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MICROBIOLOGY
Nervous travellers
The complex journey of
viruses and toxins
Protein export from
malaria parasites
Hostile takeover
REVIEWS
A hitchhiker’s guide to the nervous
system: the complex journey of viruses
and toxins
Sara Salinas*‡, Giampietro Schiavo§ and Eric J. Kremer*‡
Abstract | To reach the central nervous system (CNS), pathogens have to circumvent the wall
of tightly sealed endothelial cells that compose the blood–brain barrier. Neuronal projections
that connect to peripheral cells and organs are the Achilles heels in CNS isolation. Some
viruses and bacterial toxins interact with membrane receptors that are present at nerve
terminals to enter the axoplasm. Pathogens can then be mistaken for cargo and recruit
trafficking components, allowing them to undergo long-range axonal transport to neuronal
cell bodies. In this Review, we highlight the strategies used by pathogens to exploit axonal
transport during CNS invasion.
Tetanus
A spastic paralysis resulting
from the inhibition of
neurotransmitter release at the
level of inhibitory interneurons.
This inhibition is caused by
intoxication with TeNT
(a protein toxin produced by
Clostridium tetani), which is
taken up at neuromuscular
junctions.
*Institut de Génétique
Moléculaire de Montpellier,
CNRS UMR 5535,
34293 Montpellier Cedex 5,
France.
‡
Universités de Montpellier I
& II, 34090 Montpellier,
France.
§
Molecular NeuroPathobiology
Laboratory, Cancer Research
UK London Research Institute,
London WC2A 3LY, UK.
e-mails:
[email protected];
Giampietro.Schiavo@
cancer.org.uk;
[email protected]
doi:10.1038/nrmicro2395
Corrected 13 August 2010
In most cases, pathogen access to the central nervous
system (CNS) can be prevented by a neurovascular fil‑
tering system made up of firmly sealed endothelial cells
that create a physical barrier known as the blood–brain
barrier (BBB; see BOX 1). Nonetheless, numerous patho‑
gens find their way into the CNS. Some viruses, such
as HIV‑1, can cross the BBB using cells of the immune
system1 (BOX 1), whereas other neurotropic pathogens
can reach the CNS using long‑range axonal transport.
Indeed, an Achilles heel in the protection of the CNS is
the existence of neuronal projections that cross the BBB
and functionally connect peripheral organs and tissues
with the soma of neurons. Molecules present at nerve
terminals can serve as receptors for some pathogens,
leading to neuronal uptake and subsequent transport of
these organisms.
In many cases, neuronal infection as a result of micro‑
bial agents using axonal transport causes impaired neuro‑
nal homeostasis, leading to a range of severe pathologies.
For example, tetanus, caused by tetanus toxin (TeNT;
also known as TetX), and botulism, caused by botulinum
toxins (BoNTs; also known as Bot proteins), are neu‑
rological disorders that result from the impairment of
neurotransmission2. Rabies virus (RABV)‑induced neu‑
rodegeneration is another example of the damage that
microbial agents can cause3. Theiler’s murine encephalo‑
myelitis virus (TMEV) causes demyelination and is used
as a model for multiple sclerosis in rodents4, whereas
Borna disease virus (BDV) infections induce defects in
synaptogenesis5 as well as behavioural changes (TABLE 1).
Because inflammation is frequent in patients with
age‑related neurological diseases, a link between viral
infections of the CNS and neurodegenerative disorders
has been proposed6,7; for example, Parkinson’s disease
may be linked to infection with influenza virus or West
Nile virus (WNV)8,9.
Recent advances in cell biology and cell imaging have
led to a better understanding of the mechanisms under‑
lying the entry and transport of several neurotropic path‑
ogens. In this Review, we focus on the different strategies
used by viruses and bacterial toxins to reach the CNS by
long‑range axonal transport. The mechanisms of entry
through the BBB, such as the use of haematopoietic cells
to invade the CNS, have been reviewed elsewhere10.
Neuronal architecture and axonal trafficking
Polarization is an essential aspect of neuron biology,
as it allows neuronal networks to receive, integrate
and transmit vectorial signals. During development,
a specific neurite becomes an axon and the remaining
projections become dendrites11. Not surprisingly, the
neuronal architecture creates challenges when it comes
to delivering signals or cargoes over long distances12.
To ensure spatial and temporal control of cargo pro‑
gression along the secretory and endocytic pathways,
cytoskeletal tracks and motors must be regulated 13
(FIG. 1) . Motors move cargo in an ATP‑dependent
directional process: cytoplasmic dynein is responsi‑
ble for axonal retrograde transport from the synapses to
the cell body, whereas members of the kinesin family
ensure the delivery of cargo to nerve endings through
axonal anterograde transport13 (FIG. 1). Dynein and kinesins
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Box 1 | The blood–brain barrier and neurotropic pathogens
The blood–brain barrier (BBB) is a neurovascular filtering system that allows the brain
to be supplied with nutrients such as oxygen and glucose while being protected from
potentially toxic molecules that are present in the blood. Under ‘normal’ physiological
conditions, the BBB does not allow agents such as bacteria to invade the central
nervous system (CNS). Only small, non-polar molecules can diffuse through the BBB,
and chemical backbones of such molecules therefore represent chemical templates for
many of the drugs that are designed to cross this barrier. The BBB is composed of
microvascular endothelial cells that are firmly sealed with tight junctions, creating a
physical barrier between the bloodstream and the CNS. Other cell types, such as
astrocytes, microglia, pericytes and neurons, provide molecular support to the BBB and
form a functional neurovascular unit. The ‘immune-privileged’ status of the CNS does
not mean that it is free from immune surveillance. Macrophages and leukocytes can
actively migrate through the BBB and may, paradoxically, serve as Trojan Horses for
pathogen entry into the CNS. For example, HIV is often associated with neurological
dysfunctions that result from the migration of HIV-infected leukocytes through the
BBB followed by viral spreading. Ultimately, HIV will also lead to impairment of the BBB.
