Endocytic compartments are pleiomorphic structures that fuse with one another to promote ligand trafficking. Subsequent to internalization, endosomes often undergo complex trafficking and sorting events (reviewed by Gruenberg, 2001; Mellman, 1996; Mukhergee et al., 1997). Two principal post-internalization endocytic trafficking routes exist in the cell, which can be termed recycling or lysosome-targeted (reviewed by Gruenberg, 2001). Regulation of sorting and trafficking is determined by inherent signals on the internalized receptor and by signalling events within the cell. One set of crucial regulatory molecules are the Rab family of small GTPases and their accessory proteins (Collins & Brennwald, 2000; Somsel Rodman & Wandinger-Ness, 2000) and, in addition, the action of specific protein kinases is likely to be crucial for correct endocytic trafficking. The internalized vesicle acquires properties that are defined temporally and are thus termed 'early' and 'late' endosomes. The early endosome is an often pleiomorphic tubulo-vesicular structure (Tooze & Hollinshead, 1991) and is a major sorting station where internalized cargo can be delivered back to the plasma membrane (the recycling pathway) or can progress to the late endosome (Hopkins et al., 1990). Late endosomes, comparatively, have a mostly juxtanuclear distribution, are more spherical and contain internal vesicles – leading to the term multi-vesicular bodies (Piper & Luzio, 2001). They also differ from early endosomes in that they have a significantly lower pH (approximately pH 5.5 versus pH 6.2–6.5 in the early/recycling endosome). Late endosomes subsequently progress to lysosomes, which are characterized by the presence of degradative proteases and hydrolases, delivered by communication of endosomes with the trans-Golgi network (Kornfeld & Mellman, 1989).

Viruses not only depend on the machinery of the cell for internalization but also for trafficking within the cytoplasm and the ability to find the correct site for replication. For example, fusion of Semliki Forest virus (SFV) at the plasma membrane under extracellular low-pH conditions results in the internalization of the virus but does not necessarily result in a successful infection (Marsh & Bron, 1997). Such trafficking depends on specific post-internalization membrane sorting and, in some cases, direct interactions of virus particles with the cytoskeleton (Sodeik, 2000).

Clathrin

As befits its major role in endocytosis, clathrin has been shown to play a major role in the internalization of many viruses. Traditionally, several viruses, including influenza virus, vesicular stomatitis virus and SFV were identified in clathrin-coated vesicles at early times of internalization (for example, 5 min) based on the presence of an electron-dense coat by transmission electron microscopy (Marsh & Helenius, 1980; Matlin et al., 1981, 1982). However, non-coated vesicles were also observed in these experiments, due either to the release of clathrin or to the presence of non-clathrin-coated pathways. Subsequently, a crucial role for clathrin in SFV internalization has been shown by microinjection of anti-clathrin antibodies (Doxsey et al., 1987).

Inhibition of clathrin function traditionally relied on three principle approaches – low-pH shock treatment, potassium depletion and treatment of cells with brefeldin A (BFA) (Brodsky et al., 2001) and more recently by the use of chlorpromazine (Wang et al., 1993). However, all of these treatments are non-specific; for example, BFA targets ADP-ribosylation factor (ARF) GTPase exchange factors in the secretory pathway and chlorpromazine targets many intracellular enzymes. As such, they have multiple effects on cell function and their use to inhibit virus infection should be treated with some caution. Chlorpromazine has been employed most extensively for studies of virus entry. It has been used to demonstrate the role of clathrin-mediated endocytosis in the uptake of influenza virus (Krizanová et al., 1982), the polyomavirus JC virus (Pho et al., 2000) and the picornavirus parechovirus type 1 (Joki-Korpela et al., 2001). Interestingly, a role for clathrin in the uptake of Epstein–Barr virus into B cells of the immune system, but not epithelial cells, has also been implicated through the inhibitory effect of chlorpromazine (Miller & Hutt-Fletcher, 1992). Similarly, human cytomegalovirus has been shown to enter epithelial cells by endocytosis but fuses at the plasma membrane of fibroblasts (Bodaghi et al., 1999), indicating that internalization pathways may be cell type-dependent. Chlorpromazine also blocks entry of the picornavirus hepatitis A virus (Bishop, 1998) but in this case its effects were ascribed to non-clathrin endocytosis. The alternative (and possibly more specific) technique of potassium depletion and hypotonic shock has been used to investigate the role of clathrin in human rhinovirus infection (Bayer et al., 2001; Madshus et al., 1987).

