Tervetuloa sisään ja riisukaa takkinne!! ( Vaipallinen virus tuli sisään soluun ja riisui manttelinsa ja kävi taloksi.

• https://www.ncbi.nlm.nih.gov/pubmed/20678171

Cell Microbiol. 2010 Oct;12(10):1378-88. doi: 10.1111/j.1462-5822.2010.01510.x. "Come in and take your coat off" - how host cells provide endocytosis for virus entry.

Schelhaas M¹.

Abstract

VIRUKSET ovat solunsisäisiä loisia  ja  ne tarvitsevat isänsäsolun koneiston elinsykliisä ja virioniensa tuottoon.  Vasta kehkeytyneitten  viruspartikkeleiten  tehtävänä on välittää  genominen informaatio uusiin infektoitumattomiin soluihin ja organismeihin.  Viruksen sisäänmeno soluun pn se prosessi, jolla  se hankkii itselleen pääsyn   virusksen replikoitumiskohtiin infektoitumattoman solun sisällä, tämä on monivaiheinen tapahtumasarja, joka alkaa  kiinnittymisestä kohdesoluihin. Koska virukset ovat erittäin ykinkertaisia rakenteeltaan ja kokoomukseltaan ja niiltä puuttuu liikkumiskyky, ne tarvitsevat sadoittain isäntäsolun proteiineja  solun sisään pääsemiseen. (ENTRY)

• Viruses are intracellular parasites that rely upon the host cell machinery for their life cycle. Newly generated virus particles have to transmit their genomic information to uninfected cells/organisms. Viral entry is the process to gain access to viral replication sites within uninfected cells, a multistep course of events that starts with binding to target cells.

•  Since viruses are simple in structure and composition and lack any locomotive capacity, viruses depend on hundreds of host cell proteins during entry. Most animal viruses take advantage of endocytosis to enter cells.

 Solubiologiset , morfologiset ja biokemialliset tutkimukset, elävän solun kuvaukst ja systemaattiset lähestymistavat ovat auttaneet  tunnistamaan  erilaisia uusia endosyyttisiä mekanismeja  klatriinivälitteisen endosytoosin, makropinosytoosin ja kaveola/lipidilevy-välitteisen endosytoosin ohella. Sentakia  viruksen soluun menon tutkimus on muuttunut entistä monimutkaisemmaksi . tässä katsauksessa  on  biologinen yleisnäkymä  olemassa olevista  endosytoottisista mekanismeista ja strategioista, joita virukset käyttävät tai mahdollisesti käyttävät  solun sisäänmenossa.

•  Cell biological, morphological and biochemical studies, live cell imaging and systematic approaches have identified various new endocytic mechanisms besides clathrin-mediated endocytosis, macropinocytosis and caveolar/lipid raft-mediated endocytosis. Hence, studying virus entry has become ever more complex. This review provides a cell biological overview of the existing endocytic mechanisms and strategies used or potentially used by viruses to enter cells.

PMID: 20678171

DOI: 10.1111/j.1462-5822.2010.01510.x

 Introduction

Virus particles have a single purpose – to safely transfer their genomic information from infected to uninfected cells/organisms. Composed of nucleic acids (RNA or DNA), proteins and – for enveloped viruses – membrane lipids, viruses are rather simple in structure and lack any metabolic or motile processes. Hence, they have evolved a ‘Trojan horse’ strategy to exploit the cell machinery for entry into target cells, a complex programme that involves hundreds of cellular proteins.
During this process, two major tasks are fulfilled by the viruses through a series of complex interactions with the cell: (i) to overcome the obstacles that bar the virus from the site of replication such as the plasma membrane, a crowded cytoplasm and the nuclear membrane, and (ii) to release the genomic information at the right place within the cell to ascertain viral transcription and replication. Both tasks are coordinated in time and space and rely on a number of ‘cues’ provided by interactions of the host cell with the virus structure.

