Clathrin-mediated endocytosis is the most widely studied endocytic pathway and is used by several viruses for entry into target cells [37,38,39]. Viral entry via clathrin-mediated endocytosis involves the localization of viruses in clathrin-coated pits, which invaginate and are pinched off by dynamin. The pits then form endocytic vesicles, which fuse with early endosomes and travel further into the cytoplasm. Clathrin dissociates from these vesicles and remains in the cytoplasm until it is recruited to line newly formed pits.

Several cellular factors participate in and regulate various steps of the clathrin-mediated endocytic pathway. These include the coat protein clathrin, which assembles into a polyhedral lattice on the inner surface of the plasma membrane to form the coated pit. The PICALM protein promotes the assembly of clathrin triskelia into cages [40]. The HIP1 protein localizes with clathrin at the plasma membrane and is involved in the formation of the coated vesicle [41,42]. HIP1 can also bind to the adaptor protein AP-2 [43] (see below). LDLRAP1, which interacts with the cytoplasmic tail of the low density lipoprotein (LDL) receptor, can bind to both clathrin and AP-2 [44].

The adaptor proteins involved in the clathrin pathway initiate vesicle formation by bringing cargo molecules to the clathrin coat [45,46]. These include AP-2, which links the clathrin lattice to the cell membrane [47]; β-arrestins, which can bind to clathrin directly [48]; DAB2, which can sort the LDL receptor independently of AP-2 and LDLRAP1 [49]; and Eps15, which constitutively associates with AP-2 during clathrin-mediated endocytosis [37]. Interestingly, anthrax toxin is known to enter cells via an Eps15-, AP-2- and DAB2-independent clathrin pathway that requires AP-1 and β-arrestin [40].

In addition to Eps15, several other proteins are known to possess Eps homology (EH) domains such as Epsin 1, Intersectin 1 (ITSN1), REPS1 and REPS2 [50,51,52]. Epsin 1 is involved in clathrin‑mediated endocytosis of influenza A virus [53], whose entry was previously reported to be AP-2 independent [54]. Epsin 1 binds to both ITSN1 [55] and Eps15 [53] through the same EH‑domain binding sequence and therefore, simultaneous binding of Eps15 and ITSN1 is unlikely, suggesting that Epsin 1 and ITSN1 may act as alternate adaptors to substitute for the respective functions of AP-2 and Eps15 in the clathrin pathway. The EH domains of ITSN bind to epsin [55], while the Src homology 3 (SH3) domains bind to dynamin and synaptojanin [56]. Another adaptor protein NUMB, can bind to the α-adaptin subunit of AP-2 [57] and Eps15 [58].

Some of the regulatory proteins involved in the clathrin pathway include INPPL1, which recruits ITSN1 to clathrin-coated pits on the plasma membrane [59]. RalBP1/RLIP76, a Rac/Cdc42 guanine‑nucleotide activating protein, targets the AP-2 complex and regulates endocytosis [60]. REPS1 and REPS2 proteins associate with RalBP1 and act as molecular switches to coordinate the actions of the RalBP1-interacting Ral-GTPases [61]. Also, REPS1 forms complexes with two adaptor proteins Crk and Grb2 [61], while REPS2 can bind directly to epsin through its EH domain [62].

The scission of the clathrin-coated pits is carried out by the GTPase dynamin 2 (DYN-2), which assembles at the neck of the coated pits and functions as a constrictase to pinch off the pits [63]. BAR domain proteins such as amphiphysin and SNX9 are recruited to the sites of clathrin assembly where they induce membrane curvature, interact with actin and synaptojanin, and promote the recruitment of dynamin [64,65].

Taken together, these studies demonstrate that numerous cellular factors with overlapping functions participate in and regulate the clathrin pathway. Given the complexity of this pathway and the variations in the composition of individual clathrin pits, it would be interesting to examine how different viruses dictate the recruitment of specific adaptors and regulators to a clathrin pit.

Caveolae-mediated endocytosis involves the formation of small flask-shaped invaginations of the plasma membrane [66]. Unlike the clathrin pathway, caveolae-mediated endocytosis is usually not a constitutive process and predominantly occurs upon cell stimulation [67,68].

Caveolae are a specialized form of lipid rafts primarily composed of cholesterol and sphingolipids [69]. The shape and structure of caveolae is determined by caveolin, a protein that binds cholesterol and self associates to form a striated coat on the surface of the invaginations [70]. The cavin proteins are known to regulate caveolae structure [71]. PTRF-cavin binds to caveolin and stabilizes the membrane curvature to produce the characteristic flask shape of caveolae [69].

