Sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins

Abstract

The sedimentation behavior of transmissible gastroenteritis coronavirus (TGEV) was analyzed. Upon sucrose gradient centrifugation, the major virus band was found at a density of 1.20 to 1.22 g/cm(3). This high density was observed only when TGEV with a functional sialic acid binding activity was analyzed. Mutants of TGEV that lacked sialic acid binding activity due to a point mutation in the sialic acid binding site of the S protein were mainly recovered at a lower-density position on the sucrose gradient (1.18 to 1.19 g/cm(3)). Neuraminidase treatment of purified virions resulted in a shift of the sedimentation value from the higher to the lower density. These results suggest that binding of sialoglycoproteins to the virion surface is responsible for the sedimentation behavior of TGEV. When purified virions were treated with octylglucoside to solubilize viral glycoproteins, ultracentrifugation resulted in sedimentation of the S protein of TGEV. However, when neuraminidase-treated virions or mutants with a defective sialic acid binding activity were analyzed, the S protein remained in the supernatant rather than in the pellet fraction. These results indicate that the interaction of the surface protein S with sialoglycoconjugates is maintained after solubilization of this viral glycoprotein by detergent treatment.

FIG. 1

Sucrose gradient centrifugation of untreated (TGEV) and neuraminidase-treated (TGEV-NA) TGEV. The gradient was fractionated from the bottom. Aliquots of each fraction were used to measure the sucrose concentration (lower panels) and to analyze by SDS-PAGE for the presence of virus (upper panels). The bands were stained with Coomassie brilliant blue. Only the portion of the gel containing the S protein is shown.

FIG. 2

Sucrose gradient centrifugation of untreated and neuraminidase-treated virions. The upper right panel shows a schematic drawing of the two bands harvested. The upper left panel shows the S, N, and M protein profiles of the upper (1) and lower (2) bands of untreated (TGEV) and neuraminidase-treated (TGEV-NA) TGEV. The lower panel shows the S and N protein profiles of the upper (1) and lower (2) bands of the m9, m6, and HAD7 mutants. Lanes representing untreated virions (−) and neuraminidase-treated virions (+) are indicated. The left lane (M) shows molecular mass markers. The proteins were stained with Coomassie brilliant blue.

FIG. 3

Sedimentation of TGEV and the HAD7 mutant. Purified virions were sedimented for 1 h at 0 (lane 1), 25,000 (lane 2), 50,000 (lane 3), 75,000 (lane 4), and 100,000 × g (lane 5). Virions remaining in the supernatant were sedimented by centrifugation for 1 h at 150,000 × g. The pellet fractions of the first centrifugation (P) and the second centrifugation (S) were analyzed by Western blotting for the presence of S protein.

FIG. 4

Sedimentation of the S protein after solubilization with octylglucoside (OG). Untreated (−VCNA) and neuraminidase-treated (+VCNA) virions were solubilized with 0, 1, or 5% octylglucoside and sedimented by ultracentrifugation as described in Materials and Methods. Aliquots of the pellet (P) and the supernatant (S) fractions were analyzed by Western blotting for the presence of S protein.

FIG. 5

Sedimentation of the S proteins of TGEV, PRCoV, and mutants of TGEV (HAD2, HAD3, HAD4, HAD7, m9, and m6) after solubilization with octylglucoside (OG). The purified virus preparations were not pretreated with neuraminidase. Virions were incubated with 0 or 1% octylglucoside and sedimented by ultracentrifugation as described in Materials and Methods. Aliquots of the pellet (P) and the supernatant (S) fractions were analyzed by Western blotting for the presence of S protein. HA, hemagglutinating.

FIG. 6

Electron micrograph of the pellet fraction obtained after centrifugation of octylglucoside-treated virions. Magnification, ×150,000. Bar, 100 nm.