Dominant–negative mutant versions of cellular proteins provide a more specific way to analyse the function of defined pathways within the cell. When expressed at high levels, dominant–negative mutants act by overwhelming the wild-type protein and preventing its function. This type of approach has been used extensively in studying the function of GTPases within the cell (Feig, 1999), where a dominant–negative protein can lead to sequestration of effectors and regulatory molecules and effectively shut down the function of the endogenous wild-type protein. The only drawback is that inhibition at late time-points after initiation of the dominant–negative protein may be mediated by an indirect effect due to the inevitable network of interacting molecules.

In studies of virus entry, the most commonly applied dominant–negative approach has been to analyse the role of the dynamin, a GTPase required for the release of clathrin-coated pits (Hinshaw, 2000; Schmid et al., 1998). Dynamin exists in several forms in the cell, often with cell-type specificity. Dynamin-2 is expressed ubiquitously; however, the most-studied form of dynamin experimentally (dynamin-1) is normally found only in neurons. Although these proteins are similar, they do have distinct properties. Ideally, dominant–negative dynamin-2 should be used in most cell types, although, where examined, dominant–negative versions of dynamin-1 also appear show a strong phenotype in non-neuronal cells. The original expectation was that overexpression of a GTPase-deficient version of dynamin (for example, K44A) would result in a specific block of clathrin-mediated endocytosis. However, as the study of dynamin has progressed, it is has become clear that it functions in many different membrane-traffic events. In addition to clathrin-mediated endocytosis, dynamin plays a role in formation of caveolae (Henley et al., 1998), budding of Golgi-derived vesicles (Jones et al., 1998; Nicoziani et al., 2000), phagocytosis (Gold et al., 1999) and non-clathrin endocytosis (Lamaze et al., 2001). The overexpression of dynamin K44A also results in an increase in endosomal pH (Huber et al., 2001), posing a possible complication when examining the internalization of pH-dependent viruses. The actions of mutant dynamin, therefore, need to be treated with some caution when interpreting effects on virus infection. A particularly illuminating study of the intracellular bacterium Chlamydia clearly showed an effect of dominant–negative dynamin on bacterial replication but without any effects on internalization (Boleti et al., 1999).

Nonetheless, the use of the dominant–negative dynamin K44A has proven valuable in our understanding of virus entry. The first report using this molecular inhibitor in studies of virus entry showed effects on SFV, Sindbis virus and rhinovirus but not poliovirus (DeTulleo & Kirchausen, 1998). Subsequently, an effect on rhinovirus internalization was confirmed (Bayer et al., 2001) but the effects of dynamin K44A were attributed to a change in endosomal pH (Huber et al., 2001). Other viruses where dynamin K44A is known to inhibit internalization include parvoviruses (Bartlett et al., 2000; Duan et al., 1999; Parker & Parrish, 2000), influenza virus (Roy et al., 2000) and adenovirus (Wang et al., 1998). Of particular note is the finding that the avian leukosis virus, a retrovirus, shows a 60–80 % inhibition of infection in dynamin K44A-expressing cells (Mothes et al., 2000), although another retrovirus, Moloney murine leukaemia virus, showed no effects upon expression of dominant–negative dynamin (Lee et al., 1999). It is unclear if this apparently contradictory data for retroviruses represent differences in the individual viruses or cell types, under the assay conditions used, or if dynamin is required for other membrane-traffic events important for virus assembly.

Because of the pleiotropic effects of dynamin, investigators have searched for more specific molecular inhibitors of clathrin. One promising candidate is the Eps15 protein, which binds to the AP-2 adapter required for internalization through clathrin-coated pits (Benmerah et al., 1998). Deletion of the EH domain(s) of Eps15 produces a dominant–negative version of the protein that arrests clathrin-coated pit assembly (Benmerah et al., 1999) and prevents internalization of transferrin, a cellular marker of clathrin-mediated endocytosis. Other promising tools for clathrin inhibition are the overexpression of the clathrin hub domain, which prevents normal clathrin pit assembly (Brodsky et al., 2001), and expression of the µ2 subunit of AP-2 (Nesterov et al., 1999), which results in a very specific inhibition of AP-2-mediated endocytosis of receptors containing YXX motifs, but not di-leucine-based signals. Of these clathrin/AP-2-specific approaches, Eps15 has received most attention as a way to block virus entry. Microinjection of purified Eps15 protein lacking the EH domains effectively blocked infection by Sindbis virus, even at the relatively high m.o.i. of 50 p.f.u. per cell (Carbone et al., 1997). In addition, overexpression of dominant–negative Eps15 has been shown to inhibit uptake of both SFV (unpublished results) and adenovirus (U. Greber, personal communication).