The virus particle

During transmission, the viral genome is protected by a protein shell typically termed capsid. Capsids are mostly spherical, often icosahedral structures, which are composed of many subunits of one or more structural proteins. Enveloped viruses have an additional lipid membrane displaying viral membrane glycoproteins that engage receptors and mediate fusion events with cellular membranes (Harrison, 2008).
Virus structures are stable – crosslinked by networks of intermolecular interactions to withstand hostile conditions encountered in the extracellular space. In fact, virus structures are metastable – poised upon cellular cues to undergo major conformational changes for an eventual release of the genomic information, a stepwise process termed uncoating. It is of fundamental importance that uncoating does not prematurely expose the viral genome, since this would lead to degradation and/or failed transport to the site of replication.

Virus entry – an overview

As viruses can only infect cells to which they can bind, cell receptors largely define the organism and cell tropism of a virus. Receptors promote entry by binding, by initiating conformational changes in the virus, by activating cellular signalling, and by inducing fusion at the plasma membrane or by promoting endocytosis. In fact, some viruses bind to several different receptors simultaneously or in sequence such as Human Immunodeficiency Virus type 1 (HIV‐1), adenoviruses and coxsackie B3 virus (Nemerow, 2000; Coyne and Bergelson, 2006;Mercer et al., 2010). Receptor molecules include a wide variety of different proteins, lipids and carbohydrates (for an overview see Helenius, 2007).
After receptor binding, viruses undergo a period of diffusive or directed motion on the plasma membrane, until they become confined (Burckhardt and Greber, 2009). In addition, some viruses can move along filopodia to internalization sites by receptor interaction with the actin cytoskeleton and retrograde flow within filopodia (Lehmann et al., 2005; Schelhaas et al., 2008). 

As mentioned, virus binding to receptors and ensuing signalling induce endocytosis and/or prepare the cell for invasion. While several enveloped viruses such as HIV or herpesviruses fuse directly with the plasma membrane to release the capsids into the cytoplasm, they also can use endocytosis depending on the cell system (Nicola et al., 2003; Miyauchi et al., 2009). In fact, most viruses use endocytosis, because this mode of entry is clearly advantageous: (i) endocytosis leaves no trace of the viral presence on the plasma membrane – likely to cause a delay of the immune response, (ii) endocytic uptake and ensuing vesicular transport provide a built‐in transport mechanism across the plasma membrane, the underlying actin cytoskeleton and the crowded cytoplasm, and (iii) vesicular trafficking provides access to intracellular organelles that allow viruses to ‘sense’ their environment by gradually changing conditions such as pH, redox environment and presence of specific proteases.
For activation of membrane penetration or uncoating, structural changes in viruses are triggered by the host cell. While viruses can be already activated at the plasma membrane by, e.g. receptor binding as described above, activation also occurs often by the intraluminal environment of a particular organelle. The activated viruses then penetrate the vacuolar membrane delivering the viral structure or genome into the cytosol. Viral capsids often bind directly to microtubular motors for further cytosolic transport (Dohner and Sodeik, 2005). Most RNA viruses replicate in specific regions of the cytosol, whereas most DNA viruses must gain access to the nucleus. Nuclear import is typically achieved by cooperation with the nuclear import machinery (Whittaker et al., 2000).

Endocytosis

Endocytosis is essential in eukaryotic cells to internalize extracellular particles, fluid and ligands generally termed cargo (Conner and Schmid, 2003). Endocytosis starts with the formation of primary endocytic vesicles (PEVs), endocytic vacuoles generated by pinch‐offs from the plasma membrane. This multistep process involves activation, cargo capture/sorting, induction of membrane curvature, dilation of curvature and scission. PEVs are targeted to endosomal organelles. From there, cargo is further sorted to destination organelles.
For a long time, it has been thought that receptor‐mediated endocytosis is mainly comprised of clathrin‐mediated endocytosis (CME). However, current research indicates a complex network of diverse continuing and triggered pathways (Fig. 1). In addition to CME, macropinocytosis and caveolar/raft‐dependent endocytosis, several further mechanisms have emerged that are less well characterized. They differ primarily in the formation of PEVs and involve a large number of cellular factors. The various endocytic phenomena will be briefly described (for more detail please refer to Gruenberg, 2001; Kirkham and Parton, 2005; Bonifacino and Rojas, 2006; Mayor and Pagano, 2007; Doherty and McMahon, 2009; Hansen and Nichols, 2009).

