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Virus Entry by Endocytosis
Jason Mercer,1 Mario Schelhaas,2 and Ari Helenius1
1
ETH Zurich, Institute of Biochemistry, CH-8093 Zurich, Switzerland;
email: [email protected], [email protected]
2
University of Münster, Institutes for Medical Biochemistry and Molecular Virology,
Centre for Molecular Biology of Inflammation (ZMBE), D-48149 Münster, Germany;
email: [email protected]
Annu. Rev. Biochem. 2010. 79:803–33
Key Words
First published online as a Review in Advance on
March 2, 2010
caveolar/raft-dependent endocytosis, clathrin/caveolin-independent
endocytosis, clathrin-mediated endocytosis, endosome network,
macropinocytosis
The Annual Review of Biochemistry is online at
biochem.annualreviews.org
This article’s doi:
10.1146/annurev-biochem-060208-104626
c 2010 by Annual Reviews.
Copyright All rights reserved
0066-4154/10/0707-0803$20.00
Abstract
Although viruses are simple in structure and composition, their interactions with host cells are complex. Merely to gain entry, animal viruses
make use of a repertoire of cellular processes that involve hundreds of
cellular proteins. Although some viruses have the capacity to penetrate
into the cytosol directly through the plasma membrane, most depend on
endocytic uptake, vesicular transport through the cytoplasm, and delivery to endosomes and other intracellular organelles. The internalization
may involve clathrin-mediated endocytosis (CME), macropinocytosis,
caveolar/lipid raft-mediated endocytosis, or a variety of other still poorly
characterized mechanisms. This review focuses on the cell biology of
virus entry and the different strategies and endocytic mechanisms used
by animal viruses.
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Contents
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INTRODUCTION . . . . . . . . . . . . . . . . . .
ADVANTAGES OF USING
ENDOCYTOSIS FOR ENTRY . . .
VIRUSES AS ENDOCYTIC
CARGO . . . . . . . . . . . . . . . . . . . . . . . . . .
ATTACHMENT FACTORS
AND RECEPTORS . . . . . . . . . . . . . . .
MECHANISMS OF
ENDOCYTOSIS. . . . . . . . . . . . . . . . . .
LOGISTICS OF THE
ENDOSOME NETWORK . . . . . . .
APPROACHES TO STUDY
VIRUS ENTRY . . . . . . . . . . . . . . . . . . .
VIRUSES THAT USE THE
CLATHRIN PATHWAY . . . . . . . . . .
MACROPINOCYTOSIS AND
VIRUS ENTRY . . . . . . . . . . . . . . . . . . .
VIRUSES THAT USE
CAVEOLAR/RAFTDEPENDENT ENTRY . . . . . . . . . . .
VIRUSES THAT USE UNUSUAL
ENDOCYTIC PATHWAYS . . . . . . .
VIRUS ENTRY BY
PHAGOCYTOSIS . . . . . . . . . . . . . . . .
PERSPECTIVES . . . . . . . . . . . . . . . . . . . .
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804
805
805
806
807
812
813
818
819
821
823
823
INTRODUCTION
Viruses first bind to cell surface proteins, carbohydrates, and lipids. Interactions with virus
receptors are often specific and multivalent, and
these interactions lead to the activation of cellular signaling pathways. Cells respond by internalizing the viruses using one of several endocytic mechanisms. After arrival in the lumen of
endosomes or the endoplasmic reticulum (ER),
viruses receive cues in the form of exposure to
low pH, proteolytic cleavage and activation of
viral proteins, and/or association with cell proteins. These trigger changes in the virus particle, and the activated viruses penetrate the vacuolar membrane, delivering the viral genome,
the capsid, or the intact viral particle into the
cytosol. After penetration, most RNA viruses
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replicate in different locations within the cytosol, whereas most DNA viruses continue their
journey to the nucleus. Parallel with the movement of the virus and viral capsids deeper into
the cell, a process of stepwise disassembly and
uncoating takes place, culminating in the controlled release of the genome and accessory
proteins in a replication-competent form.
Detailed understanding of virus-host cell interactions is important for several reasons. First,
the increase in world population, the explosion in international trade and travel, global
warming, and other factors have led to an increased threat from infectious pathogens, including viruses (1). Any information that can
help in the battle against existing and emerging viruses has high priority. Virus-cell interactions provide an area that is still incompletely
explored and underexploited for antiviral
strategies.
Second, viruses are increasingly used as tools
in molecular medicine. They have evolved to
master the art of entering cells and introducing
“foreign” genes and macromolecules. Therefore, they are useful devices in gene therapy and
in the delivery of macromolecules and drugs
into cells. They also have the potential for
targeted elimination of cancer cells. Finally,
viruses continue to serve as important tools and
model systems in the discovery of new concepts
in molecular, structural, and cell biology.
ADVANTAGES OF USING
ENDOCYTOSIS FOR ENTRY
Elegant morphological, genetic, and biochemical studies of bacteriophages in the 1960s
uncovered mechanisms of infection of great
complexity and ingenuity. They showed that
coliphages T4 and T2 are constructed like
hypodermic syringes with a contractile tail
forming a DNA delivery apparatus capable
of piercing through the two membranes of a
gram-negative bacteria and injecting the DNA
into the cytosol (2). Attachment to appropriate
host cell receptors was found to serve as the
cue that triggers the injection process. Animal
viruses do not need such elaborate instruments
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for entry because their host cells lack the
outer membrane and cell wall that, in bacteria,
prevent direct access to the plasma membrane.
Also, animal cells provide endocytic mechanisms that give incoming viruses advantages
that bacteriophages do not have. Endocytic
vesicles ferry incoming viruses from the periphery to the perinuclear area of the host cell,
where conditions for infection are favorable
and distance to the nucleus minimal. This
allows viruses to bypass obstacles associated
with cytoplasmic crowding and the meshwork
of microfilaments in the cortex (3, 4). Transport
in endocytic vesicles is particularly important
for viruses that infect neurons, where long
distances separate axons from the cell body.
The maturation of endosomes with gradually
changing conditions, such as a decreasing pH
and a change in redox environment, allows
viruses, moreover, to sense their location
within the cell and the pathway and to use
this information to set the time of penetration
and uncoating (5). The presence of specific
proteases, such as furin and cathepsins, provides necessary proteolytic activation of certain
viruses (6, 7). Finally, by being endocytosed,
animal viruses can avoid leaving evidence of
their presence exposed on the plasma membrane, thus likely causing a delay in detection
by immunosurveillance. Taken together, endocytosis is so advantageous that viruses, such as
herpes simplex virus 1 and human immunodeficiency virus 1 (HIV-1), that are capable of
entering directly often prefer to use endocytic
pathways for productive entry (8–10).
The topic of viral endocytosis and related
topics have been previously reviewed and discussed (5, 11–17), together with recent reviews
on endocytic pathways (18–27). These reviews
are highly recommended for more detail and
for different points of view regarding this broad
topic.
range. Although usually roughly spherical in
shape, viruses, such as Filovirus, Paramyxovirus,
and influenza viruses, are (or can be) fibrous and
highly elongated. As they bind to cells, viruses
are not deformed; it is rather the plasma membrane that shows a tendency to invaginate to
accommodate the shape of the virus. In some
cases, such invagination is essential for endocytosis (see below).
The surface of viruses is typically covered
by receptor-binding proteins either in the form
of capsid proteins in an icosahedral grid or as
spike glycoproteins covering a viral envelope.
Individual interactions with receptors are generally weak, but contact with multiple receptors
make the avidity high and binding to cells virtually irreversible. Multivalent binding leads to
receptor clustering, which in turn may result in
association with lipid domains and activation of
signaling pathways.
Once delivered to endosomes, most virus
particles are similar in size to the intralumenal vesicles (ILVs). Too big to enter the narrow
tubular extensions, they are generally localized
to the bulbous, vacuolar domains of endosomes
and are thus sorted to the degradative pathway.
Viruses have often been used as model
cargo in endocytosis and membrane trafficking
studies. They are easily recognized by electron
microscopy (EM) and can be tagged with
fluorescent groups or proteins, allowing singleparticle detection and tracking in live cells.
By providing a single spot-like source of light,
the center of mass of fluorescent viruses can
be precisely defined through the point-spread
function (28). Owing to the amplification
caused by infection, successful entry can be
easily quantified, even with minute amounts
of virus. In addition, the virology community
has developed a large number of tools, such as
virus mutants, fluorescent viruses, antibodies,
expression systems, and modified host cells.
VIRUSES AS ENDOCYTIC CARGO
ATTACHMENT FACTORS
AND RECEPTORS
The size of animal viruses varies from about
30 nm for parvoviruses to 400 nm for
poxviruses, with most viruses in the 60–150-nm
ILV: Intralumenal
vesicle
Although some viruses take advantage of receptors with known endocytic receptor functions,
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EE: early endosome
LE: late endosome
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Clathrin-mediated
endocytosis (CME):
an endocytic process
driven by the
formation of a clathrin
coat on the
cytoplasmic leaflet of
the plasma membrane
Macropinocytosis:
actin-mediated
endocytic process
involving plasma
membrane ruffles and
internalization of fluid
and particles in large,
uncoated, endocytic
vesicles
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such as transferrin and low-density lipoprotein
(LDL) receptors, most of the molecules that
viruses bind to are involved in other functions
such as cell-cell recognition, ion transport, and
binding to the extracellular matrix (16). Often
they are glycoconjugates (glycoproteins, glycolipids, proteoglycans), and the carbohydrate
moieties can play a central role in virus binding.