In the future we expect to see more studies of virus entry involving such molecular inhibitors. One note of caution concerning the clathrin-specific approaches outlined above is that, with time, cells expressing the dominant–negative proteins tend to compensate by upregulating non-clathrin-mediated pathways. Thus, experiments using these approaches need to be carefully executed and carried out under as short a time frame as possible. Another factor is that blockage of dynamin function can lead to an arrest of virus receptors at the cell surface and result in increased virus binding (Parker & Parrish, 2000).

Caveolae

Recently, caveolae have emerged as a route of entry for the polyomavirus, simian virus 40 (SV40). The uptake of SV40 is considered somewhat unusual, based on early electron micrographs showing accumulation of incoming virions in the smooth endoplasmic reticulum (ER) (Kartenbeck et al., 1989). This localization has been reinforced by the use of green fluorescent protein (GFP)-tagged caveolin-1 (a major component of caveolae) and video microscopy, which showed the delivery of SV40 from caveolae to the ER, bypassing the traditional endosome/lysosome system (Pelkmans et al., 2001). Compared to clathrin-mediated entry, caveolae perform internalization more slowly and the resulting vesicles do not become acidified. An additional difference between clathrin and caveolae is that internalization via caveolae is not a constitutive process (Thomsen et al., 2002) and only occurs upon cell stimulation. Caveolae are known to be major initiating centres for signalling within the cell (Ceresa & Schmid, 2000; Simons & Toomre, 2000). It is possible that partitioning of bound polyomaviruses into caveolae promotes their uptake and subsequent infection by activating downstream-signalling cascades (Chen & Norkin, 1999). Indeed, it has recently been shown that SV40-induced tyrosine kinase activity leads to recruitment of both actin and dynamin to virus-containing caveolae. This recruitment is necessary for formation of caveolae-derived endocytic vesicles and for SV40 infection (A. Helenius, personal communication).

Colocalization studies with endogenous caveolin have also been used to implicate caveolae in the entry of SV40 (Norkin, 1999); however, conflicting reports for colocalization with murine polyomaviruses have been reported (Gilbert & Benjamin, 2000; Richterova et al., 2001). In these studies, murine polyomavirus was also shown to be independent of both clathrin and dynamin. Clearly, more studies are required to clarify the role of caveolae in the entry of the different members of the Polyomavirinae.

Several drugs have been used to inhibit endocytosis via caveolae selectively. The most effective way of disrupting caveolar function is with sterol-binding drugs that sequester cholesterol – a prominent component of lipid rafts involved in caveolae formation. Such drugs include nystatin, filipin and methyl--cyclodextrin (Neufeld et al., 1996; Orlandi & Fishman, 1998; Rothberg et al., 1992), none of which typically affect clathrin-mediated endocytosis. Along with colocalization studies, these drugs have been used in studies of both SV40 and murine polyomavirus (Anderson et al., 1996; Gilbert & Benjamin, 2000; Richterova et al., 2001). They have also been used to show the involvement of caveolae in the internalization of the picornavirus echovirus type 1 (Marjomaki et al., 2002) and the uptake of respiratory syncytial virus into dendritic cells (Werling et al., 1999).

The most characterized protein components of caveolae are the caveolins (Kurzchalia & Parton, 1999; Rothberg et al., 1992). For reasons that are still unclear, the caveolins require an intact N terminus for their function. Deletion of the N terminus of caveolin-3 results in a dominant–negative protein that inhibits both SV40 (Roy et al., 1999) and echovirus-1 (Marjomaki et al., 2002) when overexpressed. Similar findings were obtained with SV40 by tagging the N terminus of caveolin-1 with GFP, whereas fusion to the C terminus resulted in a wild-type protein (Pelkmans et al., 2001). It will be interesting to examine the effects of these dominant–negative caveolins on the uptake of other members of the Polyomavirinae.