Endosomes

 Primary endocytic vesicles are routed to endosomes that are responsible for sorting, recycling, degradation, storage and transcytosis of cargo. The different endosomal organelles are heterogeneous in composition. They can fuse, some homo‐ others also heterotypically, and they undergo molecular changes with time. For simplicity, endosomal organelles are described here as collective identities, i.e. early endosomes (EE), late endosomes (LE), lysosomes and recycling endosomes (RE). EE are more dispersed throughout the cytosol, whereas RE, LE and lysosomes are located mainly perinuclearly, so that molecular sorting leads cargo such as viruses to different locations within the cell. Vesicular traffic and movement of endosomes occurs along microtubules. Cargo internalized by CME is typically delivered to EE 2–5 min after internalization, reach LE by 10–15 min and lysosomes by 30–60 min (Kielian et al., 1986; Mukherjee and Maxfield, 2004; Lakadamyali et al., 2006). Endosomes contain different membrane domains often specified by different Rab GTPases and their effectors, or phosphoinositides. Rab GTPases are prenylated proteins that associate with membranes and specifically localize to domains through their interactions with effector proteins (Pfeffer and Aivazian, 2004). Different Rab GTPases are associated with sorting of cargo to further destinations, e.g. Rab5 (EE), Rab4 (fast recycling to the plasma membrane), Rab11/22 (slow recycling by RE), Rab7 (LE), Rab9/retromer (trans‐GOLGI‐network, see Fig. 1). The presence of Rab GTPases and their effectors are also used for organelle specification (Zerial and McBride, 2001). Early endosomes are the first and main sorting station for cargo. From EE, cargo is transferred to RE, LE, or back to the plasma membrane. There are at least two variants of EE, highly motile and rather static ones that differ in the rate they convert to LE exemplifying the heterogeneity of endosome identity (Lakadamyali et al., 2006). Cargo sorting can be achieved by clustering of membrane proteins into domains. Membrane proteins that have been monoubiquitinated on the cytosolic side are sorted by the ESCRT complex into smaller vesicles that bud into the endosome lumen, forming intraluminal vesicles (ILV; Piper and Katzmann, 2007). This process starts in EE and leads to the multivesicular appearance of LE. The complex structure of EE, displaying vacuolar and long narrow tubular elements, likely directs diffusion of soluble and membrane proteins and could cause concentration gradients. Cargo destined for direct recycling is often concentrated in the tubular elements (Grant and Donaldson, 2009). The dimensions of ILV, and for viruses we can assume the same, mostly excludes entry into the tubular elements, so that they remain in the vacuolar part targeted for degradation. Most ILV are degraded in lysosomes. However, some backfuse with endosomal membranes. Vesicular stomatitis virus uses this feature to initially fuse with ILV, after which backfusion releases the capsid into the cytoplasm (Le Blanc et al., 2005). Endosome communication with the secretory pathway is provided by the retromer complex and Rab9 that allow vesicles to shuttle between endosomes and the trans‐GOLGI‐network (Bonifacino and Rojas, 2006). However, there is no clear evidence for viruses using the GOLGI route for entry. Instead, viruses mostly follow the route destined for degradation, from EE to LE to lysosomes. For degradation, cargo can be transported from EE to LE by vesicular transport. Alternatively, EE mature into LE (Rink et al., 2005; Vonderheit and Helenius, 2005). In the process, intermediate compartments also called maturing endosomes (ME) are established (Braulke and Bonifacino, 2009). ME exhibit markers for both, EE and LE. Endolysosomes form, when LE fuse with lysosomes. LE are ‘consumed’ in the process. Endolysosomes display a characteristic density in electron microscopy that increases when they condense into lysosomes (Mullock et al., 1998). From EE to lysosomes, the lumen of endosomes becomes increasingly acidic mainly through the action of V‐ATPase (Lafourcade et al., 2008). Also, the amount of hydrolases/proteinase increases by incoming transport from the GOLGI, so that viruses encounter a gradually changing chemical environment. A particular chemical environment provides the cues for structural changes in the particles by, e.g. a specific pH threshold and/or the activity of hydrolases/proteinases. Changes may trigger fusion of the viral envelope with endosomal membranes, partial disassembly of viruses, pore formation in endosomal membranes or more drastic changes such as rupture of endosomes by membrane destabilizing peptides. If a virus fails to penetrate the endosomal compartment of choice, its fate is usually degradation. It is noteworthy that endosomal maturation and vesicular transport are interconnected and interdependent, so that perturbation of a single function/factor may result in drastic consequences for several organelles, e.g. blocking V‐ATPase with bafilomycin A1 results not only in decreased intraluminal acidification of endosomes but also prevents formation of ME (Clague et al., 1994).