From a conceptual point of view, it is useful
to differentiate between attachment factors that
merely bind viruses and thus help to concentrate the viruses on the cell surface, and virus
receptors, which in addition serve to trigger
changes in the virus, induce cellular signaling,
or trigger penetration. One function that many
receptors share is that they promote endocytosis and accompany the virus into the cell. Often
entry starts with binding to attachment factors,
followed by associations with one or more receptors. In practice, it is often difficult to differentiate between attachment factors and receptors because both contribute to the effectiveness
of infection.
The most commonly encountered attachment factors are glycosaminoglycan (GAG)
chains—especially heparan sulphate—in proteoglycans. Binding to these negatively charged
polysaccharides is usually electrostatic and relatively nonspecific. It has been recognized in several cases that viruses evolve to use GAGs when
adapting to growth in tissue culture (29, 30).
Sialic acids constitute another common group
of carbohydrates to which many viruses bind.
As in the case of influenza and polyomaviruses,
this binding is often highly specific and involves
defined lectin domains or lectin sites (16).
The specificity of binding is a major factor
in determining tropism and species specificity,
and thus the nature of viral diseases. Another
aspect of receptor specificity is that it determines the choice of endocytic pathway and intracellular routing that the incoming virus will
take. For example, parvoviruses that bind to the
transferrin receptor use a clathrin-mediated uptake pathway and are able to recycle to the cell
surface with their receptor (31), whereas minor group rhinoviruses that bind to the LDL
receptor dissociate from the receptor in early
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endosomes (EEs) and are transported to late
endosomes (LEs) (32).
Some receptors are responsible for inducing
changes in the virus that allow binding of the
virus to a coreceptor, induction of endocytic uptake, or conversion to a membrane fusion-active
conformation. The best-characterized case is
HIV-1, where two receptors are required to induce conformational changes that trigger fusion (33). Adenoviruses 2 and 5 have two receptors that induce conformational changes and
promote endocytosis (34). In the case of avian
leukosis virus, the cues required for penetration are receptor binding combined with low
pH (35).
The use of different receptors often correlates with the need for a virus to overcome barriers existing in the cell type or tissue that they
infect. One well-studied example is the binding of Coxsackievirus B to decay-accelerating
factor (DAF) in the apical surface of epithelial
cells, and subsequently to the Coxsackievirus
and adenovirus receptor (CAR), which is localized in the tight junction region. DAF helps to
bring the virus to the tight junctions, and CAR
induces a conformational change and promotes
endocytosis (36). For hepatitis C virus (HCV),
several different receptors are thought to have
a similar “shuttle” function during infection of
hepatocytes (37).
MECHANISMS OF ENDOCYTOSIS
Viruses typically make use of various pinocytic
mechanisms of endocytosis that serve the cell by
promoting the uptake of fluid, solutes, and small
particles (Figure 1). Best studied are clathrinmediated endocytosis (CME), macropinocytosis, and caveolar/raft-dependent endocytosis.
There are, in addition, several clathrin- and
caveolin/raft-independent mechanisms that are
less well characterized but under active investigation. The formation of primary endocytic
vesicles and vacuoles involves a large number of
cellular factors; some are listed in Table 1. The
factors are different for the various pathways
and only partially known. The involvement of
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Macropinocytosis
Caveolin/lipid raft
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CME
VSV
SFV
Dengue
Rhino
Adeno 2/5
Influenza A
Phagocytosis
Vaccinia
HSV-1
Adeno 3
SV40
SV40
mPy
Plasma membrane
Novel
pathways
LCMV
Influenza A
HPV-16
Clathrin
IL2
Mimivirus
Dynamin
GEEC
Flotillin
Rota?
Caveolin
Arf6
Cytoplasm
Flotillin
Figure 1
Endocytic mechanisms. Endocytosis in animal cells can occur via several different mechanisms. Multiple mechanisms are defined as
pinocytic, i.e., they involve the uptake of fluid, solutes, and small particles. These include clathrin-mediated, macropinocytosis,
caveolar/raft-mediated mechanisms, as well as several novel mechanisms. Some of these pathways involve dynamin-2 as indicated by the
beads around the neck of the endocytic indentations. Large particles are taken up by phagocytosis, a process restricted to a few cell
types. In addition, there are pathways such as IL-2, the so-called GEEC pathway, and the flotillin- and ADP-ribosylation factor 6
(Arf6)-dependent pathways that carry specific cellular cargo but are not yet used by viruses. Abbreviations: Adeno 2/5, adenovirus 2/5;
Adeno 3, adenovirus 3; CME, clathrin-mediated endocytosis; HPV-16, human papillomavirus 16; HSV-1, herpes simplex virus 1;
LCMV, lymphocytic choriomeningitis virus; mPy, mouse polyomavirus; SFV, Semliki Forest virus; SV40, simian virus 40;
VSV, vesicular stomatis virus.
the various pathways in virus entry is discussed
below.
It is generally assumed that viruses take
advantage of existing mechanisms. Whereas,
some are internalized by ongoing endocytic activities, many actually induce their own uptake by activating cellular signal transduction
pathways. Caveolar/raft-dependent endocytosis, for example, is preceded by the activation
of several tyrosine and other kinases (26, 38).
Macropinocytosis of vaccinia virus requires activation of kinases and GTPases that regulate
changes in actin dynamics (39). CME can also
be virus triggered (14, 40, 41). For some of the
novel virus entry pathways, the physiological
functions are not yet known.
LOGISTICS OF THE
ENDOSOME NETWORK
Once internalized within primary endocytic
vesicles, the intracellular pathways followed by
incoming viruses are the same as those used
by physiological ligands and membrane components, such as nutrients and their carriers,
hormones, growth factors, extracellular matrix
components, plasma membrane factors, and
lipids. The endosomal system in question is
responsible for molecular sorting, recycling,
degradation, storage, processing, and transcytosis of incoming substances, collectively called
cargo.
The main organelle classes are EEs and LEs
(the latter often taking the form of multivesicular bodies), recycling endosomes (REs), and
lysosomes (Figure 2) (25, 42–47). Also included
in the figure is a class of intermediate organelles
between EEs and LEs, which have been called
maturing endosomes (MEs) because they contain both Rab5 and Rab7 and serve as precursors
for LEs (42, 48, 49). They are likely to play a
role in the entry of several viruses. The endosome system is tightly connected with the secretory pathway through vesicles shuttling between endosomes and the trans-Golgi network
(TGN), and also via the plasma membrane.
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RE: recycling
endosome
ME: maturing
endosome
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Table 1
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Cellular endocytic pathways and their cellular factors
Endocytic pathway
Cellular factors
Clathrin-mediated
Coat proteins
Clathrin heavy chain, clathrin light chain
Adaptors
AP2,a eps15,a epsin1a
Scission factors
Dynamin-2
Regulatory factors
PI(3,4)P, PI(4,5)P2, cholesterol,a cortactin,a Arp2/3a
Cytoskeleton
Actin,a microtubulesa
Trafficking
Rab5, Rab7,a Rab4,a Rab11,a Rab22a
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Macropinocytosis
Coat proteins
None
Scission factors
Unknown
Regulatory factors
Tyrosine kinases, PAK1, PI(3)K, PKC, Ras, Rac1, Cdc42,a Rab34,a CtBP1, Na+/ H+ exchange, cholesterol
Cytoskeleton
Actin, microtubules,a myosinsa
Trafficking
Rab5,a Rab7,a Arf6a
CAV1 pathway
Coat proteins
Caveolin-1
Scission factors
Dynamin-2
Regulatory factors
Tyrosine kinases, phosphatases, PKC, RhoA, cholesterol
Cytoskeleton
Actin, microtubules
Trafficking
Rab5
Lipid raft
Coat proteins
None (clathrin and caveolin independent)
Scission factors
Unknown (dynamin-2 independent)
Regulatory factors
Tyrosine kinases, Rho A, cholesterol
Cytoskeleton
Actin
Trafficking
Unknown
IL-2 pathway
Coat proteins
None
Scission factors
Dynamin-2
Regulatory factors
RhoA, lipid rafts, ubiquitination
Cytoskeleton
Actin
Trafficking
Rab5,a Rab 7
GEEC pathway
Coat proteins
Unknown, GRAF1a
Scission factors
Unknown
Regulatory factors
Arf1, ARHGAP10, Cdc42, lipid rafts
Cytoskeleton
Actin
Trafficking
Rab5, PI(3)K
Flotillin pathway
Coat proteins
Flotillin-1
Scission factors
Unknown
Regulatory factors
Fyn kinase, lipid rafts
Cytoskeleton
Unknown
Trafficking
Rab5, PI(3)K
(Continued )
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(Continued )
Endocytic pathway
Cellular factors
Arf6 pathway
Coat proteins
None
Scission factors
Unknown (dynamin independent)
Regulatory factors
PIP5K, Arf6, Arf1, PI(4,5)P, PI(3)K, cholesterol, actin
Cytoskeleton
Actin
Trafficking
Rab5, Rab7 (degradation) Rab11, Rab22 (recycling only)
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Phagocytosis
Coat proteins
None (particle driven)
Adaptors
AP2a
Scission factors
Dynamin-2
Regulatory factors
Tyrosine kinases, PI(3)K, PKC, Ras, RhoA, RhoG, Rac1,a Cdc42,a Arf6,a cholesterol
Cytoskeleton
Actin, microtubules, myosins
Trafficking
Rab5, Rab7
a
Factors that are required in a cell type or system-dependent fashion.
