Enterovirus 70 enters HeLa cells by a clathrin- and dynamin-dependent route

Abstract

Endocytosis, which is normally associated with cellular processes such as nutrient uptake and ligand uptake, is used by many different viruses to gain entry into a susceptible cell and to promote infection. In earlier work it was shown that CD55 is the major attachment molecule for enterovirus 70 (EV70) on HeLa cells. EV70 is a member of the enterovirus genus. Enteroviruses use a variety of receptors and enter cells by different mechanisms. For example, poliovirus binds to the poliovirus receptor (PVR), and has been shown to enter by a novel endocytic mechanism which is clathrin-, caveolin-, and dynamin-independent but does rely on cholesterol (Danthi and Chow, 2004; DeTulleo and Kirchhausen, 1998; Kronenberger et al., 1998). Coxsackievirus A9, which binds to coxsackievirus and adenovirus receptor (CAR) and the integrin alpha5beta3, and coxsackievirus B4, which binds to CAR and CD55, have been shown to enter via lipid rafts (Triantafilou and Triantafilou, 2004; Triantafilou and Triantafilou, 2003); coxsackievirus B3 which also binds to CAR and CD55 has been shown to enter via clathrin-coated pits (Chung et al., 2005); and echovirus I which binds to the alpha2beta1 integrin, enters via a caveolin-mediated and/or alternative route that is dynamin- and lipid raft-dependent (Marjomaki et al., 2002; Pietiainen et al., 2004). The major objectives of the research described in this thesis were to determine if EV70 entered HeLa cells by endocytosis, and, if so, which endocytic pathway was exploited by this virus. In studies using drug inhibitors of endocytosis, chlorpromazine, which blocks clathrin-mediated endocytosis, reduced EV70 infection of HeLa cells, but cholesterol sequestering drugs had little or no effect on virus entry. These results suggested that EV70 entry was dependent on clathrin, but not on caveolae or lipid rafts. EV70 conjugated with Alexa fluorochrome 555 appeared to co-localize with clathrin light chains tagged with enhanced yellow fluorescent protein (EYFP), consistent with a clathrin-mediated mechanism for EV70 entry into HeLa cells. Expression of the dominant-negative mutant of epidermal growth factor receptor pathway substrate clone 15 (eps15), a protein required for recruitment of adaptor protein 2 (AP-2) and epsin (both required for targeting clathrin to the cellular membrane), and assembly of clathrin-coated pits and vesicles, reduced EV70 infection of HeLa cells by 30 % when compared to the null mutant of eps15, D3Delta2. These results seem to rule in clathrin-mediated endocytosis. However, expression of the dominant-negative mutant of caveolin-1 had little or no effect on EV70 infection of cells. These results would seem to rule out caveolin-mediated endocytosis. A dominant-negative mutant of dynamin-2 strongly inhibited EV70 infection of cells, and must play an essential role in entry. Thus, it is proposed that EV70 infection may occur through an endocytic mechanism that is dependent on both dynamin and clathrin, but independent of caveolin.