Download Come in and take your coat off how host cells

Cellular Microbiology (2010) 12(10), 1378–1388
doi:10.1111/j.1462-5822.2010.01510.x
First published online 26 August 2010
Microreview
Come in and take your coat off – how host cells
provide endocytosis for virus entry
cmi_1510
Mario Schelhaas*
Emmy-Noether Group ‘Novel endocytic mechanisms
described by viruses’, Institutes of Medical Biochemistry
and Molecular Virology, Centre for Molecular Biology of
Inflammation (ZMBE), University of Münster, Münster,
Germany.
Summary
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. 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.
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
Received 30 May, 2010; revised 11 July, 2010; accepted 12 July,
2010. *For correspondence. E-mail: [email protected];
Tel. (+49) 251 835 7182; Fax (+49) 251 835 7184.
1378..1388
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
© 2010 Blackwell Publishing Ltd
cellular microbiology
Virus endocytosis 1379
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),
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388
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 receptormediated endocytosis is mainly comprised of clathrinmediated 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).
1380 M. Schelhaas
Fig. 1. Overview of the various endocytic mechanisms. In the first row, few exemplary viruses that enter by a respective endocytic mechanism
are given. Below is a schematic depiction of the different mechanisms by which cargo can be internalized from the plasma membrane and
their respective primary endocytic vesicles (PEV). The main intracellular trafficking connections are depicted in the bottom half.
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
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388
Virus endocytosis 1381
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 transGOLGI-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. block© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388
ing 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-4,5phosphate (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,
1382 M. Schelhaas
formation of PEVs requires lipid rafts and tyrosine
kinase/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 lipidsorting 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 mm, 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.
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388
Virus endocytosis 1383
Novel cell biologically described pathways
The internalization of GPI-anchored proteins such as GPIGFP, 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 membranebound, 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 flotillin1-positive PEV fuses with EE. It is noteworthy that flotillin1 depletion may result in decreased or increased
caveolin-1 levels depending on the cell type by a nontranscriptional 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 dynamindependent 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
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388
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
1384 M. Schelhaas
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 intraendosomal 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
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388
Virus endocytosis 1385
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 virus
infections (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
I apologize to all those individuals whose work I could not cite for
space limitations. I thank C. Ehrhardt, V. Gerke, V. Lütschg, M.
Reichmann and N. Wolfrum for critical reading of the manuscript.
This review was supported by the German Science Foundation
(DFG) in the Emmy-Noether-Program.
References
Babuke, T., Ruonala, M., Meister, M., Amaddii, M., Genzler,
C., Esposito, A., and Tikkanen, R. (2009) Heterooligomerization of reggie-1/flotillin-2 and reggie-2/flotillin-1
is required for their endocytosis. Cell Signal 21: 1287–1297.
Bonifacino, J.S., and Rojas, R. (2006) Retrograde transport
from endosomes to the trans-Golgi network. Nat Rev Mol
Cell Biol 7: 568–579.
Brass, A.L., Dykxhoorn, D.M., Benita, Y., Yan, N., Engelman,
A., Xavier, R.J., et al. (2008) Identification of host proteins
required for HIV infection through a functional genomic
screen. Science 319: 921–926.
Braulke, T., and Bonifacino, J.S. (2009) Sorting of lysosomal
proteins. Biochim Biophys Acta 1793: 605–614.
Brown, F.D., Rozelle, A.L., Yin, H.L., Balla, T., and
Donaldson, J.G. (2001) Phosphatidylinositol 4,5bisphosphate and Arf6-regulated membrane traffic. J Cell
Biol 154: 1007–1017.
Burckhardt, C.J., and Greber, U.F. (2009) Virus movements
on the plasma membrane support infection and transmission between cells. PLoS Pathog 5: e1000621.
Cao, W., Henry, M.D., Borrow, P., Yamada, H., Elder, J.H.,
Ravkov, E.V., et al. (1998) Identification of alphadystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus. Science 282: 2079–2081.
