Many of the most widespread and devastating diseases in humans and livestock are caused by viruses and bacteria that enter cells for replication. Being obligate intracellular parasites, viruses have no choice. They must transport their genome to the cytosol or nucleus of infected cells to multiply and generate progeny. Bacteria and eukaryotic parasites do have other options; most of them can replicate on their own.
However, some have evolved to take advantage of the protected environment in the cytosol or in cytoplasmic vacuoles of animal cells as a niche favorable for growth and multiplication. In both cases (viruses and intracellular bacteria), the outcome is often destructive for the host cell and host organism. The mortality and morbidity caused by infectious diseases worldwide provide a strong rationale for research into pathogen–host cell interactions and for pursuing the detailed mechanisms of transmission and dissemination.
The study of viruses and bacteria can, moreover, provide invaluable insights into fundamental aspects of cell biology. Here, we focus on the mechanisms by which viral and bacterial pathogens exploit the endocytosis machinery for host cell entry and replication. Among recent reviews on this topic, dedicated uniquely to either mammalian viruses or bacterial pathogens, we recommend the following: Cossart and Sansonetti (2004); Pizarro-Cerda and Cossart (2006); Kumar and Valdivia (2009); Cossart and Roy (2010); Mercer et al. (2010b); Grove and Marsh (2011); Kubo et al. (2012); Vazquez-Calvo et al. (2012a); Sun et al. (2013).
The term “endocytosis” is used herein in its widest sense, that is, to cover all processes whereby fluid, solutes, ligands, and components of the plasma membrane as well as particles (including pathogenic agents) are internalized by cells through the invagination of the plasma membrane and the scission of membrane vesicles or vacuoles.This differs from current practice in the bacterial pathogenesis field, where the term “endocytosis” is generally reserved for the internalization of molecules or small objects, whereas the uptake of bacteria into nonprofessional phagocytes is called “internalization” or “bacterial-induced phagocytosis.”
In addition, the term “phagocytosis” is reserved for internalization of bacteria by professional phagocytes (macrophages, polymorphonuclear leucocytes, dendritic cells, and amoebae), a process that generally but not always leads to the destruction of the ingested bacteria (Swanson et al. 1999; May and Machesky 2001; Henry et al. 2004; Zhang et al. 2010).
With a few exceptions, we will not discuss phagocytosis of bacteria or the endocytosis of protozoan parasites such asToxoplasma and Plasmodium (Robibaro et al. 2001).
VIRUSES
Viruses are lifeless particles lacking metabolism and means of locomotion. Their mission (raison d’eˆtre) is to serve as carriers for their own genome during cell-to-cell spread and organism-to-organism transmission. They protect the genome (RNA or DNA) during transit, and deliver it into the cytosol or the nucleus of new host cells usually together with accessory proteins.
Although often spherical, mammalian viruses can be bullet-shaped, brick-shaped, amorphous, or filamentous ranging in diameter from 18 to 2000 nm. In enveloped animal viruses, the membrane is a de facto transport vesicle. It is formed by budding (fission) from membranes in the infected cell. It releases its cargo (a nucleocapsid and accessory proteins) by fusing with a membrane in the new host cell.
In this way, there is no need for macromolecule complexes to ever cross a membrane. To overcome the membrane barriers, nonenveloped viruses induce membrane lysis, generate pores, or rely on membranecrossing devices provided by the cell. The majority of nonenveloped and enveloped viruses depend on endocytosis for entry. This means that the penetration reaction—whether by fusion or other mechanisms—occurs in intracellular organelles and involves intracellular membranes of the host cell.
BACTERIA
Although the simplest among living organisms, bacteria are considerably more complex than viruses. They are single-celled and have different shapes (spherical, spiral, or rod shaped) and appear singly or in chains. A typical bacterium is 1–5 mm in length. It has a cell membrane and a rigid cell wall, and it lacks a nucleus. Some have an additional outer membrane, and some have appendages such as flagella that they use to move in the environment or pili that they use for adhesion to inert surfaces, host cells, and even to other bacteria. In contrast to viruses, bacteria can secrete proteins including toxins via a variety of secretion systems, classified from I to VII depending on the structure and organization of the secretion machinery.
