Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

Journal Article · Mon Oct 01 00:00:00 EDT 1990 · Gene Analysis Techniques; (USA)

DOI:https://doi.org/10.1016/0735-0651(90)90030-J· OSTI ID:5945748

Murphy, C I; Lennick, M; Lehar, S M; Beltz, G A; Young, E ^[1]

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1. Cambridge Bioscience Corporation, Worcester, MA (USA)

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4.

• OSTI ID: 5945748
• Journal Information: Gene Analysis Techniques; (USA), Vol. 7:6; ISSN 0735-0651
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
PROTEINS
GENE REGULATION
AIDS VIRUS
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DNA
DNA-CLONING
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TRACER TECHNIQUES
TRITIUM COMPOUNDS
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MAMMALS
MICROORGANISMS
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