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AnUpdateonNon-clathrin-coated
Endocytosis
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VOL. 7: 199–209 (1997)
}
An Update on
Non-clathrin-coated Endocytosis
N. E. Bishop
Department of Biochemistry, University of Manchester Medical School, Manchester M13 9PT,
UK
SUMMARY
Recent evidence has proved that in addition to the well-documented clathrin-mediated
endocytic route (vesicles of 100–150 nm), at least three distinct non-clathrin-coated endocytic
pathways function at the surface of mammalian cells. Endocytosis via these pathways is
initiated by caveolae (50–80 nm), macropinosomes (500–2000 nm) and micropinosomes
(95–100 nm). The current state of knowledge about these non-clathrin coated endocytic routes
is presented and evidence that endocytic routes other than via clathrin-coated vesicles are
utilised by viruses is discussed. The recent advances in these areas have provided us with tools
to investigate the entry of those viruses which appear to enter cells via endocytosis into
non-clathrin-coated vesicles. Data indicate that these four endocytic pathways differ in the
absence, presence and/or type of coat on the vesicles, the size of the vesicles, their sensitivity
to a variety of inhibitors, and in the ligands endocytosed. A historical perspective of the
discovery of these non-clathrin-coated endocytic pathways is provided and recent information
is summarised and discussed. The entry of viruses via non-clathrin-coated pits is destined to be
an exciting new area of viral-cell entry, as has been indicated recently by the finding that entry
of simian virus type 40 into cells occurs via caveolae. ? 1997 by John Wiley & Sons, Ltd.
Rev. Med. Virol. 7: 199–209 (1997)
No. of Figures: 4 No. of Tables: 2
Accepted 9 July 1997
INTRODUCTION
Viruses generally enter cells either by endocytosis or by
direct penetration of the plasma membrane (PM), the
former mode of entry being the topic of this review.
Endocytosis is a term covering cellular uptake of both
ligands (receptor-mediated endocytosis) and solutes
(fluid-phase endocytosis or pinocytosis) into vesicles
which bud into the cytoplasm from the PM. Endocytosis
can also include phagocytosis, which only occurs in
specialised cells and is not covered here. Often the term
‘endocytosis’ is used interchangeably with ‘clathrinmediated endocytosis’ as this is the best characterised
endocytic pathway, however, this review aims to concentrate on the information available on the role of nonclathrin-coated vesicles in endocytosis. The existence of
more than one endocytic pathway into cells has been at
times controversial, but compelling data have now accumulated supporting the existence of these non-clathrin
coated endocytic pathways1–3 and will be discussed in
further detail here. The review will summarise the current
general knowledge of these non-clathrin-coated endoAbbreviations used: DAF or CD55, decay-accelerating factor; EGF,
epidermal growth factor; GPI, glycophosphatidylinositol; HRV2,
human rhinovirus type 2; IP3, inositol 1,4,5-triphosphate; PM, plasma
membrane; PMA, phorbol-12-myristate-13-acetate; PV, poliovirus;
SV40, simian virus type 40; VLP, virus-like particle; WNV, West Nile
virus.
CCC 1052–9276/97/070199–11 $17.50
? 1997 John Wiley & Sons, Ltd.
No. of References: 79.
cytic pathways, and explain why knowledge of these
areas is of relevance and importance for virologists. The
clathrin-coated vesicle pathway (see Figure 1) has become
increasingly well documented at the expense of noncoated endocytic pathways. Such endocytic mechanisms
have now been described in a variety of cell types in vitro
and in situ for a range of markers. The implications of
such pathways on the entry of viruses and their uncoating therefore needs to be considered. There have been
many reviews on clathrin-mediated endocytosis and this
pathway will be discussed only briefly.