Abbreviations: Arf1, -6, ADP-ribosylation factor-1 and 6; ARHGAP10, Rho GTPase-activating protein 1; Arp2/3, actin-related proteins 2/3; Cdc42, cell
division control protein 42; CtBP1, C-terminal-binding protein 1; PI(3)K, phosphoinositide 3-kinase; PI(3,4)P, phosphoinositol-3,4 phosphate; PI(4,5)P2,
phosphatidylinositol 4,5-bisphosphate; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; PKC, protein kinase C; Rab4, -5, -7, -11, -22, Ras-related in
brain; Rac1, Ras-related C3 botulinum toxin substrate 1; Ras, Rat sarcoma viral oncogene homolog; RhoA, Ras homolog gene family, member A;
RhoG, Ras homolog gene family, member G.
b
Cargo delivered from the surface by CME
typically reaches EEs in less than 2 min after internalization, MEs and LEs in the perinuclear region after 10–12 min, and the lysosomes within 30–60 min (50, 51). The different
classes of endosomes are heterogeneous in composition, and their function and transit through
the pathway are highly asynchronous (52). Recent studies in live cells suggest that there are
at least two populations of EEs: highly motile
and rapidly maturing as well as more static and
slowly maturing (50).
To manage their various molecular sorting and trafficking functions, EEs have a complex structure with vacuolar elements and many
long, narrow, often-branched tubes (53, 54).
The membrane is composed of a patchwork
of functionally different domains (20, 23, 44).
These differ in composition, location, and
structure (tubular or vacuolar), and EEs are
often defined by different Rabs and their effectors. The domains are responsible for selective vesicular transport to distinct targets:
the plasma membrane (Rab4), LEs (Rab7), REs
(Rab22), and the TGN (Rab9 and the retromer
complex). Most of the domains are located in
the tubular part of the endosomes.
It is important to recognize that each endosome class corresponds to a heterogeneous collection of organelles and that they go through a
program of changes with time. The conversion
of EEs to LEs is a particularly complex process
and the subject of a long-standing discussion:
Does it represent maturation or vesicle transport (45, 48, 55)? The issue is partly semantic
with current experimental data suggesting that
both models apply (42, 48, 56). The process involves the formation of an ME, a hybrid endosome with Rab5 and Rab7 domains (Figure 2).
The ME mainly contains the vacuolar component of EEs and the ILVs. It undergoes a maturation program of considerable significance for
viruses such as influenza and simian virus 40
(SV40), which use MEs and LEs for entry. The
repertoire of changes include the following:
1. There is a loss of recycling receptors, such
as those for transferrin and LDL, and
other membrane proteins and lipids targeted for recycling to the plasma membrane. These leave the EEs either by
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direct vesicle transport or via the REs, often located in the perinuclear region. It
has been found that lipids with short and
unsaturated alkyl chains tend to sort to
recycling endosomes, whereas lipids that
have long and saturated alkyl chains, such
as gangliosides, preferentially enter LEs
(57).
Plasma membrane
Cytoplasm
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Rab4
EE
Rab11
pH 6.5–6.0
Rab5
ESCRT
Rab22
RE
pH 6.0
ME
pH 6.0–5.0
LE
Rab5
Rab7
ESCRT
Rab7
Rab9
LAMP-1
TGN
Golgi
Rab7
Rab9
LAMP-1
Endolysosome
pH 5.0–4.5
ER
?
ESCRT complex
Intralumenal vesicles (ILV)
Microtubular transport
RE Recycling endosome
EE Early endosome
ME Maturing endosome
LE Late endosome
TGN Trans-Golgi network
ER Endoplasmic reticulum
Nuclear pore complex
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Lysosome
Nucleus
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2. A gradual drop in internal pH occurs from
mild acidity (pH 6.5–6.0) in EEs to values
below 5. This change is most likely caused
by variation in subunit composition and
possibly concentration of the v-ATPase,
as well as different isoforms of chloride
channels (58, 59).
3. The formation of ILVs occurs. This is
a function carried out by the endosomal sorting complex required for transport (ESCRT) complexes associated with
the membrane of EEs and MEs (60).
Monoubiquitin-tagged membrane proteins are selectively included in the ILVs.
The result is the formation of LEs filled
with vesicles (multivesicular bodies) destined for lysosomal degradation.
4. There is a switch of Rab subsets (i.e.,
Rab5 and Rab4 to Rab7 and Rab9)
and their effectors. This also results in
a change in predominant phosphatidylinositides from PI(3)P to PI(3,5)P2 (61,
62). Together, these changes have many
consequences in defining associated peripheral proteins.
5. A change in the interaction with cytoskeletal elements (mainly microtubules
and their motors) leads to endosome migration from the peripheral to the perinuclear area of the cell and, eventually, to
fusion with lysosomes.
The elements in the maturation program are
interconnected and interdependent in complex
ways. This is seen when one of the functions is
perturbed. The effect is often a disruption or
retardation of the whole program. Disruptions
can be induced by overexpressing different Rabs
and their mutants, by preventing the formation
of ILVs, by inhibiting endosome movement to
the perinuclear region using inhibitors of microtubule assembly, by inhibiting PI(3)-kinases,
by preventing acidification, by blocking maturation with a drop in temperature, and by other
perturbations. For example, in HeLa cells, inhibition of the v-ATPase with bafilomycin A1
blocks formation of MEs (63–65), and the addition of nocodazole blocks LE formation and
transport of cargo to REs (64). Wortmannin [a
PI(3)-kinase inhibitor] causes a delay in EE to
LE transport, and incubation at 20◦ C blocks LE
fusion with lysosomes (64, 66). How useful such
perturbations can be in the analysis of virus entry is exemplified by recent work on major and
minor group human rhinoviruses (67).
It is important to recognize that the
endosome network is a continuum with heterogeneous classes of organelles continuously
undergoing changes through retrieval, recycling, dissociation, entrapment, processing,
fusion, fission, and recruitment of molecular
components (68). To a considerable degree,
it is a self-organizing system able to adjust to
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 2
The endosome network. The main organelles of the endocytic pathway are the early endosomes (EEs), maturing endosomes (MEs),
late endosomes (LEs), recycling endosomes (REs), and lysosomes. EEs are usually located in the periphery of the cytoplasm. They are
complex organelles with several different domains (tubular and vacuolar). The tubular domains 50–90 nm in diameter and up to four
microns in length contain most of the endosomal membrane and give rise to recycling vesicles and vesicles for transport to the REs and
to the trans-Golgi network (TGN). The vacuolar domains 200–1000 nm in diameter contain most of the volume, the intralumenal
vesicles (ILVs), and larger endocytosed particles. The vacuolar domains dissociate (with their contents) and undergo microtubulemediated, dynein-dependent movement to the perinuclear region. These MEs contain markers of both early and late endosomes, such
as Rab5 and Rab7. They also undergo further acidification and conversion to mature LEs, which can fuse with each other and
eventually with lysosomes, generating endolysosomes in which active degradation takes place. The dense core lysosomes correspond to
the end points of such degradation processes; they serve as a depository for lysosomal enzymes and membrane proteins awaiting fusion
with incoming LEs. The majority of incoming membrane components undergo recycling to the plasma membrane. However,
membrane proteins destined for degradation are first tagged with monoubiquitins, then recognized by ubiquitin-binding components
of the endosomal sorting complex required for transport (ESCRT) machinery, and finally sequestered into ILVs. ILVs are formed by
inward budding of EEs and LEs. They fill the lumen of the vacuolar domains, forming multivesicular bodies, and are eventually
degraded in lysosomes. Abbreviation: LAMP-1, lysosomal-associated membrane protein.
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changes in physiological conditions, the nature
and quantity of incoming cargo, perturbations,
and cell function. It is regulated by complex
feed-back and feed-forward regulatory cycles
involving numerous kinases, phosphatases,
GTPases, and other factors (69, 70).
APPROACHES TO STUDY
VIRUS ENTRY
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Early studies of virus entry relied extensively on
EM, a technology of continued importance because of the new techniques of great promise,
such as cryo-EM, tomography, and focused
ion beam-scanning electron microscopy (FIBSEM) (71, 72). The newer techniques allow better organelle preservation, three-dimensional
imaging, and correlative analysis with light microscopy. Light microscopy is also increasingly
important because of its enhanced resolution
and the possibility to follow incoming fluorescent viruses in live cells (56, 73, 74). With host
components tagged with green fluorescent protein (GFP) and other fluorescent proteins, it
is possible to track the fate of individual particles and determine which cell proteins are involved. Studies using single-particle tracking
have greatly expanded our understanding of
virus entry (73, 75–78). By taking advantage of
the point-spread function, one can, under favorable conditions, define the center of a virus
particle with a resolution of less than 5 nm (79).
