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Two adjacent papainlike cysteine protease (PCP) domains, PCP alpha and PCP beta, were identified in the N-terminal region of the open reading frame 1a replicase proteins of the arteriviruses porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus. The replicase of the related virus equine arteritis virus contains only one active PCP in the corresponding region. Sequence comparison revealed that the equine arteritis virus PCP alpha counterpart probably was inactivated by loss of its catalytic Cys residue. For both porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus, the generation of two processing products, nsp1 alpha and nsp1 beta, was demonstrated by in vitro translation. Site-directed mutagenesis and sequence comparison were used to identify the putative active-site residues of the PCP alpha and PCP beta protease domains and to show that they mediate the nsp1 alpha/1 beta and nsp1 beta/2 cleavages, respectively.

Cytochalasin B has been shown to potently inhibit the transport of glucose, deoxyglucose, and glucosamine by Novikoff hepatoma cells in suspension culture without affecting their intracellular phosphorylation and metabolism. Deoxyglucose transport is inhibited by cytoehalasin B in a simple competitive manner. Although this inhibition is not sufficient to explain the biological action of the drug on cytokinesis, it does explain earlier observations on inhibition by cytochalasin B of the incorporation of glucose and glucosamine, and probably of other extracellular precursors, into macromolecules by various types of cells.Cytochalasin B (CB) was originally noted by Carter (1) to inhibit cytoplasmic division (cytokinesis) without inhibiting nuclear division (karyokinesis), to inhibit cell movement, and to cause dramatic changes in cell shape. Subsequent reports have indicated a wide range of biological effects, including inhibition of phagocytosis (2-4), pinocytosis (5), secretion of thyroid (6) and growth hormone (7), and the inhibition of morphogenesis (8, 9). It has been suggested that the effects of CB may be due to an inhibition of microfilament function (10-12). While the effects on the morphology of microfilaments is apparent, such effects may not account for the primary action of the drug (13). Direct interaction of CB with the cell membrane, on the other hand, could account for all the biological effects of the chemical.One of us (14) has previously observed that CB inhibits the incorporation of uridine and thymidine, but not of choline, into macromolecules by cultured Novikoff rat hepatoma cells. Preliminary experiments indicated that glucose incorporation is also inhibited. Similarly, the inhibition by CB of phagocytosis by leukocytes is accompanied by an inhibition of glycolysis and respiration (3, 4) as measured by the formation of lactate and CO2, respectively, from glucose. Glucosamine incorporation into mucopolysaccharides by various types of cells is also inhibited by CB, and it has therefore been suggested that CB inhibits mucopolysaccharide synthesis (15). However, the finding that nucleoside incorporation into nucleic acids by Novikoff cells is inhibited by CB without significantly affecting the increase in cell mass (14), suggested that CB might inhibit the incorporation of extracellular metabolites into macromolecules by inhibiting their uptake into the cells. This conclusion is supported by the present results and is in agreement with results of previous studies (16,17) that indicate that transport may be the rate-limiting step in the metabolism of various substances. MATERIALS AND METHODSNovikoff rat hepatoma cells (subline NlS1-67), propagated in suspension culture (18,19), were suspended to 2 X 106 cells/ ml in basal medium 42 (18), or glucose-free basal medium 42 (17), or HEPES-buffered, glucose-free basal medium 42 (17). Suspensions of cells were supplemented with CB by addition of the appropriate volume of an 8.2-mM stock solution of CB in dimethyl sulfoxide or absolute ethanol. ...