 Clathrin‐mediated endocytosis

 Clathrin‐mediated endocytosis is the endocytic pathway that is best understood (Doherty and Mcmahon, 2009). CME occurs constitutively in most mammalian cells. In addition, cargo such as viruses can also induce the formation of clathrin‐coated pits (CCPs) (Rust et al., 2004; Johannsdottir et al., 2009). For PEV formation, clathrin is recruited to the plasma membrane in response to internalization signals and forms a characteristic coat that is visible in electron microscopy. Many proteins are involved in coat assembly, although their functions remain in part elusive (Robinson, 2004). Typically, localized formation of phosphoinositide‐4,5‐phosphate (PI4,5P2) leads to recruitment of the adaptor protein AP2 that, in turn, recruits clathrin, AP180 and Eps15 followed by further adaptors involved in cargo selection/immobilization (Ungewickell and Hinrichsen, 2007). However, it has become clear that CCPs have not always the same composition (Robinson, 2004). AP2, for example, is, contrary to the early model, not always required for CME (Motley et al., 2003). The varying requirement for adaptor proteins indicates a common mechanism combined with a diverse degree of regulation. Fission of clathrin‐coated vesicles from the plasma membrane involves the GTPase dynamin (Hinshaw, 2000). From recruitment of clathrin to scission, PEV formation is fast and takes about 1 min for individual pits. Then, synaptojanin, auxilin and HSC70 help to disassemble the clathrin coat from the vesicle. PEVs are transported to EE. The first virus described to enter cells via CME was Semliki Forest Virus (SFV) (Helenius et al., 1980). Many other viruses from diverse virus families use CME for entry including adenovirus 2 and 5, hepatitis C virus, dengue virus and influenza A virus (Mercer et al., 2010). In addition to entry by CME, viruses may also use alternate pathways in the same cell, at the same time, as has been described for influenza A viruses (Rust et al., 2004).

