Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

Abstract

The fusogenic properties of Semliki Forest virus (SFV) and its mutants were used to follow the kinetics of acidification during the endocytic uptake of virus by BHK‐21 cells. It has previously been shown that the low pH of endocytic vacuoles triggers a conformational change in the SFV spike glycoprotein, activating membrane fusion and initiating virus infection. This conformational alteration was here shown to occur in endosomes and to follow the same time course as the intracellular fusion reaction, demonstrating that fusion occurs rapidly after virus exposure to endosome acidity. The kinetics of endosome acidification were monitored using wild type (wt) SFV and fus‐1, an SFV mutant with a lower fusion pH threshold. The results presented here demonstrated that wt and mutant virus were internalized with a t1/2 of 10 min, and that endosomes were acidified to the wt threshold of pH 6.2 with a t1/2 of 15 min. In contrast, endosome pH reached the fus‐1 threshold of 5.3 with a much longer t1/2 of 45 min. The subsequent degradation of SFV in lysosomes had a t1/2 of 90 min. It was found that after the initial uptake of virus from the plasma membrane, its transit through the endocytic pathway, exposure to endosome acidity and eventual delivery to lysosomes were markedly asynchronous.

Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells.

“…Endosomes of the CHO mutants DTG 1-5-4 and DTF 1-5-1 (13, 27, 28) also show differences in pH, along with a marked decrease in early acidification (40). These and other studies on the kinetics of endosome acidification (12,29,32) show that endosomes within the same cell can vary in their pH.…”

“…Endosomes of the CHO mutants DTG 1-5-4 and DTF 1-5-1 (13, 27, 28) also show differences in pH, along with a marked decrease in early acidification (40). These and other studies on the kinetics of endosome acidification (12,29,32) show that endosomes within the same cell can vary in their pH.Differences in acidification may be important in determining the function of an endocytic compartment, because endocytic processes vary in their acidification requirements. Low density lipoprotein (LDL) and ct2M need only mildly acidic conditions (pH 6.5-6.7) to dissociate from their receptors (1, 17), whereas lysosomal enzyme dissociation (8), iron release (3,15,26), and penetration of diphtheria toxin (5, 31) all require a pH of <6.0.…”

“…Low density lipoprotein (LDL) and ct2M need only mildly acidic conditions (pH 6.5-6.7) to dissociate from their receptors (1, 17), whereas lysosomal enzyme dissociation (8), iron release (3,15,26), and penetration of diphtheria toxin (5, 31) all require a pH of <6.0. Therefore, measuring the pH of a specific type of endosome may help elucidate the role that endocytic compartment has in a particular physiologic process.From kinetic studies (12,29,32,40) it is not clear whether 9 The Rockefeller University Press, 0021-9525/87/12/2723/11 $2.00 …”

“…From kinetic studies (12,29,32,40) it is not clear whether 9 The Rockefeller University Press, 0021-9525/87/12/2723/11 $2.00…”

Lentiviruses with trastuzumab bound to their envelopes can target and kill prostate cancer cells

“…[20][21][22] The outer protein shell contains 240 copies of glycoproteins, E1 and E2. 20,23,24 E1 causes cell fusion, whereas E2 is predominantly the protein responsible for cell membrane attachment. 19,[25][26][27][28][29] Importantly, the Sindbis E1 protein can fuse to cells independently of the receptor binding E2 protein.…”