Similarly, measles virus is thought to use lymphoid cells to cross the BBB.
Botulism
A flacid paralysis resulting from
the inhibition of acetylcholine
release at neuromuscular
junctions, mediated by BoNTs.
These toxins act by cleaving
the synaptic components that
mediate fusion of synaptic
vesicles to the plasma
membrane.
Synaptogenesis
Formation of synapses in the
central and peripheral nervous
systems. This process initiates
early in development and
continues throughout
adulthood.
Axonal retrograde transport
Traffic of molecules and
organelles from nerve terminals
to cell bodies. This mechanism
is mainly microtubuledependent and involves the
molecular motor cytoplasmic
dynein.
Axonal anterograde
transport
Traffic of molecules and
organelles from cell bodies
to nerve terminals. This
mechanism is mainly
microtubule-dependent and
involves molecular motors of
the kinesin family.
Neurotrophin
One of a family of growth
factors involved in neuronal
growth, differentiation and
survival. A classical example is
NGF, which is involved in the
survival of specific neurons
during development.
coordinate the transport of several organelles (for exam‑
ple, mitochondria and endoplasmic reticulum), under‑
lining the complex interactions between motor proteins
during intracellular transport 14,15. Axonal transport is
also modulated by microtubule‑interacting proteins
and neuronal subcompartments such as the axon initial
segment (FIG. 1), which regulates the targeting of cargo
to the axons16. Moreover, to ensure fast and restricted
control of protein synthesis in rapidly expanding areas
such as growth cones or during synaptic plasticity and
injury responses, mRNAs and microRNAs are spe‑
cifically transported in neurites, allowing neurons to
synthesize proteins on demand locally17–19.
Axonal retrograde transport also allows peripheral
signals to be translated into nuclear responses. For
example, receptors that are activated by target‑derived
neurotrophins during development create ‘signalling
endosomes’, which contain neurotrophin receptor
complexes as well as downstream‑activated molecules
such as extracellular signal‑regulated kinase 5 (ERK5;
also known as MAPK7) and the cyclic AMP‑responsive
element‑binding (CREB) transcription factors20. The
delivery of signalling organelles to the soma ensures the
survival of neurons that have reached a physiological
target, whereas cells that do not connect with appropri‑
ate targets undergo apoptosis21. During adulthood, this
mechanism is maintained in specific neuronal popula‑
tions that rely on neurotrophins for survival. It is there‑
fore not surprising that environmental factors or genetic
mutations resulting in impaired axonal transport lead to
neuronal death and that disrupted transport is associ‑
ated with several neurodegenerative disorders, including
Huntington’s disease, Alzheimer’s disease, amyotrophic
lateral sclerosis and hereditary spastic paraplegia14,22.
Neurotropic viruses and toxins
Given the essential role of axonal transport, it seems logi‑
cal that viruses and bacterial toxins would take advantage
of this pathway to access the CNS. owing to their impact
on human health, poliovirus23, RABV24, alphaherpes‑
viruses25 and TeNT26 are among the best characterized
pathogenic agents that undergo axonal transport. In
addition, axonal transport is used by WNV27,28, TMEV29,
measles virus30, BDV31, human enterovirus 71 (REF. 32)
and influenza A virus H5N1 (REF. 9), among others
(TABLE 1). Moreover, BoNT type A (BoNT/A), which
was thought to act mainly at neuromuscular junctions
(NMJs) and other peripheral nerve terminals, also
undergoes retrograde transport in motor neurons,
unlike the other BoNTs 33 (TABLE 1) . Interestingly,
WNV27 and canine distemper virus34 can enter the CNS
through both axonal transport and the BBB, giving rise
to distinct neuronal pathologies. other microbial agents
— such as measles virus30 — exploit the axonal transport
route after passing the BBB.
Numerous studies on axonal transport of patho‑
gens have used direct intravitreous, intramuscular or
intrasciatic injections of the pathogen; for example, this
has been carried out for WNV28, TMEV29 and several
adenoviruses35. These sites of delivery may differ from
the physiological route of pathogen entry, bypassing
some endogenous internalization mechanisms or forc‑
ing ‘unnatural’ tropism. Nonetheless, these approaches
have helped to dissect the molecular mechanisms behind
the transport of these pathogens and have opened new
avenues for their use in gene therapy and neuronal‑
network tracing. Indeed, viral vectors can be delivered
into almost any site of the CNS, making them promising
tools for gene therapy of the CNS36 as well as for address‑
ing questions about more complex cognitive behav‑
iours (BOX 2). Because of their transynaptic spreading,
herpesviruses and rabies viruses have also been used to
map transneuronal circuits37,38. Several laboratories use
viruses and toxins to characterize factors that are also
involved in the regulation of axonal dynamics and that
are defective in neurodegenerative diseases.
Finally, the advent of ‘cellular microbiology’ in the
mid 1990s39 instigated the study of intracellular traffick‑
ing of viruses, bacteria and virulence factors. To date,
most studies have focused on epithelial‑like cells40, irre‑
spective of the cellular tropism of the infectious agent.
Several seminal studies have identified key cellular
proteins and their functions in pathogen trafficking.
Interestingly, some of these ubiquitous cellular players
also have specialized roles in axonal transport.
Entering the central nervous system
Binding to nerve terminals is a crucial step in the neu‑
ronal uptake of some microbial agents, but the pathways
of entry can be quite different. RABV41 and alphaherpes‑
viruses25 must replicate and spread in non‑neuronal cells
before entering peripheral nerves, possibly to boost the
chances of a viral particle accessing the CNS. other
pathogens and virulence factors, such as BDV 42 and
clostridial neurotoxins, can enter neurons directly using
endocytic and intracellular trafficking pathways coupled
to the axonal machinery (see below).