Highly relevant for this review are the type III and the type IV secretion systems, which appear as nanomachines on the bacterial surface (Backert and Meyer 2006; Thanassi et al. 2012). They are dedicated to the transport of effector proteins directly from the interior of the bacteria into the interior of the host eukaryotic cell. These effector proteins are either enzymes that use host components as substrates or proteins that structurally or functionally mimic eukaryotic proteins and thus interfere with cellular mechanisms (Galan and Wolf-Watz 2006; Agbor and McCormick 2011).
Many pathogenic bacteria multiply extracellularly, but it is increasingly recognized that many bacteria that were long considered extracellular can also reside, replicate, or persist inside cells (Pizarro-Cerda and Cossart 2006). Depending on the type of host cells (phagocytic or nonphagocytic) with which bacteria interact, there are two possible scenarios. In phagocytic cells such as macrophages, cells are the active players in the internalization process; they engulf bacteria and internalize them. In contrast, in nonphagocytic cells, only a few bacterial species—qualified as “invasive”—enter as the result of a process that the bacteria initiate.
Some bacteria have obligate intracellular life styles like viruses; that is, they cannot replicate outside a eukaryotic cell. Generally, these bacteria have lost genes required for independent replication in broth medium and have small genomes. Nevertheless, as recently shown for Coxiella burnetii, the use of complex media may provide nutrients for extracellular replication of pathogens up to now considered as obligate intracellular pathogens (Omsland et al. 2009).
ENDOCYTOSIS OF VIRUSES AND BACTERIA: THE GENERAL PICTURE
There are many reasons why incoming pathogens enter host cells by endocytosis for replication. In the case of bacteria, entry into cells and replication therein are believed to protect them from circulating antibodies and complementinduced destruction. Yet, inside cells, pathogens have to confront a variety of cellular defense mechanisms. They have evolved numerous sophisticated mechanisms to counteract these bactericidal processes. Specifically, they may secrete proteins or components that either allow modification of the internalization vacuole to permit an intravacuolar lifestyle with concomitant replication or trigger escape from the vacuole (Kumar and Valdivia 2009).
Of the bacteria that escape from vacuoles, some are able to recruit actin and move intra- and intercellularly (Gouin et al. 2005). Others simply multiply without further movement within the nutrient-rich environment of the cytosol. In this case, cell lysis leads to pathogen dissemination. It is to be noted that for intracytosolic bacteria, entry into the cytosol involves lysis of intracellular vacuoles. This is likely to be less damaging for the cell than if it occurred directly through the plasma membrane.
For viruses, endocytic vesicles help to ferry the incoming particles deep into the cytoplasm unobstructed by cytoplasmic crowding and obstacles such as the cytoskeleton. In the process of intracellular maturation of endocytic vacuoles, the host cell exposes the viruses to changing conditions including a drop in pH that many viruses use as a cue to activate penetration (Helenius et al. 1980; Vazquez-Calvo et al. 2012a).
Exposure to proteases and processing of viral proteins is critical for some viruses because it allows activation of viral penetration and uncoating mechanisms (Danthi et al. 2010; Hunt et al. 2012; Kubo et al. 2012). By fusing their envelope with membranes of internal organelles, enveloped viruses can, moreover, avoid exposing their glycoproteins on the cell surface, and can thus presumably delay detection by immune surveillance. Some enveloped viruses belonging to the retro-, paramyxo-, pox-, and herpesviruses can, however, release their capsids into the cytosol by fusing their envelope membrane with the plasma membrane.
In most cases, it is not clear whether such fusion results in infection because virus particles are also being endocytosed in the same cells, and these could be the ones causing infection.
Unlike many of the invasive bacteria, incoming viruses cannot manipulate the endocytic machinery by prior delivery of effector proteins into the cytosol of host cells. To induce endocytosis and prepare the host cell for invasion, they make use of the cell’s signaling and regulatory pathways, but they do so indirectly from the plasma membrane.
VIRUS BINDING TO HOST CELLS
Endocytic entry of viruses occurs in a stepwise manner involving attachment to the cell surface, clustering of receptors, activation of signaling pathways, formation of endocytic vesicles and vacuoles, delivery of viral cargo to endosomal compartments, sorting, and escape into the cytosol. Depending on the virus, capsid escape occurs from early endosomes, late endosomes, lysosomes, macropinosomes, or the endoplasmic reticulum (ER) (Fig. 1; Table 1).