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388
Chandran, K., Sullivan, N.J., Felbor, U., Whelan, S.P., and
Cunningham, J.M. (2005) Endosomal proteolysis of the
Ebola virus glycoprotein is necessary for infection. Science
308: 1643–1645.
Clague, M.J., Urbe, S., Aniento, F., and Gruenberg, J. (1994)
Vacuolar ATPase activity is required for endosomal carrier
vesicle formation. J Biol Chem 269: 21–24.
Clement, C., Tiwari, V., Scanlan, P.M., Valyi-Nagy, T., Yue,
B.Y., and Shukla, D. (2006) A novel role for phagocytosislike uptake in herpes simplex virus entry. J Cell Biol 174:
1009–1021.
Conner, S.D., and Schmid, S.L. (2003) Regulated portals of
entry into the cell. Nature 422: 37–44.
Coyne, C.B., and Bergelson, J.M. (2006) Virus-induced Abl
and Fyn kinase signals permit coxsackievirus entry through
epithelial tight junctions. Cell 124: 119–131.
Daaka, Y., Luttrell, L.M., Ahn, S., Della Rocca, G.J.,
Ferguson, S.S., Caron, M.G., and Lefkowitz, R.J. (1998)
Essential role for G protein-coupled receptor endocytosis
in the activation of mitogen-activated protein kinase. J Biol
Chem 273: 685–688.
Damm, E.M., Pelkmans, L., Kartenbeck, J., Mezzacasa, A.,
Kurzchalia, T., and Helenius, A. (2005) Clathrin- and
caveolin-1-independent endocytosis: entry of simian virus
40 into cells devoid of caveolae. J Cell Biol 168: 477–
488.
Diederich, S., Thiel, L., and Maisner, A. (2008) Role of
endocytosis and cathepsin-mediated activation in Nipah
virus entry. Virology 375: 391–400.
Doherty, G.J., and McMahon, H.T. (2009) Mechanisms of
endocytosis. Annu Rev Biochem 78: 857–902.
Dohner, K., and Sodeik, B. (2005) The role of the cytoskeleton during viral infection. Curr Top Microbiol Immunol 285:
67–108.
Ghigo, E., Kartenbeck, J., Lien, P., Pelkmans, L., Capo, C.,
Mege, J.L., and Raoult, D. (2008) Ameobal pathogen mimivirus infects macrophages through phagocytosis. PLoS
Pathog 4: e1000087.
Glebov, O.O., Bright, N.A., and Nichols, B.J. (2006) Flotillin-1
defines a clathrin-independent endocytic pathway in mammalian cells. Nat Cell Biol 8: 46–54.
Golden, J.W., Bahe, J.A., Lucas, W.T., Nibert, M.L., and
Schiff, L.A. (2004) Cathepsin S supports acid-independent
infection by some reoviruses. J Biol Chem 279: 8547–
8557.
Grant, B.D., and Donaldson, J.G. (2009) Pathways and
mechanisms of endocytic recycling. Nat Rev Mol Cell Biol
10: 597–608.
Gruenberg, J. (2001) The endocytic pathway: a mosaic of
domains. Nat Rev Mol Cell Biol 2: 721–730.
Hansen, C.G., and Nichols, B.J. (2009) Molecular mechanisms of clathrin-independent endocytosis. J Cell Sci 122:
1713–1721.
Harrison, S.C. (2008) Viral membrane fusion. Nat Struct Mol
Biol 15: 690–698.
Helenius, A. (2007) Virus entry and uncoating. In Fields Virology, 5th edn. Knipe, D.M. (ed.). Philadelphia: Wolters
Kluwer/Lippincott Williams, pp. 99–118.
Helenius, A., Kartenbeck, J., Simons, K., and Fries, E. (1980)
On the entry of Semliki forest virus into BHK-21 cells. J Cell
Biol 84: 404–420.
