INTRODUCTION

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A rotavirus nonstructural glycoprotein, NSP4, and a synthetic peptide, NSP4(114-135), corresponding to residues 114 to 135 of this protein, both have been shown to induce age- and dose-dependent diarrhea in young rodents (5). Because the induction of diarrhea and alterations in chloride secretion, unaccompanied by any histological lesions, occurred within a period of about 3 h, similar to those induced by the heat-stable toxin  of Escherichia coli, NSP4 was proposed to be a viral enterotoxin which, by activating a Ca²⁺-dependent signal transduction pathway, impairs intestinal epithelial transport (5). However, it is known that the ionic concentrations in the stools of rotavirus-infected animals, although high, are considerably less than those occurring in the secretory diarrheas caused by secretagogues such as the enterotoxins of Vibrio cholerae and E. coli (11, 15). Since the mechanisms by which rotavirus and NSP4 cause diarrhea are not completely understood, investigating possible, alternative mechanisms might prove useful. Recently, others have shown that rotavirus infection alters regulation of the expression of digestive enzymes (13).

Independently, we have demonstrated that both natural and experimental infection by a lapine group A rotavirus, La/RR510 strain, isolated in our laboratory impairs Na⁺-D-glucose (SGLT1) and Na⁺-L-leucine symport activity across intestinal brush border membrane (BBM) vesicles isolated from young rabbits. Because infection reduces D-glucose transport capacity (V_max) without affecting the density of phlorizin-binding sites and of SGLT1 protein antigen present in the BBM vesicles, we concluded that the rotavirus effect on this symporter is direct (10). Transport inhibition preceded viral shedding into the lumen and the onset of diarrhea. No intestinal lesion was seen in the experimentally infected rabbits, confirming that diarrhea is not necessarily a consequence of the intestinal lesion but can precede it, as if cell dysfunction were the cause, not the consequence, of the histological damage (5, 6, 10, 17, 18, 22).

Since SGLT1 is involved in the reabsorption of large volumes of water under physiological conditions, we proposed that the mechanism of rotavirus diarrhea involves a generalized inhibition of symport systems and hence of water reabsorption. The resulting massive water loss through the intestine would eventually contribute to overwhelming the organ's capacity for water reabsorption, thereby facilitating establishment of the main symptom of enteritis, diarrhea.

In this work, we investigated the hypothesis that NSP4 acts, at least in part, by specifically inhibiting sodium-coupled solute transport across the intestinal BBM. In particular, we examined whether NSP4(114-135) and certain related peptides directly affect the kinetics of Na⁺ cotransport with either D-glucose or L-leucine. To avoid possible problems of interpretation, due for instance to indirect metabolic effects, changes in intestinal structure, and/or changes in rotavirus protein synthesis, transport was assayed with a rapid in vitro technique, based on using jejunal BBM vesicles isolated from specific-pathogen-free (SPF) young rabbits.

(A preliminary account of this work has been presented elsewhere [2].)

	MATERIALS AND METHODS

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BBM vesicle preparation. SPF 4- and 7-week-old New Zealand albino hybrid rabbits were obtained from Charles River. Jejunal segments from 7-week-old rabbits and the entire small intestine from 4-week-old rabbits were removed, rinsed with saline at room temperature, everted, and distributed into plastic bags for storage at 80°C as described elsewhere (21).

Viral protein and synthetic peptides. The SA11 NSP4 protein and all peptides used in this work were synthesized and purified as described elsewhere (5). They were dissolved in the appropriate volume of membrane buffer to obtain either 8 µM protein or 10 mM peptide stock solutions, which were stored at 20°C until use. The peptides used were Norwalk virus NV(464-483) (DTGRNLGEFKAYPDGFLTCV), NSP4(114-135) (DKLTTREIEQVELLKRIYDKLT), and mNSP4(131K) (DKLTTREIEQVELLKRIKDKLT).

Transport assay and expression of results. Transport was assayed by using a rapid filtration technique and either D-[¹⁴C]glucose or L-[¹⁴C]leucine as the substrate (7). BBM vesicle aliquots suspended in membrane buffer were mixed with appropriate amounts of the same buffer containing either the intact protein or a given peptide. After preincubation for 5 min at 35°C (unless stated otherwise), 10-µl aliquots were used to carry out transport measurements by mixing with 40 µl of transport buffer formed by membrane buffer supplemented with either constant or variable (see below) concentrations of unlabeled substrate, ¹⁴C-labeled substrate as a tracer, and 100 mM sodium cyanide (all concentrations given as the final ones in the incubation mixtures). Initial uptake rate measurements were then carried out for 2.6 s at 35°C in an automatic, temperature-controllable short-time incubation apparatus constructed in our laboratory. Uptake data were statistically compared by applying a global one-way analysis of variance (19). Results are shown as absolute uptake rates, v, in picomoles per second per milligram of protein ± standard deviation (SD).