Figure 1. Viruses in the endocytic network. After binding to cell-surface receptors, viruses are internalized through a variety of endocytic processes including macropinocytosis, clathrin-mediated endocytosis, caveolae, and clathrin- and caveolin-independent mechanisms. Some of the viruses that use these mechanisms of endocytosis are listed. The primary endocytic vesicles and vacuoles formed ferry incoming virus particles into the endocytic network, where they undergo sorting and eventually penetration into the cytosol from different locations within the vacuolar network. Five different locations are indicated by the numbered gray arrows. Pathways followed by influenza A virus, SV40, Uukuniemi virus, and vaccinia virus are shown. Of these, SV40 is transported to the endoplasmic reticulum for uncoating and penetration, whereas influenza A and vaccinia virus undergo acid-activated membrane fusion in maturing and late endosomes and macropinosomes, respectively. The location and timing of escape are often determined by the pH threshold for penetration of the virus particles as indicated. LCMV, lymphocytic choriomeningitis virus.
Possible additional locations include the trans-Golgi network (TGN), the Golgi complex, recycling endosomes, or amphisomes (Suikkanen et al. 2002; Berryman et al. 2012; Day et al. 2013; Lipovsky et al. 2013).
Viruses only infect cells to which they can bind. The choice of receptors and the specificity of binding contribute to the choice of entry mechanism, to species and tissue specificity of infection, to cell tropism, and ultimately to the course of the disease. Enveloped viruses bind via spike glycoproteins, whereas nonenveloped viruses usually attach via fibers, spikes, or surface indentations. Ranging from proteoglycans to glycolipids and glycoproteins, the overall spectrum of cellular molecules identified as virus receptors is extremely broad (Helenius 2007).
Some serve merely for attachment and concentration of viruses to the cell surface, whereas others have additional roles in signaling, endocytosis, and in the modification of the bound virus. In many cases, virus–cell interactions involve carbohydrates (Vasta 2009; Kamhi et al. 2013). Although well-known endocytic receptors such as transferrin and LDL receptors occur in the list, the majority of virus receptors are not directly involved in the uptake of physiological ligands. By inducing clustering, viruses can generate a receptor-rich membrane microdomain that differs from the rest of the plasma membrane in protein and lipid composition (English and Hammer 2005; Lozach et al. 2011b). Such domains may serve as a platform for transmembrane signaling, for recruitment of cytoplasmic coats, and for assembly of endocytic machinery. In some instances, antibodies, complement factors, and other proteins in body fluids can play a bridging function between the virus and its receptors (Flipse et al. 2013).
Receptors usually follow the virus into endocytic vesicles and into the cell. In addition to the virus particle, the endocytic “cargo” thus comprises a complex of receptors and lipids. Importantly, virus–receptor interactions can result in alterations in the structure of viral proteins or in the virus particle as a whole (Lonberg-Holm and Philipson 1974; Raff et al. 2013). Simultaneous binding to a mobile and an immobile receptor was, for example, recently shown to subject adenovirus particles to forces that initiate uncoating by detaching some of the viral fibers (Burckhardt et al. 2011). A receptorinduced conformational change in the glycoprotein of HIV-1 is essential for triggering penetration by membrane fusion (Wilen et al. 2012). In recent years, a new class of interaction partners on the cell surface has been recognized for viruses. These could be called “accessory factors” because they are primarily needed to activate signaling pathways and thus trigger endocytosis. They include receptor tyrosine kinases, phosphatidylserine (PS) receptors (TIM and TAM receptors), and integrins (Mercer and Helenius 2010; Morizono et al. 2011; Meertens et al. 2012).
The interaction of viruses with these can be mediated by adaptors such as Gas6, a soluble protein that bridges between PS in the viral membrane and receptor tyrosine kinase Axl during activation of virus uptake by macropinocytosis (Morizono et al. 2011; Meertens et al. 2012). The physiological role of Gas6 is to serve as a soluble adaptor protein between PS in the membrane of cell remnants after apoptosis and Axl on the surface of cells (Lemke and Burstyn-Cohen 2010). It is often observed that viruses use more than one type of receptor either in parallel, in sequence, or when interacting with different cell types. Rotaviruses, hepatitis C virus, HIV-1, and coxackie B virus are examples of viruses that interact sequentially with different cell-surface molecules (Coyne and Bergelson 2006; Lopez and Arias 2006). Other viruses such as many herpesviruses carry multiple receptor-binding glycoproteins with different specificity and can therefore enter different cell types (Eisenberg et al. 2012). That viruses can exploit multiple endocytic mechanisms and pathways is also clear. Herpes simplex virus I uses different pathways in different cell types (Campadelli-Fiume et al. 2012). Influenza Aviruses can, for example, use two or three different pathways (Matlin et al. 1981; Sieczkarski and Whittaker 2002; Lakadamyali et al. 2006; de Vries et al. 2011). Considering the underlying redundancy and complexity in virus–cell interactions, it is not surprising that the literature on the entry of many viruses is inconsistent and confusing.
