Biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport

Abstract

When human immunodeficiency virus type 1 envelope glycoproteins were expressed in 293 cells by using a recombinant adenovirus expression vector, the envelope precursor (gpl60) was initially glycosylated by cotranslational addition of N-linked high-mannose oligosaccharide units to the protein backbone and then cleaved to gpl20 and gp4l. The subunits gpl20 and gp4l were then further modified by the addition of fucose, galactose, and sialic acid, resulting in glycoproteins containing a mixture of hybrid and complex oligosaccharide side chains. A fraction of glycosylated gpl60 that escaped cleavage was further modified by the terminal addition of fucose and galactose, but the addition of sialic acid did not occur, consistent with the notion that it is compartmentalized separately from the gpl20 envelope protein. Processing and transport of gpl60 were blocked by the monovalent ionophore monensin, which at high concentrations (25 ,uM and above) was a potent inhibitor of the endoproteolytic cleavage of gpl60; at lower concentrations (1 to 10 ,uM), it selectively blocked the secondary glycosylation steps so that smaller products were produced. Monensin (1 ,uM) treatment also resulted in a reduction in syncytium formation, which was observed when recombinant infected cells were cocultivated with CD4-bearing HeLa cells. The infectivity of human immunodeficiency virus type 1 was also reduced by monensin treatment, a decrease that may be due to incompletely glycosylated forms of gpl20 that have a lower affinity for the CD4 receptor.

The Role of CD4 and its Downmodulation in Establishment and Maintenance of HIV‐1 Infection

“…No effect of the ionophore nonensin was demonstrated on gpl20/gp41 production, indicating that gpl60 cleavage probably takes place in a neutral environment (Stein et al 1987). Other workers have reported, however, that nonensin can inhibit gpl60 cleavage (Dewar et al 1989, Pal et al 1988. Although these results might be attributed to a perturbation of gpl60 transport to the Golgi apparatus by nonensin, neutralization of an acidic compartment by NH4CI was also shown to impair production ofgp!20/gp41 (Willeyetal.…”

“…Endoproteolytic processing of gpl60 takes place in the cis or medial compartment of the Golgi appartus (Stein & Engleman 1990, Dewar et al 1989) and correlates with the trimming of mannose-rich oligosaccharide chains by a-mannosidase 1. Whether the cis-median Golgi compartment involved in gpl60 cleavage has a neutral or acidic pH is still controversial.…”

“…Transport of gpl60 from the ER to the Golgi apparatus, in which cleavage takes place, may thus depend on the removal of the glucose residues of the high mannose structure by glucosidase 1. Cleavage of gpl 60 may consequently take place before addition of complex oiigosaccharide chains (Dewar et al 1989, Stein & Engleman 1990.…”

The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

“…Gp160 cleavage occurs in the trans-Golgi network (TGN) or after it exits the TGN (Bultmann et al, 2000;Pal et al, 1991;Pfeiffer et al, 1997;Stein and Engelman, 1990). Brefeldin A, A1Fn, monensin, and tunicamycin inhibit gp160 cleavage even when gp160 is allowed to accumulate in the TGN, suggesting that cleavage occurs in the late compartment of the Golgi or after gp160 has exited from the Golgi (Dewar et al, 1989;Kantanen et al, 1995;Pal et al, 1991Pal et al, , 1988. Cleavage may occur in an acidic compartment, as it was inhibited by NH 4 Cl and partially inhibited by chloroquine (Courageot et al, 1999;Willey et al, 1988).…”

Biophysical characterization of recombinant proteins expressing the leucine zipper-like domain of the human immunodeficiency virus type 1 transmembrane protein gp41

“…Envelope multimerization is thought to be critical for replication of fusogenic viruses such as human immunodeficiency virus type 1 (HIV-1) (for reviews, see references 17, 40, and 50). Oligomerization of the viral envelope (Env) polypeptide precursor gp160 is required for the processing and intracellular transport (16,17,21) of a functional Env complex (composed of the surface glycoprotein gp120 and the transmembrane protein gp41). This process has been termed assembly oligomerization and is mediated through gp41 (19,20).…”