1386 M. Schelhaas
Hewlett, L.J., Prescott, A.R., and Watts, C. (1994) The coated
pit and macropinocytic pathways serve distinct endosome
populations. J Cell Biol 124: 689–703.
Hinshaw, J.E. (2000) Dynamin and its role in membrane
fission. Annu Rev Cell Dev Biol 16: 483–519.
Johannsdottir, H.K., Mancini, R., Kartenbeck, J., Amato, L.,
and Helenius, A. (2009) Host cell factors and functions
involved in vesicular stomatitis virus entry. J Virol 83: 440–
453.
Kalia, M., Kumari, S., Chadda, R., Hill, M.M., Parton, R.G.,
and Mayor, S. (2006) Arf6-independent GPI-anchored
protein-enriched early endosomal compartments fuse with
sorting endosomes via a Rab5/phosphatidylinositol-3′kinase-dependent machinery. Mol Biol Cell 17: 3689–
3704.
Karlas, A., Machuy, N., Shin, Y., Pleissner, K.P., Artarini, A.,
Heuer, D., et al. (2010) Genome-wide RNAi screen
identifies human host factors crucial for influenza virus
replication. Nature 463: 818–822.
Kartenbeck, J., Stukenbrok, H., and Helenius, A. (1989)
Endocytosis of simian virus 40 into the endoplasmic reticulum. J Cell Biol 109: 2721–2729.
Kerr, M.C., and Teasdale, R.D. (2009) Defining macropinocytosis. Traffic 10: 364–371.
Kielian, M.C., Marsh, M., and Helenius, A. (1986) Kinetics of
endosome acidification detected by mutant and wild-type
Semliki Forest virus. EMBO J 5: 3103–3109.
Kirkham, M., and Parton, R.G. (2005) Clathrin-independent
endocytosis: new insights into caveolae and non-caveolar
lipid raft carriers. Biochim Biophys Acta 1746: 349–
363.
Kirkham, M., Fujita, A., Chadda, R., Nixon, S.J., Kurzchalia,
T.V., Sharma, D.K., et al. (2005) Ultrastructural identification of uncoated caveolin-independent early endocytic
vehicles. J Cell Biol 168: 465–476.
Konig, R., Zhou, Y., Elleder, D., Diamond, T.L., Bonamy,
G.M., Irelan, J.T., et al. (2008) Global analysis of host–
pathogen interactions that regulate early-stage HIV-1 replication. Cell 135: 49–60.
Kumari, S., and Mayor, S. (2008) ARF1 is directly involved in
dynamin-independent endocytosis. Nat Cell Biol 10:
30–41.
Lafourcade, C., Sobo, K., Kieffer-Jaquinod, S., Garin, J., and
van der Goot, F.G. (2008) Regulation of the V-ATPase
along the endocytic pathway occurs through reversible
subunit association and membrane localization. PLoS
ONE 3: e2758.
Lajoie, P., and Nabi, I.R. (2007) Regulation of raft-dependent
endocytosis. J Cell Mol Med 11: 644–653.
Lakadamyali, M., Rust, M.J., and Zhuang, X. (2006) Ligands
for clathrin-mediated endocytosis are differentially sorted
into distinct populations of early endosomes. Cell 124:
997–1009.
Lamaze, C., Dujeancourt, A., Baba, T., Lo, C.G., Benmerah,
A., and Dautry-Varsat, A. (2001) Interleukin 2 receptors
and detergent-resistant membrane domains define a
clathrin-independent endocytic pathway. Mol Cell 7: 661–
671.
Le Blanc, I., Luyet, P.P., Pons, V., Ferguson, C., Emans, N.,
Petiot, A., et al. (2005) Endosome-to-cytosol transport of
viral nucleocapsids. Nat Cell Biol 7: 653–664.
