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Journal of Virology, June 2008, p. 5815-5824, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02719-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca²⁺ Homeostasis Induced by Infection of Cultured Cells

José Luis Zambrano,¹ Yuleima Díaz,² Franshelle Peña,² Esmeralda Vizzi,¹ Marie-Christine Ruiz,² Fabián Michelangeli,² Ferdinando Liprandi,¹ and Juan Ernesto Ludert^1*

Center for Microbiology and Cell Biology,¹ Center for Biochemistry and Biophysics, Venezuelan Institute for Scientific Research (IVIC), Caracas 1020-A, Venezuela²

Received 21 December 2007/ Accepted 2 April 2008

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Rotavirus infection of cells in culture induces major changes in Ca²⁺ homeostasis. These changes include increases in plasma membrane Ca²⁺ permeability, cytosolic Ca²⁺ concentration, and total cell Ca²⁺ content and a reduction in the amount of Ca²⁺ released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca²⁺ homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca²⁺ permeability of the plasma membrane and cytosolic Ca²⁺ concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca²⁺ permeability and Ca²⁺ pools released by agonist (ATP). In addition, the increase of total Ca²⁺ content (as measured by ⁴⁵Ca²⁺ uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca²⁺ homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca²⁺ homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects.

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Viral-associated diarrhea remains one of the most common causes of morbidity and mortality among infants and young children. Worldwide estimations indicate that rotaviruses are the leading viral agent associated with severe diarrhea in children younger than 5 years old (20). In addition, rotavirus infections are also a main cause of diarrhea in calves, piglets, and the young of other animals of economic importance (20). Thus, further knowledge of the virus-cell interactions and the events leading to pathogenesis are necessary to improve or develop new strategies that may prevent or reduce the health and economic impact caused by rotavirus infections.

Rotaviruses are members of the Reoviridae family. The rotavirus virion is icosahedral, nonenveloped, and composed of three concentric layers of proteins and a genome of 11 segments of double-stranded RNA. Each genomic segment, with the exception of segment 11, encodes one viral protein for a total of six structural (VP1 to VP7) and six nonstructural proteins (NSP1 to NSP6). The inner layer of the virion is formed by VP2 and also contains the RNA-dependent RNA polymerase VP1 and the guanylyltransferase/methylase VP3. The middle capsid is composed of the major virion protein VP6, and the outer capsid is composed of VP7, which is a glycoprotein, and by VP4, which forms trimeric spikes that project from the surface of the virus. For the virion to be fully infectious, VP4 must undergo proteolytic cleavage into two polypeptides, namely VP8* and VP5* (15, 20).

The enterocyte is the main target cell of rotavirus infection in vivo. However, most studies of the rotavirus replication cycle have been made on cells in culture. Rotavirus replication takes place in the cytoplasm, and its life cycle is closely associated with the endoplasmic reticulum (ER). Particularly, rotavirus utilizes the ER for assembly and maturation during morphogenesis (15). RNA replication and assembly of the double-layer particle (DLP) particles take place in the cytoplasm in electron-dense structures known as viroplasms. Subsequently, DLPs bud into the ER through the interaction between VP6 and NSP4, which act as a viral receptor to dock the viroplasm to the ER (7). NSP4 is a glycosylated integral ER membrane protein. During the budding process, the immature virion acquires VP7 and a transient envelope. Once inside the ER, the virion acquires VP4, selectively retains VP7, and loses the lipid envelope and NSP4 by a yet unknown process. Mature virions are thought to be retained in the ER and finally released by cell lysis (15, 20). However, for differentiated polarized cells, alternative modes of virion release without cell lysis have been proposed (11).

Ca²⁺ is known to control many key cell processes, and thus, its concentration within the cell is tightly regulated (6). During rotavirus replication in cultured cells, profound changes in Ca²⁺ homeostasis have previously been observed (31). Rotavirus-infected cells show a progressive increase in plasma membrane permeability to Ca²⁺, which in turn leads to an increase in cytosolic Ca²⁺ concentration and to an enhancement of sequestered Ca²⁺ pools releasable with thapsigargin, an inhibitor of ER Ca²⁺ ATPase (4, 23). In addition, an increase in the total cell Ca²⁺ pools, as measured by ⁴⁵Ca²⁺ uptake, has been observed. The onset of the changes in Ca²⁺ homeostasis is concomitant with the onset of TPL assembly in the ER (24, 28). Alterations in cell Ca²⁺ homeostasis have been related to cytotoxicity and cell death, since the prevention or reduction of these changes results in a delay in cell death (28). Furthermore, alterations in Ca²⁺ homeostasis may be part of the basis of physiologic alterations in the intestine caused by rotavirus infection which finally lead to diarrhea (16, 26). Changes in Ca²⁺ homeostasis appear to be mediated by the synthesis of viral proteins, since they are abolished when infected cells are treated with cycloheximide, but not with actinomycin D (23, 24).