Several regulatory factors are known to be involved in various steps of the caveolae pathway. These include Cdc42 and RhoA, which facilitate caveolae formation by promoting actin polymerization and binding to caveolin 1 [72]; tyrosine kinases and phosphatases, which trigger downstream signaling pathways after cargo binding; integrins, which regulate trafficking of caveolae; and protein kinase C (PKC), which stimulates uptake by caveolae [73,74].

Dynamin can directly bind to caveolin [75] and is suggested to act as a scission factor by triggering fission of the caveolae [76]. Intersectin 2 localizes at the neck of the caveolae and regulates the activity of dynamin. An adaptor protein, NOSTRIN, recruits dynamin to the caveolae [69].

Phosphorylation of caveolin 1 by Src leads to caveolar internalization. The cytoskeletal components associated with the caveolae pathway include actin, filamin and microtubules, which facilitate formation as well as internalization of caveolae [69,74].

Upon internalization, caveolae form characteristic grape-like, multi-caveolar complexes of heterogeneous morphology known as caveosomes. Rab 5 regulates the fusion of caveosomes with early endosomes. The endosomal route taken by different ligands internalized by the caveolae pathway is regulated by various Rabs, kinases and phosphatases [69].

Viruses such as SV40 enter through caveolae [77] which are approximately 60 nm in size [66]. Since filoviruses have also been reported to enter via caveolae-mediated endocytosis [78,79], it is conceivable that caveolae can adapt to fit the size of their cargo.

Several sterol-binding chemicals such as methyl-β-cyclodextrin and filipin can block caveolae‑mediated endocytosis [80,81] but their inhibitory effects are not restricted to the caveolar pathway alone [82].

Macropinocytosis is a transient actin-dependent endocytic process that is typically employed for cellular uptake of fluids and large solutes via large (0.5–10 µm diameter) irregular-shaped vacuoles or macropinosomes.

Macropinocytosis is generally initiated by external stimuli (growth factor-mediated) resulting in the formation of actin-driven cellular protrusions called membrane ruffles that can fuse to form macropinosomes. Rho family GTPases (Rac1, Cdc42) and p21-activated kinase (Pak1) are known important mediators of ruffle formation in addition to Na⁺ influx and H⁺ efflux. Membrane ruffles also contain several regulators of actin polymerization, disassembly, stabilization and cytoskeletal membrane attachment such as Arp2/3, VASP, WAVE, PKC and several classes of myosins, all of which are known to play a role in ruffle extension and macropinosome formation [83,84].

Several adaptors and regulators have been reported to be involved in macropinocytosis. These include the adaptor complex-1 (AP-1), which is required for macropinosome formation [85], Abi1 [86], TBC1D3 [87], c-Cbl [88] and NHE1 which is required to achieve the necessary H⁺ concentration to promote actin polymerization during macropinocytosis [89]. Other cellular factors involved in this pathway include epidermal growth factor (EGF) receptor, phosphatidylinositol (PI) 3‑kinase (PI3K), Phospholipase C (PLC), the ARF-family of GTPases, and CtBP1 (carboxyl-terminus binding protein 1), which is associated with macropinosome closure [83].

Several viruses are known to enter via macropinocytosis and distinct cellular factors have been shown to be capable of inducing macropinocytosis of these virus particles. For example, human adenovirus serotype 3 requires alpha v integrins [90], vaccinia virus uses phosphatidyl serine exposed on its surface to induce its uptake via macropinocytosis [91,92], Kaposi's sarcoma-associated herpesvirus requires the adaptor protein c-Cbl and myosin IIA [88,93], coxsackievirus requires occludin and Rab34 [94] and Nipah virus induces macropinocytosis via a signaling cascade involving Rac1 and Cdc42 [95].

Several chemical agents such as dimethyl amiloride [96], cytochalasin D and PI3K inhibitors [97] have been shown to block macropinocytosis but they are not specific inhibitors of this pathway [82].

Phagocytosis is a receptor-mediated form of endocytosis that includes a number of closely related yet distinct mechanisms. It is carried out by specialized cells such as neutrophils, monocytes and macrophages and is typically used by cells to clear large pathogens and debris. Like macropinocytosis, phagocytosis is also associated with actin-dependence, large vacuole size and cellular factors such as RhoA, Cdc42, Rac-1 and PI3K. However, unlike macropinocytosis, phagocytosis involves cargo‑specific receptor interactions resulting in a signaling cascade that triggers cytoskeletal rearrangements. This causes formation of cell surface extensions that specifically zipper up around the cargo and form a cargo-sized vacuole called the phagosome [66]. Dynamin-2 has been reported to be required for the closure of phagosomes whereas macropinosome closure is associated with CtBP1 [98].