Perturbations using inhibitors, dominant
negative (D/N) or constitutively active (C/A)
constructs of cellular proteins, siRNA silencing, and cell mutants provide another general
approach commonly used. When adequately
controlled and combined with specific, quantitative assays for infection, endocytosis, and
penetration, these perturbations are quite powerful. The challenges and potential pitfalls include cell toxicity, cell type variability, off-target
effects, unwanted side effects of protein tagging, and poor transfection or silencing efficiency. The compensatory activation of alternative pathways when one pathway is closed down
is another problem, especially in long-lasting
experiments (19).
812
Mercer
·
Schelhaas
·
Helenius
Chemical inhibitors used today in virus
entry span a wide array of effects, including
signaling, regulation, endocytic machinery,
cytoskeleton function, lipid composition of
membranes, and others. Here, we will only
mention a couple of inhibitor classes commonly
employed because their use is not as straightforward as often thought. Lysosomotropic weak
bases (such as ammoniun chloride, chloroquine, methylamine), carboxylic ionophores
(monensin, nigericin), and v-ATPase inhibitors
(bafilomycin A1 and concanamycin) are often
employed to determine whether viruses require
low pH for infection. The inhibition of virus
infection by one or more of these pH perturbants does not automatically mean that the
virus undergoes a pH-induced conformational
change. Inhibition can also be caused by
secondary effects, such as defective receptor
recycling, inhibition of endosome maturation, inhibition of enzymes with a low pH
optimum, inhibition of Ca2+ efflux from endosomes, and pH neutralization of nonendocytic
compartments, such as the TGN.
Weak bases raise the pH almost instantly after addition, which makes them useful agents
for determining the time course of the acid activation step (80). It is, however, important to
note that they raise only endosomal and lysosomal pH reliably and inhibit virus penetration if
the pH in the medium remains above 7.0 (81).
Some of the confusion regarding the acid dependency of certain viruses is likely explained
by the use of poorly buffered media in experiments involving lysosomotropic weak bases.
Inhibitors that cause cholesterol depletion
are often used to test whether viruses enter by
caveolar/raft-mediated pathways. The results
depend on which agents and concentrations
are used (19). Methyl-β-cyclodextran extracts
the cholesterol from the plasma membrane
rapidly and efficiently and inhibits not only
caveolar/raft-mediated pathways but often
clathrin-mediated pathways as well (82, 83).
The effects of nystatin, an agent that binds
cholesterol (often used in combination with
progesterone, a cholesterol synthesis inhibitor),
are generally milder.
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Transfection with D/N and C/A constructs
and siRNA allows targeted analysis of individual host cell proteins and pathways. The use of
controls is critical, and poor cell viability can
be a problem. The results obtained by silencing of individual genes using siRNA should be
validated using immunoblotting or other techniques to quantify the amount of the target protein remaining in the cells. Multiple siRNA sequences are recommended against each gene to
avoid off-target effects. Because the knockdown
of proteins is never complete, negative data are
only interpretable with proper controls.
Finally, it is important that viruses be properly stored. For acid-activated viruses, it is essential that they not be frozen in phosphatebuffered media such as MEM without added
Hepes or Tris buffer, because the drop in pH
during freezing may preacidify the virus, leading to losses in infectivity.
VIRUSES THAT USE THE
CLATHRIN PATHWAY
Early EM studies revealed that certain viruses,
such as adenovirus 2 and 5 and vesicular stomatitis virus (VSV), were present in thickened
regions of the plasma membrane (84, 85), i.e.,
in regions that we now know as clathrin-coated
pits (CCPs). That CME can be part of the productive infectious pathway was first shown for
Semliki Forest virus (SFV), an enveloped animal virus (86), and this was followed by similar observations for several other viruses. The
pathway is today the most commonly observed
in virus entry. Table 2 contains a partial list
of viruses that have been reported to use this
pathway. In some cases, CME serves as one of
several pathways used by a virus.
The molecular information about the various steps in clathrin coat assemblies, induction of membrane curvature, cargo recruitment, vesicle fission, and coat disassembly is
extensive, and there are several recent reviews (21, 24, 148). It is increasingly apparent that the composition of CCPs in the
plasma membrane is not always the same. Once
thought to be an integral part of all plasma
membrane-coated pits, the adaptor complex
AP2, for example, has turned out not to be
an absolute requirement for the formation of
coated pits and for the internalization of certain cargo (24, 149–151). This is also true for
viruses; some depend on AP2, and others do not
(Table 1).
The formation of clathrin-coated vesicles
was initially thought to be a constitutive process that occurred whether cargo was present
or not. It is now evident that cargo can actively
initiate the formation of CCPs. This is particularly clear in the case of some viruses (reovirus,
VSV, Influenza A) that induce de novo formation of a CCP at their site of binding (40, 41,
74, 88). Of clathrin-internalized influenza virus
particles on BS-C-1 cells, 94% induce de novo
assembly of a CCP centered under the virus
(74). The adaptor protein required in this case
is epsin-1 (111).
Similarly, clathrin, dynamin-2, and AP2 can
be seen to accumulate transiently under or in
close proximity to surface-bound VSV particles (41, 88). The assembly phase lasts about
110 s, which is somewhat longer than for normal coated pits (50 s). If the VSV particle cannot
be internalized because it is fixed to the substratum, several cycles of “frustrated” CCP formation can be observed in the same spot. What
causes coat assembly is not clear, but receptor
clustering induced by the virus may give rise to
a microdomain with properties different from
that of the surrounding membrane.
CME of viruses is generally a rapid process,
with surface-bound viruses entering within
minutes after attachment, or after cell warming if binding was performed in the cold. Delivery to EEs follows within 1–2 min. For
VSV and SFV, which have a high fusion pH
threshold adjusted to the pH of EEs, the acidactivated step occurs within 1–5 min after internalization. Viruses that have a pH of fusion
in the LE range, typically fuse 10–20 min after
internalization.
A recent single-particle tracking study with
dengue virus (serotype 2, strain 1) has allowed
visualization of the various steps in the clathrinmediated entry process (56). After binding,
www.annualreviews.org • Virus Entry by Endocytosis
Dynamin-2: a large
GTPase required for
various types of
endocytosis and
intracellular scission
events. Dynamin-2 is
indispensable for
clathrin-mediated
endocytosis
813
814
Mercer
·
Schelhaas
·
Alpha (ssRNA+)
Alpha (ssRNA+)
Flavi (ssRNA+)
Flavi (ssRNA+)
Adeno (dsDNA)
Picorna
(ssRNA+)
Aphtho
CME
CME
CME
CME
CME
Rhabdo
(ssRNA−)
Helenius
Cyto
Nuc
Cyto
Cyto
Cyto
Cyto
Cyto
Hand, foot, and
mouth disease (−)
Adenovirus 2 and
5 (−)
Hepatitis C (+)
Dengue (+)
Sindbis (+)
Semliki Forest (+)
Vesicular
stomatitis (+)
Example virus
envelope ( ± )
GAGs ?
GAGs
GAGs,
DC-SIGN
GAGs,
LDLR,
SRB1, CD81
CAR, αV
integrins
αV integrins
+ (<6.2)
+
+
+ (<6.0)
+
?
Receptor
molecules
+
+ (<6.4)
Low pH ±
dependency
Comments
Can enter EEs and REs;
infection requires Rab 5 but
not Rab 7, 4, or 11
Uptake into CCVs in parallel
with induction of
macropinocytosis (required
for infection); penetration
in EE
Fusion requires proteolytic
activation
Uses preformed CCPs; fuses
at LE; entry similar to West
Nile virus; in Vero cells
clathrin, CAV1, and actin
independent; dynamin
dependent
Data on CME and acid
activation contradicts
previously published direct
penetration model
Induces CCPs de novo;
transported to EEA1/Rab5
peripheral endosomes; fuses
5–8 min after internalization
Induces CCPs de novo; role
of AP2 controversial; can
fuse at EE; suggested fusion
with ILVs
References
102, 103
92, 100, 101
37, 99
56, 97, 98
93–96
86, 90–92;
A. Vonderheit
& A. Helenius,
unpublished
41, 87–89
27 April 2010
CME
CME
Virus family
(DNA/RNA)
Replication
nucleus
(Nuc) or
cytoplasm
(Cyto)
Endocytic internalization of mammalian viruses. Listed are the primary endocytic mechanisms used by a variety of virus families
ARI
Primary
pathway
Table 2
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ANRV413-BI79-28
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Picorna
(ssRNA+)
Picorna
(ssRNA+)
Macro
Macro
Myxo (ssRNA−)
CME/Novel
pathway
Adeno (dsDNA)
Retro (ssRNA)
CME
Macro
Parvo (ssDNA)
CME
www.annualreviews.org • Virus Entry by Endocytosis
Cyto
Cyto
Nuc
Cyto
Nuc
Nuc
Nuc
Cyto
Coxsackievirus B
(+)
Echovirus 1 (−)
Adenovirus 3 (−)
Vaccinia MV (+)
Influenza A (+)
Avian leukosis (+)
Canine parvo (−)
Reovirus(+)
Human rhino 2
minor group (−)
α2β1
integrins
CAR
+
±
Sialoglycoproteins
+ (<5.6)
CD46, αV
integrins
Transferrin
receptor
+
+ (<6.0)
Transferrin
receptor
+
GAGs?