The viral proteins associated with changes in cell Ca²⁺ homeostasis in rotavirus-infected cells and the operating mechanisms are not well known. It has recently been shown that in tunicamycin-treated, rotavirus-infected cells, the changes in Ca²⁺ permeability of the plasma membrane are inhibited, thus suggesting that a glycosylated viral product, NSP4 or VP7, is responsible for such changes (32). NSP4 appears as a most likely candidate, given its several effects in Ca²⁺ homeostasis. The expression of recombinant NSP4, but not of any other rotavirus protein, in insect Sf9 cells results in a fourfold increase in the cytosolic Ca²⁺ concentration without changes in plasma membrane permeability to Ca²⁺ (38). Moreover, the expression of the recombinant NSP4-enhanced green fluorescent protein (EGFP) fusion protein in mammalian HEK 293 cells results in a twofold increase in basal intracellular Ca²⁺ concentration compared to the level in nonexpressing cells (2).

To further understand rotavirus pathogenesis and the molecular and cellular basis of the changes in Ca²⁺ homeostasis observed in rotavirus-infected cells, the effects of inhibiting the expression of NSP4, VP7, and VP4 by using short interfering RNAs (siRNAs) on several Ca²⁺-related parameters were studied. The data reported here indicate that the expression of NSP4 accounts for most of the alterations in Ca²⁺ homeostasis observed in rotavirus-infected cells. Nevertheless, VP7 may contribute to these effects.

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Cell cultures and virus infections. Cells of the epithelial monkey cell lines MA-104 and Cos-7 were grown in minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS). The reassortant rotavirus strain DxRRV, used throughout the experiments, was propagated in MA-104 cells in the presence of trypsin. This reassortant strain, kindly provided by H. B. Greenberg (Stanford University, Stanford, CA), contained the gene encoding VP7 from the human strain D and the rest of the genes from the simian strain RRV and was chosen based on the specificity of the siRNAs to be used. Typically, cells were infected with strain DxRRV at a multiplicity of infection (MOI) of 10 after trypsin activation and the experiments were performed at 7 h postinfection (h.p.i.).

Silencing of rotavirus gene expression. All the siRNAs used in this study have previously been described in the literature. The siRNA used to silence the expression of NSP4 (siRNA^NSP4) has previously been described by Cuadras et al. (7), that of VP7 (siRNA^VP7) by Silvestri et al. (35), and that of VP4 (siRNA^VP4) by Dector et al. (12). The siRNA^VP4 and siRNA^NSP4 were designed based on strain RRV genes, while the siRNA^NSP4 was designed based on strain D genes. Duplex siRNAs specific for NSP4, VP7, and VP4 as well as the irrelevant siRNA used to silence lamin A/C were purchased from Dharmacon Research, Inc. (Lafayette, CO). Transfection experiments with the siRNAs were performed basically as described previously by Cuadras et al. (7). In brief, MA-104 or Cos-7 cells were grown in six-well plates and, when the monolayers reached about 90% confluence, the medium was removed, the cells were washed twice with phosphate-buffered saline (PBS), and the transfection mixture consisting of 71.5 pmol of siRNA and 1% Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 600 µl of OptiMEM (Gibco-Invitrogen, Carlsbad, CA) was added to each well. Cells were incubated for 30 min at room temperature and subsequently at 37°C for additional 4 to 5 h. At this time, 600 µl of MEM supplemented with 20% FCS was added to each well. Medium was replaced by fresh MEM supplemented with 10% FCS at 15 h posttransfection (h.p.t.). Typically cells were infected with rotavirus at 36 h.p.t.

Immunofluorescence. MA-104 cells grown on coverslips (24 by 24 cm; L&M, Germany) placed inside six-well plates were transfected and infected as described above. Cells were fixed with 4% paraformaldehyde in PBS for 20 min at 37°C, permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature, and finally blocked by incubation with 3% BSA in PBS for 1 h at 37°C. Between each of these steps, coverslips were washed twice with PBS. Cells were incubated with the appropriate monoclonal antibodies diluted in 1% BSA in PBS for 1 h at 37°C as primary antibodies. After washing the coverslips three times with PBS, we incubated the cells with anti-mouse Alexa Fluor 488-conjugated antibodies diluted in 1% BSA in PBS for 1 h at 37°C as secondary antibodies. Finally, coverslips were washed three times with PBS, mounted with Mowiol (Calbiochem, Germany), and observed in an inverted fluorescence microscope (Eclipse TE2000-S; Nikon, Japan).

Metabolic labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To radiolabel viral proteins, MA-104 or Cos-7 cells grown in six-well plates were transfected with specific siRNAs as described above. At 36 h.p.t., cells were infected with DxRRV at an MOI of 10. At 7 h.p.i., the medium was replaced with methionine-cysteine-free MEM and 1 h later 50% of the medium was again replaced with methionine-cysteine-free MEM containing 200 µCi of [³⁵S]methionine-cysteine mix (Amersham Bioscience, United Kingdom). The cells were labeled for 1 h; after that time, the medium was discarded and the cells were washed twice with cold PBS, detached from the plates with a rubber policeman, and pelleted by low-speed centrifugation. Lysates were obtained by incubating the cells for 10 min in ice with a buffer containing 10 mM Tris-Base, pH 7.8, 100 mM NaCl, 1% NP-40, and 2 mM phenylmethylsulfonyl fluoride. The cell lysates were recovered after low-speed centrifugation to pellet the nuclei, and 5-µl aliquots taken to measure radioactivity by liquid scintillation counting. The lysates were analyzed by 12% PAGE under denaturing conditions. Finally, ³⁵S-labeled proteins resolved on gels were visualized by autoradiography.