Binding the axonal membrane. To reach the CNS,
viruses and toxins can enter different types of nerve
endings, such as sensory‑nerve endings and NMJs (after
dermal or muscle wounds, respectively). NMJs are spe‑
cialized synapses that connect motor neurons to muscles,
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Table 1 | The toolkit of neurotropic viruses and toxins
Agent
Viral family
Entry into the cNS
Pathology
Herpesvirus
Herpesviridae
Sensory endings
Oral and genital herpes,
encephalitis, and keratitis
25
Poliovirus
Picornaviridae
BBB and NMJs
Paralysis
23
Rabies virus
Rhabdoviridae
NMJs
Encephalitis
24
Measles virus
Paramyxoviridae
BBB and peripheral
nerves?
Encephalitis
30
West Nile virus
Flaviviridae
BBB and peripheral
nerves
Encephalitis and flacid
paralysis
27
Bornavirus
Bornaviridae
Nose and olfactory
neuroepithelia
Behavioural changes
31
Influenza A virus H5N1
Orthomyxoviridae Peripheral nerves
Enterovirus 71
Picornaviridae
Peripheral nerves
Encephalitis and flacid
paralysis
Theiler’s murine
encephalomyelitis virus
Picornaviridae
Transfer to
oligodendrocytes after
axonal transport
Inflammation,
demyelination and axonal
damage
Adenovirus
Adenoviridae
NMJs and occular
infections?
Brain tumours and
encephalitis
Tetanus toxin (a clostridial
neurotoxin)
N/A
NMJs
Tetanus (spastic paralysis)
Botulinum toxin A (a clostridial
neurotoxin)
N/A
NMJs
Botulism (flaccid
paralysis)
gp120 (an HIV envelope protein)
and Tat (the HIV transactivator
protein)
N/A
BBB (through
HIV-infected leukocytes)
Dementia
Encephalitis
refs
9
33
4
45
2, 26
2
101,
102
BBB, blood–brain barrier; CNS, central nervous system; NMJ, neuromuscular junction.
Neuromuscular junction
Specialized synapse that
connects motor neurons to
muscle fibres. Axon terminals
contact muscle fibres through
motor end plates, which
are specialized regions that are
responsible for the transmission
of electrical signals.
Lipid raft
Microdomain of the plasma
membrane that is enriched in
cholesterol and sphingolipids.
Certain classes of GPI-anchored
and transmembrane proteins
acting as virus and toxin
receptors seem to be
concentrated in these
structures.
forming a functional motor unit (FIG. 1). NMJs have been
intensively studied as a model for both synaptic connec‑
tion and pathogen invasion of the CNS. uptake of patho‑
gens or their agents at NMJs has been described in mice
and non‑human primates for poliovirus23,43, RABV44 and
clostridial neurotoxins2. Moreover, intramuscular injec‑
tion of canine adenovirus type 2 (CAdV‑2; also known
as CAV‑2)35,45 in leg muscles gives rise to preferential
motor neuron transduction. Although RABV transport
was reported in motor and sensory neurons, data from
rodents and primates suggest that NMJs may be the
main point of RABV entry 46. This is probably due to
the presence of RABV receptors — the neural cell adhe‑
sion molecules (NCAMs) and nicotinic acetylcholine
receptors (nAChRs) — at NMJs46. By contrast, sensory‑
nerve endings are the main gateway for herpesvirus
infection of the CNS25.
Receptors for many pathogens seem to be con‑
centrated at synapses. Synapses are regions with high
membrane dynamics owing to the exo‑endocytosis of
synaptic vesicles that occurs during stimulation (FIG. 1).
Exocytosis of synaptic vesicles and granules occurs
mainly at active zones, which typically occupy the centre
of synapses47. By contrast, synaptic vesicle endocytosis is
thought to occur mainly outside active zones47 and may
regulate internalization of the receptors that pathogens
exploit to reach the CNS. Specialized regions of the syn‑
apse also concentrate specific molecules. For example,
lipid rafts contain several lipids and proteins that interact
with viruses (such as cholesterol, which interacts with
pseudorabies virus48) or with bacterial toxins (such as
polysialogangliosides, which interact with BoNTs and
TeNT49). In addition to binding polysialogangliosides,
BoNTs and TeNT bind other synaptic‑vesicle proteins,
including synaptotagmins, bound by BoNT/B and
BoNT/G, and synaptic vesicle 2 proteins (SV2s), bound
by BoNT/A, BoNT/E and BoNT/F2 (FIG. 1) (see below).
Given the high rate of fusion and recycling of these
organelles and their high concentration at nerve termi‑
nals, it is easy to understand why membrane proteins
associated with synaptic vesicles might be preferential
targets for neurotropic viruses and toxins.
In addition to synaptic vesicle components, other
classes of molecules found at nerve endings are recog‑
nized by infectious agents. For example, highly con‑
served cell adhesion proteins act as pathogen receptors
in numerous species (FIG. 1). This is illustrated by the
broad host range of alphaherpesviruses50 and RABV24.
Furthermore, members of the same protein family can
act as receptors for different viruses. CD155, a member
of the immunoglobulin superfamily, serves as a recep‑
tor for poliovirus, and nectin 1 (also known as PVRl1)
and nectin 2 (also known as PVRl2), which also belong
to the immunoglobulin superfamily, are receptors for
members of the Herpesviridae family 51. The interaction
of herpesvirus glycoprotein D with nectin 1 initiates viral
uptake and is consistent with preferential targeting of the
virus to dorsal root ganglia neurons, because nectin 1
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Dendrite
Somatodendritic
compartment
Nucleus
Axon initial
segment
Microtubules
Axon
Motor
neuron
Dynein
Kinesin
Retrograde
Anterograde
–
Dynactin
Node
of Ranvier
+
Myelin
Synaptic
vesicle
Nectin 1
CD155
NCAM
G SV2 CAR
Muscle
nAChR
Herpesvirus
Glycoprotein D
RABV
Sensory-nerve ending
(DRG axon)
TeNT BoNT
Poliovirus
CAdV-2
Neuromuscular junction
Figure 1 | Neuronal architecture and axonal transport. Neurons are the most
Nature
Reviews
| Microbiology
polarized cells of the body, with extensions (axons) that exceed
a metre
in length
in
large animals. They are the functional cellular unit of the nervous system. Axons can be
enclosed in myelin sheaths (consisting of several layers of cellular membranes originating
from oligodendrocytes or Schwann cells), which provide enhanced propagation
of electrical signals. Intracellular transport has an essential role in the distribution of
neuronal proteins and organelles. Microtubules are polymers of α-tubulin and β-tubulin
and form major cytoskeletal tracks that are involved in intracellular transport. Microtubules
display mixed polarity in proximal dendrites and are unipolar in axons and distal
dendrites. Cytoplasmic dynein is the motor responsible for retrograde transport of cargo
from nerve terminals to the cell body, whereas members of the kinesin family move
cargo in the opposite direction. Specific subcompartments, such as the axonal initial
segment, regulate polarized transport. Neuromuscular junctions and sensory-nerve
endings contain membrane molecules that can serve as receptors for viruses and toxins.