Caveolar/raft‐mediated endocytosis 

 The term caveolar/raft‐mediated endocytosis will be applied to caveolar and caveolin‐independent mechanisms of endocytosis that are lipid raft‐dependent (Pelkmans et al., 2001; Pelkmans et al., 2002; Parton and Richards, 2003; Damm et al., 2005). As suggested by Kirkham and Parton (2005), they may share a congeneric core mechanism somewhat similar to CME requiring different sets of molecular factors. Characteristically, formation of PEVs requires lipid rafts and tyrosinekinase/phosphatase‐regulated, ligand‐triggered signalling events. Uptake can involve caveolae and dynamin‐2, but may occur without. While it has been previously thought that a novel organelle, the caveosome, acts as an intermediate station in intracellular trafficking, recent work from the Helenius laboratory indicates that this organelle corresponds to modified late endosomes/lysosomes (Mercer et al., 2010; A. Helenius, pers. comm.). From there, cargo is often routed to the endoplasmic reticulum (ER). How transit from endosomes to the ER occurs is not entirely clear yet, but the process seems to involve lipid‐sorting events (Qian et al., 2009). Cellular cargo includes glycolipids and some glycosyl‐phosphatidylinositol‐(GPI)‐anchored proteins and their ligands (Lajoie and Nabi, 2007). Studying the entry of polyomaviruses has contributed much to our understanding of this pathway. Polyomaviruses such as Simian Virus 40 (SV40) and mouse polyomavirus (mPy) are small non‐enveloped DNA viruses with capsids assembled from 72 pentamers of the major capsid protein VP1. Polyomaviruses use different gangliosides as receptors. As an example, SV40 associates with detergent‐resistant microdomains in the plasma membrane and enter uncoated, tight‐fitting pits (Kartenbeck et al., 1989; Damm et al., 2005). Caveolae and pits formed in caveolin‐deficient cells are morphologically indistinguashable. Internalization and vesicular trafficking is asynchronous and slow, e.g. SV40 reaches the ER roughly 6 h post infection (Kartenbeck et al., 1989). Internalization is inhibited by cholesterol depletion, by inhibitors of actin dynamics and by inhibitors of tyrosine kinases (Pelkmans et al., 2002; Damm et al., 2005). Conversely, internalization is accelerated by inhibitors of serine/threonine or tyrosine phosphatases. RhoA seems to regulate the actin polymerization events. SV40 entry into caveolin‐deficient cell lines is mechanistically similar to entry by caveolar endocytosis but does not require dynamin‐2 (and obviously not caveolin‐1), and is less dependent on actin dynamics (Damm et al., 2005). Internalization is also faster in caveolin‐deficient cells suggesting that caveolin/dynamin introduce an additional level of regulation. Depending on the cellular system, it is likely that both variants of caveolar/lipid raft‐mediated endocytosis operate in parallel.
  

            Macropinocytosis/phagocytosis      

Macropinocytosis and phagocytosis differ from other endocytic mechanisms in that they require extensive actin cytoskeletal reorganization. These rearrangements are coupled to an outward‐directed formation of plasma membrane extensions, whereas in other pathways the plasma membrane ‘buds’ into the cell. Under normal conditions, macropinocytosis is induced by growth factors. Depending on cell type and stimulation, formation of membrane ruffles, filopodia, or ‘bleb’ formation precedes the generation of macropinosomes. Macropinosomes form by backfolding of membrane extensions and fusion with the plasma membrane (Swanson and Watts, 1995). Depending on the extent of membrane extension, resulting macropinosomes vary in shape and size. With diameters of up to 10 µm, they are relatively large. Macropinocytosis transiently increases fluid uptake up to 10‐fold. The activation of macropinocytosis and formation of PEVs requires sodium/proton exchanger, the RhoGTPases Rac1 and/or Cdc42, various cellular kinases (e.g. PAK1, PKC), CtB1 and cholesterol (Kerr and Teasdale, 2009; Mercer and Helenius, 2009). There is a significant degree of cell type dependence for further requirements such as the GTPases Rab34, Arf6 or actin modulatory factors. Macropinosomes can be recycled to the plasma membrane, undergo intraluminal acidification, homotypic fusion and heterotypic fusion with EE (Racoosin and Swanson, 1993; Hewlett et al., 1994; Swanson and Watts, 1995). Viruses that use macropinocytosis for entry include, e.g. vaccinia virus and Kaposi's sarcoma‐associated herpesvirus (Mercer and Helenius, 2009; Raghu et al., 2009). Another group of viruses induces macropinocytosis and requires it for entry, yet does not use it for internalization. This group includes, e.g. species C human adenoviruses 2 and 5 and rubella virus. For adenovirus 2, macropinocytosis is required for penetration of endosomal membranes after CME (Meier et al., 2002). The mechanism is not entirely understood but seems to require lysis of macropinosomes.