Virus yield and rotavirus titer determination. MA-104 or Cos-7 cells grown in six-well plates were either not transfected or transfected with specific siRNAs and infected with DxRRV as described above. At 7 h.p.i., aliquots were taken from the supernatant medium and treated with 10 µg of trypsin per ml for 30 min at 37°C for virus activation. The supernatants were serially diluted (10-fold) in MEM, and 100 µl of each dilution was inoculated in triplicate onto confluent monolayers of MA-104 cells grown in 96-well plates. Cells were washed twice with PBS before inoculation. The virus inoculum was removed after 1 h of incubation at 37°C, the cells were washed again twice with PBS, and 100 µl of fresh MEM was added to the cells. The infection was left to proceed overnight at 37°C, and the next day (16 to 18 h.p.i.), cells were fixed with ice-cold methanol for 15 min. Cells were immunostained for focus-forming units with monoclonal antibody O-4B2 (VP6) as primary antibody and an anti-mouse peroxidase-conjugated antibody as secondary antibody. Rotavirus-infected cells were counted, and infectivity was obtained from siRNA-transfected cells expressed as a percentage of the infectivity obtained from nontransfected cells.

Determination of intracellular Ca²⁺ concentration in cell suspensions. MA-104 and Cos-7 cells were seeded in six-well plates and were either not transfected or transfected with specific siRNAs and infected with DxRRV as described above. At 7 h.p.i., cells were detached from the plates by trypsin treatment, washed by centrifugation and resuspended in a medium containing 130 mM NaCl, 5 mM KCl, 20 mM HEPES (pH. 7.2), 1 mM CaCl₂, 0.4 mM MgCl₂, and 0.1% (wt/vol) albumin at an approximate concentration of 8 x 10⁶ cells/ml. Cell suspensions were incubated with 10 µM fura-2 AM with Pluronic (2%) for 30 min, washed twice with PBS by centrifugation, resuspended in the medium described above, and maintained at room temperature until used. Intracellular Ca²⁺ measurements were taken as previously described (32). Briefly, aliquots of the cell suspensions were washed and resuspended in the same medium but without albumin. Fluorescence was measured at 37°C in a spectrofluorometer (Photon Technology International, United Kingdom) equipped with stirrer and temperature control. Excitation wavelengths were 340 and 380 nm, and emission was fixed at 510 nm. The intracellular free Ca²⁺ concentration [Ca²⁺]_i, was calculated using the equation described previously by Grynkiewicz et al. (18). Maximal and minimal fluorescence ratios (R_max and R_mim) were determined by the sequential addition of digitonin and EDTA. Data was acquired and processed using the Felix program (Photon Technology International, United Kingdom).

Determination of ⁴⁵Ca²⁺ uptake. MA-104 cells were grown in six-well plates and were either not transfected or transfected with specific siRNAs and infected with DxRRV as described above. At 7 h.p.i., the medium was removed and replaced with 250 µl/well of fresh MEM containing 1 µCi/well of ⁴⁵Ca²⁺ (Amersham Bioscience, United Kingdom). Cells were incubated with the label for 10 min at 37°C. Uptake was stopped by washing the plates four times in ice-cold PBS. Cells were left to air dry and then dissolved with 200 µl of 0.1 N NaOH. After neutralization with 100 µl of 0.1 N HCl, radioactivity in 50-µl aliquots was determined by liquid scintillation counting. As the number of cells per well was found to be rather constant, uptake values for single experiments were expressed as counts per minute per well. Results were processed using Origin (version 6.0; Microcal Software, Inc., Northampton, MA) software.

Statistical analysis. Differences in [Ca²⁺]_cyto and ⁴⁵Ca²⁺ uptake between cells transfected with specific or irrelevant siRNAs or nontransfected cells were tested for significance by the Student's t test.

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Silencing of rotavirus gene expression. The expression level of rotavirus proteins NSP4, VP7, and VP4 after transfection with specific or irrelevant siRNA lamin A/C was evaluated by SDS-PAGE of rotavirus proteins after metabolic labeling with ³⁵S-amino acids in MA-104 and Cos-7 cells. In addition, virus yield in transfected cells was also measured. Figure 1 shows by analysis by gel electrophoresis and autoradiography that viral proteins in lysates recovered from infected MA-104 (Fig. 1A) and Cos-7 cells (Fig. 1B) were either not transfected or transfected with specific siRNAs. Transfection with siRNA^NSP4 reduced the synthesis of NSP4 in infected cells to almost undetectable levels compared to the levels of the control in both MA-104 and Cos-7 cells. Specifically, a reduction of more than eightfold in the expression of this protein in relation to VP6 was observed. However, as previously reported by other authors (7, 21), the transfection with siRNA^NSP4 also affected the synthesis of other viral proteins as well. Particularly in these experiments, the synthesis of VP7 and VP4 was affected. On the other hand, transfection with siRNA^VP7 and siRNA^VP4 resulted in a marked reduction of the synthesis of VP7 and VP4, respectively, to levels barely detected by autoradiography, but without visible effect on the expression of the other viral proteins.

