The rabies virus glycoprotein (G protein) has several important functions and is a major antigenic stimulus of the host immune system following rabies virus infection or vaccination. We developed a model system for studying the role of N-linked glycosylation in the intracellular transport and antigenicity of this molecule. The full-length cDNA of the G protein of the ERA strain of rabies virus was inserted into the eukaryotic shuttle vector pSG5 and then stably transfected into wild-type Chinese hamster ovary (CHO) cells and mutant CHO cell lines defective in glycosylation. Transfected wild-type CHO cells expressed the G protein (detected by immunofluorescence) on the cell surface in a manner similar to rabies virus-infected cells. The transfected wild-type CHO cells were shown by immunoprecipitation to produce a protein of 67K that comigrated with the fully glycosylated G protein isolated from virus-infected cells or purified virions. Treatment of the transfected cell lines with tunicamycin completely blocked surface expression and resulted in the intracellular accumulation of the G protein, suggesting that the presence of N-linked oligosaccharides is important for transport of this glycoprotein to the plasma membrane. The G protein cDNA was also expressed in the lectin-resistant CHO cell lines Lec 1, Lec 2 and Lec 8. In these cells initial Nlinked glycosylation does occur, but later steps in processing of the oligosaccharides are blocked. In each case, the G protein was expressed on the surface of lectin-resistant CHO cells in a similar manner to expression on wild-type CHO cells. This suggests that various different N-linked oligosaccharide structures support intracellular transport of this glycoprotein. Thus, stably transfected CHO cell lines will provide a useful model system for further studies of the role of Nlinked glycosylation in trafficking and antigenicity of the rabies virus G protein.

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Isolates of aflatoxin-producing strains of Aspergillus can grow on Glycine max beans. Furthermore, both mixed aflatoxins and ariatoxin BI inhibit the elongation of both attached and excised G. max roots. In addition, the toxin inhibits the capacity of the excised roots to take up [14C]leucine. This suggests that the toxin may inhibit the activity of an uptake system responsible for uptake of low-molecular-weight (MW) compounds by possibly altering membrane structure and/or function. In this connection, incubation of excised roots in media containing either mixed ariatoxins or ariatoxin B a results in a diminution of acid-insoluble protein, but neither sterol nor lipid phosphorus levels, of an 80,000 • g pellet (purported "crude" plasmalemma fraction). To provide preliminary evidence that mixed aflatoxins can decrease the amount of a specific plasmalemma protein (which might regulate the uptake of low-MW compounds), we incubated roots with and without mixed aflatoxins and then gel-filtrated integral proteins which were released by detergents from 80,000 X g pellets that had been obtained by differential centrifugation of Miracloth filtrates. The released proteins were gelfiltrated on Sephadex G-100 columns. Sodium dodecyl sulfate, Triton X-IO0 and Tween 20 each solubilized greater than 85% of the pellet's protein. Gel fdtration yielded 280 nm absorbing void volume and retarded peaks for substances which were either precipitated by trichloroacetic acid (total protein) or solubilized by detergents (integral protein) from pellets that were derived from ariatoxin-treated and nontreated roots. The amounts of protein which were recovered within column void volumes following gel fdtration of 80,000 X g, acid-precipitable protein were not significantly different for treated and nontreated roots, suggesting that incubation of roots with aflatoxins does not reduce the pellet's content of an acidprecipitable, high-MW (100,000 or more) protein. However, this conclusion is highly tentative as less than 15% of the acid-insoluble protein which was layered onto the column was recovered. The amplitude of the void volume peaks for detergent-released proteins from treated roots was consistently less than that for nontreated roots, suggesting that treatment of roots with aflatoxins diminishes the "crude" plasmalemma fraction's content of high-MW protein(s). This suggestion was supported through calculation of the amounts of protein which were recovered within both the void volume and retarded peaks and comparing these to the total protein levels that were recovered from the columns. The amount of protein which was found within the void volume peak following gel filtration of either Triton X-IOO or Tween 20-released proteins from pellets that were derived from treated roots was less than that for nontreated roots.