HISTORICAL PERSPECTIVE
Endocytosis is considered to be responsible for both
fluid-phase and receptor-mediated uptake of markers by
cells. Clathrin-coated vesicles (Figure 1) have been documented to mediate uptake of various ligands, including
viruses such as retroviruses, herpesviruses, orthomyxoviruses, paramyxoviruses, alphaviruses, rhabdoviruses,
togaviruses, adenoviruses, papovaviruses, reoviruses and
picornaviruses. Subsequent passage of such ligands
through endosomes and lysosomes leads to the exposure
of virions to low pH conditions. The acidic conditions
encountered in endosomes can trigger conformational
changes in some viruses, leading to the fusion of the
200
Figure 1. Schematic intepretation of the first of the four endocytic
pathways in mammalian cells. Receptor-bound ligands, membrane
markers, and fluid-phase molecules are taken up by cells via the wellcharacterised clathrin-coated pit pathway. The clathrin-mediated endocytic pathway is initiated when clathrin-coated vesicles bud into the
cytoplasm, lose their clathrin coat and become acidified. Virus particles
can exploit the acidic conditions to gain entry to the cytoplasm, use
the membrane of the endosomes to initiate replication-complex
formation, and/or use endosomes to deliver them to the correct
location within the cell for replication to occur. Vesicles have a
diameter of between 100–150 nm. Markers following this pathway
can be recycled to the cell surface or ultimately reach lysosomes for
degradation. It is possible that a link exists between this pathway and
the Golgi complex.
endosomal membrane with the viral envelope or capsid.
The precise time and location of virus entry may therefore be determined by the pH-dependence of the fusion/
penetration activity. If a virus can fuse with or penetrate
membranes at neutral pH, or due to interactions with its
receptor alone, virus entry may occur in early endosomes
or at the PM. For viruses, such as Sendai virus, fusion is
pH-independent, and can occur in acidic, neutral or mildly
alkaline conditions and may occur either at the PM or in
early or late endosomes. For other viruses, however, the
site at which uncoating occurs may determine whether
replication ensues.4
Early data indicated that the fluid-phase pathway had
at least two different mechanisms: via coated pits and
vesicles, and via smaller non-coated pits and vesicles.
Initially, knowledge of at least two endocytic pathways
was based on morphological studies and the use of
experimental approaches where internalisation of various
ligands was selectively inhibited.
Inhibition of coated pit formation in Hep2, Vero, and
WI38 cells was achieved by hypertonic shock and
potassium depletion, which blocked the entry of transferrin and diphtheria toxin, but not ricin toxin into cells.5
Hep2 cells with active coated pits internalised twice as
much ricin toxin as Hep2 cells without coated pits. Entry
of ricin toxin was a slow process when compared with
transferrin internalisation. This demonstrated that while
diphtheria toxin and transferrin entered by clathrinRev. Med. Virol. 7: 199–209 (1997)
N. E. BISHOP
coated pits, another system of endocytosis was used by
ricin.
Further convincing evidence for the existence of noncoated endocytic pathways came from studies of cells
transfected with a dominant-negative mutant of dynamin.