BACTERIAL BINDING TO HOST CELLS
Endocytosis of bacteria starts by an irreversible interaction. Two main mechanisms may take place. In the first type, the adhesion between a bacterial ligand and a specific host surface receptor causes receptor clustering, the zippering of the plasma membrane around the bacterium, and the initiation of signaling events that culminate in entry. It is often difficult to separate the steps of adhesion and endocytosis during entry by this “zipper mechanism.”
In the second mechanism of bacterial endocytosis, the initial contact is a relatively transient event that essentially permits the direct delivery into the cell cytosol of active T3SS effectors that trigger cytoskeleton rearrangements and membrane remodeling, which lead to a macropinocytosislike event. This second way of entry is called “the trigger mechanism” (Fig 2; Table 2) (Cossart and Sansonetti 2004).
Figure 2. Pathways of endocytic entry by invasive bacteria. Schematic representation of bacterial endocytosis mechanisms and intravacuolar or intracytosolic lifestyles. In epithelial cells, Listeria enters via two internalins; it then resides transiently in a vacuole that is lysed allowing intracytosolic replication and actin-based motility. The obligate intracellular bacterium Chlamydia trachomatis resides in a vacuole that intercepts the pathway involved in the transport of shingomyelin from the Golgi apparatus to the plasma membrane. Shigella is first taken up by filopodia present on the surface of epithelial cells. The type 3SS then injects effectors that trigger entry, formation of the vacuole, and escape from this compartment, leading to intracytosolic replication and actin-based motility. Salmonella appears to enter after a “near-surface swimming” mechanism. The T3SS-1 (or SPI-1) triggers entry and formation of the replicative vacuole, which acquires markers of endosomes and lysosomes owing to the secretion of effectors of the T3SS-2 (or SPI-2). In some circumstances, Salmonella can reach the cytosol and rapidly replicate therein. The Brucella replicative vacuole is derived from the endocytic vacuole and matures in an ER-derived vacuole. Coxiella burnetii is the only bacterium that has evolved to survive and replicate in a lysosome-derived vacuole. Bartonella henselae can enter into endothelial cells as a single bacterium or as a group, leading to the formation of an invasome. In macrophages, Legionella and Mycobacterium tuberculosis reside in a vacuole. The Legionella vacuole acquires markers of the ER. It is not the case for M. tuberculosis, which blocks the maturation of the internalization vacuole.
Binding to cells occurs via proteins called “adhesins” located either on the bacterial surface or at the tip of appendages called “pili” or “fimbriae,” which form hair-like fibers that protrude from the surface. Because pili can be used for the transfer of genetic material during conjugation, the term “fimbriae” has in the past been used to describe those pili whose specific function is adhesion. However, the nomenclature remains fuzzy.
Interestingly, some pili (e.g., type IV pili) not only mediate bacteria–host cell or bacteria–bacteria adhesion but also perform complex functions such as force-driven contraction providing bacteria with powerful tools to enhance their contact with target surfaces.
One of the best-characterized fimbriae is the pyelonephritis-associated (P) pilus, expressed by uropathogenic Escherichia coli that colonize the urinary tract and then infect the kidney. P pili bind through the PapG adhesin, located at the tip of the complex pilus rod, to the aD-galactopyranosyl(1-4)-b-D-galactopyranoside moiety of glycolipids of upper urinary tract cells (Waksman and Hultgren 2009).
