Macropinocytotic Uptake and Infection of Human Epithelial Cells with Species B2 Adenovirus Type 35

Abstract

Human adenovirus serotype 35 (HAdV-35; here referred to as Ad35) causes kidney and urinary tract infections and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence, which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here we show that infectious entry of Ad35 into HeLa cells, human kidney HK-2 cells, and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180, which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chemicals against serine/ threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin, and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3, or the sodiumproton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked endocytic uptake of Ad35. Dominant-negative proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1, or the Pak1 effector C-terminal binding protein 1 (CtBP1), potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy, and live cell imaging showed that Ad35 colocalized with fluid-phase markers in large endocytic structures that were positive for CD46, ␣ integrins, and also CtBP1. Our results extend earlier observations with HAdV-3 (Ad3) and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells.

Identification of Adenovirus-Derived Cell-Penetrating Peptide

“…Kälin et al reported that the interaction of type 35 Ad with the host-cell receptor CD46 and integrins, but not with heparin sulfate, facilitates the internalization of the vector via receptor-mediated macropinocytosis in K562 cells. 13) We speculate that in certain cell types, such as the K562 cell, the shaft region of type 35 Ad seems to be involved in the transduction of Ad35 vector. This speculation might be supported by the finding that recombinant Ad35 shaft protein inhibits transduction of type 35 Ad vector in K562 cells but not in HepG2 cells (data not shown).…”

“…Greber and colleagues reported that internalization of type 35 Ad vector in K562 does not involve binding to heparin sulfate on the cell surface. 13) We speculated that peptide C induces receptor-dependent endocytosis in K562 cells, and analyzed the mechanism of intracellular penetration of peptide C into K562 cells. FITC-labeled peptide C entered into K562 cells at 37°C, but not at 4°C (Fig.…”

“…In contrast, species B Ad viruses, such as type 3 Ad and type 35 Ad, are internalized by macropinocytosis. 12,13) Furthermore, species C Ad viruses, such as types 2 and 5 Ad, induce macropinocytosis and leakage of macropinosomal contents into the cytosol, as shown by codelivery assays. 14) This uptake pathway could be delivery of the cointernalized dextran into the cytosol, but at least for type 2 Ad macropinocytosis is not the infectious entry pathway.…”

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

“…Amiloride inhibits macropinocytosis by reducing cytosolic pH and inhibiting Rac1 and Cdc42 signaling. 36,37 The multimechanism involved in the cellular uptake thus explained the non-compartmentalized ( Figures S2 and S3) and diffused nature of the fluorescence-loaded nanocarrier upon uptake as demonstrated in Figure 9.…”

Virus entry at a glance

“…The signaling pathways are best characterized for viruses that trigger plasma membrane ruffling and macropinocytosis for endocytic uptake. These include vaccinia virus (VACV), Ebola virus, Kaposi's sarcoma-associated herpesvirus (KSHV), adenovirus and influenza A virus (IAV) (Amstutz et al, 2008;Brindley et al, 2011;Chakraborty et al, 2011;de Vries et al, 2011;Eierhoff et al, 2010;Hunt et al, 2011;Kälin et al, 2010;Meertens et al, 2012;Meier et al, 2002;Mercer and Helenius, 2008;Nanbo et al, 2010;Raghu et al, 2009;Saeed et al, 2010;Schmidt et al, 2011;Shimojima et al, 2006;Valiya Veettil et al, 2010).…”