Macropinocytosis

Macropinocytosis is considered generally to be a non-specific mechanism for internalization, in that it is not reliant on ligand binding to a specific receptor. Instead, formation of endocytic vesicles occurs as a response to cell stimulation, resulting in the closure of lamellipodia at the sites of membrane ruffling to form the large, irregular vesicles known as macropinosomes. Membrane ruffling is primarily actin-driven and macropinocytosis is, in terms of mechanics, similar to the process of phagocytosis that occurs in specialized immune system cells such as neutrophils and macrophages. The finding that macropinosomes have the ability to become acidified and can intersect with endocytic vesicles (Hewlett et al., 1994), makes them possible routes of entry for a wide variety of viruses.

Because of its strict requirement for actin, the most commonly used inhibitors of macropinocytosis are the cytochalasins, especially cytochalasin D (Maniak, 2001). Macropinocytosis is also highly dependent on the activity of phosphatidylinositol (PI) 3-kinase (PI3K) and the activity of Rho family small GTPases, which regulate actin rearrangements. Inhibitors of PI kinases, such as wortmannin and LY294002 (Araki et al., 1996; West et al., 2000), and Rho GTPases, such as toxin B (Just et al., 1995), along with amiloride, an inhibitor of Na⁺/H⁺ exchange (West et al., 1989), have all been employed to inhibit macropinocytosis in cells. As with the use of drugs to study other endocytic pathways, their specificity for macropinosomes may be limited. In particular, PI3K activity clearly affects early endosome dynamics (Corvera, 2001). It is also becoming increasingly apparent that actin is crucial for clathrin-dependent endocytosis, especially in polarized epithelial cells (Apodaca, 2001) – the in vivo target of many viruses.

Several promising molecular approaches in targeting macropinocytosis specifically have emerged recently, which target ARF- and Rho-family GTPases. Overexpression of ARF6 locked in its GTP-bound form (Nichols & Lippincott-Schwartz, 2001), dominant–negative forms of the Rho family GTPases (West et al., 2000) and the autoinhibitory domain of the Rac-dependent kinase PAK1 (Dharmawardhane et al., 2000), all result in an inhibition of macropinocytosis. However, none of these approaches have been used to study the role of macropinocytosis in virus entry and their usefulness in specifically targeting macropinocytosis remains to be established. At present, traditional approaches based on the visualization of virus particles at sites of membrane ruffles, visible by light or electron microscopy, or within large irregular vesicles may still be the best approach to demonstrate a role for macropinocytosis in virus entry. Such experiments have been used recently to demonstrate macropinocytic uptake of human immunodeficiency virus type 1 into macrophages (Marechal et al., 2001). Whereas most of the virions internalized in this way were degraded, some could in fact infect the cell.

Although macropinocytosis per se was not implicated, expression of dominant–negative Rho family members, as well as cytochalasin D, reduced significantly the entry of the intracellular mature virus (IMV) form of vaccinia virus (VV) (Locker et al., 2000). In these studies, the other infectious form of VV (the extracellular enveloped virus or EEV) was not affected. Other investigators, however, found that both EEV and IMV were sensitive to cytochalsin D, with differences occurring in the exposure of the two virus forms to a low-pH compartment (Vanderplasschen et al., 1998). The very large size and different morphological forms of VV (with possibly spatially restricted entry into conventional coated pits and vesicles), combined with the complex signalling network elicited upon infection, raises special challenges for a study of virus entry.

The fact that macropinocytosis appears to require cell stimulation raises issues of how a virus might induce signalling, and subsequently macropinocytosis, upon binding to the cell surface. Such virus-mediated signalling has recently been shown for adenovirus, where integrin-dependent virus binding activates macropinocytosis. However, in this case, macropinocytosis in not used for virus uptake but for escape from the endosome (U. Greber, personal communication). Another factor to bear in mind is the apparent lack of a specific receptor for macropinocytosis – a problem perhaps unimportant for viruses without a specific receptor like the sialic acid-binding influenza virus.

Finally, macropinocytosis is known to be a major route of entry into antigen-presenting cells like dendritic cells (DCs) and in this case it is constitutive in that it does not require cell stimulation (Garrett et al., 2000; West et al., 2000). As DCs are one of the first immune system cells to encounter an invading virus, they have special importance for virus pathogenesis. The virus could productively infect the DC or could internalize the virus and then 'regurgitate' it to infect other cells at a distant site. Such regurgitation could occur via a process involving the release of so-called exosomes, derived from specialized multilamellar endocytic vesicles present in DCs (Denzer et al., 2000; Kleijmeer et al., 2001). The ultimate importance of macropinocytosis in virus infection may therefore turn out to be in the presentation of the invading virus to the immune system and in the spread of virus through the host, rather than in its primary infection of epithelial surfaces.