In addition, the pili mediate the generation of biofilm-like structures on and inside host cells, contributing to the persistence of the bacteria in the bladder. Type IV pili are essentially composed of a homopolymer of a single pilin subunit such as pilA in Pseudomonas aeruginosa, PilE in Neisseria spp., or TcpA in Vibrio cholerae (Craig et al. 2004). As stated above, type IV pili have been implicated in other functions including biofilm formation. Interestingly, a posttranslational modification of the Neisseria meningitidis pilin subunit has been shown to change the conformation of the pilin and lead to loss of interbacterial interactions at sites of adhesion leading to Neisseria dissemination through epithelial cells and bacterial access to blood vessels (ChamotRooke et al. 2011).
Recently a novel type of pilus made of a single type of pilin subunit—polymerized via sortases that covalently link the subunits together—was identified in clinically important pathogens such as Streptococci and Pneumococci and shown to be critical for infection paving the way to new vaccine strategies (Ton-That et al. 2004; Dramsi et al. 2006; Falker et al. 2008). A variety of nonpolymeric adhesins have been reported that mediate bacterial adhesion to host surface components, in particular, to host membrane adhesion receptors such as integrins, cadherins, selectins, and CEACAMs. In the case of invasive bacteria, these adhesins often serve as invasion proteins, that is, proteins that mediate the endocytosis process itself (Fig. 2).
It is the case for Internalin (InlA) of Listeria monocytogenes (Gaillard et al. 1991) or invasin of Yersinia pseudotuberculosis (Isberg et al. 1987) (see below). A unique situation has been described in the case of enteropathogenic E. coli. These bacteria inject a type III effector called Tir into mammalian cells. Tir inserts in the plasma membrane and acts as a receptor for the bacterial protein intimin (Rosenshine et al. 1996; Kenny et al. 1997). Tir also mediates the recruitment of several proteins including clathrin, dynamin, and Dab2 before the recruitment of components of the actin cytoskeleton and, in particular, the Arp2/3 complex, which mediates the polymerization of actin filaments (Unsworth et al. 2007; Veiga et al. 2007; Bonazzi et al. 2011). The result is the formation of a pedestal on which bacteria are located and tightly adhere. Hence, tight adherence in the presence of typical endocytic molecules such as clathrin or dynamin does not necessarily lead to endocytosis.
ENDOCYTOSIS OF VIRUSES
The rate and efficiency of viral endocytosis are variable. After binding to the cell surface, internalization can occur with a half-time as short as a few minutes or as long as several hours. Uptake is usually nonsynchronous, but in the end quite efficient. Viruses exploit the capacities of the host cell by making use of different cellular endocytosis mechanisms, which they can activate and modify for their specific purposes when needed (Fig. 1). Although the dependence of viruses on cellular processes in all stages of entry and uncoating makes possible the stripped-down economy in the structure of viral particles, it also means that viruses and their components must interact directly or indirectly with a multitude of cellular factors.
Clathrin-Mediated Endocytosis
Clathrin-mediated endocytosis (CME) is probably the most common mechanism for endocytosis of small and medium-size viruses (Figs. 1 and 3; Table 1) (Dales 1978; Helenius et al. 1980; Matlin et al. 1981; DeTulleo and Kirchhausen 1998; Sun et al. 2013). Time-lapse movies show two general behaviors. After a period of lateral movement along the membrane, some particles diffuse into preexisting clathrin-coated areas, and others induce clathrin assembly at the site of binding (Ehrlich et al. 2004; Rust et al. 2004; van der Schaar et al. 2008; Johannsdottir et al. 2009). In the latter case, the assembly of a clathrin coat takes somewhat longer than for clathrin pit formation during uptake of endogenous protein ligands (1–6 min). The coated vesicles pinch off with one or more viruses. The coat dissociates within 5–20 sec, and the viruses are delivered within minutes to early endosomes in the periphery of the cell. Like CME of physiological cargo, uptake of viruses depends on PI(4,5)P2 and dynamin-2 and is usually inhibited by chlorpromazine, an inhibitor of CME (Wang et al. 1993; Sieczkarski and Whittaker 2002). Vesicular stomatitis virus (VSV) belongs to the viruses that use CME for productive infection (Matlin et al. 1982). In human cells, the receptor for VSV was recently identified as the LDL receptor and its family members (Finkelshtein et al. 2013). Light microscopy in live cells combined with single-particle tracking shows that the clathrin coat is recruited to sites of VSV binding (Cureton et al. 2009; Johannsdottir et al. 2009). Because of the bullet shape and large size of VSV particles, the clathrin vesicles form slowly, they deviate from the round shape, and some carry only a partial clathrin coat (Cureton et al. 2009). Although VSV endocytosis is dependent on plasma membrane PI(4,5)P2, dynamin, and actin, it is unclear whether AP2 is the adaptor protein required for endocytosis and infection (Cureton et al. 2009; Johannsdottir et al. 2009; Vazquez-Calvo et al. 2012b). A general lesson from the observations with VSV and other viruses is that although the viruses differ in size, they have evolved to use CME very efficiently. They can induce the formation of clathrin-coated vesicles (CCVs) locally, and they can adapt the size and shape of the clathrincoated pits (CCPs) for their needs. It will be interesting to determine how VSV and other viruses guide and regulate the coat assembly process.