JAM1
+
± (<5.0)
LDL receptor
+ (<5.6)
Enters at tight junctions;
does not require Rac1;
requires Rab34
Entry requires PAK1, CtBP1;
cellular factors consistent
with macropinocytosis
Entry requires PAK1, CtBP1;
cellular factors consistent
with macropinocytosis
Entry linked to membrane
blebbing; apoptotic mimicry
strategy; cellular
requirements vary with
strain
About 50% of particles or less
use CME; induces CCPs de
novo; epsin dependent,
fuses at LEs. The rest enter
clathrin independently
Colocalizes with CCPs;
chlorpromazine sensitive
CME is main pathway for
parvo. Virus found in EEs
and REs; penetration from
LEs. Exception AAV5 (goes
to Golgi)
Penetrates from EEA1
positive endosomes
Virus and receptor separate
in EEs; viral RNA deposited
into cytosol from MEs;
empty capsids degraded in
LE/LY
121, 122
119, 120
116–118
(Continued )
39, 114, 115
74, 111–113
35, 110
31, 107–109
40, 106
104, 105
27 April 2010
Pox (dsDNA)
Reo (dsRNA)
CME
Cyto
ARI
Macro
Picorna
(ssRNA+)
CME
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ANRV413-BI79-28
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815
816
Mercer
·
Schelhaas
·
Retro (ssRNA+)
Herpes (dsDNA)
Polyoma
(dsDNA)
Papilloma
(dsDNA)
Polyoma
(dsDNA)
Reo (dsRNA)
Macro
CAV1/Lipid
raft
CAV1/Lipid
raft
Lipid raft
IL-2
tentative
Herpes (dsDNA)
Helenius
Cyto
Nuc
Nuc
Nuc
Nuc
Nuc
Nuc
Replication
nucleus
(Nuc) or
cytoplasm
(Cyto)
Rhesus rotavirus
(−)
Mouse
polyomavirus (−)
Human
papillomavirus
type 31 (−)
Simian virus 40 (−)
Human herpes
virus 8/(KSHV)
(+)
HIV-1 (+)
Herpes simplex
virus 1 (+)
Example virus
envelope ( ± )
CD4, CCR5,
CXCR4,
CCR2
GAGs?
GM1
GAGs?
GD1a
Sialic acid,
integrins,
Hsc70
+
+
+
+
−
GAGs?
Receptor
molecules
+
+ (<6.0)
Low pH ±
dependency
Comments
Clathrin and CAV1
independent; cholesterol
and dynamin dependent
Transport via EE and RE to
ER, where penetration
occurs
Exceptional pathway for
HPVs; depends on CAV1,
lipid rafts, dynamin; routed
to EE
Parallel CAV1-dependent
and –independent entry;
transport via EE and LE to
ER where penetration
occurs
Cell type specific; Rab34;
cellular factors consistent
with macropinocytosis
Cell type specific; Na+/ H+
dependent; virions
colocalize with fluid
markers; needs further
experimentation
Cell type specific; RTK and
PI3K dependent; virions
colocalize with fluid
markers; needs further
experimentation
References
137, 138
134–136
133
130–132; S. Engel,
T. Heger, R. Mancini,
J. Kartenbeck,
F. Herzog, et al., in
preparation
129
125–128
8, 123, 124
27 April 2010
Macro
Macro
Virus family
(DNA/RNA)
(Continued )
ARI
Primary
pathway
Table 2
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Cyto
Nuc
Nuc
Nuc
Mimivirus (+)
Herpes simplex
virus 1 (+)
Porcine circovirus
2 (−)
Human
papillomavirus
type 16 (−)
Lymphocytic
choriomeningitis
virus (+)
?
?
+
+
GAGs?
+
HS, HVEM,
Nectin1
Alphadystroglycan
+
+
DC-SIGN
?
Amoebal pathogen; can only
enter professional
phagocytes; entry requires
dynamin-2 and PI3K
Cell type specific;
dynamin-2; cellular factors
consistent with phagocytosis
Cell-type dependent; in
porcine monocytes by
CME; in HeLa dependent
on actin and Rho GTPase
Noncoated vesicles; clathrin,
CAV1, flotillin, lipid-raft,
and dynamin independent;
escape likely in
LE/lysosomes
Independent of clathrin,
CAV1, flotillin, lipid rafts,
actin; routed directly to LE;
minor fraction by CME
Clathrin, CAV1, Rho
GTPase independent;
cholesterol and dynamin
dependent. IL-2 pathway?
147
146
144, 145
143; M. Schelhaas, M.
Holzer, P. Blattmann,
P.M. Day, J.T.
Schiller, A. Helenius,
submitted
141, 142
139, 140
Abbreviations: +, low pH dependent; −, low pH independent; AAV5, adeno-associated virus 5; AP2, adapter protein 2; CAR, coxsackievirus and adenovirus receptor; CAV1, caveolin-1;
CCR2, -5, chemokine (CC motif) receptor 2 and 5; CCV, clathrin-coated vesicles; CD4, -46, cluster of differentiation 4 and 46; CCP, clathrin-coated pit; CD81, cluster of differentiation 81;
CME, clathrin-mediated endocytosis; CtBP1, C-terminal-binding protein 1; CXCR4, CXC chemokine receptor 4; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing
non-integrin; EE, early endosome; EEA1, early endosome antigen 1; ER, endoplasmic reticulum; GAGs, glycosaminoglycans; GD1α, alpha series ganglioside; HHV8/KSHV, human
herpesvirus 8/Kaposi’s sarcoma-associated herpesvirus; HIV-1, human immunodeficiency virus 1; HPV, human papillomavirus; HS, heparan sulfate; Hsc70, human heat shock protein 70;
HVEM, herpesvirus entry mediator; IL-2, interleukin-2; ILV, intralumenal vesicle; JAM1, junctional adhesion molecule-1; LDLR, low-density lipoprotein receptor; LY, lysosome; LE, late
endosome; Macro, macropinocytosis; Mimi, Mimivirus; MV, mature virion; PI3K, phosphoinositide 3-kinase; PAK1, p21-activating kinase 1; Phago, phagocytosis; Rab5, -34, Ras-related in
brain; RE, recycling endosome; RTK, receptor tyrosine kinase; SARS, severe acute respiratory syndrome; SRB1, scavenger receptor class B -1; SV40, simian virus 40.
Mimi (dsDNA)
Circo (ssDNA−)
Novel
pathway
Phago
Papilloma
(dsDNA)
Novel
pathway
Cyto
Feline infectious
peritonitis virus,
SARS (+)
27 April 2010
Herpes (dsDNA)
Arena (ssRNA−)
Novel
Pathway
Cyto
ARI
Phago
Corona
(ssRNA+)
IL-2
tentative
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817
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Endosome network:
a complex collection of
membrane organelles
responsible for sorting,
recycling, degradation,
storage, and
processing of
endocytic membranes
and cargo
21:57
the virus particles move randomly along the
plasma membrane for an average time of 110 s
before associating with preexisting, clathrincontaining domains, where their movement is
constrained. In the presence of chlorpromazine,
an inhibitor of clathrin assembly, the movement continues without stopping. The viruses
are next delivered to endosomes; 86% to Rab5positive EEs and 14% to Rab5- and Rab7positive MEs. The majority (80%) fuse within
an average of 12.5 min after virus addition and
5.5 min after entry into Rab7-positive LEs. Of
the fusion events, the majority occurred in peripherally located endosomes.
In Table 2, we summarize some of the information on a number of viruses that use
CME. Some seem to depend exclusively on
the clathrin pathway. Others, such as Influenza
A virus (a myxovirus) and lymphocytic choriomeningitis virus (LCMV, an arena virus) can
also exploit other pathways.
MACROPINOCYTOSIS AND
VIRUS ENTRY
Under normal conditions, macropinocytosis
can be defined as a transient, growth factorinduced, actin-dependent endocytic process
that leads to internalization of fluid and membrane in large vacuoles. It is unique among
the pinocytic processes in involving dramatic,
cell-wide plasma membrane ruffling (152).
Macropinocytic vacuoles (macropinosomes)
are formed when membrane ruffles fold back
onto the plasma membrane to form fluid-filled
cavities that close by membrane fusion (153,
154). The ruffles can take the form of lamellipodia, filopodia, or blebs.
Macropinosome formation is not guided by
a particle or a cytoplasmic coat, and this gives
rise to their irregular size and shape. With a diameter up to 10 μm, they are large compared
to other pinocytic vacuoles and vesicles. Their
formation is associated with a transient 5- to
10-fold increase in cellular fluid uptake. After
formation, macropinosomes move deeper into
the cytoplasm, where they can undergo acidification as well as homo- and heterotypic fusion
818
Mercer
·
Schelhaas
·
Helenius
events (155). Depending on the cell type, they
either recycle back to the cell surface or feed
into the endosome network and mature with
the gain and loss of classic EE and LE markers before fusing with lysosomes (155, 156).
