Rotavirus NSP4 114-135 peptide has no direct, specific effect on chloride transport in rabbit brush-border membrane

Abstract

The direct effect of the rotavirus NSP4 114-135 and Norovirus NV464-483 peptides on 36Cl uptake was studied by using villus cell brush border membrane (BBM) isolated from young rabbits. Both peptides inhibited the Cl-/H+ symport activity about equally and partially. The interaction involved one peptide-binding site per carrier unit. Whereas in vitro NSP4 114-135 caused nonspecific inhibition of the Cl-/H+ symporter, the situation in vivo is different. Because rotavirus infection in young rabbits accelerated both Cl- influx and Cl- efflux rates across villi BBM without stimulating Cl- transport in crypt BBM, we conclude that the NSP4 114-135 peptide, which causes diarrhea in young rodents, did not have any direct, specific effect on either intestinal absorption or secretion of chloride. The lack of direct effect of NSP4 on chloride transport strengthens the hypothesis that NSP4 would trigger signal transduction pathways to enhance net chloride secretion at the onset of rotavirus diarrhea.

Figure 1

Dose-dependent effects of the NSP4_114-135 and NV_464-483 peptides on the initial rate of pH gradient-activated chloride uptake by rabbit intestinal BBM vesicles. Fixed amounts of BBM vesicles were mixed with variable amounts of peptide in the appropriate volume of membrane (Tris gluconate) buffer to give the indicated (final) peptide concentrations. After preincubation for 5 min at 22°C, aliquots were used to measure the initial rate of 15 mM chloride uptake in the presence of an initial pHo/pHi = 5.0/7.5 gradient. Symbols: (▲) for NSP4_114-135 and (△) for NV_464-483. Results are shown as absolute uptake rates in pmoles·mg membrane protein^-1·s^-1 ± standard deviation with 6 – 15 determinations per point. Because the two peptides gave statistically indistinguishable results, the two sets of data were pooled to obtain the overall fits in Table 1. The continuous line illustrates the best overall fit obtained by fixing the n_i value to 1.