Clathrin- and Caveolin-Independent Endocytosis
The existence of a clathrin-, caveolin-, and dynamin-independent pathway for virus endocytosis was first described for members of the polyomaviruses: mouse polyoma virus, and SV40 (Gilbert and Benjamin 2000; Damm et al. 2005). These are small nonenveloped DNA viruses (diameter 50 nm) that replicate in the nucleus of host cells. The VP1 proteins that form the icosahedral viral coat bind to the sialic-acid-containing carbohydrate moiety of specific gangliosides that serve as cell-surface receptors (Tsai et al. 2003). Present as 72 homopentamers, VP1 constitutes the main building block in the capsid shell (Stehle et al. 1996). By binding to multiple GM1 molecules, SV40 actively generates membrane curvature (Ewers et al. 2010). In a process that in EM sections looks as if the virus particle would be budding into the cell, the PM wraps itself tightly around the virus particle (Fig. 4F). The same can be seen when viruses are added to giant unilamellar liposomes containing GM1 with long acyl chains. When many viruses are present, long, narrow, tight-fitting, tubular invaginations with multiple viruses can form in the PM of cells and in liposomes. Interestingly, pit and tube formation does not require the whole virus; isolated VP1 homopentamers are sufficient. For the fission reaction that detaches the virus-containing indentations, the virus alone is not sufficient. Energy in the form ATP is required, as well as cellular tyrosine kinases, cholesterol, and a change in actin dynamics. Contrary to CCVs, vesicle fission is independent of dynamin (Damm et al. 2005). The viruscontaining vesicles formed are delivered to early endosomes, and the virus is eventually transported via late endosomes to the ER (Fig. 1) (Qian et al. 2009; Engel et al. 2011). It is noteworthy that a similar mechanism is used by pentameric, bacterial toxins (cholera and Shiga) that bind to glycolipids (Ro¨mer et al. 2010). In all these cases, the receptors are glycosphingolipid molecules concentrated in lipid rafts. They differ from most other receptors in that they do not span the bilayer.
Caveolar Endocytosis
Caveolar endocytosis has been proposed for a variety of viruses. One of the problems in assigning viruses to this pathway is that the endocytic role of caveolae in cell life is still poorly defined. No single characteristic such as cholesterol dependence alone is sufficient as a criterion to define this pathway. Although much less dynamic than CCVs, it is clear, however, that a fraction of the caveolae is mobile and can undergo endocytosis especially when activated by a ligand (Kirkham and Parton 2005; Pelkmans and Zerial 2005; Tagawa et al. 2005). The best studied among the viral candidates for caveolar uptake is SV40. Morphological evidence from several laboratories using immune-electron and immunofluorescence microscopy shows colocalization of the viruses and caveolin-1 in plasma membrane spots and pits (Anderson et al. 1996; Stang et al. 1997; Pelkmans et al. 2001). Addition of SV40 to cells causes a dramatic, tyrosine-phosphorylationdependent elevation in caveolar vesicle formation and caveolar vesicle motility (Tagawa et al. 2005). shRNA- and siRNA-mediated depletion of caveolin-1 results in a decrease in SV40 endocytosis and infection (Pelkmans et al. 2004; Stergiou et al. 2013). Recently, it was shown that depletion of EHD2 (a peripheral caveolar ATPase) leads to a dramatic increase in caveolar vesicle trafficking and increases the efficiency of SV40 infection (Stoeber et al. 2012; M Stoeber, G Balustreri, and A Helenius, unpubl.). Thus, in summary, it seems that SV40 can make use of two parallel endocytic mechanisms. One corresponds to the caveolar pathway and relies on caveolin-1. The other is caveolin independent, with the virus particle serving as the curvature-generating principle. To what extent viruses other than SV40 can use these pathways is not clear. There are reports suggesting that other polyomaviruses such as BK virus and mouse polyoma virus may also exploit caveolae (Richterova et al. 2001; Neu et al. 2009).