Non-clathrin, non-caveolae-dependent endocytosis

Beyond the established roles of clathrin and caveolae, and of macropinocytosis in stimulated cells, there exists an ill-defined route of non-clathrin, non-caveolae-dependent endocytosis in resting cells (Bishop, 1997; Nichols & Lippincott-Schwartz, 2001). Although the entry of many viruses has been implicated to occur through these pathways, based mainly on the presence of viruses in non-coated vesicles by electron microscopy (for example, influenza and Sendai viruses) (Marsh & Helenius, 1989), the paucity of specific information or cellular markers for these pathways has not allowed unequivocal conclusions to be drawn.

Recent reports, however, have begun to clarify some of these non-clathrin, non-caveolae pathways. As with caveolae, it is likely that such pathways are intimately linked to the presence of detergent-resistant microdomains on the plasma membrane. The interleukin-2 (IL-2) receptor is perhaps the best-characterized marker for these pathways. Based on dominant–negative approaches in caveolin-negative T cells, IL-2 receptor uptake and delivery to late endosomes and lysosomes is clearly independent of clathrin, yet, like caveolae, requires dynamin and specialized membrane domains (Lamaze et al., 2001). Another pathway has been studied using GFP-tagged glycosyl PI-anchored proteins. This cholesterol-sensitive pathway delivers material directly to the Golgi, bypassing both clathrin and early endosome markers (Nichols et al., 2001).

Save for a report on picornaviruses that utilized physiological means of inhibiting clathrin (Madshus et al., 1987), little specific information is available on non-clathrin routes of virus entry. Potentially complicating factors include the likelihood that non-clathrin-dependent uptake of a particular ligand may well be occurring simultaneously with clathrin-mediated endocytosis and that multiple non-clathrin pathways exist (Dautry-Varsat, 2001). Overall, non-clathrin routes of entry may only be able to be examined once clathrin is inhibited specifically – a finding observed with uptake of certain lipid analogues (Puri et al., 2001) and should be carefully examined alongside known cellular markers. The availability of such markers, along with specific molecular targets for clathrin and caveolae (see above) now makes an examination of non-clathrin-dependent virus entry more meaningful.

Rab proteins and endocytosis

A major class of molecule that regulate membrane-traffic events is the Rab family of small GTPases (Collins & Brennwald, 2000). For endocytosis, four principal Rab proteins are involved – Rab5 (needed for formation of early endosomes), Rab7 (required for early to late endosome/lysosome traffic) and Rab4 and Rab11 (involved in the recycling pathway) (Somsel Rodman & Wandinger-Ness, 2000). Rab9 GTPase is also part of the endocytic network as it controls traffic between the endosome and the trans-Golgi network. For all of these Rabs, well-characterized point mutations exist which can be overexpressed as dominant–negative proteins to block a specific point in the endocytic pathway.

Such dominant–negative Rabs are becoming utilized in studies of virus entry. As with the use of many other molecular tools, the adenovirus system has led the field in this regard. Adenovirus uptake was increased by overexpression of wild-type Rab5 and decreased by dominant–negative Rab5 (Rauma et al., 1999). Rab proteins are also convenient markers for colocalization studies, as they are organelle-specific in their localization. For example, Miyazawa et al. (2001) used Rab7 to show the localization of adenovirus subgroup B (Ad7) to late endosomes. In contrast, these authors showed that the related (and more commonly studied) adenovirus subgroup C does not enter the late endosome but seems to penetrate from the early endosome. Thus, very similar viruses may show selective endocytic traffic, which could have profound implications for their pathogenesis. One unusual feature of Ad7 is that endosomal trafficking is very slow (Miyazawa et al., 2001). Other non-enveloped viruses, such as canine parvovirus, also show much delayed trafficking through endosomes (Parker & Parrish, 2000). Whether or not these viruses enter any kind of recycling compartment before finally penetrating into the cytoplasm remains to be seen.