Some adenoviruses bind the coxsackievirus and adenovirus receptor (CAR; also known
as CXADR) at neuromuscular junctions to enter the central nervous system. Similarly,
poliovirus binds CD155, and rabies virus (RABV) binds neural cell adhesion molecules
(NCAMs) and nicotinic acetylcholine receptors (nAChRs). Some botulinum toxins (BoNTs;
also known as Bot proteins) bind synaptic vesicle 2 (SV2) proteins to enter neurons.
Sensory-nerve endings of dorsal root ganglia (DRG) neurons contain nectin 1 (also
known as PVRL1), a surface protein recognized by glycoprotein D of alphaherpesviruses.
CAdV-2, canine adenovirus type 2 (also known as CAV-2); G, ganglioside; TeNT, tetanus
toxin (also known as TetX).
is present at sensory‑nerve endings but not at NMJs52,53
(FIG. 1). Similarly, binding of poliovirus to NMJ‑localized
CD155 may be the first step in the neuronal spread of
this virus23,54. The coxsackievirus and adenovirus recep‑
tor (CAR; also known as CXADR), another member
of the immunoglobulin superfamily, and NCAM are
found at NMJs and act as receptors for adenoviruses35,45
and RABV55, respectively (FIG. 1). In addition, molecules
involved in neuronal homeostasis, such as the neuro‑
trophin receptor p75NTR (also known as NGFR), which
is recognized by RABV glycoprotein G56, may be ideal
targets for pathogen recognition and uptake.
Some receptors are involved in transcytosis, a mecha‑
nism that allows the transfer or targeting of ligands from
axon terminals to the somatodendritic compartment and
then to other juxtaposed cells. This axodendritic path‑
way is mirrored by a somatodendritic‑to‑axonal target‑
ing mechanism that is used by some newly synthesized
proteins, such as neuronal‑glial cell adhesion molecule
(NgCAM) and tropomyosin‑related kinase B (TrkB;
also known as NTRK2)57,58. Both proteins are targeted
to axons following ligand‑independent internalization
or recycling at the somatodendritic membrane57,58. It is
tempting to speculate that similar mechanisms exist to
target axonal receptors back to the cell body. Pathogens
using these molecules as receptors may exploit this proc‑
ess for their transport to the soma. For example, CAR
seems to be constitutively transported in the sciatic
nerve without exogenous ligand45 and so may be an ideal
means for some adenoviruses and for coxsackievirus B
to reach the CNS and cause pathogenesis59,60.
Entering neurons through endosomes. Receptor‑
mediated entry provides pathogens with an efficient way
of exploiting the regulatory mechanisms that control
endocytosis and trafficking. For the most part, as men‑
tioned above, pathogen internalization and sorting have
been characterized in epithelial‑like cells61, in which dis‑
tinct endocytic mechanisms have been described. These
mechanisms include clathrin‑dependent and clathrin‑
independent uptake, caveolae‑dependent internaliza‑
tion, phagocytosis and macropinocytosis62. In these
cells, endocytic vesicles can be retrogradely trans‑
ported to the trans‑Golgi network or recycled to allow
internalized molecules to be redirected to the plasma
membrane, or they can deliver cargo to lysosomes for
degradation62.
Although these vesicle transport pathways are
mostly conserved in neurons, some notable exceptions
exist. For example, the tethering factor early‑endosome
antigen 1 (EEA1) is absent in axons, suggesting a dif‑
ference in the mechanisms of early‑endosome sort‑
ing in epithelial‑like cells and axons63. Furthermore,
synaptic vesicles are distinct from the early‑endocytic
structures found in fibroblasts and vary depending on
whether the vesicles are recruited to the readily releasable
or the reserve pool64. The contribution of these pools
to the uptake and intracellular traffic of pathogens is
still unclear. As synaptic vesicles mainly undergo cycles
of local exo‑endocytosis, a mechanistic understand‑
ing of the link between these organelles, their trans‑
synaptic shuttling 65 and their long‑range axonal
transport needs to be addressed. For example, BoNT/A,
which interacts with SV2 proteins to enter NMJs (FIG. 1),
is transported to the soma of motor neurons33. Whether
the transported BoNT/A is still in the lumen of a syn‑
aptic vesicle, sorted to another endocytic organelle or
transported directly in the axoplasm is unknown2.
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Box 2 | Viral vectors for gene transfer in the central nervous system
There are more genes expressed in the mammalian central nervous system (CNS) than in any other tissue, creating a
formidable temporal and spatial complexity that confounds functional and therapeutic questions. The choice of gene
transfer vector, which can be derived from pathogens of various origins, depends on many parameters, including the
required duration of expression, the cell type to be targeted and the cloning capacity of the vector36. Four major CNS
vector platforms are briefly described below.
