Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation

Abstract

Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH 2 ), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH 2 was uncovered by examining Env expression and maturation in treated cells. GPG-NH 2 treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH 2 negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH 2 , which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.

Assembly of Subtype 1 Influenza Neuraminidase Is Driven by Both the Transmembrane and Head Domains

“…The resulting pellet containing the membrane fraction was resuspended with reducing Laemmli sample buffer and the supernatant containing the soluble fraction was precipitated with 20% TCA before dissolving in sample buffer. Endoglycosidase H (Endo-H) and peptide-n-glycosidase F (PNGase F) treatments were performed on 20 l of transfected cell post-nuclear supernatants according to the manufacturer's instructions (New England Biolabs) except that 5% SDS was used in place of 10ϫ glycoprotein denaturing buffer (32).…”

Anti-human immunodeficiency virus type 1 agent alpha-hydroxy glycineamide enters the target cells via a mechanism of passive diffusion

“…Further electron microscopy studies indicated a possible interaction of GPG‐NH 2 derivatives with viral capsid assembly [16]. We have also shown that αHGA has an effect on HIV‐1 envelope precursor protein, gp160, through the endoplasmic reticulum associated protein degradation pathway [17,18]. Thus, this compound affects two steps in the virus replication cycle.…”

Human Ubc9 Is Involved in Intracellular HIV-1 Env Stability after Trafficking out of the Trans-Golgi Network in a Gag Dependent Manner

“…Proteins targeted by ERAD are partially deglycosylated, exported out the ER, and targeted for degradation in a proteasome dependent manner (reviewed in 71–73. It has been previously demonstrated that treatment of Env expressing cells with GPG-NH 2 causes Env to undergo ERAD, which results in gp160 migrating at a molecular weight (MW) similar to wild type gp120 due to gp160 partial deglycosylation [67,74]. If a population of gp160 was partially deglycosylated via ERAD, it could affect the identification and quantitation of the gp160 and gp120 levels in the Ubc9 knockdown cells.…”

“…Activation of the unfolded protein response was analyzed by XBP-I splicing as previously described with slight modifications [67]. Briefly, transfected and untransfected 293T cells were treated with 5mM dithiothreitol (DTT) for 3 hours or left untreated.…”