MUTATIONS IN ROTAVIRUS NONSTRUCTURAL GLYCOPROTEIN NSP4 ARE ASSOCIATEDWITH ALTERED VIRUS VIRULENCE

Rotaviruses are major pathogens causing life-threatening dehydrating g astroenteritis in children and animals. One of the nonstructural prote ins, NSP4 (encoded by gene 10), is a transmembrane, endoplasmic reticu lum specific glycoprotein. Recently, our laboratory has shown that NSP 4 causes diarrhea in 6- to 10-day-old mice by functioning as an entero toxin, To confirm the role of NSP4 in rotavirus pathogenesis, we seque nced gene 10 from two pairs of virulent and attenuated porcine rotavir uses, 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, W e next expressed the cloned gene 10 from the OSU virulent (OSU-v) and OSU attenuated (OSU-a) viruses by using the baculovirus expression sys tem and compared the biological activities of the purified proteins, N SP4 from OSU-v virus increased intracellular calcium levels over 10-fo ld in intestinal cells when added exogenously and 6-fold in insect cel ls when expressed endogenously, whereas NSP4 from OSU-a virus had litt le 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 mutat ed OSU-v NSP4 with deletion or substitutions in the region of aa 131 t o 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.