Kinetic analyses. Uncorrected uptake rates as a function of either the substrate (v = f[S] at [I] constant) or inhibitor (v = f[I] at [S] constant) concentration were fitted by nonlinear least-squares regression analysis (23) to the appropriate equation as indicated. To perform each fit, the procedure of Fletcher and Powell as modified by van Melle and Robinson (23) was used. To test the fit of data to each relevant equation, we used the commercial program Stata (Integral Software, Paris, France). For statistical evaluation, fits were compared either within each given condition (F test) or between pairs of conditions (F' test) as described elsewhere (23). For each given data set, comparison between a lack-of-fit and a pure-error component, with the degrees of freedom given in that order in parentheses in the tables, yielded F values that provide a quantitative assessment of the goodness of fit. All individual F values listed in the tables were found to be not significant, meaning that the data points did not differ statistically (P < 0.01) from the theoretical fit of the equation used to perform the fit. This was true for each of the curves forming each set, as well as for the overall fit of the set. All calculations were done on Apple Macintosh microcomputers.

	RESULTS

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Effect of rotavirus NSP4 on intestinal BBM Na⁺-solute symport activities in young rabbits. Jejunal BBM vesicles from 7-week-old rabbits were mixed with appropriate amounts of purified NSP4. After incubation for 5 min at 35°C, aliquots were used to assay uptake under standard conditions that included a constant (0.1 mM) substrate concentration and a zero-trans sodium cyanide gradient. Because of its low solubility, the maximal protein concentration that could be reached in the incubation mixtures was 1 µM. This dose, however, was found to be too low to have any significant effect on the uptake of either D-glucose or L-leucine (results not shown). The question posed having remained unanswered, we turned our attention to more soluble peptides derived from NSP4, some of which have been shown to induce diarrhea in young rodents, with kinetics similar to that of the intact protein (5).

Comparative effects of NSP4-related peptides on the kinetics of D-glucose and L-leucine uptake by intestinal BBM vesicles from young rabbits. In preliminary assays, we established that doses in the order of 1 mM peptide were necessary to cause significant effects on these symporters. This information was then used to investigate the possible mode of action of each of these peptides on intestinal transport, using first the v = f [S] approach, at constant [I].

(1)

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Kinetic effects of NSP4-related peptides on D-glucose uptake by intestinal BBM vesicles from 4-week-old rabbits. Initial rates of D-glucose uptake, expressed as the uncorrected, absolute uptake rates, are plotted as a function of substrate concentration in either the absence () or presence of 1 mM NV(464-483) (), mNSP4(131K) (), and NSP4(114-135) (). Solid lines represent theoretical fits computed by using the kinetic parameters listed in Table 1. However, because the control, NV(464-483), and mNSP4(131K) results were all statistically indistinguishable, they have been pooled and are shown as a single curve corresponding to the overall fit for 4-week-old rabbits in Table 1. The insert shows the results obtained by using only the lowest [D-glucose] range used, 0.1 to 10 mM.

Dose-dependent peptide effects on the Na⁺-solute symporters. To confirm that the NSP4(114-135) effect on D-glucose (but not on L-leucine) transport is indeed specific, experiments were performed by using the v = f[I] approach, at a constant (0.1 mM) concentration of each substrate.

(i) Effects on D-glucose uptake. With the NSP4(114-135) peptide we found a progressive effect, approaching 78% inhibition of the D-glucose symporter at 1 mM peptide (Fig. 2). A significant but lesser inhibition was observed when the NV(464-483) peptide was used (29% inhibition at 1 mM peptide [Fig. 2]). However, the two curves have different shapes, which can be taken as evidence that different transport pathways are involved, as discussed below.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Dose-dependent effects of the NSP4(114-135) and NV(464-483) peptides on the initial rate of D-glucose uptake by jejunal BBM vesicles from 7-week-old rabbits. Fixed amounts of vesicles were mixed with variable amounts of peptide in the appropriate volume of membrane buffer to give the indicated (final) peptide concentrations. These mixtures were either used directly (cis peptide conditions) or subjected first to two cycles of freezing and thawing to permit equilibration of the extra- and intravesicular contents (cis-plus-trans conditions), as described elsewhere (24). After incubation for 5 min at 35°C, aliquots were used to measure the initial rate of 0.1 mM D-glucose uptake under standard conditions. Because the results at all given cis and cis-plus-trans peptide concentrations were statistically indistinguishable, they have been pooled and fitted again to obtain relevant, overall fits. These are illustrated by the same symbol,  for NSP4(114-135) and  for NV(464-483). Results are shown as absolute substrate uptake rates ± SD. The solid lines represent theoretical fits of either set of data to equation 2, computed by using the kinetic parameters listed in Table 2.