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FIG. 1. Effect of siRNAs on the synthesis of rotavirus proteins NSP4, VP4, and VP7. MA-104 and Cos-7 cells were grown in six-well plates and transfected with specific siRNA for the silencing of rotavirus proteins NSP4, VP4, and VP7. At 32 h posttransfection, cells were infected with rotavirus DxRRV at an MOI of 10. At 7 h.p.i., the medium was replaced with MEM without methionine. After 1 h of methionine starvation, viral proteins were marked with 35S-labeled methionine-cysteine mix for 1 h. The monolayers were lysed, and cell lysates were clarified by low-speed centrifugation and subjected to SDS-PAGE. Viral proteins were visualized by autoradiography. (A) MA-104 cells. (B) Cos-7 cells. Lanes 1 show the migration of rotavirus DxRRV proteins in control infected cells transfected with siRNA lamin A/C. Lanes 2 of panels 1, 2, and 3 show the inhibition of the expression of rotavirus proteins NSP4 (siNSP4), VP7 (siVP7), and VP4 (siVP4), respectively, obtained with the use of specific siRNAs.

The virus yields recovered at 7 h.p.i. in MA-104 and Cos-7 cells transfected with specific and control siRNA lamin A/C were measured using a focus forming assay on MA-104 cells. As shown in Table 1, transfection with any of the specific siRNAs for NSP4, VP7, and VP4 resulted in a reduction of viral yield in MA-104 cells of more than 98% compared to control cells transfected with siRNA lamin A/C. Virus yield reduction in transfected Cos-7 cells ranged from 91% with siRNA^VP4 to 99% with siRNA^NSP4 compared to control cells. In addition, experiments carried out in parallel showed no significant reduction in virus yield in cells transfected with siRNA lamin A/C in relation to the level of control nontransfected cells, thus suggesting that transfection with siRNA lamin A/C has no effect on rotavirus replication (data not shown).

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TABLE 1. Virus yield recovered from MA-104 and Cos-7 cells transfected with specific siRNA for NSP4, VP4, and VP7

Effect of silencing rotavirus gene expression on changes in Ca²⁺ homeostasis in infected cells. It has been shown that rotavirus infection of cells in culture induces a progressive increase in plasma membrane Ca²⁺ permeability concomitant with an increase in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto) and an enhancement of the total cell Ca²⁺ content as measured by ⁴⁵Ca²⁺ uptake (23, 24). In addition, infection also leads to a progressive depletion of agonist-mobilizable Ca²⁺ from ER pools which have been proposed to be compatible with an increase of Ca²⁺ buffering capacity within the ER (32). The roles of rotavirus proteins NSP4, VP7, and VP4 on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto), plasma membrane Ca²⁺ permeability, and agonist-releasable Ca²⁺ pools were investigated in fura-2 loaded infected Cos-7 cells, where the expression of the individual proteins was silenced (Fig. 2, 3, and 4). Cos-7 cells were preferentially used to measure changes in free [Ca²⁺]_cyto because previous results indicated that under the required experimental conditions, they are more stable and resistant than MA-104 cells (data not shown).

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FIG. 2. Effect of silencing NSP4 expression on [Ca2+]cyto, plasma membrane Ca2+ permeability, and agonist-releasable Ca2+ pools in rotavirus-infected Cos-7 cells. Cos-7 cells grown in 75-cm2 flasks were transfected with specific NSP4 siRNA. At 32 h posttransfection, monolayers were infected with rotavirus DxRRV at an MOI of 10. At 7 h.p.i., the cells were harvested with trypsin and cell suspensions were loaded with fura-2 for the measurement of the cytosolic Ca2+ concentration ([Ca2+]cyto). (A and B) Permeability to Ca2+ in rotavirus-infected cells transfected with siRNAs to NSP4 or lamin A/C and mock-infected cells was evaluated by the changes in [Ca2+]cyto induced by the addition of 5 mM CaCl2 to the extracellular medium, which initially contained 1 mM Ca2+, before or after the addition of ATP (250 µM). The state of filling of agonist-sensitive sequestered Ca2+ pools was evaluated using ATP (250 µM) before or after the addition of 5 mM Ca2+. (C) Quantitative analysis of the initial rate of change in [Ca2+]cyto (dCa2+/dt) induced by the addition of 5 mM CaCl2. (D) Quantitative analysis of the variation of the cytosolic Ca2+ concentration induced by the addition of 5 mM CaCl2 ([Ca]max – [Ca]basal). (E) Quantitative analysis of the variation of the cytosolic Ca2+ concentration induced by the addition of 250 µM ATP. For panels C, D, and E, arithmetic means ± standard deviations (error bars) from four independent experiments are shown. NS, not significant; *, P 0.05.