Adenoviruses
Adenoviruses are non-enveloped DNA viruses. Adenovirus vectors have cloning capacities of ~30 kb, can be purified
into pure high titres (>1013 physical particles per millilitre) and lead to long-term expression (in the order of years) in vivo
without integration. In vivo, human adenovirus serotype 5 vectors can transduce ependymal cells, oligodendrocytes and
neurons, but they preferentially transduce astrocytes in the brain parenchyma. Limited retrograde transport has been
reported. Vectors derived from canine adenovirus type 2 preferentially transduce neurons, and axonal retrograde
transport of these vectors is particularly efficient.
Adeno-associated viruses
Adeno-associated viruses (AAVs) are non-enveloped single-stranded-DNA viruses of the family Parvoviridae. Most
serotypes have a cloning capacity of ~5 kb. Human and non-human primate serotypes transduce a wide range of cells in
the CNS, including neurons. The ability to be transported by axonal retrograde transport varies depending on the
serotype. The duration of transgene expression also seems to vary depending on the serotype, the target cells and
the host. The serotypes that preferentially transduce neurons can lead to long-term expression in vivo.
Herpes simplex virus
Herpes simplex virus type 1 (HSV-1) is an enveloped DNA virus. Replication-incompetent vectors (~108 infectious
particles per millilitre) efficiently express transgenes in central and peripheral neurons. Amplicon vectors have a
theoretical cloning capacity of 150 kb and efficiently express transgenes in central and peripheral neurons. The
enormous potential of HSV vectors has been slow to come to fruition, because of their immunogenicity and cytotoxicity.
lentiviruses
Lentiviruses are enveloped retroviruses belonging to the family Retroviridae. They have a cloning capacity of ~8 kb and
can readily be pseudotyped with surface glycoproteins from numerous other viruses. Pseudotyping with envelops from
vesicular stomatitis virus leads to a preferential infection of neurons in the rodent brain. Lentiviruses pseudotyped with
rabies virus protein G undergo efficient retrograde transport in neurons after intramuscular injections.
Specific endocytic pathways at nerve terminals can
provide direct access to axonal retrograde transport.
The binding fragment of TeNT (TeNT HC)66 (BOX 3) is
internalized along with CAdV‑2 and CAR45 in clathrin‑
coated vesicles and progresses from Rab5‑positive to
Rab7‑positive endocytic compartments, which undergo
axonal transport (see below). Similarly, poliovirus and
CD155 are co‑internalized in endocytic organelles that
become coupled to the axonal transport machinery 43,67,
whereas p75NTR, which is used by RABV, can be taken up
through a similar pathway involving clathrin68.
Microtubule-organizing
centre
Site of microtubule nucleation
in eukaryotic cells; it organizes
flagella, ciliae and spindle
poles, and it is closely
associated with the Golgi
apparatus.
Direct membrane fusion and endosomal escape. The
internalization of pathogens does not always involve
entry or progression through the endocytic pathway.
Some enveloped viruses, such as alphaherpesviruses,
undergo pH‑independent fusion with the plasma
membrane25,69 and enter the cytoplasm ‘naked’. In the
axoplasm, the exposed tegument proteins may recruit
adaptor proteins and motors to be transported along
microtubules. other viruses, such as RABV, undergo
pH‑dependent fusion with the membrane at pH <6.4
(REF. 70). This observation implies that certain viruses
escape endocytic organelles at later stages of transport,
when the pH is optimal for their fusion (see below).
Indeed, neuronal transport of fully enveloped RABV in
endosomes has been described71. Non‑enveloped viruses
can also trigger endosomal lysis. In epithelial cells, some
adenoviruses rapidly escape early endosomes after acidi‑
fication and are then transported to the microtubuleorganizing centre by direct recruitment of dynein72. It is
likely that the exit point from the endosomal pathway
is an important difference between viral transport in
epithelial cells and neurons, because most of the axonal
transport of CAdV‑2 occurred in intact endosomes45.
However, this does not exclude the possibility that a
minority of virions escape axonal endosomes and traf‑
fic by interacting directly with motors, as suggested by
studies in epithelial cells73,74.
Long-range axonal transport of pathogens
Why is motor‑based axonal transport a key mecha‑
nism for CNS invasion by pathogens? The large dis‑
tances separating cell bodies from nerve terminals
mean that neither viruses nor endogenous molecules
can rely on passive diffusion. Indeed, it was calcu‑
lated that viral transport through diffusion would take
hundreds of years per centimetre of cytoplasm75. By
contrast, molecular motors ensure the transport of
cargoes at speeds exceeding several micrometres per
second (equivalent to 3–10 millimetres per hour)14.
It is likely that neurotropic viruses and toxins were
therefore selected, in evolutionary terms, owing to
their ability to take advantage of the axonal transport
machinery.
Recruitment of motors. Members of the Herpesviridae
family — for example, herpes simplex virus (HSV) and
pseudorabies virus — are classical examples of pathogens
that can directly recruit molecular motors. Early studies
showed the crucial role of microtubules in herpesvirus
transport76, and live‑cell imaging in the giant squid axon
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Box 3 | Clostridium spp. toxins
Tetanus toxin (TeNT; also known as TetX) and botulinum toxins (BoNTs; also known as
Bot proteins) are the most potent neurotoxins affecting humans. They are produced by
several Clostridium species and cause spastic (TeNT) and flaccid (BoNTs) paralysis. The
high toxicity is due to their high-affinity binding to neuronal membranes and their
specific cleavage of synaptic SNARE (soluble NSF (N-ethylmaleimide-sensitive factor)
attachment protein (SNAP) receptor) proteins. TeNT and the seven serotypes of BoNT
(types A–G) share sequence and structure homologies. They are each synthesized as a
single-chain, inactive polypeptide of 150 kDa, which is cleaved by cellular proteases to
give rise to a 100 kDa heavy chain (H chain) and a 50 kDa light chain (L chain). The
H chain is functionally divided in two 50 kDa parts, the amino-terminal (HN) fragment,
which is involved in pH-dependent membrane translocation, and the carboxy-terminal
(HC) fragment, which is responsible for neuronal binding. The L chain bears an
endopeptidase activity specific for members of the SNARE family, which are key
regulators of the fusion of synaptic vesicles with the plasma membrane.
allowed one of the first visualizations of viral axonal
transport77,78. Several HSV‑1 and pseudorabies virus pro‑
teins can interact with dynein or dynactin25. Incoming
capsids are associated with the dynein–dynactin com‑
plex, and their transport is blocked by destabilization of
this complex, triggered by dynamitin overexpression79.