 Phagocytosis is not commonly used for viral entry but for uptake of large particles such as bacteria. Actin rearrangement and protuberance of the plasma membrane is induced and guided by an external particle bound to the cell. A tight fitting endocytic vacuole is formed around the particle with no or little fluid uptake (Swanson, 2008). Factors commonly involved include actin, RhoA, tyrosine kinases, cholesterol and dynamin‐2. A number of cell type‐dependent factors can be required, e.g. AP2, Arf6, Cdc42 and Rac1. The entry of mimivirus, an amoebal pathogen, and the entry of herpes simplex virus into corneal fibroblasts were found to be consistent with phagocytosis (Clement et al., 2006; Ghigo et al., 2008). For the purpose of this review, further novel endocytic pathways can be subdivided into (i) pathways described cell biologically but not for virus entry, and (ii) virus entry pathways by endocytic mechanisms inconsistent with any of the above. Both have in common that we have to learn much more about them to understand their use and their relationship to other pathways, e.g. whether some may share similar core mechanisms with a variation of factors. Novel cell biologically described pathways The internalization of GPI‐anchored proteins such as GPI‐GFP, decay‐accelerating factor and folate receptor GPI occurs by tubular endocytic pits resulting in the generation of ‘GPI‐anchored enriched endocytic compartments’ (GEECs; Sabharanjak et al., 2002). GEEC endocytosis requires lipid rafts but not clathrin, caveolin or dynamin. During pit formation, the GTPase Arf1 is recruited to the plasma membrane (Kumari and Mayor, 2008). Arf1 recruits ARHGAP10, a GAP for Cdc42, which regulates and precedes internalization of cargo by actin polymerization dynamics. GRAF1 may be a coat factor. The resulting GEEC, a tubular PEV, is not readily observed, since it is fragile and very sensitive to fixation conditions (Kirkham et al., 2005). Subsequent fusion of GEECs with the early endosome occurs in a Rab5/PI3 kinase‐dependent manner (Kalia et al., 2006). Flotillin‐1 has been implicated in the internalization of GPI‐anchored proteins (e.g. CD59), cholera toxin B and proteoglycans (Glebov et al., 2006; Payne et al., 2007; Hansen and Nichols, 2009). Flotillins are membrane‐bound, ubiquitously expressed, highly conserved proteins that share a similar membrane topology with caveolins. Flotillin‐1 heterooligomerization with flotillin‐2 to form microdomains at the plasma membrane depends on cholesterol and is required for assembly and function (Babuke et al., 2009). Internalization by flotillin microdomains is regulated by fyn kinase. The resulting flotillin‐1‐positive PEV fuses with EE. It is noteworthy that flotillin‐1 depletion may result in decreased or increased caveolin‐1 levels depending on the cell type by a non‐transcriptional mechanism (Vassilieva et al., 2009, M. Schelhaas and A. Helenius, unpubl. results). Endocytosis of MHCI and CD59 into HeLa cells is sensitive to cholesterol depletion and is associated with membrane ruffling initiated by the GTPase Arf6, the defining factor for this pathway (Naslavsky et al., 2004). Arf6 recruits and stimulates PI5 kinase to produce PI4,5P2 (Brown et al., 2001). After internalization, PI4,5P2 hydrolysis occurs and results in an Arf6‐positive compartment that fuses with the early endosome 5–10 min after PEV formation (Naslavsky et al., 2003). It is noteworthy that this pathway may be specific to HeLa cells, as Arf6 has been additionally implicated in macropinocytosis and CME for other cargos and/or cell types. The uptake of IL‐2 and gamma‐c cytokine receptor is independent of clathrin but occurs by a dynamin‐dependent process (Lamaze et al., 2001; Sauvonnet et al., 2005). IL‐2 binding to its receptor leads to prominent association of the receptor with lipid rafts, which is coupled to internalization by non‐coated, non‐tubular pits. Actin polymerization dynamics regulated by RhoA are also required for internalization. Cortactin – a potential cofactor for CME – participates in this process independently of clathrin. The PEV is routed to EE. Viruses that use unusual endocytic pathways Lymphocytic choriomeningitis virus (LCMV) entry illustrates an endocytic pathway that has not been observed previously, but which has been defined on negative terms. LCMV, an arenavirus, uses alpha‐dystroglycan as major receptor for cell entry (Cao et al., 1998). Endocytosis of LCMV occurs in smooth, non‐coated