Dynamin is required for pinching-off of clathrin-coated
vesicles at the PM but, when nonfunctional, fluid-phase
uptake still occurs, although cells can no longer internalise markers typically entering cells via clathrin-coated
vesicles.6 Again the kinetics of internalisation for receptors utilising non-coated vesicles appeared to be slower
than that of receptors entering cells via the coated vesicle
pathway.7,8
The non-clathrin-mediated internalisation pathways
detected were functionally distinct, since manipulations
which disrupted internalisation through coated pits did
not disrupt uptake via the non-coated pathways.7
Clathrin-independent endocytic pathways were also demonstrated in unperturbed cells and these pathways were
not simply artifacts of the inhibition process.8–10
Coated pits occupy only 0·4% of the adipocyte cell
surface, one fifth to one tenth that reported on fibroblasts
and hepatocytes, cell types in which receptor-mediated
endocytosis has been extensively studied. Non-coated
pits, in contrast, are numerous and occupy 13·1% of the
adipocyte cell surface, 7–12 times greater than has been
reported on fibroblasts.11 The coated pit pathway
accounts for around 50% of total endocytosis in some,12
but not all fibroblasts.13 The coated pit pathway appears
to contribute to only 16% of total endocytosis in
polymorphonuclear leukocytes.14 In other cells, such as
macrophages or hepatocytes, the contribution of the
coated pit pathway to the ability of the cell to internalise
fluid volume is small or negligible.14
Studies on insulin entering cells via smooth nonclathrin-coated vesicles, found ligand subsequently
co-localised with a marker entering cells via clathrincoated pits.15 Further studies likewise detected markers,
initially entering from coated and non-coated regions,
subsequently in common intracellular vesicles.8,14,16
However, in some situations the intracellular destination
of ligand internalised via non-coated regions of the PM
differed from that of ligand internalised via coated pits.7
One clathrin-independent mechanism of ligand internalisation was reported to lead directly to the ER.17 In BHK
cells, approximately 5% of ricin internalised can be found
in the Golgi complex, most of this in the transGolgi network. This is also true for polyvalent ricin
conjugates in MCF-7 and Vero cells, but not for monovalent ricin conjugates.18 For some ligands, therefore, a
link appears to exist between the endocytic and exocytic
pathways.
Although clathrin-coated pits are clearly involved in
entry of many enveloped and non-enveloped viruses,
other endocytic mechanisms have been encountered (see
Table 1). Influenza virus and Sendai viruses appear to use
both coated and non-coated vesicles to enter cells,19
whilst EBV has also been observed entering cells via
non-coated vesicles20 and uptake has been reported to be
sensitive to cytochalasins.21 Papovaviruses22 and simian
? 1997 John Wiley & Sons, Ltd.
NON-CLATHRIN COATED ENDOCYTOSIS
201
Table 1. Examples of viruses entering cells via non-clathrin coated vesicles
Virus group
Enveloped viruses
Orthomyxo
Retro
Toga
Herpes
Non-enveloped viruses
Papova
Papova
Picorna
Papilloma
Virus
Receptor
Influenza virus
Sialic acid
HIV
HIV
Semliki Forest Virus
Galactosyl ceramide
GPI-linked CD4
Galactosyl ceramide
West Nile virus
EBV
Fc receptor
CR2/CD21
SV 40
Polyomavirus
MHC-1
Glycoproteins
Poliovirus
HRV 2
Coxsackieviruses
Echoviruses
Enterovirus type 70
Human papillomavirus
Immunoglobulin receptor family
LDL family
DAF or CD55
DAF or CD55
DAF or CD55
á6 integrin subunit
virus type 40 (SV40)23 are known to be internalised
predominantly into non-coated vesicles, and these vesicles are very small and tight-fitting. In the picornavirus
family, polioviruses may enter by both coated-vesicles24
and another endocytic pathway.25,26 Human rhinovirus
type 2 has also been reported to infect cells in clathrinindependent manner.24 Until recently, it has not been
possible to investigate the process of viral entry via
non-clathrin coated vesicles, because of a lack of information about their basic biology. Cell biologists are now
providing further information on these pathways, much
of which will be of relevance to virologists, who can now
study the viral entry process via these mechanisms in
more detail.
Thus, for a number of cell types and markers, a
growing pool of evidence shows that uptake can occur
from clathrin and non-clathrin coated regions of the PM,
and these other endocytic pathways could serve
additional cellular functions.
ENDOCYTOSIS VIA CAVEOLAE
Caveolae are non-coated, smooth, flask-shaped vesicles of
the PM, which have been noted since some of the earliest
ultrastructural studies of cells. Caveolae are smaller than
clathrin-coated vesicles, with a diameter of 50–90 nm
(Figure 2) compared with 100–150 nm, for clathrincoated vesicles. In the past, caveolae have been referred
to using the more general term of non-coated ‘plasmalemmal vesicles’.