Several alphaherpesvirus proteins associate with mem‑
bers of the dynein complex: pul34 interacts with cyto‑
plasmic dynein 1 intermediate chain 1 (REF. 80), whereas
the viral helicase pul9 contains the dynein light chain
DlC8 (also known as DYNll1) binding site, KSTQT,
and associates with DlC8 in vitro81. However, the rel‑
evance of these interactions for the retrograde transport
of herpesviruses is unclear, because these viral proteins
are not found in mature virions25. A more relevant find‑
ing, in the context of transported virions, is the demon‑
stration that HSV‑1 capsid protein pul35 (also known
as VP26) interacts with dynein axonemal light chain 4
(RP3; also known as DNAl4) and dynein light chain
TCTEX1 (also known as DYNlT1)82. Interestingly, cap‑
sids lacking pul35 or most of the tegument proteins
retained their retrograde transport capacity83,84, suggest‑
ing that other tegument proteins associated with entering
capsids, such as pul25, pul36 and pul37 (REFS 25,85),
may mediate retrograde transport.
Dynein is not the only motor recruited by alphaherpes‑
viruses: the HSV‑1 tegument protein uS11 can bind
conventional kinesin heavy chain (KIF5B) in vitro 86.
Whereas retrograde transport is the key process in CNS
invasion by herpesviruses, bidirectional transport was
observed during both entry and egress in sensory neu‑
rons87,88. Not surprisingly, the average speed of retrograde
traffic is higher during viral entry, whereas anterograde
transport becomes dominant during viral egress, sug‑
gesting an efficient regulation of motor coordination
and/or recruitment (see below). Although the mecha‑
nism underlying this phenomenon is still unclear, it may
be due to a difference in the capsid composition dur‑
ing these two phases25,88,89. Interestingly, DlC8‑binding
domains are also present in other viral components81,
such as RABV phosphoprotein81,90. However, depend‑
ing on the immune status of mice, mutations in this
domain of the viral proteins had contrasting effects on
the pathogenicity of the virus24,91. A study using RABV
with a deletion in the DlC8‑binding domain of phos‑
phoprotein showed that this mutant had normal CNS
access, but early transcription of viral genes in neurons
was strongly impaired, suggesting a role for DlC8 during
neuronal replication of the virus rather than in axonal
transport 92. Moreover, RABV protein G, which binds to
p75NTR (REF. 56), induced axonal retrograde transport of
pseudotyped lentivirus93; it is therefore unlikely that the
interaction between RABV phosphoprotein and host
DlC8 has a crucial role in the neuronal transport of
the virus.
Linking receptors to motor complexes. one of the sim‑
plest ways for a pathogen to access the CNS is to bind a
synaptic receptor that can be internalized and directly
recruit motors. The poliovirus protein CD155 has a
TCTEX1‑binding site that allows the protein to recruit
the dynein–dynactin complex and results in the trans‑
port of endosomes containing the virus54,94. In PC12
cells, direct interaction between TCTEX1 and the cyto‑
plasmic domain of CD155, as well as transport studies
with CD155 mutants that cannot bind TCTEX1, sug‑
gested that this interaction is required for the retrograde
transport of poliovirus and its receptor 54. However, a
recent study showed that some CD155‑independent
transport of poliovirus may occur in mice expressing
human CD155, suggesting that parallel trafficking routes
may be exploited by pathogens in vivo67.
Pathogens hitchhiking on long-range vesicular transport pathways. Endocytic and exocytic vesicles are
constantly shuttled in axons and therefore provide a
continuous source of membranes and signals. Some
pathogens can access these organelles to spread in the
CNS. For example, TeNT exploits the vesicular pathway
used by neurotrophins such as β‑nerve growth factor
(NGF) and brain‑derived neurotrophic factor (BDNF),
and their receptors p75NTR and TrkB, to reach the cell
body 66,95 (FIG. 2). These endocytic structures are also
responsible for the receptor‑dependent axonal trans‑
port of poliovirus67 and CAdV‑245 (FIG. 2). Importantly,
these axonal endosomes have a lumenal pH of close to
neutral45,96, which allows cargo to be transported over
long distances in a protective environment, precluding
pH‑induced conformational changes and avoiding deg‑
radation. This finding could also explain why CAdV‑2
is found in the lumen of endosomes in axons but in the
cytoplasm of epithelial cells, in which the pH drop that
occurs in early endosomes is linked to viral escape72,97.
RABV could also use neutral endosomes to be trans‑
ported over long distances, as the pH required for viral
fusion with the membrane is pH <6.4 (REF. 70). Neutral
axonal endosomes could therefore provide a multi‑
functional transport pathway for ligands with distinct
somatodendritic fates.
Pathogenic proteins and axonal transport. Endogenous
pathogenic proteins also traffic in the CNS. The glycosyl
phosphatidylinositol‑anchored prion protein (PrP) can
switch from a non‑pathogenic, protease‑sensitive form
(PrPC) to a pathogenic, protease‑resistant, aggregated
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REVIEWS
p75NTR TrkB
CD155
Herpesviridae
BDNF
Egress
Entry
Dynein
Kinesin
CAdV-2
RABV
or
Poliovirus
TeNT
CAR
+
G
?