Although typically associated with growth
factor–induced fluid uptake, a variety of particles, including apoptotic bodies, necrotic cells,
bacteria, and viruses, can induce ruffling and
thus promote their macropinocytic internalization together with fluid (157–162).
Like the factors involved in other endocytic
mechanisms, the cellular components required
for macropinocytosis are numerous, complex,
interconnected, and somewhat variable depending on the cell type, internalized cargo,
mode of stimulation, and cellular conditions.
Activation involves cellular lipids, kinases,
GTPases, Na+/ H+ exchangers, adaptor
molecules, actin, actin modulatory factors,
myosins, as well as fusion and fission factors
(see Table 1). For the numerous functions
and molecular details, we recommend several
recent reviews that focus on different aspects
of the process (22, 26, 27, 163–167). The role
of macropinocytosis in virus entry has been
recently reviewed (17, 168).
Viruses from several different families make
use of macropinocytosis either as a direct means
of internalization or as an indirect mechanism to assist penetration of particles that
have entered by some other form of endocytosis. Viruses reported to use macropinocytosis as a direct route of entry include vaccinia virus mature virions, species B human
adenovirus serotype 3, echovirus 1, group
B Coxsackieviruses, herpes simplex virus 1,
Kaposi’s sarcoma-associated herpesvirus, and
HIV-1 (17, 129, 168).
The use of macropinocytosis by these
viruses has been assessed using EM for the
presence of particles in macropinosomes, light
microscopy for membrane ruffling and colocalization of virus with macropinocytic markers,
induction of fluid uptake, as well as perturbation analyses focusing on actin, microtubules,
Na+/ H+ exchange, Rho GTPases (Rac1 or
Cdc42), and various families of cellular kinases,
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including tyrosine, serine, threonine, and PI(3)
kinases. Although all of these viruses depend on
actin and Na+ /H+ exchange for entry, each has
been analyzed to varying degrees of completeness. Many require further investigation before
conclusive assignment to macropinocytic internalization can be made.
The best-characterized example of virus entry by macropinocytosis is the vaccinia virus
(39). Although the cellular requirements of the
canonical macropinocytosis pathway are conserved, vaccinia-induced macropinocytosis differs in several ways. Notably, the association
of mature virions with cells triggers the formation of large transient plasma membrane blebs
rather than classical lamellipodial ruffles. FIBSEM suggests that uptake of vaccinia and fluid
occurs as part of the bleb retraction process
(72). Additionally, the membrane of vaccinia
is enriched in phosphatidylserine, a phospholipid required for the macropinocytic clearance
of apoptotic debris (160, 169). The requirement of phosphatidylserine for vaccinia virus
entry suggests that vaccinia virions elicit the
macropinocytic response in host cells by mimicking apoptotic bodies (39).
There are several reasons why viruses may
have evolved to use macropinocytosis for internalization and entry. In the case of vaccinia
and herpes simplex virus 1, the most obvious
reason is particle size. These viruses are likely
too large for uptake by most other forms of
endocytosis. For other viruses, macropinocytic
entry may serve as a mechanism to broaden
their host range or tissue specificity. The
macropinosomes undergo acidification, and
many of the viruses are acid activated.
Some viruses that induce macropinocytosis require it for infectivity but do not
use macropinosomes as a direct internalization pathway. They include species C human adenoviruses 2 and 5 and rubella virus.
These are internalized by CME, do not enter
macropinosomes, and do not colocalize with
macropinosome markers. Yet, the cellular requirements for infection indicate that they need
macropinocytosis to be infectious. For adenoviruses 2 and 5, it has been demonstrated
that induction of macropinocytosis and rupture of newly formed macropinosomes are required for escape of the virus from EEs (92,
170). Thus, the induction of macropinocytosis,
formation of macropinosomes, or the release of
some macropinosomal component upon rupture is somehow required for productive penetration and infection.
VIRUSES THAT USE CAVEOLAR/
RAFT-DEPENDENT ENTRY
Lipid raft: specialized
cholesterol- and
sphingolipid-enriched
membrane
microdomains that can
influence membrane
fluidity, receptor
clustering, and
assembly of signaling
molecules
A characteristic of the caveolar/raft-dependent
pathways is that formation of primary endocytic
vesicles depends on cholesterol, lipid rafts, and
a complex signaling pathway involving tyrosine
kinases and phosphatases. The process is ligand
triggered. Uptake may involve caveolae but can
also occur without these lipid raft-containing
microdomains. After internalization, the cargo
passes through EEs and LEs, often followed by
vesicle-mediated transport to the ER. Many of
the viruses that use these pathways make use of
glycosphingolipids as their receptors. Once in
the ER, some exploit specific ER thiol oxidoreductases and related proteins to initiate uncoating and use components of the ER-associated
protein degradation pathway for penetration
into the cytosol (171–174).
Uptake and forward movement in this pathway are slow and asynchronous. Depending
on the virus and the cell type, the penetration event in the ER can occur as late as 6–
12 h after internalization (171). Nonviral ligands include bacterial toxins, such as cholera
toxin and shiga toxin, which also bind to glycolipids, and some glycosylphosphatidylinositol (GPI)-anchored proteins and their ligands,
including the autocrine motility factor and
cytokines (175).
The best-studied viruses that make use of
caveolar/raft-dependent pathways belong to
the polyomavirus family (176). They include
SV40, mouse polyomavirus (mPy), and two human pathogens, BK and JC viruses. All have different gangliosides as receptors (177–179). The
association with the glycan moieties is highly
specific but of relatively low affinity (180). With
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multiple receptors, the avidity of virus binding
is high. Viruses from other families have also
been reported to use caveolar/raft-dependent
pathways, but information about them is
incomplete (181–183).
Polyomaviruses are nonenveloped DNA
viruses that replicate in the nucleus. The main
structural components are 72 homopentamers
of the major capsid protein, VP1, icosahedrally
arranged (184). Because each VP1 has a binding
site for the carbohydrate moiety of a ganglioside
(180, 185), the virus offers 320 receptor-binding
sites about 9 nm apart in a fixed pattern over the
curved surface of the 50-nm-diameter particle.
The use of gangliosides as receptors is of great
significance for the polyomaviruses as it determines their behavior on the cell surface, their
mechanism of endocytosis, and the intracellular
pathway from endosomes to the ER. Whereas
SV40 needs the ganglioside GM1 for binding,
internalization, and infection (186), recent evidence for mPy indicates that the receptor ganglioside GD1α is not required for binding and
endocytosis but only for guiding the virus from
LEs to the ER (134).
The viruses associate with detergentresistant microdomains in the plasma membrane of cells and with liquid-ordered phases
in giant unilamellar vesicles (186). Immediately
after binding to cells, mPy particles undergo
rapid, random, lateral movement for 5–10 s
before stopping or changing to a slow drift
(77, 134). Electron micrographs show that cellassociated viruses enter uncoated, tight-fitting
indentations and pits of variable depth (187–
190). The tight wrapping of the membrane
around the particle gives the impression that
the virus actually “buds” into the cell. Recent
studies show that SV40, in fact, induces membrane curvature and thus promotes the formation of what later becomes the primary endocytic vesicle (186). For the final fission reaction
generating a tight-fitting small vesicle, the virus
depends, however, on cellular sources of energy
and cellular factors recruited in response to activation of signaling pathways.
Several observations have led to a model in
which SV40 entry involves caveolae and caveo-
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CAV1: caveolin-1
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lar vesicles (38, 191–196). The association with
caveolae is probably explained by the higher
concentration of GM1 in caveolae (197) and
possibly by a tendency of virus particles to take
advantage of the already existing curvature, particularly because the dimensions of caveolae are
close to those of the virus. Upon careful analysis, it has, however, become apparent that in
many cell types the majority of surface-bound
SV40 and mPy, although present in indentations of the plasma membrane, do not associate
with caveolae (134, 136, 198). If colocalization
with caveolin-1 (CAV1) occurs, it is usually at
a later time when most of the viruses have already been internalized. Evidence for a second, closely related mechanism of SV40 uptake
has emerged from studies with CAV1-deficient
cell lines and primary fibroblasts from CAV1
knockout mice. These cells are efficiently infected by SV40 and mPy, and the viruses entered via virus-containing invaginations morphologically indistinguishable from those in
CAV1-containing cells (136, 198). Although
CAV1 and dynamin independent, the alternate
mechanism shares many of the features described for caveolar endocytosis. It is, for example, cholesterol and tyrosine kinase dependent
(198). The fraction of virus using caveolar entry
may vary with cell type and virus because when
CAV1 was expressed in caveolin-deficient Jurkat cells, infection by mPy was increased (136).
Taken together, the results indicate that only
a fraction of SV40, mPy, and probably other
polyomaviruses enter via caveolae. The rest
use a related, CAV1-independent mechanism.