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FIG. 3. Effect of silencing VP7 expression on [Ca2+]cyto, plasma membrane Ca2+ permeability and agonist-releasable Ca2+ pools in rotavirus-infected Cos-7 cells. Cos-7 cells were transfected with specific VP7 siRNA, infected with rotavirus and loaded with fura-2 for [Ca2+]cyto measurements as described in the legend for Fig. 2. (A and B) Permeability to Ca2+ in infected cells transfected with siRNAs to VP7 or lamin A/C and mock-infected cells after the addition of 5 mM CaCl2 to the extracellular medium. The state of filling of agonist-sensitive sequestered Ca2+ pools was evaluated using ATP (250 µM) before or after addition of 5 mM Ca2+. (C) Quantitative analysis of the initial rate of change in [Ca2+]cyto (dCa2+/dt) induced by the addition of 5 mM CaCl2. (D) Quantitative analysis of the variation of the cytosolic Ca2+ concentration induced by the addition of 5 mM CaCl2 ([Ca]max – [Ca]basal). (E) Quantitative analysis of the variation of the cytosolic Ca2+ concentration induced by the addition of 250 µM ATP. For panels C, D, and E, arithmetic means ± standard deviations (error bars) from five independent experiments are shown. NS, not significant; *, P 0.05.

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FIG. 4. Effect of silencing VP4 expression on [Ca2+]cyto, plasma membrane Ca2+ permeability, and agonist-releasable Ca2+ pools in rotavirus-infected Cos-7 cells. Cos-7 cells were transfected with specific VP4 siRNA, infected with rotavirus, and loaded with fura-2 for [Ca2+]cyto measurements as described in the legend for Fig. 2. (A and B) Permeability to Ca2+ in infected cells transfected with siRNAs to VP4 or lamin A/C and in mock-infected cells after the addition of 5 mM CaCl2 to the extracellular medium. The state of filling of agonist-sensitive sequestered Ca2+ pools was evaluated using ATP (250 µM) before or after addition of 5 mM Ca2+. (C) Quantitative analysis of the initial rate of change in [Ca2+]cyto (dCa2+/dt) induced by the addition of 5 mM CaCl2. (D) Quantitative analysis of the variation of the cytosolic Ca2+ concentration induced by the addition of 5 mM CaCl2 ([Ca]max – [Ca]basal). (E) Quantitative analysis of the variation of the cytosolic Ca2+ concentration induced by the addition of 250 mM ATP. For panels C, D, and E, arithmetic means ± standard deviations (error bars) from four independent experiments are shown. NS, not significant; *, P 0.05.

Basal [Ca²⁺]_cyto measured at 7 h.p.i. began to increase in rotavirus-infected cells compared to the level in mock-infected cells (Fig. 2, 3, and 4, panels A). Basal [Ca²⁺]_cyto was calculated to be 40.2 ± 4.8 and 58.2 ± 5.6 nM (means ± standard deviations of the means) in mock- and virus-infected cells transfected with siRNA lamin A/C, respectively. This increase was already statistically significant (P < 0.01; n = 15).

In Fig. 2, we present the effect of silencing NSP4 expression in infected Cos-7 cells. The addition of 5 mM Ca²⁺ to the extracellular medium induced a marked increase of [Ca²⁺]_cyto in virus-infected cells, but not in mock-infected cells (Fig. 2A). In the case of rotavirus-infected cells silenced for NSP4, the addition of 5 mM Ca²⁺ provoked only a small increase of [Ca²⁺]_cyto, which was of a magnitude similar to that observed in mock-infected cells (Fig. 2A).

To evaluate the change of plasma membrane permeability, we have calculated the initial rate of change of [Ca²⁺]_cyto in the cytoplasm (dCa²⁺/dt) and the amplitude of [Ca²⁺]_cyto increase induced by the addition of 5 mM Ca²⁺ in a series of experiments (Fig. 2C and D). These two parameters were significantly increased in infected cells (P < 0.01; n = 11), confirming that infection increases plasma membrane Ca²⁺ permeability (29).

When the infected cells were transfected with siRNA^NSP4, the initial rate (dCa²⁺/dt) and the amplitude of change of [Ca²⁺]_cyto were significantly smaller than the rate and amplitude observed in rotavirus-infected cells and not different from those observed in mock-infected cells (Fig. 2A, C, and D). These results reveal a reversion by silencing of NSP4 expression of the effects of rotavirus infection on plasma membrane Ca²⁺ permeability.

As Cos-7 cells express a specific purinergic receptor, we evaluated the state of filling of agonist-sensitive Ca²⁺ pools in these cells by the addition of ATP (250 µM) to the extracellular medium. ATP induces a fast biphasic [Ca²⁺]_cyto response, corresponding to a fast Ca²⁺ release from intracellular stores, followed by Ca²⁺ regulation and entry. Figure 2B shows that the peak corresponding to the Ca²⁺ released in infected cells is reduced in comparison to the peak obtained in mock-infected cells. This effect was statistically significant (P < 0.01; n = 6) (Fig. 2E). In infected cells where the expression of NSP4 was silenced, the peak Ca²⁺ response to ATP attained the same level as that in the infected cells transfected with siRNA lamin A/C (Fig. 2B and E). Thus, siRNA^NSP4 transfection did not reverse the effects of rotavirus infection on agonist-sensitive Ca²⁺ pools. The addition of 5 mM Ca²⁺ to the suspension medium in the continuous presence of the agonist induced a new increase in [Ca²⁺]_cyto in the three conditions (Fig. 2B), which was higher than that observed in the absence of ATP (Fig. 2A). Ca²⁺ entry in rotavirus-infected cells is due to the activation of the virus-induced pathway plus an additional cellular component, called capacitative Ca²⁺ entry, through store-operated channels located at the plasma membrane (6, 32). Ca²⁺ entry observed in mock-infected cells is mostly due in this case to the capacitative pathway. The increase in Ca²⁺ entry was abrogated in infected cells when the expression of NSP4 was silenced, and only the capacitative entry was observed.

