Subversion of CtBP1-controlled macropinocytosis by human adenovirus serotype 3

Abstract

Endocytosis supports cell communication, growth, and pathogen infection. The species B human adenovirus serotype 3 (Ad3) is associated with epidemic conjunctivitis, and fatal respiratory and systemic disease. Here we show that Ad3 uses dynamin-independent endocytosis for rapid infectious entry into epithelial and haematopoietic cells. Unlike Ad5, which uses dynamin-dependent endocytosis, Ad3 endocytosis spatially and temporally coincided with enhanced fluid-phase uptake. It was sensitive to macropinocytosis inhibitors targeting F-actin, protein kinase C, the sodium-proton exchanger, and Rac1 but not Cdc42. Infectious Ad3 macropinocytosis required viral activation of p21-activated kinase 1 (PAK1) and the C-terminal binding protein 1 of E1A (CtBP1), recruited to macropinosomes. These macropinosomes also contained the Ad3 receptors CD46 and av integrins. CtBP1 is a phosphorylation target of PAK1, and is bifunctionally involved in membrane traffic and transcriptional repression of cell cycle, cancer, and innate immunity pathways. Phosphorylation-defective S147A-CtBP1 blocked Ad3 but not Ad5 infection, providing a direct link between PAK1 and CtBP1. The data show that viruses induce macropinocytosis for infectious entry, a pathway used in antigen presentation and cell migration.

Molecular mechanisms of clathrin-independent endocytosis

“…Endocytosis is perturbed by the overexpression of dominant-negative Arf1, Cdc42, Arf6 and Rac1, and these mutant proteins provide a useful means of differentiating between different endocytic mechanisms under specific conditions of cell type and GTPase expression level (Kumari and Mayor, 2008;Lamaze et al, 2001;Naslavsky et al, 2004;Sabharanjak et al, 2002). However, extrapolation to more general conclusions about the specific involvement of any of these GTPases in just one type of endocytosis is hampered by reports of different effects in different experiments -for example, overexpression of mutants of Arf6 blocks both uptake via clathrincoated pits and a clathrin-independent endocytic pathway (D' SouzaSchorey et al, 1995;Naslavsky et al, 2004;Palacios et al, 2002), and overexpression of mutant Cdc42 blocks clathrin-independent uptake of GPI-linked proteins under some conditions (Sabharanjak et al, 2002) but can also perturb clathrin-mediated uptake of E-cadherin (Izumi et al, 2004) and macropinocytosis (Amstutz et al, 2008;Dharmawardhane et al, 1997;Garrett et al, 2000). The identification of effectors downstream of small GTPases that regulate different types of endocytosis, and an understanding of how such effectors mediate vesicle formation, would clearly be a major step forward.…”

“…Several papers published in the last 2 years suggest that such information is starting to be available (Amstutz et al, 2008;Frick et al, 2007;Karjalainen et al, 2008;Liberali et al, 2008;Lundmark et al, 2008;Romer et al, 2007). From these studies (coupled with ultrastructural information), a classification of endocytic pathways on the basis of functionally important proteins that localize to forming transport intermediates at the plasma membrane can be derived.…”

“…It was originally proposed that CtBP1 mediates fission through the acylation of lysophosphatidic acid and the associated alterations in membrane curvature (Weigert et al, 1999), but further experiments revealed that CtBP1 is, in fact, unlikely to have acyltransferase activity (Gallop et al, 2005). Recently, a role for CtBP1 in macropinocytosis has been demonstrated (Amstutz et al, 2008;Liberali et al, 2008). CtBP1 is recruited to macropinosomes that are induced by high concentrations of epidermal growth factor (EGF), and loss of CtBP1 function reduces the frequency of formation of these structures .…”

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).…”

Identification of Adenovirus-Derived Cell-Penetrating Peptide

“…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.…”