Mutations in rotavirus nonstructural glycoprotein NSP4 are associated with altered virus virulence

Abstract

Rotaviruses are major pathogens causing life-threatening dehydrating gastroenteritis in children and animals. One of the nonstructural proteins, NSP4 (encoded by gene 10), is a transmembrane, endoplasmic reticulum-specific glycoprotein. Recently, our laboratory has shown that NSP4 causes diarrhea in 6- to 10-day-old mice by functioning as an enterotoxin. To confirm the role of NSP4 in rotavirus pathogenesis, we sequenced gene 10 from two pairs of virulent and attenuated porcine rotaviruses, the OSU and Gottfried strains. Comparisons of the NSP4 sequences from these two pairs of rotaviruses suggested that structural changes between amino acids (aa) 131 and 140 are important in pathogenesis. We next expressed the cloned gene 10 from the OSU virulent (OSU-v) and OSU attenuated (OSU-a) viruses by using the baculovirus expression system and compared the biological activities of the purified proteins. NSP4 from OSU-v virus increased intracellular calcium levels over 10-fold in intestinal cells when added exogenously and 6-fold in insect cells when expressed endogenously, whereas NSP4 from OSU-a virus had little effect. NSP4 from OSU-v caused diarrhea in 13 of 23 neonatal mice, while NSP4 from OSU-a caused disease in only 4 of 25 mice (P < 0.01). These results suggest that avirulence is associated with mutations in NSP4. Results from site-directed mutational analyses showed that mutated OSU-v NSP4 with deletion or substitutions in the region of aa 131 to 140 lost its ability to increase intracellular calcium levels and to induce diarrhea in neonatal mice, confirming the importance of amino acid changes from OSU-v NSP4 to OSU-a NSP4 in the alteration of virus virulence.

FIG. 1

Amino acid sequences of NSP4 from OSU and Gottfried virulent and attenuated porcine rotaviruses. Amino acid residues are shown in a single-letter format. Dashes represent the same amino acid residue as in the top line. Residues in bold indicate differences between NSP4 from attenuated virus and NSP4 from virulent virus in either the OSU or Gottfried strain. OSU-v, NSP4 from OSU virulent virus; OSU-a, NSP4 from OSU attenuated virus; Gott-v, NSP4 from Gottfried virulent virus; Gott-a, NSP4 from Gottfried attenuated virus.

FIG. 2

[Ca²⁺]_i in Sf9 cells expressing NSP4 and NSP4 mutants. Fura-2/AM-loaded cells were superfused continuously with Na-HEPES buffer, and intracellular calcium was measured by ratio imaging. The averaged ratio signal obtained from each cell was digitally saved as a log file. The collected values from cells imaged within a single experiment (6 to 10 cells) were then averaged to give an experimental observation of 1 (n = 1). WT, cells infected with wild-type baculovirus (n = 3); OSU-a, cells infected with recombinant baculovirus expressing OSU-a NSP4 (n = 5); OSU-v, cells infected with recombinant baculovirus expressing OSU-v NSP4 (n = 3); D131-140, cells infected with recombinant baculovirus expressing the deletion mutant; VVP3, cells infected with recombinant baculovirus expressing the substitution mutant; P138S, cells infected with recombinant baculovirus expressing the point mutant. Standard errors of the means are shown by the bars.

FIG. 3

Silver staining of purified NSP4 from OSU virulent and attenuated viruses. Five microliters of infected Sf9 cell lysate (6 × 10⁷ cells/ml; lanes L) or 1 μg of purified protein (lanes P) was analyzed by SDS-PAGE (12% gel). M, molecular weight markers. Arrows indicate that two different glycosylated forms of NSP4 are present in the purified material.

FIG. 4

Effects of NSP4 and NSP4 mutant on [Ca²⁺]_i in HT-29 cells. Fura-2/AM-loaded cells were superfused continuously with Na-HEPES buffer, and intracellular calcium was measured by ratio imaging. The averaged ratio signal obtained from each cell was digitally saved as a log file. The collected values from cells imaged within a single experiment (6 to 10 cells) were then averaged to give an experimental observation of 1 (n = 1). Seven experimental observations from different dye loadings were averaged and presented. (A) NSP4 from OSU-a virus, (NSP4-a) or OSU-v virus (NSP4-v) was added as indicated. (B) NSP4 point mutant (P138S) or NSP4 from OSU virulent virus (NSP4-v) was added as indicated.