Virus-Triggered Macropinocytosis
Macropinocytosis offers incoming viruses an altogether different type of endocytic experience (Fig. 1). Transiently triggered by external ligands such as growth factors and PS-containing particles, this mechanism is known to induce internalization of fluid, membrane, and whatever happens to be associated with the membrane such as viruses (Swanson and Watts 1995). The process is important for the clearing of cell remnants after apoptotic cell death. The PS exposed on cell remnants constitutes an “eatme” signal that secures efficient uptake and degradation of cellular garbage without triggering an inflammatory response (Hoffmann et al. 2001). The key events in macropinocytosis include the activation of receptor molecules such as receptor tyrosine kinases, integrins, and PS receptors (Swanson and Watts 1995). This triggers downstream signaling cascades resulting in transient, global changes in actin dynamics that lead to cell-wide plasma membrane ruffling in the form of filopodia, lamellopodia, circular ruffles, or blebs. Large, uncoated vacuoles (macropinosomes) are formed. After moving deeper into the cytoplasm, these usually end up fusing with late endosomes or lysosomes. Important distinguishing features for macropinocytosis include activation Rho GTPases (Rac1 and/or Cdc42), myosins, and kinases such as PAK1, PI3 kinase, and PKC. The process is sensitive to inhibitors of Naþ/Hþ exchangers. Because there are many variations of the process, macropinocytosis must be viewed as a collective term for an assortment of related mechanisms. Macropinocytosis plays a role in the infection of viruses of different families including large viruses such as pox-, filo-, paramyxo, and herpesviruses (Lim and Gleeson 2011; Mercer and Helenius 2012). Some smaller enveloped and nonenveloped viruses such as influenza A virus seem to use pathways that share many properties with macropinocytosis (Khan et al. 2010; de Vries et al. 2011). By exposing PS in their envelope, vaccinia and some other enveloped viruses make use of “apoptotic mimicry” (Mercer and Helenius 2008). The emerging role of PS receptors of the TIM and TAM families and adaptors such as Gas6 in this process was already discussed above. Epidermal growth factor receptor (EGFR) and other growth factor receptors are often activated and essential as accessory factors in infectivity (Eierhoff et al. 2010; Mercer et al. 2010a; Krzyzaniak et al. 2013).
Other Endocytic Pathways for Virus Internalization
There are viruses for which endocytic entry does not fall into the categories listed above (Table 1). One of them is human papilloma virus 16 (HPV-16), which in HeLa and HaCaT cells uses a mechanism with similarities to macropinocytosis (Schelhaas et al. 2008). However, the vesicles are small, Rho GTPases are not activated, and there is no elevation in fluid uptake. Some of these characteristics are shared by human rhinovirus 14 and one of the alternate pathways described for influenza A virus (Matlin et al. 1981; Rust et al. 2004; Khan et al. 2010; de Vries et al. 2011). In addition, two Old World arenaviruses, LCMV and Lassa virus, enter by a clathrin-, caveolin-, and dynamin-independent pathway via multivesicular bodies bypassing early endosomes (Quirin et al. 2008; Kunz 2009). In the case of Acanthamoeba polyphaga mimivirus, a giant enveloped virus (750 nm in diameter) that uses amoeba as a host, uptake into human macrophages seems to involve a phagocytosis-like mechanism (Ghigo et al. 2008). This uptake process resembles the phagocytosis of bacteria. Of the various endocytic mechanisms described in mammalian cells, the CLIC/GEEC, IL2, flotillin, and CIE/Arf 6 pathways do not seem to support virus entry (Lamaze et al. 2001; Mayor and Pagano 2007; Donaldson et al. 2009; see also Mayor et al. 2014). However, because virus entry studies and the classification of endocytic mechanisms are not always straightforward, the situation may change. Viruses may, in fact, provide one of the most informative systems for further classification of endocytic processes.
ENDOCYTOSIS OF BACTERIA
The rate and efficiency of entry of bacteria into cells vary greatly with the cell type and other parameters such as the temperature or the growth phase at which bacteria have been harvested (Fig. 5).