In the experiments reported here, we found that internalization of CVB occurs by a process that combines aspects of caveolar endocytosis with features more characteristic of macropinocytosis. We found that entry of CVB depended on the TJ protein occludin, and that virus induces internalization of occludin within macropinosomes. Both CVB entry (Coyne and Bergelson, 2006
) and occludin internalization (this report) required caveolin but were independent of dynamin; both were blocked by drugs that inhibit macropinocytosis; and both required Rab34, a GTPase implicated in macropinosome formation, and Rab5, a GTPase known to stimulate macropinocytosis.
The molecular mechanisms underlying macropinocytosis in polarized epithelia are poorly defined. Although macropinocytosis from the apical membrane has been linked to Src kinase activity (Mettlen et al., 2006
), we found that a Src siRNA had no effect on CVB entry (Coyne and Bergelson, 2006
) or occludin internalization (data not shown) from the TJ, suggesting that different mechanisms control macropinocytosis from the apical and lateral domains.
In fibroblasts, Rab34-mediated macropinocytosis has been shown to require the activity of Rac and the actin nucleation factor WAVE2 (Sun et al., 2003
). In Caco-2, cells, we found that Rab34-induced macropinocytosis was Rac independent () and was unaffected by a WAVE2 siRNA (data not shown). Although Rac was not required for macropinocytosis, CVB-induced activation of Ras was essential for virus entry and infection as well as for macropinocytosis induced by Rab34 (). Ras-mediated signals have also been implicated in Rab5-dependent macropinocytosis (Lanzetti et al., 2004
Few viruses appear to utilize macropinocytosis to gain entry into host cells. Human immunodeficiency virus-1 (HIV-1) enters both macrophages and brain endothelium by macropinocytosis (or a macropinocytosis-like process) (Liu et al., 2002
; Marechal et al., 2001
). Adenovirus type 2 (Ad2) utilizes Rac-dependent macropinocytosis not for entry into the host cell, but rather for pH-activated escape from endosomes into the cytoplasm (Meier et al., 2002
). Although both Ad2 and CVB induce macropinocytosis, and both interact with CAR, CAR itself does not appear to be essential for induction. In the case of Ad2, macropinocytosis depends on virus interaction with its αv
-integrin coreceptor (Meier et al., 2002
), and we have found that CVB-induced macropinocytosis of occludin was not inhibited by CAR siRNA (data not shown). It may be that CAR and occludin provide different, but equally important, functions in CVB entry—CAR is required for CVB uncoating (Coyne and Bergelson, 2006
), and occludin may serve as a scaffold to recruit and anchor signaling or regulatory molecules in the vicinity of virus entry.
If macropinocytosis is required for CVB and occludin entry, why is caveolin also necessary? The pinching off of caveolar vesicles to internalize caveolar cargoes such as SV40 and cholera toxin depend on dynamin (reviewed in Parton and Simons, 2007
), but the internalization of both occludin and CVB occurred even in the presence of dominant-negative dynamin (; Coyne and Bergelson, 2006
). Caveolae have been implicated in the internalization of cargos such as bacteria and lymphocytes that are too large (>1 µm for bacteria and > 7 µm for lymphocytes) to be contained within a single caveolar vesicle (60–80 nm) (Millan et al., 2006
; Mulvey and Hultgren, 2000
). The internalization of large cargoes likely requires membrane rearrangements on a scale characteristic of macropinosome or phagosome formation. Caveolin-1 knockout mice display phagocytic defects, consistent with a role for caveolin-1 in endocytic processes distinct from internalization of typical caveolar vesicles (Li et al., 2005
). Taken together, these observations have led to the suggestion that caveolin may serve to regulate a variety of endocytic processes (Parton and Simons, 2007
The mechanism of CVB entry may be distinct from those described for other caveolin-dependent viruses because the virus enters through the TJ. TJs themselves share characteristics of lipid rafts, and TJ proteins have been shown to associate with caveolin-rich membranes (Nusrat etal., 2000b
). However, the flask-like invaginations typical of caveolae are located primarily at the basolateral surface of resting polarized epithelial cells and are not generally observed at the TJ (Lahtinen et al., 2003
; Mora et al., 1999
; Vogel et al., 1998
). In previous experiments, we did not detect a concentration of caveolin-1 at the TJ of resting Caco-2 cells. However, CVB interaction with DAF on the cell surface rapidly induced the phosphorylation of caveolin-1 on tyrosine 14 and the appearance of phosphorylated caveolin at the TJ, which was necessary for CVB entry (Coyne and Bergelson, 2006
). Phosphorylation of tyrosine 14 of caveolin-1 has been associated with the formation and fusion of caveolin-derived vesicles (Aoki et al., 1999
). Nonetheless, it remains unclear whether TJ-associated caveolin-1 leads to the formation of typical caveolae, or whether it may serve another function. The physical interaction of caveolin with cellular membranes may facilitate membrane perturbations associated with the formation and/or closure of macropinosomes at the junction. Alternatively, caveolin-1 may act as an anchor to concentrate signaling molecules or other effector molecules in the area of the TJ.
The TJ is a complex of transmembrane proteins (such as occludin) and cytoplasmic scaffolding proteins, both of which serve to link the cell membrane to the actin cyto-skeleton and an array of signaling molecules. With such an elaborate network of proteins concentrated at the TJ, why is occludin specifically required for CVB infection? Occludin is not involved in CVB attachment, or in the relocalization of CVB to the TJ, but an occludin siRNA trapped virus at the TJ, suggesting that occludin regulates a key aspect of CVB entry. Occludin interacts directly with signaling molecules (Basuroy et al., 2006
; Chen et al., 2002
; Nusrat et al., 2000a
), including the p85 subunit of PI3K, a known mediator of phagocytosis and macropinocytosis (reviewed in Lindmo and Stenmark, 2006
). In addition, occludin interacts with caveolin-1 (Nusrat et al., 2000b
), suggesting a possible functional connection. Although occludin has never been implicated as a regulator of endocytosis, it is possible that it promotes CVB entry by recruiting caveolin or other regulatory molecules to the TJ.
CVB enters polarized epithelial cells by a complex mechanism. The complexity may be due to the particular properties of polarized intestinal cells, or to the fact that CVB uses a receptor that is itself a component of the TJ. At least three viruses initiate infection by attaching to receptors within the junctional complex of polarized epithe-lia. Like CVB (and adenoviruses, which also bind to CAR), reoviruses (Barton et al., 2001
) and members of the herpesvirus family (Geraghty et al., 1998
) interact with receptors that are normally sequestered within junctions. In addition, recent data support a role for claudin-1 (a transmembrane component of the TJ with structural similarities to occludin) in a postattachment event associated with the entry of hepatitis C virus (HCV) (Evans et al., 2007
). Claudin-1 is not known to bind HCV, and HCV infection may also require interaction of the virus with two coreceptors—the tetraspanin CD81 (Pileri et al., 1998
) and the scavenger receptor class B member I (Scarselli et al., 2002
)—neither of which is sufficient to promote infection. It is therefore conceivable that claudin-1 and occludin facilitate virus internalization by similar mechanisms. Defining the mechanism of CVB entry is likely to provide insights into the physiology of tight junction regulation as well as the entry mechanisms of other viral pathogens.