed by quantitative RT-PCR in the presence or absence of calpeptin (140 μM) at the indicated time points p.i. are shown. Negative-strand (D) and positive-strand (E) EV1 RNA copies in the presence or absence of calpain inhibitors 1 and 2 (130 μM) at the indicated time points p.i. also are shown. (F) The number of genomic-strand (positive) EV1 RNA copies analyzed by quantitative RT-PCR in the presence or absence of calpeptin (Calp; 140 μM) and protease inhibitors antipain (Ant; 500 μM), leupeptin (Leu; 500 μM), aprotinin (Apr; 5 U/ml), trypsin inhibitor (Try; 2 mM), and elastatinal (Ela; 250 μM) 6 h p.i. (G) The effect of calpeptin on in vitro cell-free translation of EV1 RNA in the rabbit reticulocyte system. The control (C) sample does not contain EV1 RNA.
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