Both mechanisms are inhibited—or considerably reduced—by genistein (a general tyrosine
kinase inhibitor), by nystatin and progesterone
(for depletion of cholesterol), by brefeldin A
(an inhibitor of Arf1, a guanine nucleotidebinding protein), by bafilomycin A (an inhibitor
of the vacuolar ATPase), by lactrunculin A or
jaspakinolide (inhibitors of actin dynamics), and
by reduction of temperature below 20◦ C (26,
192, 194, 199). Internalization is accelerated by
okadaic acid and orthovanadate (inhibitors of
Ser and Thr, or Tyr phosphatases, respectively)
(196, 198, 200). All these agents and conditions
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also block infectivity. Internalization through
caveolae is generally slower and dependent on
dynamin-2 and also more dependent on actin
dynamics than the noncaveolar pathway (194).
Kirkham & Parton (26) have suggested that
the underlying mechanisms for the lipid raftdependent, caveolar, and CAV1-independent
endocytic pathways are fundamentally similar. They suggest that the basic pathway is a
lipid raft-dependent, cargo-activated process to
which CAV1 and dynamin provide an additional level of regulation. The regulatory effect
is most likely inhibitory, as caveolae are normally highly stationary (19, 196, 200), and as
expression of CAV1 generally tends to suppress
lipid raft-mediated endocytosis (175, 201). For
more detailed discussion of the field of caveolae
and their endocytosis, the reader is referred to
recent reviews (13, 19, 26, 202).
For these viruses, the site of penetration
is the ER, which they reach after three hours
or longer depending on cell type. Viruses accumulate in tubular, smooth membrane networks with complex geometry enriched for the
smooth ER markers syntaxin 17 and BiP (188,
190, 198). What happens during the intervening hours when the virus is en route to the ER is
not entirely clear. Recent studies with mPy and
SV40 indicate that most of the time is spent in
the organelles of the classical endocytic pathway, especially in LEs (134, 136; S. Engel, T.
Heger, R. Mancini, F. Herzog, A. Hayer, & A.
Helenius, in preparation).
That they can enter endosomes has been
documented using EM, immunofluorescence
microscopy, and live cell video microscopy
(134–136, 188, 203). Quantitation indicates
that 15% to 20% of the viruses colocalize with
EE markers 1–2 h after cell warming. Starting
at ∼60 min, the virus begins to colocalize with
LE markers, and after 2–4 h, SV40 enters
tubular, vesicular extensions that detach from
the LEs and move along microtubules (132,
134; S. Engel, T. Heger, R. Mancini, F. Herzog,
A. Hayer, & A. Helenius, in preparation). The
viruses then accumulate in the smooth ER.
How the viruses move from LEs to the ER
remains unclear. The formation of tubular,
SV40-containing vesicles has been observed in
live cells, followed by microtubule-mediated
plus and minus end-directed movement (132,
198). Recent studies with mPy indicate that
this step requires the presence of the GD1α
receptor (134).
Our recent experiments indicate that in most
cell types, the so called caveosomes that we described as an intermediate station in the pathway of SV40 entry correspond to modified LEs
or endolysosomes (132; A. Hayer, D. Ritz, S.
Engel, & A. Helenius, submitted; S. Engel,
T. Heger, R. Mancini, F. Herzog, A. Hayer,
& A. Helenius, in preparation). Although organelles with the characteristics of caveosomes
are seen in cells overexpressing CAV1-GFP
or other CAV1 constructs, the corresponding
CAV1-containing organelles are not present in
control cells with endogenous levels of CAV1.
Instead, the distribution of CAV1 in the cytoplasm corresponds to much smaller structures,
often associated with EEs (203). The generation of caveosome-like structures is caused by
saturation of the caveolar assembly machinery
in the Golgi complex and by release of unassembled CAV1 complexes to the cell surface from
which they move into LEs (A. Hayer, D. Ritz,
S. Engel, & A. Helenius, submitted).
VIRUSES THAT USE UNUSUAL
ENDOCYTIC PATHWAYS
In addition to the established endocytic mechanisms described above, there is a growing body
of data indicating involvement of additional
mechanisms in the internalization of some
viruses. These have in common the lack of
detectable coats by EM, the lack of dependency
on clathrin and caveolin, and the transfer of
viruses to the endosomal network. However,
there are also clear differences, implying the
existence of variations of a common theme
or multiple independent mechanisms. So far,
the viruses that use these pathways include
rotavirus, LCMV, human papillomavirus 16
(HPV-16), and Influenza A virus. In the case
of influenza virus, the process operates in
parallel with CME and serves as a pathway of
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productive entry (74, 111, 113), but there is
little detailed information available.
Rotavirus is a nonenveloped RNA virus of
the reovirus family. It is the leading cause
of severe diarrhea among infants and young
children. The particles interact sequentially
on the cell surface with sialic acid-containing
molecules, integrin α2β1, Hsc70, and finally
integrins α5β3 and αXβ2 (204). These interactions trigger conformational changes in
the capsid preparing the virus for uncoating and membrane penetration. The endocytic
mechanism is dynamin-2 dependent and somewhat sensitive to cholesterol depletion, suggesting a role for lipid rafts. These features
suggest a possible relationship with the uptake of interleukin 2, which is internalized by
clathrin- and caveolin-independent pathways
(205, 206).
Membrane penetration of rotavirus occurs
by lysis or pore formation in endosomes, mediated by the trypsin-activated VP4 protein. Infection is not blocked by lysosomotropic, weak
bases, suggesting that it is independent of acid
activation (138). However, bafilomycin A1, an
inhibitor of the endosomal proton pump, does
block infection, possibly through a decrease in
Ca2+ concentration (137).
LCMV is an enveloped RNA virus of the
arenavirus family (207, 208). For cell entry, it binds to the carbohydrate moiety of
α-dystroglycan (209). Endocytosis occurs in
smooth, noncoated pits (142, 210). Cholesterol is required, but clathrin, caveolin, flotillin,
Arf6, dynamin-2, and actin are not (141, 142).
The available evidence suggests that a large
fraction of the viruses are directly transferred
to the LEs, where membrane fusion is activated by the low pH (142). Bypassing conventional EEs may provide cell surface components a fast route to degradation without
recycling.
HPV-16 is a nonenveloped virus of the papillomavirus family that infects mucosal epithelia and causes warts as well as cervical and
anogenital tumors (211). Virus particles contain two structural proteins (L1 and L2) that
form an icosahedral (T = 7) particle of ∼55 nm
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in diameter. Cell entry involves an initial interaction with heparan sulfate proteoglycans
(HSPGs), followed by a sequence of structural changes caused by interaction with the
sugar moiety of HSPGs, by cyclophilin B, and
by activation by furin, a proprotein convertase
(212–214).
Although HPV-16 was originally proposed
to enter by CME, recent studies indicate
endocytosis occurs by a clathrin-, caveolin-,
flotillin-, lipid raft-, dynamin-independent
mechanism distinct from macropinocytosis and
phagocytosis (143; M. Schelhaas, M. Holcer, P.
Blattmann, P.M. Day, J.T. Schiller, & A. Helenius, submitted). Uptake depends on actin
polymerization but is independent of Rho-like
GTPases. Endocytosis via small tubular pits requires PI3-kinase and protein kinase C activities, as well as a sodium-proton exchanger (M.
Schelhaas, M. Holcer, P. Blattmann, P.M. Day,
J.T. Schiller, & A. Helenius, submitted). Intracellular trafficking occurs through the endosomal network, where HPV-16 seems to follow
the canonical transport from EEs and LEs to
lysosomes. The virus is acid activated. Taken
together, the requirements for infectious endocytosis and the morphology of vesicular carriers
suggest an endocytic mechanism that combines
features of macropinocytosis and the mechanism used by LCMV.
Although the information is still incomplete,
it is likely that viruses have evolved to use mechanisms of endocytosis that have not yet been
described in the cell biology literature. It will
be interesting to learn more about these pathways and to identify their physiological ligands
and functions. In the cell biological literature,
there are, however, pathways with which no
virus has yet been associated. These include the
so called GEEC pathway involved in the endocytosis of GPI-anchored proteins, the flotillin
pathway for GPI-anchored proteins and proteoglycans, the Arf6 pathway for the internalization of major histocompatibility antigens,
and as mentioned above, the IL-2 pathway for
the internalization of some cytokine receptors
(Figure 1 and Table 1) (summarized in
References 21, 26, and 215).
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VIRUS ENTRY BY
PHAGOCYTOSIS
Phagocytosis is an endocytic mechanism classically considered distinct from the pinocytic
mechanisms discussed above. It is used by
specialized cell types, such as macrophages
and amoeba, for the uptake of large particles,
such as bacteria. It shares several features with
macropinocytosis, including large vacuole size,
transient activation, actin dependency, cellular
factors, and regulatory components (Table 1).
However, the molecular mechanism is fundamentally different because the attachment of
the particle surface to the plasma membrane not
only triggers the process but guides the actindependent formation of a tight-fitting endocytic vacuole around the particle (27, 216, 217).
The process involves a major transient reorganization of the plasma membrane, which is localized only to the region in contact with the
cargo particle.
