Expression and function of viral glycoproteins in insect cells

Recombinant baculoviruses were used to express the gp160 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) and a truncated variant, designated gp160(t), which lacks a transmembrane domain. Glycosylation, proteolytic cleavage, secretion, and biological activities of gp160 and gp160(t) were studied in Spodoptera frugiperda cells. Both proteins were rapidly glycosylated and initially were found to be totally endo-{dollar}beta{dollar}-N-acetyl-D-glucosaminidase H (endo-H) sensitive. However, partial resistance to endo-H was gradually acquired by both molecules. Gp160 was found to remain cell-associated, whereas gp160(t) was secreted into the culture medium in large amounts. A fraction of gp160 and gp160(t) appeared to be proteolytically cleaved, and a cleavage product corresponding in size to gp120 was identified in the culture medium. Gp160(t) was found to interact specifically with CD4 receptors without any requirement for proteolytic cleavage. The gp160 protein was shown to be expressed on the surface of S. frugiperda cells by indirect immunofluorescence. These surface molecules were biologically active, as demonstrated by their ability to induce syncytium formation when co-cultivated with HeLa T4 cells.; The gene encoding the major envelope glycoprotein complex, gp55-116 (gB), of human cytomegalovirus (HCMV) was expressed at high levels in insect cells utilizing a recombinant baculovirus. The mature intracellular form of the insect-derived gp55-116 was a protein of Mr 150,000 which contained approximately 50,000 daltons of N-linked oligosaccharides. The oligosaccharide linkages were found to be almost exclusively endo-H sensitive. The Mr 150,000 protein was processed, presumably by proteolytic cleavage, to yield at least one of the previously defined cleavage products of gp55-116. This processing step was significantly less efficient in insect cells than the analogous step in mammalian cells. Finally, the insect derived gp55-116 was found to be highly immunogenic in experimental animals and readily recognized by antibodies contained within HCMV immune human serum, suggesting that this recombinant protein warrants further study as a potential HCMV subunit vaccine candidate.; The syncytium-inducing ability of Spodoptera frugiperda cells infected with the baculovirus-HIV envelope gene recombinant was used to develop a sensitive assay for identification of potential inhibitors of HIV-induced syncytium formation. This assay was used to analyze the effects of cloned human interferon upon HIV-induced syncytium formation. It was observed that pretreatment of HeLa T4 cells with either alpha, beta, or gamma interferons completely prevented HIV-induced syncytium formation, whereas addition of interferon at the time of cocultivation had no such inhibitory activity. These results indicate that human interferons induce a fusion-resistant state in HIV-susceptible target cells, and suggest that they may serve as effective inhibitors of the early stages of virus infection.