1

1

(ii) Hill equation analysis. Because of the shape of the curves obtained, the results in Fig. 2 suggest strongly that an empirical equation of the Hill type might be appropriate to describe the observed peptide effects on D-glucose uptake (although not necessarily those on L-leucine uptake). We therefore refitted this set of data by using an equation adapted from Alvarado and Vasseur (3),

(2)

where V is an apparent maximal velocity; K_i is the apparent affinity constant for the inhibitor, I (the peptide); and n_i is the Hill number. Nonlinear regression analysis of these data gave the numerical results in Table 2. Both NV(464-483) and NSP4(114-135) results could be fit very well by using equation 2, but the two curves were statistically different according to an F' test. The NSP4(114-135) peptide gave a Hill number larger than 1 (n_i = 2.8), indicating the participation of at least three peptide-binding sites in the interaction, each with an average K_i of 0.67 mM. In contrast, the NV(464-483) peptide yielded a Hill number of 1, with a lower affinity (K_i = 1.7 mM), suggesting that although NV(464-483) inhibits D-glucose uptake, the effect is relatively small and probably involves a different mechanism that might be nonspecific.

(iii) Effects on L-leucine uptake. The most salient feature of the L-leucine results is that both the NV(464-483) and NSP4(114-135) peptides cause a practically identical, partial inhibition of L-leucine uptake (Fig. 3; Table 2). Consequently, because they are statistically indistinguishable, these data have been pooled and fitted again to obtain the overall fit listed in Table 2. As illustrated in Fig. 3, this result strongly suggests that L-leucine symport inhibition is nonspecific and is not mediated by NSP4(114-135), a specific inhibitor of SGLT1. Analysis of the L-leucine results according to the Hill equation (Table 2) reveals that the two peptides used, NV(464-483) and NSP4(114-135), gave the same Hill number, 0.4. The fact that these n_i values are smaller than unity strongly indicates deviation from simple Michaelian kinetics, as further discussed below. Interestingly, such differences in the peptide effects on the sodium-coupled transport of either D-glucose or L-leucine strongly suggest that the two symporters do not use the same mechanism.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Dose-dependent effects of the NSP4(114-135) and NV(464-483) peptides on the initial rate of L-leucine uptake by jejunal BBM vesicles from 7-week-old rabbits. Details are as for Fig. 2, except for the use of L-leucine as the substrate and peptides only in the cis side. Symbols: , NSP4(114-135); , NV(464-483). Because the two peptides gave statistically indistinguishable results, the two sets of data have been pooled to obtain the overall fit in Table 2 (heavy middle line).

Testing whether a three-site symport model can explain the L-leucine and D-glucose transport inhibitions caused by NSP4-related peptides. To test the preceding hypothesis, we reanalyzed the relevant data to see whether they can be explained in terms of the three-site symport model recently proposed by Alvarado and Vasseur to explain the interaction of another BBM symport system, that of the enterocyte H⁺ and Cl symporter, with the amphipatic inhibitor carbonyl cyanide m-chlorophenylhydrazone (4). According to this model, certain Na⁺-solute cotransporters may involve a carrier, C, with three distinct, specific binding sites: one for the substrate, S; one for the allosteric activator, A (the Na⁺ ion); and a third for the inhibitor, I (a peptide in the present paper). In the general model, it is further assumed that all of the possible substrate-bound carrier complexes, C-S, I=C-S, A=C-S, and IA=C-S, can form and translocate. To use well-established jargon, the model includes slippage; i.e., there is no need to propose that the C-S complex is not mobile. Kinetically, the system is defined by equation A1 in reference 4, that is,

(3)

where v is the total substrate uptake, V₁ and V₂ are two distinguishable maximal velocities resulting when [I] tends to infinity and zero, respectively; and K_I is the apparent affinity constant for I.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Effect of the NSP4(114-135) peptide on the kinetics of L-leucine uptake, according to the full, nonobligatory three-site symport model. Experimental L-leucine data (taken from Fig. 3) were fitted by nonlinear regression analysis to equation 3. The theoretical fits thus computed show the overall result to be the sum (ct) of two distinct hyperbolas: one that is Michaelian (concave; c1) and another that is convex (c2).