pits (Quirin et al., 2008). Internalization is largely independent of clathrin, caveolin, dynamin, actin dynamics and lipid rafts but requires membrane cholesterol (Quirin et al., 2008; Rojek et al., 2008). LCMV internalization is also independent of Arf6 and flotillin‐1. After internalization, LCMV particles mostly bypass EE and are routed to LE directly from the plasma membrane (Quirin et al., 2008). Entry of Human papillomavirus type 16 (HPV‐16), a non‐enveloped virus of the papillomavirus family, occurs likewise by a novel endocytic mechanism. Virus particles consist of two structural proteins (L1, L2) that form an icosahedral (T = 7) particle of about 55 nm in diameter. After an initial interaction with heparan sulfate proteoglycans followed by a sequence of structural changes, the virus is released and transferred to an unknown co‐receptor (Selinka et al., 2007; Sapp and Day, 2009). Endocytosis occurs by a clathrin‐, caveolin‐, flotillin‐, lipid raft‐, dynamin‐independent mechanism that is distinct from macropinocytosis and phagocytosis (Spoden et al., 2008; M. Schelhaas et al., unpublished). Actin polymerization events independent of classical Rho‐like GTPases drive scission of wide, uncoated pits of up to 100 nm in diameter leading to PEV formation. Endocytosis further depends on PI3 kinase, protein kinase C and sodium/proton exchange (M. Schelhaas et al., unpublished). Intracellular trafficking occurs through endosomes. Why did viruses evolve to use a particular pathway? With multiple endocytic pathways to choose from, viruses must have evolved to follow a particular route for specific reasons. What are these? To date, very little evidence exists to substantiate answers to this question. One simple reason may be the standard size of endocytic pits/PEVs. Clathrin‐coated pits are somewhat variable in size and can accommodate larger cargo, and the same may be true for other pathways. However, large viruses such as herpesviruses, poxviruses and mimivirus with particles bigger than 150 nm in diameter may have evolved to use macropinocytosis or phagocytosis that easily provide a big enough container for such a large cargo. Another trivial but unsubstantiated reason may be that the primary target cells/tissues for certain viruses more actively deploy a certain endocytic mechanism. However, some evidence for a role of different endocytic organelles in virus entry is provided by the viruses' necessity for membrane penetration and uncoating. Many enveloped viruses require a low intra‐endosomal pH for activation of the fusiogenic activity of the viral glycoproteins to release their capsids into the cytoplasm. Depending on the pH threshold and the intraluminal pH of endosomes, they likely fuse in EE (e.g. VSV, SFV; Marsh et al., 1983; Johannsdottir et al., 2009), ME/LE (e.g. influenza A; Matlin et al., 1981; 1982), or potentially endolysosomes/lysosomes (e.g. vaccinia; Mercer et al., 2008). LCMV may have chosen a route that mostly bypasses EE for a quick rather than a gradual drop in pH, when it is delivered to LE (Quirin et al., 2008). Similarly, non‐enveloped viruses can be pH‐dependent for membrane penetration and uncoating. In addition, viruses may require a low pH in endosomes for the activity of pH‐dependent proteases within endosomes that results in shedding of membrane penetration factors/partial uncoating. In particular cathepsins, endosomal cysteine proteases, were found to proteolytically cleave viral surface proteins of a variety of different viruses/virus families (e.g. Golden et al., 2004; Chandran et al., 2005; Simmons et al., 2005; Schornberg et al., 2006; Diederich et al., 2008; Regan et al., 2008). Most of the proteolytically or pH‐induced structural changes in endosomes occur within minutes, once the threshold has been reached. In contrary, HPV‐16 requires hours in the endosomes for membrane penetration (M. Schelhaas et al., unpubl. results). Hence, we have to learn much more about the kinetics of endocytic events, about timing or retaining of virus trafficking in combination with the associated structural consequences for viruses to understand the role of particular organelle environments in entry. Polyomaviruses such as SV40 and mPy seem to use caveolar/raft‐dependent endocytosis, since it can lead cargo to the endoplasmic reticulum. There, the viruses use molecular chaperones of the biosynthetic machinery to partially uncoat: ERp57 isomerizes intermolecular disulfide bonds in the SV40 capsid