Intracellular degradation of the HIV-1 envelope glycoprotein. Evidence for, and some characteristics of, an endoplasmic reticulum degradation pathway.

Analysis of the fate of HIV-1 envelope protein gp160 (Env) has shown that newly synthesized proteins may be degraded within the biosynthetic pathway and that this degradation may take place in compartments other than the lysosomes. The fate of newly synthesized Env was studied in living BHK-21 cells with the recombinant vaccinia virus expression system. We found that gp160 not only undergoes physiological endoproteolytic cleavage, producing gp120, but is also degraded, producing proteolytic fragments of 120 kDa to 26 kDa in size, as determined by SDS/PAGE in non reducing conditions. Analysis of the 120-kDa proteolytic fragment, and comparison with gp120, showed that it is composed of peptides linked by disulfides bonds and lacks the V3-loop epitope and the C-terminal domain of gp120 (amino acids 506-516). A permeabilized cell system, with impaired transport of labeled Env from the endoplasmic reticulum (ER) to Golgi compartments, was developed to determine the site of degradation and to define some biochemical characteristics of the intracellular degradation process. In the semipermeable BHK-21 cells, there was: (a) no gp120 production (b), a progressive decrease in the amount of newly synthesized gp160 and a concomitant increase in the amount of a 120-kDa proteolytic fragment. This fragment had the same biochemical characteristics as the 120-kDa proteolytic fragment found in living nonpermeabilized cells, and (c) susceptibility of the V3 loop. This degradation process occurred in the ER, as shown by both biochemical and indirect immunofluorescence analysis. Furthermore, there was evidence that changes in redox state are involved in the ER-dependent envelope degradation pathway because adding reducing agents to permeabilized cells caused dose-dependent degradation of the 120-kDa proteolytic fragment and of the remaining gp160 glycoprotein. Thus our results provide direct evidence that regulated degradation of the HIV-1 envelope glycoprotein may take place in the ER of infected cells.