Cholesterol Is Required for Endocytosis and Endosomal Escape of Adenovirus Type 2

Abstract

The species C adenovirus type 2 (Ad2) and Ad5 bind the coxsackievirus B Ad receptor and ␣v integrin coreceptors and enter epithelial cells by clathrin-mediated endocytosis. This pathway is rapid and efficient. It leads to cell activation and the cholesterol-dependent formation of macropinosomes. Macropinosomes are triggered to release their contents when incoming Ad2 escapes from endosomes. Here, we show that cholesterol extraction of epithelial cells by methyl-␤-cyclodextrin (m␤CD) treatment reduced Ad5-mediated luciferase expression ϳ4-fold. The addition of cholesterol to normal cells increased gene expression in a dose-dependent manner up to threefold, but it did not restore gene expression in m␤CD-treated cells. m␤CD had no effect in the presence of excess cholesterol, indicating that the inhibition of gene expression was due specifically to cholesterol depletion. Cholesterol depletion inhibited rapid Ad2 endocytosis, endosomal escape, and nuclear targeting, consistent with the notion that clathrin-dependent endocytosis of Ad2 is cholesterol dependent. In cholesterol-reduced cells, Ad2 internalized at a low rate, suggestive of an alternative, clathrin-independent, low-capacity entry pathway. While exogenous cholesterol completely restored rapid Ad2 endocytosis, macropinocytosis, and macropinosome disruption, it did not, surprisingly, restore viral escape from endosomes. Our results indicate that macropinosome disruption and endosomal escape of Ad2 are independent events in cells depleted of and then refilled with cholesterol, suggesting that viral escape from endosomes requires lipidcontrolled membrane homeostasis, trafficking, or signaling.

Stochastic Model of Acidification, Activation of Hemagglutinin and Escape of Influenza Viruses from an Endosome

“…The steady-state pH is pH early ∞ = 6.0 and pH late ∞ = 5.5 for early and late endosomes respectively [23]. Thus, we calibrated the permeability constant L and Rab conversion kinetics by solving numerically Equation 6 and fitting the experimental acidification curve (Figure 2). We found that the permeabilities of early and late endosomes are L early = 3.5 10 −3 N A cm s −1 and L late = 0.1N A cm s −1 , respectively, and the half-maturation time is t 1/2 = 10 min.…”

“…Several modeling approaches, including kinetics rate equations [1], stochastic modeling [2,3] and mechanics of molecular binding [4] have been developed to describe how membrane receptors are activated and engaged into endosomal pathways. However, little attention has been devoted to study viral trafficking inside an endosome, which is a critical and limiting step in replication and more generally to gene delivery [5][6][7][8].…”

PCI-enhanced adenoviral transduction employs the known uptake mechanism of adenoviral particles

“…37 Our study therefore questions the efficacy of adenoviral entry into the cytosol at low viral concentration and suggests that PCI can be used to realize the biological activity of these viral particles. Imelli et al 42 showed recently that adding cholesterol to the medium enhanced the transgene expression after adenoviral infection more than threefold, demonstrating that the efficiency of adenovirus infection can be enhanced using different methods.…”

Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization

“…The role of endogenous cholesterol (involved in macropinocytosis and both clathrin-mediated endocytosis [CME] and clathrin-independent pathways [CIP]) in the cellular uptake of the PCL/ MD nanocarrier was explored using MβCD. [28][29][30][31] As shown in Figure 9, this resulted in the highest decline of uptake in all the three PCa cell lines studied. There was a 51% decrease in the uptake of PCL/MD nanocarrier in DU145 cell line and about 65% and 75% decrease in LNCaP and PC3 cell lines, respectively.…”