Dynactin
Microtubules
–
Retrograde
Mitochondria
Anterograde
+
Figure 2 | Axonal transport of viruses and toxins. Microbial agents exploit several of the mechanisms used by
endogenous organelles such as mitochondria and endocytic vesicles to be transported in the nervous system. Despite
Nature Reviews | Microbiology
some unresolved controversies, studies addressing the transport of members of the Herpesviridae family are shedding
light on how the viruses can access and spread in the central nervous system after entry at peripheral nerve endings. Viral
proteins — in particular, tegument components — can bind cytoplasmic dynein and play a part during axonal transport or
virus assembly. Viral egress may involve fully formed, enveloped virions, or it may take place with non-enveloped particles
and separate capsids (consisting of tegument proteins and viral membranes), with the assembly of virions occurring in
growth cones. Tetanus toxin (TeNT; also known as TetX) enters a dynein-dependent vesicular pathway that is regulated
by the small GTPase Rab7 and used by neurotrophins and their receptors; the question mark indicates the possible
involvement of kinesin in this pathway. Poliovirus, canine adenovirus type 2 (CAdV-2; also known as CAV-2) and their
respective receptors, CD155 and coxsackievirus and adenovirus receptor (CAR; also known as CXADR), are also found
in these multifunctional transport vesicles. Rabies virus (RABV) is transported in axonal vesicles, although some of its
glycoproteins bind dynein, suggesting a direct recruitment of motors to ‘naked’ virions. However, mutations in the
dynein-binding sites of RABV did not abrogate axonal retrograde transport of this virus. BDNF, brain-derived neurotrophic
factor; G, ganglioside; TrkB, tropomyosin-related kinase B (also known as NTRK2).
form (PrPSc) that will spread in the CNS and, ultimately,
cause neurodegeneration98. Prions undergo retrograde
transport in axons after the injection of PrPSc‑containing
brain extracts into the tongue muscles of hamsters99.
Anterograde movement is also detected on intracerebral
injection99, suggesting a complex interaction between
prions and motors of different polarities. Finally, direct
impairment of axonal transport in motor neurons was
also observed after inoculation of muscles with PrPSc
(REF. 100).
Similarly to PrPSc, viral proteins can be transported in
axons independently of fully assembled viruses. Although
HIV is often found in the brains of patients with AIDS,
the virus itself does not seem to infect neurons6. AIDS‑
associated dementia may be due, at least in part, to two HIV
proteins, gp120 and Tat, which trigger neuronal apopto‑
sis101 (TABLE 1). Axonal transport is involved in the spread
of gp120 and Tat and their associated neurotoxic effects.
The envelope protein gp120 undergoes microtubule‑
dependent axonal retrograde transport in the rat CNS
and is responsible for neurotoxic activity 102,103, and Tat‑
induced apoptosis was detected at distal sites following
injection of Tat‑producing astrocytes104. To date, the
molecular mechanisms underlying the transport of Tat
and gp120 remain largely uncharacterized.
Transport efficiency and redundancy. From an evolution‑
ary perspective, natural selection favours microbial agents
that can use several pathways to reach their final destina‑
tion. Viruses engaging the trafficking machinery in mul‑
tiple ways (such as poliovirus, which can be transported
in either a CD155‑dependent or CD155‑independent
manner 67) will have an advantage in CNS invasion and
propagation. In the same vein, HSV and pseudorabies
virus contain several proteins that bind directly to the
dynein complex and, as a result, mutations in individual
viral components have only a mild effect on retrograde
spread of the pathogens24,83. This redundancy is probably
one of the main mechanisms allowing these and other
viruses to be transported efficiently in the CNS25.
NATuRE REVIEWS | Microbiology
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REVIEWS
Different pathogens also recruit motors with a dis‑
tinct polarity to ensure their efficient axonal transport.
Alphaherpesviruses and CAdV‑2 undergo bidirectional
transport with a bias for the retrograde direction dur‑
ing their first phase of infection45,88. Inhibition of either
dynein or kinesin‑mediated trafficking led to a strong
reduction of the overall transport of CAdV‑2, suggesting
a
b TeNT transcytosis
Axon
TeNT
VAMP2
SV
c Measles virus synaptic microfusion
Microfusion
formation
MV
HSV
NK1
HSV episome
heterochromatin
d Rabies virus trans-synaptic spread
RABV
Figure 3 | Somatodendritic sorting of microbial agents. After microorganisms
Reviews
| Microbiology
reach the neuronal cell body, they can have distinct fates. Nature
Cell-to-cell
transfer
is
called transcytosis and can be due to different molecular mechanisms, such as local
synaptic fusion events, or release of material from the presynaptic compartment into
the synaptic cleft and subsequent uptake by endocytosis at postsynaptic sites.
a | Viruses of the Herpesviridae family, such as herpes simplex virus (HSV), can
package their DNA genome into a chromatin-like structure and establish latency in
the nucleus. b | Tetanus toxin (TeNT; also known as TetX) is transcytosed from the
somatodendritic compartment of motor neurons in the spinal cord to adjacent
inhibitory neurons, where a portion of its heavy chain (BOX 3) triggers the
cytoplasmic translocation of the active subunit (the light chain). In the cytoplasm of
nerve terminals, the endopeptidase activity of the light chain cleaves VAMP2 (also
known as synaptobrevin 2), which is a SNARE (soluble NSF (N-ethylmaleimidesensitive factor) attachment protein (SNAP) receptor) protein, and so inhibits the
fusion of synaptic vesicles (SVs) with the plasma membrane, preventing
neurotransmitter release. c | Measles virus (MV) continues its journey in a new
neuron by trans-synaptic spread, possibly involving microfusion events requiring the
specific interaction of the MV glycoprotein F with neurokinin 1 (NK1; also known as
TACR1). d | Rabies virus (RABV) is released in the synaptic lumen and re-enters at the
postsynaptic level to disseminate in the central nervous system.
a possible coordination between these two motors45.
Interestingly, motors of different polarities are found
on organelles undergoing bidirectional transport, such
as mitochondria. How the coordination between these
large complexes is regulated is only starting to emerge105.
In this light, viruses and toxins may be ideal tools to
understand the regulation between different motor
complexes.