Lehmann, M.J., Sherer, N.M., Marks, C.B., Pypaert, M., and
Mothes, W. (2005) Actin- and myosin-driven movement of
viruses along filopodia precedes their entry into cells. J Cell
Biol 170: 317–325.
Li, Q., Brass, A.L., Ng, A., Hu, Z., Xavier, R.J., Liang, T.J.,
and Elledge, S.J. (2009) A genome-wide genetic screen for
host factors required for hepatitis C virus propagation. Proc
Natl Acad Sci USA 106: 16410–16415.
Lilley, B.N., Gilbert, J.M., Ploegh, H.L., and Benjamin, T.L.
(2006) Murine polyomavirus requires the endoplasmic
reticulum protein Derlin-2 to initiate infection. J Virol 80:
8739–8744.
Magnuson, B., Rainey, E.K., Benjamin, T., Baryshev, M.,
Mkrtchian, S., and Tsai, B. (2005) ERp29 triggers a conformational change in polyomavirus to stimulate membrane binding. Mol Cell 20: 289–300.
Marsh, M., Bolzau, E., and Helenius, A. (1983) Penetration of
Semliki Forest virus from acidic prelysosomal vacuoles.
Cell 32: 931–940.
Matlin, K.S., Reggio, H., Helenius, A., and Simons, K. (1981)
Infectious entry pathway of influenza virus in a canine
kidney cell line. J Cell Biol 91: 601–613.
Matlin, K.S., Reggio, H., Helenius, A., and Simons, K. (1982)
Pathway of vesicular stomatitis virus entry leading to infection. J Mol Biol 156: 609–631.
Mayor, S., and Pagano, R.E. (2007) Pathways of clathrinindependent endocytosis. Nat Rev Mol Cell Biol 8: 603–
612.
Meier, O., Boucke, K., Hammer, S.V., Keller, S., Stidwill, R.P.,
Hemmi, S., and Greber, U.F. (2002) Adenovirus triggers
macropinocytosis and endosomal leakage together with its
clathrin-mediated uptake. J Cell Biol 158: 1119–1131.
Mercer, J., and Helenius, A. (2008) Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells.
Science 320: 531–535.
Mercer, J., and Helenius, A. (2009) Virus entry by macropinocytosis. Nat Cell Biol 11: 510–520.
Mercer, J., Schelhaas, M., and Helenius, A. (2010) Virus
entry by endocytosis. Annu Rev Biochem 79: 803–833.
Miyauchi, K., Kim, Y., Latinovic, O., Morozov, V., and
Melikyan, G.B. (2009) HIV enters cells via endocytosis and
dynamin-dependent fusion with endosomes. Cell 137:
433–444.
Motley, A., Bright, N.A., Seaman, M.N., and Robinson, M.S.
(2003) Clathrin-mediated endocytosis in AP-2-depleted
cells. J Cell Biol 162: 909–918.
Mukherjee, S., and Maxfield, F.R. (2004) Lipid and cholesterol trafficking in NPC. Biochim Biophys Acta 1685:
28–37.
Mullock, B.M., Bright, N.A., Fearon, C.W., Gray, S.R., and
Luzio, J.P. (1998) Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density
and is NSF dependent. J Cell Biol 140: 591–601.
Naslavsky, N., Weigert, R., and Donaldson, J.G. (2003) Convergence of non-clathrin- and clathrin-derived endosomes
involves Arf6 inactivation and changes in phosphoinositides. Mol Biol Cell 14: 417–431.
Naslavsky, N., Weigert, R., and Donaldson, J.G. (2004)
Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements. Mol Biol Cell 15:
3542–3552.
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388
Virus endocytosis 1387
Nemerow, G.R. (2000) Cell receptors involved in adenovirus
entry. Virology 274: 1–4.
Nicola, A.V., McEvoy, A.M., and Straus, S.E. (2003) Roles for
endocytosis and low pH in herpes simplex virus entry into
HeLa and Chinese hamster ovary cells. J Virol 77: 5324–
5332.