To distinguish between phagocytic and
macropinocytic entry of viruses, multiple experimental approaches can be used. These include EM and light microscopy, colocalization and internalization of phagocytic and fluid
phase tracers, and inhibitor profiling. Using
these techniques, the entry mechanism of herpes simplex virus 1 into nonprofessional phagocytes and of the amoebal pathogen mimivirus
into macrophages were found to be consistent
with phagocytosis (146, 147). As suggested for
macropinocytic internalization, the huge size of
mimiviruses may explain why they have evolved
to make use of this unusual mechanism.
PERSPECTIVES
The viruses themselves may be simple, but the
complexity of the pathways involved in endocytic membrane trafficking and their regulation
pose major challenges for understanding virus
entry and infection. Because the plasma membrane and its components provide the boundary of the cell to the outside world, any functions that it may have, including endocytosis,
are highly regulated and carefully controlled.
Powerful new technologies, such as siRNA silencing screens, provide unprecedented access
to the underlying network of cellular factors involved. When applied to virus infection, validated, and followed up, the information obtained using such screening approaches will lay
a new and much more complete foundation for
understanding the cellular basis for infectious
disease (218, 219).
To move forward, however, it is important
to take advantage of novel technologies, including single-virus tracking, quantitative live cell
imaging, and advanced EM techniques, combined with improved assays for following a
virus step-by-step through its replication cycle. What is needed is the ambitious use of
a full spectrum of cell biological approaches.
The effort will be important and worthwhile
because it will lead to new antiviral approaches
and new defenses against existing and emerging viral diseases. The outcome will be new
drugs that target critical functions in the host
cell. Host-directed inhibitors have advantages
in being less likely to generate resistance and in
allowing inhibition of multiple viruses. However, to be realistic, a detailed understanding of
virus-cell interactions is required.
Most of the studies on virus entry and endocytosis are currently performed in tissue culture
cells with purified virus added to the medium.
However, infection in situ in the tissues and live
animal hosts raises many additional questions.
How do viruses penetrate tissue barriers, and
how do they target specific cells for infection?
How do they avoid immune surveillance and
other host defenses? To what extent do they
take advantage of cells and cell mobility for
transmission?
In this context, it is increasingly evident that
for local infection virus particles do not need to
be released from the surface of infected cells.
The extracellular particles of poxviruses can,
for example, induce the formation of actincontaining mobile protrusions that are thought
to push the newly formed, surface-associated
viruses into neighboring cells (220, 221). Other
viruses can take advantage of filopodia and nanotubes as bridges for moving between cells
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(222, 223). There is also evidence that motile
cells, such as dendritic cells, carry viruses with
them and deliver them in a targeted fashion to
new host cells through formation of specialized
cell contact regions, sometimes called virological synapses (222, 223). The cell biology of these
types of processes is of great interest for future
studies of viral pathogenesis.
SUMMARY POINTS
1. Endocytic internalization provides several advantages to incoming viruses. Most importantly, endocytosis leaves no evidence of virus entry, and primary endocytic vesicles and
endosomes serve as intracellular transporters for the virus.
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2. To define and compare the cell factors required for different endocytic pathways, viruses
offer many advantages over traditional endogenous ligands. Individual virus particles can
be monitored by different microscopy techniques, and infection provides a clear, highly
sensitive, quantitative end point assay.
3. Eukaryotic cells possess numerous tightly regulated endocytic mechanisms that vary in
regard to the cargo internalized, mechanisms of initiation, molecular factors utilized,
intracellular trafficking, and cargo destination.
4. It is becoming increasingly clear that many viruses can activate cellular signaling processes
to trigger their own endocytosis and prepare the cell for invasion.
5. After internalization, viruses are channeled to the endosome network, where they take
advantage of the various sorting and trafficking functions of the system.
6. Recent evidence suggests that lipid raft-dependent endocytosis can be either CAV1 dependent or independent.
7. Several novel clathrin-independent mechanisms have been described, and some of them
serve as important entry pathways for viruses. They are still poorly characterized.
FUTURE ISSUES
1. What is the molecular basis and advantage for viruses that make use of multiple endocytic
mechanisms? Will this cause a problem in future antiviral efforts based on inhibiting virus
entry?
2. siRNA silencing screens and other systems-based approaches used to identify cell proteins
for the development of cell-based antiviral agents will require further development.
3. The influence of ligand type, ligand availability, and regulatory factors on the heterogeneity and interrelationship of different endocytic pathways and endosome populations
must be addressed.
4. The cellular factors and roles of the novel endocytic pathways in normal cell life need to
be further defined.
5. Virus endocytosis studies need to be moved into tissue explants and model organisms in
order to provide detailed information about in vivo cell type specificity, virus cell-cell
transfer, and the effectiveness of potential antiviral agents.
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DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank members of the Helenius laboratory for thoughtful discussions. A.H. is supported by
LipidX, the Swiss National Science Foundation, and the European Research Council, M.S. by the
German Science Foundation, and J.M. by the European Molecular Biology Organization.
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Contents
Volume 79, 2010
Preface
The Power of One
James E. Rothman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p pv
Prefatory Article
Frontispiece
Aaron Klug p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p xiv
From Virus Structure to Chromatin: X-ray Diffraction
to Three-Dimensional Electron Microscopy
Aaron Klug p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Recent Advances in Biochemistry
Genomic Screening with RNAi: Results and Challenges
Stephanie Mohr, Chris Bakal, and Norbert Perrimon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p37
Nanomaterials Based on DNA
Nadrian C. Seeman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p65
Eukaryotic Chromosome DNA Replication: Where, When, and How?
Hisao Masai, Seiji Matsumoto, Zhiying You, Naoko Yoshizawa-Sugata,
and Masako Oda p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p89
Regulators of the Cohesin Network
Bo Xiong and Jennifer L. Gerton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 131
Reversal of Histone Methylation: Biochemical and Molecular
Mechanisms of Histone Demethylases
Nima Mosammaparast and Yang Shi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 155
The Mechanism of Double-Strand DNA Break Repair by the
Nonhomologous DNA End-Joining Pathway
Michael R. Lieber p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 181
The Discovery of Zinc Fingers and Their Applications in Gene
Regulation and Genome Manipulation
Aaron Klug p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 213
vii
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Origins of Specificity in Protein-DNA Recognition
Remo Rohs, Xiangshu Jin, Sean M. West, Rohit Joshi, Barry Honig,
and Richard S. Mann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 233
Transcript Elongation by RNA Polymerase II
Luke A. Selth, Stefan Sigurdsson, and Jesper Q. Svejstrup p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 271
Biochemical Principles of Small RNA Pathways
Qinghua Liu and Zain Paroo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 295
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Functions and Regulation of RNA Editing by ADAR Deaminases
Kazuko Nishikura p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 321
Regulation of mRNA Translation and Stability by microRNAs
Marc Robert Fabian, Nahum Sonenberg, and Witold Filipowicz p p p p p p p p p p p p p p p p p p p p p p p p 351
Structure and Dynamics of a Processive Brownian Motor:
The Translating Ribosome
Joachim Frank and Ruben L. Gonzalez, Jr. p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 381
Adding New Chemistries to the Genetic Code
Chang C. Liu and Peter G. Schultz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 413
Bacterial Nitric Oxide Synthases
Brian R. Crane, Jawahar Sudhamsu, and Bhumit A. Patel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 445
Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective
Olga Khersonsky and Dan S. Tawfik p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 471
Hydrogenases from Methanogenic Archaea, Nickel, a Novel Cofactor,
and H2 Storage
Rudolf K. Thauer, Anne-Kristin Kaster, Meike Goenrich, Michael Schick,
Takeshi Hiromoto, and Seigo Shima p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 507
Copper Metallochaperones
Nigel J. Robinson and Dennis R. Winge p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 537
High-Throughput Metabolic Engineering: Advances in
Small-Molecule Screening and Selection
Jeffrey A. Dietrich, Adrienne E. McKee, and Jay D. Keasling p p p p p p p p p p p p p p p p p p p p p p p p p p 563
Botulinum Neurotoxin: A Marvel of Protein Design
Mauricio Montal p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 591
Chemical Approaches to Glycobiology
Laura L. Kiessling and Rebecca A. Splain p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 619
Cellulosomes: Highly Efficient Nanomachines Designed to
Deconstruct Plant Cell Wall Complex Carbohydrates
Carlos M.G.A. Fontes and Harry J. Gilbert p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 655
viii
Contents
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Somatic Mitochondrial DNA Mutations in Mammalian Aging
Nils-Göran Larsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 683
Physical Mechanisms of Signal Integration by WASP Family Proteins
Shae B. Padrick and Michael K. Rosen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 707
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Amphipols, Nanodiscs, and Fluorinated Surfactants: Three
Nonconventional Approaches to Studying Membrane Proteins in
Aqueous Solutions
Jean-Luc Popot p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 737
Protein Sorting Receptors in the Early Secretory Pathway
Julia Dancourt and Charles Barlowe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 777
Virus Entry by Endocytosis
Jason Mercer, Mario Schelhaas, and Ari Helenius p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 803
Indexes
Cumulative Index of Contributing Authors, Volumes 75–79 p p p p p p p p p p p p p p p p p p p p p p p p p p p 835
Cumulative Index of Chapter Titles, Volumes 75–79 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 839
Errata
An online log of corrections to Annual Review of Biochemistry articles may be found at
http://biochem.annualreviews.org
Contents
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