The observations obtained in infected cells transfected with siRNA^NSP4 indicated that when the expression of NSP4 was knocked down, Cos-7 cells infected with rotavirus showed plasma membrane Ca²⁺ permeability [Ca²⁺]_cyto and capacitative Ca²⁺ entry levels that were similar to those observed in mock-infected cells. However, the Ca²⁺ response to ATP was still reduced in infected cells silenced for NSP4. Of note, the results obtained with Cos-7 cells were all reproduced when experiments were carried out in MA-104 cells transfected with siRNA^NSP4 (data not shown).

To evaluate the role of proteins VP7 and VP4 in the alterations in Ca²⁺ homeostasis observed in infected cells, experiments similar to those carried out with cells transfected with siRNA^NSP4 were also performed with Cos-7 cells transfected with siRNA^VP7 and siRNA^VP4. In the absence of VP7 protein expression, infected cells showed an increase of [Ca²⁺]_cyto after the addition of the Ca²⁺ pulse to the extracellular medium, whose amplitude and rate of change were significantly smaller than those recorded for infected cells transfected with siRNA lamin A/C (Fig. 3A, C, and D). On the other hand, the amplitude of change in silenced cells was significantly higher than the one calculated for mock-infected cells, although the difference in amplitude did not reach statistical significance (Fig. 3D). These results suggest a partial reversion (by the silencing of VP7) of the increase of plasma membrane Ca²⁺ permeability induced by rotavirus infection (Fig. 3A, C, and D). In contrast with the results obtained with siRNA^NSP4-transfected cells, the amount of Ca²⁺ released from the intracellular pools after the addition of the agonist ATP to siRNA^VP7-transfected cells was not significantly different from the levels observed in control mock-infected cells (Fig. 3B and E).

Finally, no significant differences were observed in any of the Ca²⁺ parameters studied between infected cells and infected cells in which the expression of VP4 was silenced (Fig. 4).

Effect of the silencing of rotavirus gene expression on ⁴⁵Ca²⁺ uptake. Rotavirus infection also induces a progressive increase in total cellular Ca²⁺ pools, which comprise both cytosolic and sequestered Ca²⁺ compartments (23, 24). To investigate the role of NSP4, VP7, and VP4 in these changes, total cellular Ca²⁺ pools were evaluated using ⁴⁵Ca²⁺ uptake at 7 h.p.i. in infected MA-104 cells transfected with specific as well as the irrelevant siRNA lamin A/C. Figure 5 shows that in infected cells transfected with irrelevant siRNA, the ⁴⁵Ca²⁺ uptake was significantly elevated in relation to the uptake for mock-infected cells (P < 0.01). Meanwhile, in siRNA^NSP4-transfected cells and also in siRNA^VP7-transfected cells, ⁴⁵Ca²⁺ uptake was reduced to levels quite similar to those observed in mock-infected cells. Similar levels of reduction were obtained when double-transfection experiments, intended to silence both NSP4 and VP7 expression, were carried out (data not shown). On the other hand, the silencing of VP4 did not modify the effect of virus infection.

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FIG. 5. Effect of silencing NSP4, VP7, and VP4 expression on 45Ca2+ uptake in rotavirus-infected MA-104 cells. MA-104 cells grown in 24-well plates were transfected with specific or irrelevant siRNA lamin A/C. At 24 h posttransfection, cells were mock-infected or infected with rotavirus at an MOI of 10. Intracellular Ca2+ pools were evaluated at 7 h.p.i. by 45Ca2+ uptake. The time of 45Ca2+ uptake was 10 min. White bar, mock-infected cells. Black bar, cells infected with DxRRV and transfected with an irrelevant siRNA. Gray bars, cells infected with DxRRV and transfected with siRNAs specific for indicated rotavirus proteins. Arithmetic means ± standard deviations (error bars) from seven independent experiments are shown. NS, not significant; *, P 0.05.

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Changes in the intracellular concentration of free Ca²⁺ are known to regulate a great variety of fundamental physiological processes. Thus, the control of Ca²⁺ concentration in the cytoplasm and organelles of eukaryotic cells is finely regulated (6). The free [Ca²⁺]_cyto is maintained at a level 10⁴-fold lower than that in the extracellular medium, by the operation of a concert of on and off mechanisms of Ca²⁺ entry and extrusion. Ca²⁺ entry down its electrochemical gradient is carried out through different types of Ca²⁺ channels in the plasma membrane and ligand-operated channels in the ER (IP₃ channels). Among the mechanisms for Ca²⁺ removal from the cytosol are the Ca²⁺ pump and the Na⁺/Ca²⁺ exchange at the plasma membrane and the SERCA pump in the ER.