Parton, R.G., and Richards, A.A. (2003) Lipid rafts and
caveolae as portals for endocytosis: new insights and
common mechanisms. Traffic 4: 724–738.
Payne, C.K., Jones, S.A., Chen, C., and Zhuang, X. (2007)
Internalization and trafficking of cell surface proteoglycans
and proteoglycan-binding ligands. Traffic 8: 389–401.
Pelkmans, L., Kartenbeck, J., and Helenius, A. (2001)
Caveolar endocytosis of simian virus 40 reveals a new
two-step vesicular-transport pathway to the ER. Nat Cell
Biol 3: 473–483.
Pelkmans, L., Puntener, D., and Helenius, A. (2002) Local
actin polymerization and dynamin recruitment in SV40induced internalization of caveolae. Science 296: 535–
539.
Pelkmans, L., Fava, E., Grabner, H., Hannus, M.,
Habermann, B., Krausz, E., and Zerial, M. (2005) Genomewide analysis of human kinases in clathrin- and caveolae/
raft-mediated endocytosis. Nature 436: 78–86.
Pfeffer, S., and Aivazian, D. (2004) Targeting Rab GTPases
to distinct membrane compartments. Nat Rev Mol Cell Biol
5: 886–896.
Piper, R.C., and Katzmann, D.J. (2007) Biogenesis and function of multivesicular bodies. Annu Rev Cell Dev Biol 23:
519–547.
Qian, M., Cai, D., Verhey, K.J., and Tsai, B. (2009) A lipid
receptor sorts polyomavirus from the endolysosome to the
endoplasmic reticulum to cause infection. PLoS Pathog 5:
e1000465.
Quirin, K., Eschli, B., Scheu, I., Poort, L., Kartenbeck, J., and
Helenius, A. (2008) Lymphocytic choriomeningitis virus
uses a novel endocytic pathway for infectious entry via late
endosomes. Virology 378: 21–33.
Racoosin, E.L., and Swanson, J.A. (1993) Macropinosome
maturation and fusion with tubular lysosomes in macrophages. J Cell Biol 121: 1011–1020.
Raghu, H., Sharma-Walia, N., Veettil, M.V., Sadagopan, S.,
and Chandran, B. (2009) Kaposi’s sarcoma-associated
herpesvirus utilizes an actin polymerization-dependent
macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial
cells. J Virol 83: 4895–4911.
Regan, A.D., Shraybman, R., Cohen, R.D., and Whittaker,
G.R. (2008) Differential role for low pH and cathepsinmediated cleavage of the viral spike protein during entry of
serotype II feline coronaviruses. Vet Microbiol 132: 235–
248.
Rink, J., Ghigo, E., Kalaidzidis, Y., and Zerial, M. (2005) Rab
conversion as a mechanism of progression from early to
late endosomes. Cell 122: 735–749.
Robinson, M.S. (2004) Adaptable adaptors for coated
vesicles. Trends Cell Biol 14: 167–174.
Rojek, J.M., Perez, M., and Kunz, S. (2008) Cellular entry of
lymphocytic choriomeningitis virus. J Virol 82: 1505–1517.
Rust, M.J., Lakadamyali, M., Zhang, F., and Zhuang, X.
(2004) Assembly of endocytic machinery around individual
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388
influenza viruses during viral entry. Nat Struct Mol Biol 11:
567–573.
Sabharanjak, S., Sharma, P., Parton, R.G., and Mayor, S.
(2002) GPI-anchored proteins are delivered to recycling
endosomes via a distinct cdc42-regulated, clathrinindependent pinocytic pathway. Dev Cell 2: 411–423.
Sapp, M., and Day, P.M. (2009) Structure, attachment and
entry of polyoma- and papillomaviruses. Virology 384:
400–409.