Reaching the neuronal cell body
The last step in the transfer of cargo from an axon to
the cell body is still poorly understood. In epithelial‑like
cells, numerous viruses that are targeted to the nucleus
transit through the MToC40. Although the MToC may
be a potential sorting platform for axonally‑transported
cargoes, no clear role has been reported for this struc‑
ture. Pathogens follow different intracellular fates
after they have arrived in the soma (FIG. 3). In the case
of DNA viruses, the nucleus is their final destination.
Alphaherpesviruses establish latency by ‘silencing’ the
expression of their episomal DNA, partly owing to their
genome being packaged into chromatin‑like structures106
(FIG. 3a). Periodic reactivation of latent HSV leads to DNA
replication and de novo viral‑protein synthesis, resulting
in new viruses travelling along axons back to the primary
infection site. The mechanisms responsible for antero‑
grade transport and egress of herpesviruses remain con‑
troversial25. live‑cell imaging with fluorescently labelled
pseudorabies viruses and electron microscopy analyses
showed anterograde transport of fully formed viruses
(with teguments and an envelop)107–109, whereas other
studies found mostly unenveloped capsids in axons110,111
(FIG. 2). In the unenveloped or ‘subassembly’ transport
model, assembly of virus particles with a full envelope
would only occur at distal axonal sites before release25.
However, recent ultrastructural data suggest that the
anterograde transport of pseudorabies virus occurs in
vesicles109. Interestingly, glycoprotein E, which is needed
for replication in epithelial cells and subsequent retro‑
grade transport of HSV‑1 in neurons112, is also crucial for
the anterograde spread of HSV‑1 (REF. 113).
In contrast to DNA viruses, other viruses can repli‑
cate in the cytoplasm. RABV, an RNA virus, uses protein
aggregates called Negri bodies, which contain the innate
immune response receptor Toll‑like receptor 3 (TlR3),
as a viral factory to enhance its replication in neuronal
cells114. Whether axoplasmic replication of RABV can
also occur remains to be seen.
Transcytosis. Some toxins (for example, TeNT) and
viruses (for example, HSV and RABV) undergo transcy‑
tosis and continue their journey in connecting neurons
(FIG. 3b–d). In this case, the mechanisms used by infectious
agents seem to differ: neuron‑to‑neuron spread can occur
across synapses, as has been shown for measles virus30,
WNV27 and RABV3, or by direct cell‑to‑cell transfer, as is
the case for pseudorabies virus115. However, the molecular
mechanisms involved in transneuronal transfer remain
mostly uncharacterized. It is tempting to speculate that
viruses which bind to receptors undergoing transcytosis
may be efficiently transferred to adjacent neurons. Data
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REVIEWS
Microfusion event
Local membrane fusion
triggered by the interaction
between certain viral proteins
and surface receptors.
addressing the trans‑synaptic spread of measles virus
shed light on how viruses can force this cellular cross‑
ing: in this example, synaptic transfer depends on the
interaction between viral proteins and neurokinin 1 (also
known as TACR1), leading to microfusion events30 (FIG. 3c).
In another model system, it was shown that TMEV was
transferred to myelin sheets of oligodendrocytes after
axonal transport in the optic nerve to allow persistent
infection29. In mice expressing mutants of myelin com‑
ponents, TMEV could not induce persistent infection
after intravitreous injections. Clostridial neurotoxins
can also reach secondary neurons (BOX 3; FIG. 3b). TeNT
is transcytosed from motor neurons to the synapses of
inhibitory neurons, where it cleaves the synaptic protein
VAMP2 (also known as synaptobrevin 2), thus inhibiting
neurotransmitter release2. on intramuscular injection of
high doses of BoNT/A, this toxin undergoes a similar
transcytosis process after axonal retrograde transport in
motor neurons33.
Conclusions and perspectives
understanding the function of neuronal networks
remains one of the exciting challenges of modern neuro‑
biology. Many pathogens undergo axonal transport to
reach and damage the CNS, but we are only just start‑
ing to understand the mechanisms responsible for their
neuronal targeting. Many questions concerning axonal
transport of viruses and pathogenic proteins remain.
For example, there is increasing evidence that local
translation is a key process for ensuring a fast response
to acute stimuli in axons, such as injury or local growth.
local translation of importin‑α is required for the retro‑
grade transport of signalling complexes containing
nuclear localization signals18. These targeting motifs are
found in many viral proteins (for example, pul36 of
alphaherpesviruses) and numerous endosomal proteins
that are involved in the axonal transport of pathogens.
Therefore, signals that are activated during the entry
of infectious agents at nerve terminals might stimulate
local translation, leading to enhanced axonal transport.
Along these lines, little is known about the signalling
molecules that may be activated during entry and may
regulate axonal transport of pathogens. Whether these
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Acknowledgments
We thank the members of the G.S. and E.J.K. laboratories
and J. Schwarz for constructive comments. Work in the G.S.
laboratory is supported by Cancer Research UK, the Motor
Neurone Disease Association, the Jean Coubrough Charitable
Trust and funding from the European Community’s 7th
Framework Programme (FP7/2007–2013; grant 222992
— BrainCAV). The E.J.K. laboratory is supported by BrainCAV,
the French Agence National de la Recherche, the Region
Languedoc Roussillon, the Fondation de France and the
Association Française contre les Myopathies. E.J.K. and S.S.
are Institut National de la Santé et de la Recherche
Médicale (INSERM) fellows.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
Entrez Genome: http://www.ncbi.nlm.nih.gov/genome
BDV | canine distemper virus | CAdV-2 | RABV
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
genomeprj
HIV-1 | WNV
UniProtKB: http://www.uniprot.org
BDNF | BoNT/A | CAR | CD155 | glycoprotein D | nectin 1 |
p75NTR | TeNT | TrkB
FURTHER INFORMATION
Eric J. Kremer’s homepage: http://www.igmm.cnrs.fr/spip.
php?rubrique35
Giampietro Schiavo’s homepage: http://london-researchinstitute.co.uk/research/loc/london/lifch/schiavog/?view=L
RI&source=research_portfolio
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