During its replication, rotavirus induces a progressive and profound change in the Ca²⁺ homeostasis of the infected cell, an increase of plasma membrane Ca²⁺ permeability, thereby inducing an increase of free cytoplasmic concentrations and sequestered Ca²⁺ in the ER (23). These changes may in turn lead to cell death (29, 31) and also may be ultimately related to pathogenesis. The synthesis de novo of viral proteins is required for these alterations to take place (24). In addition, viroplasm formation, rotavirus maturation, and particle stabilization are all Ca²⁺-dependent processes (23, 30, 33, 34, 39). Evidence indicates that NSP4 may be responsible for the increases of [Ca²⁺]_cyto. Thus, the expression of NSP4 in insect or vertebrate cells resulted in the elevation of basal [Ca²⁺]_cyto independent of a phospholipase C-IP₃ signaling pathway (2, 38). Aside from the changes in homeostasis in cells expressing NSP4, exogenous addition of the protein or the peptide fragment NSP4_(114-135) also disturbs Ca²⁺ balance in noninfected cells. However, in this case, the increase in cytoplasmic Ca²⁺ levels is dependent on a phospholipase C-IP₃ signaling pathway (2, 26).

When recombinant proteins are expressed, these may be overexpressed or compartmentalized in a manner different from that of natural infection. In this work, we have taken advantage of the siRNA technology to evaluate the role of NSP4 and other viral proteins (VP7 and VP4) in the changes in Ca²⁺ homeostasis occurring in infected cells. These techniques have successfully been used to study rotavirus biology (1, 3, 5, 7, 12, 21, 22, 25, 35, 36). The results presented here indicate that NSP4 is the rotavirus protein mainly responsible for the changes in Ca²⁺ homeostasis observed in infected cells.

Inhibition of NSP4 expression totally ablated the progressive increase in plasma membrane Ca²⁺ permeability observed in infected cells. On the other hand, neither infection nor the silencing of NSP4 affected cellular capacitative Ca²⁺ entry. These results clearly indicate that the silencing of NSP4 inhibits the activation of plasma membrane Ca²⁺ permeability induced by rotavirus infection. This observation is in accordance with the increase in basal [Ca²⁺]_cyto in cells expressing NSP4 or NSP4-EGFP (2, 38). Moreover, the transient expression of NSP4 or NSP4-EGFP in Cos-7 and MA-104 cells increases the basal [Ca²⁺]_cyto associated with an increase in Ca²⁺ permeability of the plasma membrane Ca²⁺ (13).

The activation of Ca²⁺ permeability induced by infection was also reduced by the silencing of VP7; however, it was reduced to a lesser extent. This result may suggest that VP7 may contribute in some way to the effect of infection on Ca²⁺ permeability. It is worth noticing that NSP4 silencing induced a decreased accumulation of VP7 as well as VP4. Silencing the expression of NSP4 affected the synthesis of other viral proteins, as has been reported previously by others (7, 21). This effect may be related to the multiple roles played by NSP4 in rotavirus replication (2, 36). In this case, particularly affected were the levels of the two outer capsid proteins VP7 and VP4. One possible explanation for this phenotype is that the interruption in the translocation of DLPs to the ER results in the accumulation and subsequent degradation of unassembled forms of VP7 and VP4. However, since the silencing of VP4 has no effect whatsoever on Ca²⁺ homeostasis, the reduction of VP7 expression might explain partially the effect of NSP4 silencing on Ca²⁺ permeability. However, we can rule out this interpretation since in the total absence of VP7 expression (silencing VP7), there was still a sizable increase in plasma membrane Ca²⁺ permeability, whereas in the case of NSP4 silencing, this effect was totally eliminated.

With respect to the mechanism by which the silencing of VP7 inhibits the increase of permeability induced by infection, a different hypothesis can be proposed. VP7 might directly activate an ionic channel. However, in contrast with the case for NSP4, the expression of VP7 in insect cells did not modify basal [Ca²⁺]_cyto (38). Alternatively, the silencing of VP7 might indirectly inhibit the NSP4 effect on Ca²⁺ permeability of the plasma membrane. As the silencing of VP7 did not change the level of expression of NSP4, this effect may take place at a postsynthesis stage, such as the processing or trafficking of NSP4 to the plasma membrane. Further experiments, such as an evaluation of the NSP4 distribution within the cell in the absence of VP7, are required to differentiate between these possibilities.

In a recent study, it was reported that rotavirus infection leads to a progressive depletion of ER pools released by agonists (32). Interestingly, in this work, such depletion was still observed when NSP4 was silenced, suggesting that this protein is not directly responsible for this effect. Other viruses, such as coxsackievirus, have been shown to cause Ca²⁺ leakage from intracellular stores, thereby reducing agonist-releasable Ca²⁺ pools in infected HeLa cells (40). In the case of rotavirus infection, the reduction of Ca²⁺ release from the ER by ATP does not seem to be due to an emptying of the ER deposits. Instead, rotavirus infection induces an increase of ER Ca²⁺ pools sensitive to thapsigargin, as measured by ⁴⁵Ca²⁺ uptake (23, 24).