Sauvonnet, N., Dujeancourt, A., and Dautry-Varsat, A. (2005)
Cortactin and dynamin are required for the clathrinindependent endocytosis of gammac cytokine receptor.
J Cell Biol 168: 155–163.
Schelhaas, M., Malmstrom, J., Pelkmans, L., Haugstetter, J.,
Ellgaard, L., Grunewald, K., and Helenius, A. (2007)
Simian Virus 40 depends on ER protein folding and
quality control factors for entry into host cells. Cell 131:
516–529.
Schelhaas, M., Ewers, H., Rajamaki, M.L., Day, P.M.,
Schiller, J.T., and Helenius, A. (2008) Human papillomavirus type 16 entry: retrograde cell surface transport along
actin-rich protrusions. PLoS Pathog 4: e1000148.
Schornberg, K., Matsuyama, S., Kabsch, K., Delos, S.,
Bouton, A., and White, J. (2006) Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein.
J Virol 80: 4174–4178.
Selinka, H.C., Florin, L., Patel, H.D., Freitag, K., Schmidtke,
M., Makarov, V.A., and Sapp, M. (2007) Inhibition of transfer to secondary receptors by heparan sulfate-binding drug
or antibody induces noninfectious uptake of human papillomavirus. J Virol 81: 10970–10980.
Simmons, G., Gosalia, D.N., Rennekamp, A.J., Reeves, J.D.,
Diamond, S.L., and Bates, P. (2005) Inhibitors of
cathepsin L prevent severe acute respiratory syndrome
coronavirus entry. Proc Natl Acad Sci USA 102: 11876–
11881.
Snijder, B., Sacher, R., Ramo, P., Damm, E.M., Liberali, P.,
and Pelkmans, L. (2009) Population context determines
cell-to-cell variability in endocytosis and virus infection.
Nature 461: 520–523.
Sorkin, A., and von Zastrow, M. (2009) Endocytosis and
signalling: intertwining molecular networks. Nat Rev Mol
Cell Biol 10: 609–622.
Spoden, G., Freitag, K., Husmann, M., Boller, K., Sapp, M.,
Lambert, C., and Florin, L. (2008) Clathrin- and
caveolin-independent entry of human papillomavirus type
16 – involvement of tetraspanin-enriched microdomains
(TEMs). PLoS ONE 3: e3313.
Swanson, J.A. (2008) Shaping cups into phagosomes and
macropinosomes. Nat Rev Mol Cell Biol 9: 639–649.
Swanson, J.A., and Watts, C. (1995) Macropinocytosis.
Trends Cell Biol 5: 424–428.
Ungewickell, E.J., and Hinrichsen, L. (2007) Endocytosis:
clathrin-mediated membrane budding. Curr Opin Cell Biol
19: 417–425.
Vassilieva, E.V., Ivanov, A.I., and Nusrat, A. (2009) Flotillin-1
stabilizes caveolin-1 in intestinal epithelial cells. Biochem
Biophys Res Commun 379: 460–465.
Vieira, A.V., Lamaze, C., and Schmid, S.L. (1996) Control of
EGF receptor signaling by clathrin-mediated endocytosis.
Science 274: 2086–2089.
1388 M. Schelhaas
Vonderheit, A., and Helenius, A. (2005) Rab7 associates
with early endosomes to mediate sorting and transport
of Semliki forest virus to late endosomes. PLoS Biol 3:
e233.
Whittaker, G.R., Kann, M., and Helenius, A. (2000) Viral entry
into the nucleus. Annu Rev Cell Dev Biol 16: 627–651.
Zerial, M., and McBride, H. (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2: 107–117.
Zhou, H., Xu, M., Huang, Q., Gates, A.T., Zhang, X.D.,
Castle, J.C., et al. (2008) Genome-scale RNAi screen for
host factors required for HIV replication. Cell Host Microbe
4: 495–504.
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 12, 1378–1388