Several adaptor proteins are involved in phagocytosis, which include Syk, Grb2, Gab2, and CrkII. Lipids are also actively involved in phagocytosis and anionic phospholipids such as phosphatidyl serine and phosphoinositides are known to make the inner leaflet of the plasma membrane negatively charged during the early steps of this process [99]. Other cellular factors that are recruited to phagosomes include PI3K, Phospholipase D, IQGAP1, amphiphysin1 and adhesion proteins [74].

Herpes simplex virus 1 [100] and foot-and-mouth disease virus [79] are known to enter cells via phagocytosis. PI3K inhibitors [97] and filamentous actin depolymerizing agents can block phagocytosis but their inhibitory effects are not restricted to this pathway [82].

Several clathrin- and caveolae-independent endocytic pathways involving internalization of ligands in non-coated vesicles have been reported, including two distinct pathways of lipid transport to the Golgi apparatus [101]. The IL-2 receptor is suggested to be a marker for these pathways [102], whose entry requires Rac1, Paks, and cortactin [103].

Each of these clathrin- and caveolae-independent pathways uses distinct cellular factors for internalization. Feline infectious peritonitis virus was shown to enter via a dynamin-dependent pathway [104]. Dynamin is also implicated in the entry of coxsackievirus A9 through a pathway that requires β2-microglobulin and Arf6 [105]. By contrast, lymphocytic choriomeningitis virus (LCMV) entry requires cholesterol but is independent of dynamin, Arf6 and actin [106,107]. Similarly, human papillomavirus type 16 entry is also independent of dynamin and Rho GTPases but requires tetraspanin-enriched microdomains, PI3K, PKC, and actin [108]

In addition to these clathrin- and caveolae-independent pathways that are known to be utilized by different viruses for entry, several other pathways exist that are not currently associated with viral entry. These include the GEEC pathway involved in endocytosis of GPI-anchored proteins [109], the flotillin-1-dependent pathway utilized by GPI-anchored proteins and proteoglycans [110], the Arf6‑dependent pathway used by MHC antigens [111] and the IL-2 pathway, which is utilized for internalization of cytokine receptors [74].

Thus, there are many distinct clathrin- and caveolae-independent pathways that have been partially characterized, and perhaps several more that are yet to be uncovered. It would be interesting to explore if any of these additional pathways are utilized by viruses for entry.

The average size of a clathrin-coated pit is approximately 120 nm. However Listeria monocytogenes, which is usually 2 μm in length, has been shown to enter target cells using the clathrin pathway [120]. More recently, Cureton and co-workers showed that bullet-shaped VSV particles (~200 nm in length) are internalized through vesicles containing partial clathrin coats, and whose formation requires actin polymerization in a cargo size-dependent manner [121,122]. These findings led to the premise that clathrin-coated pits adapt to accommodate the size of their cargo and could therefore play a role in filovirus entry.

A study with chemical inhibitors proposed that wild type filoviruses could enter Vero cells by clathrin as well as caveolar pathways [123]. However, chemical agents such as chlorpromazine and sucrose, which are known to prevent recycling of clathrin to the plasma membrane [124], do not specifically block the clathrin pathway and can also inhibit other endocytic pathways [82]. Therefore, data obtained using chemical inhibitors must be substantiated with other approaches to establish the specificity of target inhibition.

Using multiple approaches to inhibit several cellular factors involved in the clathrin pathway, Bhattacharyya and co-workers showed that retrovirus pseudotypes containing EBOV GP utilized clathrin-mediated endocytosis to enter several cell lines, including human endothelial cells [117]. A more exhaustive follow-up study revealed a differential requirement for several key cellular components of the clathrin pathway in cell entry by retrovirus pseudotypes bearing MARV GP versus EBOV GP. Importantly, EBOV GP-mediated entry required Eps15, AP-2 and DAB2, whereas MARV GP-mediated entry was independent of these cellular factors and instead requires the adaptor protein β‑arrestin-1 [116] Subsequent studies have also corroborated the involvement of clathrin-mediated endocytosis in EBOV entry in a human glioblastoma cell line using feline immunodeficiency virus pseudotyped with EBOV GP [79], and in HeLa cells using EBOV GP VLPs [118].

