Intracellular accumulation of punta toro virus glycoproteins expressed from cloned cDNA

Abstract

The Punta Toro virus (PTV) middle size (M) RNA encodes two glycoproteins, G1 and G2, and possibly a nonstructural protein, NS M. A partial cDNA clone of the M segment which contains G1 and G2 glycoprotein coding sequences but lacks most of the NS M sequences was inserted into the genome of vaccinia virus under the control of an early vaccinia promoter. Cells infected with the recombinant virus were found to synthesize two polypeptides with molecular weights of 65,000 (G1) and 55,000 (G2) that reacted specifically with antibody against PTV. Studies using indirect immunofluorescence microscopy revealed that these proteins accumulated intracellularly in the perinuclear region. The results of endoglycosidase H digestion of these glycoproteins suggested that both G1 and G2 glycoproteins were transported from the RER to the Golgi complex. These proteins were not chased out from the Golgi region during a 6-hr incubation in the presence of cycloheximide. Surface immune precipitation and 125I-protein A binding assays also demonstrated that the majority of the G1 and G2 glycoproteins are retained intracellularly. These results indicate that the PTV glycoproteins contain the necessary information for retention in the Golgi apparatus.