It should be noted that the amount of Ca²⁺ released from the ER by ATP and the fraction of ⁴⁵Ca²⁺ uptake sensitive to thapsigargin are measuring different ER pools (23). Agonists acting through IP₃ liberate readily accessible free Ca²⁺ from the ER, whereas ⁴⁵Ca²⁺ uptake estimates Ca²⁺ content both free and buffered in all cell compartments. The increase of ⁴⁵Ca²⁺ uptake induced by infection is released by thapsigargin, indicating that the Ca²⁺ is sequestered inside the ER. The two pools inside the ER, free and buffered, would be in equilibrium. It has previously been proposed that rotavirus infection would increase Ca²⁺ buffers inside the ER, which would decrease free Ca²⁺ (32). Such buffers may be directly linked to the rotavirus replication cycle which is closely associated with the ER, where two of the most abundantly produced viral proteins, NSP4 and VP7, are heavily modified ER membrane proteins (15). Thus, an explanation is that rotavirus infection results in an increase in the amount of Ca²⁺ buffered (and therefore not readily releasable) inside the ER through the induction of Ca²⁺ binding chaperones, such as those expressed during ER stress. Noteworthy, upregulation of chaperones Bip and GRP94, which is a hallmark of the unfolded protein response, has been observed during rotavirus infection (8, 41).

Knocking down NSP4 or VP7 expression causes a reduction of the amount of total Ca²⁺ content (⁴⁵Ca²⁺ uptake) in infected cells to mock-infected cell levels. An explanation for this effect is that NSP4 and VP7 may both be responsible for the upregulation of chaperones associated with the unfolded protein response and therefore of the buffering capacity. Furthermore, VP7, which is accumulated during infection inside the ER, is known to bind Ca²⁺ (14) and it has been proposed to act as a buffer (32). In addition to the increase in buffering capacity, the reduction of the amount of total Ca²⁺ content may be also influenced by the reduction in plasma membrane Ca²⁺ permeability in NSP4 or VP7 silenced cells. One limitation of this study is that the effect of the studied proteins on Ca²⁺ homeostasis was generally asserted one by one, and the possibility exists that complexes formed by VP7-NSP4-VP4 present in the ER membrane of infected cells would be more efficient in releasing Ca²⁺ than NSP4 or VP7 is alone. However, the results obtained when NSP4 and VP7 were simultaneously silenced suggest that this is not the case.

It has been well established that the replication of a number of RNA as well as DNA viruses results in disruptions of Ca²⁺ homeostasis in the infected cell (9). These alterations are part of the viral strategies aimed at promoting viral replication (9). The effects on Ca²⁺ homeostasis as well as the strategies used vary from virus to virus. The mechanism by which NSP4 modifies plasma membrane permeability to Ca²⁺ is currently unknown; however, rotavirus NSP4 may be acting as a viroporin (17). NSP4 presents features, such as highly hydrophobic domains (10), the ability to interact and destabilize membranes (19, 27, 37), and the ability to form oligomeric structures, that are compatible with viroporin structures (17). The notion that NSP4 was a viroporin was first proposed by Ruiz et al. (32), who observed that in infected cells treated with tunicamycin or brefeldin A, the changes in plasma membrane Ca²⁺ permeability were inhibited. However, the possibility that NSP4 is exerting its permeabilizing activity through interactions with resident plasma membrane cellular proteins cannot be excluded at this time.

In summary, our results suggest that NSP4 expression can account for most of the changes in Ca²⁺ homeostasis and, in particular, for a key event, such as the increase in plasma membrane Ca²⁺ permeability. Nonetheless, research is still needed to fully understand the basis for these alterations and to understand the role that other viral proteins, including VP7, may play. Changes in Ca²⁺ homeostasis are required by the virus along its maturation process and no doubt are part of the basis of the cytopathological effects caused by infection. But more importantly, these changes most likely are also part of the basis of the pathophysiological processes that end in diarrhea.

 
We acknowledge Mariela A. Cuadras, Stanford University School of Medicine, for her extensive help in setting up the siRNA transfection protocols.

This project was partially supported by the Fondo Nacional de Investigaciones Científicas y Tecnológicas (FONACIT), Venezuela through FONACIT grants (S1-2001000329 and G2001000637), the FONACIT-ECOSNORD international program (PI 2002000905), BID-FONACIT II (Subproyecto de Biotecnología N° 2004000386), and TOTAL Venezuela S.A (LOCTI Fund).

 
* Corresponding author. Present address: Department of Experimental Pathology, Center for Research and Advanced Studies (CINVESTAV-IPN), Mexico City, 07360 Mexico. Phone: 52-57473800, ext. 5647. Fax: 52-55-57473377. E-mail: jeludert{at}gmail.com

Published ahead of print on 9 April 2008.

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Journal of Virology, June 2008, p. 5815-5824, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02719-07
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