	DISCUSSION

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Our results show that in vitro, the NSP4(114-135) peptide strongly and specifically inhibits the SGLT1 activity of intestinal BBM vesicles from young rabbits. The Na⁺-L-leucine symporter also is inhibited (Fig. 3), but this effect is much weaker and probably nonspecific. Indeed, it seems conceivable that this effect involves nonspecific, lipophilic interactions of the amphipathic peptides with the membrane.

Interestingly, in in vivo experiments with young rabbits infected either naturally or experimentally with a lapine rotavirus, La/RR510 strain, the Na⁺-L-leucine symporter was inhibited strongly and to an extent similar to that of SGLT1. In both cases, V_max was the only kinetic parameter impaired after rotavirus infection (10). Because with D-glucose qualitatively and quantitatively similar effects are obtained, either during rotavirus infection in vivo (10) or in in vitro experiments with isolated NSP4 peptides (this report), the in vitro effects exhibited by certain NSP4-related peptides on SGLT1 might explain the rotavirus transport effects observed in vivo.

The case of L-leucine appears to be special. In contrast with the in vivo experiments just mentioned, the Na⁺-L-leucine symport activity was not significantly inhibited when the isolated NSP4(114-135) peptide was used directly (Table 1). Such a result strongly suggests that if rotavirus infection results in inhibition of the Na⁺-L-leucine symporter, this inhibition, in contrast with that of the SGLT1, is unlikely to be mediated by the NSP4(114-135) peptide. More work will be required to establish whether or not the Na⁺-L-leucine symporter inhibition is caused by some other rotavirus protein or by some NSP4 peptide distinct from NSP4(114-135).

The concentrations of NSP4(114-135) that cause full inhibition of SGLT1, about 1 mM according to the data presented here, are of the same order of magnitude as those used by Ball et al. (5) to induce diarrhea in young rodents. In these experiments, both intraperitoneal and intraduodenal NSP4(114-135) peptide were effective, but only in young (6- to 8-day-old) mice. Peptide administration to slightly older (11- to 13-day-old) animals was much less effective. Our experiments were carried out by using vesicles from either 4- or 7-week-old rabbits. The possibility clearly exists that stronger effects may have been obtained if younger rabbits had been used. But such work was not attempted at this time, mainly because working with SPF rabbits younger than 4 weeks would have posed practical problems of availability.

The very low concentration of the NSP4 protein that can be maintained in the incubation mixtures (1 µM) was found to have no significant effect on the uptake of either D-glucose or L-leucine. Such a result was not surprising because to induce diarrhea in young rodents (5), doses as high as 2 µM were necessary. However, the validity of comparing biologically effective doses derived from either in vivo or in vitro work appears to be limited (25).

The specific and strong inhibition caused in vitro by the NSP4(114-135) peptide on SGLT1 strongly suggests that this effect can be one (but not necessarily the only) important factor in the pathogenesis of rotavirus diarrhea. Based on our in vivo work with rotavirus-infected rabbits (10), we proposed earlier that a generalized inhibition of Na⁺-solute cotransport systems might be at the basis of the inhibition of intestinal water reabsorption caused by rotavirus in the absence of any histological damage to the intestinal mucosa. The subsequent massive water loss might eventually overwhelm the intestinal capacity for water reabsorption, thereby contributing to establishment of the main symptom of enteritis, diarrhea (10).

The fact that the mNSP4(131K) peptide does not inhibit SGLT1 constitutes perhaps the strongest evidence for our conclusion that the specific inhibition of this cotransporter is a key factor in the pathogenesis of rotavirus diarrhea. In closing, we have established the existence of the following correlation: NSP4(114-135) inhibits SGLT1 and causes diarrhea, whereas mNSP4(131K) neither inhibits SGLT1 nor causes diarrhea.

Concerning the possible role of NSP4 in the pathogenesis of rotavirus diarrhea, the present in vitro work strongly suggests that NSP4 is at least one among other effectors directly causing SGLT1 inhibition during rotavirus infection in vivo. Whereas in vitro NSP4(114-135) inhibits SGLT1 practically instantaneously, the situation in vivo is different. Being absent from the mature, infective virion particle, the nonstructural protein NSP4 needs to be synthesized after infection, and time will be required for the newly synthesized protein to be released into the cytoplasm and/or the intestinal lumen and migrate to its target, the symporter molecule.