The capacity of different types of particles containing filovirus glycoproteins to use clathrin‑mediated endocytosis for cell entry raises the possibility that the filamentous morphology of native filovirus virions is not a critical determinant of the route of viral internalization. A similar case has been made for filovirus entry via macropinocytosis (see below) [18,119]. Therefore, a systematic examination of the relationship between filovirus particle size and the requirement for clathrin‑mediated endocytosis, analogous to the studies with VSV, is warranted.

Two studies used the chemical inhibitor EIPA, co-localization with fluid phase markers (high‑molecular-weight dextrans), and dominant-negative Pak-1 (known to inhibit macropinocytosis), to show that EBOV entered Vero cells and HEK-293T cells via a pathway resembling macropinocytosis [17,18]. These studies also showed that inhibition of clathrin, caveolin and DYN-2 did not affect entry of EBOV and morphologically-similar VLPs. Additionally, CtBP1 was shown to be important in this process, presumably as a macropinosome closure factor, although its precise role remains to be determined [17]. Hunt and coworkers [79] showed that infectious EBOV, VLPs and pseudotyped viruses bearing EBOV or MARV GP could use multiple endocytic routes including pathways dependent upon clathrin and resembling macropinocytosis to enter a human glioblastoma cell line and primary human foreskin fibroblasts, Similar results were shown using VLPs in Vero and HeLa cells [118]. Consistent with these findings, Mulherkar and coworkers reported that EBOV enters human peripheral monocyte-derived macrophages and Vero cells through a macropinocytosis-like pathway [119]. Surprisingly, DYN-2 (generally considered dispensable for macropinocytosis [98]) was also found to play an important role in EBOV entry, an observation at odds with two previous reports [17,18]. The basis of these apparent differences in DYN-2 utilization during macropinocytic uptake of viral particles observed among studies using similar viral reagents and cell lines remains unclear and is worthy of further investigation.

The large size and distinctive filamentous morphology of filovirus particles may provide one explanation for why they exploit macropinocytosis-like pathways to enter cells. However, filovirus uptake through macropinocytosis was shown to depend on interactions between components of the viral envelope and cell surface molecules, and not on viral size/morphology per se [18,119]. Recent work suggests that direct interactions between phosphatidylserine (PtdSer) in the outer leaflet of the viral membrane and members of two classes of cell-surface receptors can mediate the macropinocytic uptake of multiple enveloped viruses in tissue culture [125,126]. Specifically, members of the TAM receptor tyrosine kinase family (e.g., Axl, Tyro3) enhance viral uptake and entry via their PtdSer‑binding ligands Gas6 and Protein S, which bridge the virus particle and TAM [125]. PtdSer‑binding members of the TIM family are proposed to enhance viral entry by directly binding to the viral envelope and inducing particle uptake [126]. Both TAM and TIM proteins enhance filovirus entry [127,128,129], and the former do so by stimulating macropinocytic uptake of virus particles [79]. Also using multiple cell lines and primary cells, Axl was shown to facilitate endosomal uptake and membrane fusion of EBOV in a cell type-specific manner. Importantly, Axl did not interact directly with EBOV GP [130]. Whether filoviruses exploit TAM and TIM proteins through PtdSer binding (as seems likely), or through a distinct PtdSer-independent mechanism (see below), remains to be explored.

Current evidence also points to a role for filovirus GP in inducing uptake through macropinocytosis: VSV pseudotypes bearing EBOV GP induce plasma membrane ruffling upon attachment and are internalized into cells via macropinocytosis; in contrast, VSV pseudotypes bearing VSV G are internalized via clathrin-coated vesicles, as shown previously [18,119]. The simplest hypothesis to explain this observation is that GP interacts with cell-surface receptors that induce macropinocytosis. These putative internalization receptors may include members of the TIM family. The molecular determinants within GP that trigger macropinocytic uptake remain to be defined.

The use of multiple endocytic pathways by filoviruses may influence the outcome of viral infection in vivo in several ways, as suggested previously [97,98]. First, viral replication in tissues crucial for the development of filovirus disease might require specific pathways of viral internalization that differ depending upon the cell type. Second, differential use of endocytic pathways by viral isolates, presumably determined by differences in viral interactions with cellular host factors, might contribute to strain-specific patterns of filovirus tissue tropism and virulence. Therefore, future studies dissecting the interactions between filovirus GP (and other components of the viral envelope) and key components of cellular endocytic pathways could prove insightful.