to release specifically the vertex capsomers (Schelhaas et al., 2007), ERp29 externalizes the stabilizing C‐terminal arm of the major capsid protein VP1 from intercapsomer connections in mPy (Magnuson et al., 2005). In addition, both seem to use the ER‐associated degradation machinery for transport across the ER membrane to the cytosol for eventual import into the nucleus (Lilley et al., 2006; Schelhaas et al., 2007). The viruses most likely have evolved to use the endocytic route to the ER, since the required factors/functions are unique to this organelle. Finally, a largely speculative thought deals with viral requirements for signal transduction events. As mentioned, virus endocytosis often requires signal transduction. In a systematic siRNA screen for the kinase requirements of VSV and SV40 endocytosis, a high number (208 of 590) was found to regulate the entry of these viruses (Pelkmans et al., 2005). Many of the kinases directly fine tune endocytic events or are important for a certain cell state that allows infection (e.g. migratory versus non‐migratory cells; Snijder et al., 2009). In addition, it is likely that several of the required kinases are needed for viral signal transduction events to prepare the cell for invasion and/or omit anti‐viral responses such as apoptosis. In general, signalling during endocytosis is compartmentalized (Sorkin and von Zastrow, 2009). It can occur at the plasma membrane, it may occur at the plasma membrane and from within endosomes (sustained signalling; Vieira et al., 1996), or it may occur solely from within endosomes (Daaka et al., 1998) depending on the cell system or signalling components. This compartmentalization of signalling provides for a variety of cellular effects that viruses may take advantage of by choosing a particular endocytic pathway. Conclusions Studying virus endocytosis is as complex as the choice of endocytic mechanisms/vesicular traffic and their numerous factors for entry are. An additional level of complexity is provided by existing and potential interconnections and interdependences between endocytic phenomena. The complexity increases even further, since viruses potentially use multiple routes in different cell types and/or within a single cell. Also, it is now clear that low pH‐activated viruses do not necessarily use CME, so that for some viruses it may be required to carefully re‐evaluate their entry pathway. Several considerations need to be made when studying the cell biology of virus endocytosis: virus inocula may contain only a small fraction of particles that enter productively. Microscopic or biochemical methods to follow viruses on their way into the cell do not allow a distinction between productive and non‐productive particles, so that investigations have to be complemented by techniques relying on infection. Cellular perturbation methods include the use of inhibitors, dominant negative mutants, small interfering RNA‐mediated silencing and mutant cells lines. As some of these cellular perturbations cause drastic changes in endocytosis beyond the intended, analysis of the perturbation effects must include morphological/biochemical techniques, and a careful use of cell biological and/or viral controls. Again, only a combination of biochemical, morphological, visual and functional methods allow the pathway(s) of entry to be analysed with confidence. Since viruses potentially use several different endocytic entry mechanisms in different cells or even a single cell, it will be necessary to relate the information obtained in tissue culture systems to in vivo infection models. This will be one of the major challenges for the future, as these model systems may be not available for certain viruses, and as cell biological perturbation studies in these model systems can be extremely difficult. Studies on virus endocytosis can provide new insights into endocytic mechanisms, as viruses can be easily followed by morphological techniques and provide access to reliable endocytosis endpoint detection methods, i.e. infection. These features are already exploited by siRNA screens aimed to determine host cell factors of virusinfections (e.g. Brass et al., 2008; Konig et al., 2008; Zhou et al., 2008; Li et al., 2009; Karlas et al., 2010). The information obtained may in addition to valuable information on the cell biology of virus infections provide new targets for antiviral therapy that would target the host instead of the virus. Acknowledgements