Rho proteins and endocytosis

In addition to the established roles of the Rho family members, Rac, RhoA and Cdc42, in macropinocytosis, the Rho B and D proteins are important players in clathrin-mediated endocytosis (Ellis & Mellor, 2000). In contrast to the Rab proteins, which control membrane fusion and remodelling events, the endosomal Rho proteins apparently regulate the rate of vesicle traffic along cytoskeletal tracks (Murphy et al., 1996). Rho D localizes to early/recycling endosomes (Murphy et al., 1996), while Rho B and its associated kinase PRK-1 are likely to control entry into multivesicular bodies (Gampel et al., 1999). As an example of the convergence of endocytic pathway regulatory molecules, the effects of dominant–negative Rac and Cdc42, as well as toxin B (which targets Rho GTPases) and cytochalasin D, have all been shown to inhibit adenovirus endocytosis (Li et al., 1998a). On the one hand, this set of experimental conditions could suggest macropinocytosis as a route of entry for the virus, yet the effects of these reagents are attributed to PI3K-related effects on clathrin-mediated endocytosis (see below).

Kinase-mediated regulation of endocytosis

As befits a role as a major signalling event in the cell, virus endocytic trafficking can be regulated by phosphorylation. Attention has focussed on two kinase families, PI3K and protein kinase C (PKC). For adenovirus, the initial interaction of the virus with cell surface integrins activates PI3K, which in turn modulates virus endocytosis. Addition of the PI3K inhibitors wortmannin or LY294002 reduced adenovirus internalization to approximately 30 % of control samples (Li et al., 1998b). It has not been shown at which point in internalization adenovirus internalization is arrested by PI3K inhibition but the kinases are believed to act upstream of small GTPases in the signalling cascade (Kotani et al., 1995). Similarly, endocytic trafficking of adeno-associated virus type 2 is dependent on integrin/Rac-dependent PI3K activation (Sanlioglu et al., 2000).

For many years, PKC has also been implicated in the processes of virus entry. The entry of several enveloped viruses, including rhabdoviruses, alphaviruses, poxviruses and herpesviruses, have been proposed to require PKC based on the action of protein kinase inhibitors, such as H7 and staurosporine (Constantinescu et al., 1991). More recently, it has been shown that the successful entry of adenovirus type 2 requires PKC. In the presence of calphostin C, an inhibitor of the classical and novel PKC isoforms, adenovirus is prevented from escaping endosomes and accumulates in cytoplasmic vesicles near the cell periphery (Nakano et al., 2000). Influenza virus has the ability to activate PKC upon binding to host cell-surface receptors (Kunzelmann et al., 2000) and we have described previously that bisindolymaleimide I, a specific PKC inhibitor, prevented influenza virus entry and subsequent infection (Root et al., 2000). A role for PKC in virus entry is reinforced by recent data from our laboratory using cells overexpressing a phosphorylation-deficient form of the PKCII isotype. In these cells, influenza virus entry was arrested at the level of the late endosome (S. B. Sieczkarski, H. A. Brown and G. R. Whittaker, unpublished data), without any apparent defect in endosome acidification. It remains to be seen which isoforms of PKC are involved in the entry of different viruses and at which point in endocytic trafficking they act.

As research into the mechanisms of endocytosis progresses, the tools to study these processes are becoming increasingly precise. The development of molecular inhibitors in the form of dominant–negative molecules has surpassed the use of chemical inhibitors in terms of decreasing non-specific effects (Table 1). The use of molecular tools with such precise specificity to individual cellular functions allows the defined examination of endocytic pathways. Such techniques are invaluable in our study of virus infections. However, even with these advances, ambiguity and a reason for caution still exist. The plasticity of many of cellular pathways means that virus entry may be impacted by an indirect mechanism rather than by direct inhibition. Additionally, it is possible that viruses make use of multiple internalization pathways. The next advancements in the study of virus entry are the developments in real-time single molecule imaging of virus infections, examples of which may be seen in Seisenberger et al. (2001). Such experiments allow an extra level of sophistication; for instance, it is now possible to view, in real-time, the entry and subsequent trafficking of viruses into live cells expressing dominant–negative molecules and to monitor each component by double-label microscopy. Such techniques enable virus endocytosis and subcellular trafficking to be examined with exquisite clarity.

Table 1. Toolbox of reagents targeting the endocytic pathway

We thank Ruth Collins for critical reading of the manuscript and for help with illustrations. Work in the author's laboratory is supported by the American Lung Association, the American Heart Association and the National Institutes of Health.

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