? 1997 John Wiley & Sons, Ltd.
Cell line
References
Canine kidney
CD4-negative human cells
(e.g.) human colon
epithelial HT29 cells)
Transfected CHO
BHK- 21
Macrophage-like
cell line (P388D1)
Human B lymphocytes
19
50
CV-1 and HeLa
Mouse kidney
Mouse embryo
HeLa
HeLa
Various
Various
HeLa
CV-1
53
51,70
62
60,61
27, 28
22, 71
25,26
24
73,74
47–49
49
72
Caveolae are dynamic structures, form free intracellular
vesicles, and can incorporate both fluid-phase and
receptor-bound molecules,29,30 albeit at a slow rate compared with clathrin-coated vesicles.8,27 Studies using
compounds that selectively inhibit caveolae function
provided the first data on which ligands are internalised
and transcytosed via this endocytic route,31–33 and the
role these vesicles play in cellular signalling.34,35
Caveolae possess a granular, spiral cytoplasmic coat
structure composed of fine concentric striations or ridges,
linked together by strands. This coat structure is not
detectable by conventional EM and can only be visualised using high-resolution scanning and rapid-freeze
deep-etch techniques. Caveolin, a phosphorylated integral membrane protein of Mr 21 000, is a major structural
component of this coat.36 One of the best understood
examples of uptake by caveolae is the receptor-mediated
entry of folate in MA104 cells, a monkey kidney epithelial cell line following binding to a glycophosphatidylinositol(GPI)-anchored membrane receptor which clusters
in caveolae, despite the presence of fully functional
clathrin-coated pits.37 The dissociation of folate from its
receptor within cells is thought to be stimulated by low
pH, as agents that dissipate pH gradients in acidic
compartments inhibit folate release.38
Caveolae have recently been demonstrated to mediate
productive uptake of Simian virus type 40.27,28 Caveolae
are enriched in gangliosides, glycosphingolipids, GPIanchored proteins and cholesterol.30,39 Lowering the
cholesterol content of cells inhibits the internalisation
of ligands clustered in caveolae without significantly
Rev. Med. Virol. 7: 199–209 (1997)
202
Figure 2. Uptake of markers via caveolin-coated caveolae. Caveolae
appear as flask-shaped vesicles at the PM, which subsequently bud
into the cytoplasm. These vesicles are the smallest of the endocytic
vesicles, with a diameter of 50–90 nm. GPI-anchored proteins concentrate over these regions of the PM, and the caveolar membrane also
contains a Ca2+ channel and pump, and molecules involved in cellsignalling. Vesicles are surrounded by a striated caveolin-containing
coat, which unlike the clathrin coat, is not typically visible by EM.
Budded vesicles may acidify on internalisation, at least in some cell
types. Current evidence suggests that markers entering this pathway
can be targeted to the ER and subsequently the Golgi, be recycled or
transcytosed, or meet with markers entering by the clathrin-coated
pathway (Figure 1) in late endosomes. SV40 is the first virus
conclusively proven to productively infect cells using this pathway.
inhibiting internalisation of ligands via clathrin-coated
vesicles.40,41 Similarly drugs that bind cholesterol (e.g.
filipin, nystatin), or exposure of cells to cholesterol
oxidase, inhibit caveolar endocytosis, including the
uptake of SV40, without affecting endocytosis via
clathrin-coated pits.27,30,32,42 In addition, the internalisation cycle of caveolae is known to be regulated by kinase
activity and requires an intact actin network.30,43,44
Therefore, phorbol-12-myristate-13-acetate (PMA) or
cytochalasin D, inhibit ligand entry via caveolae, including that of SV40, without affecting clathrin-mediated
endocytosis.27,45,46 In contrast, however, okadaic acid
stimulates caveolae-mediated entry and has an inhibitory
effect on the clathrin-mediated endocytic process.30
These reagents should be useful for identifying further
ligands, including viruses, entering cells by this route
and in determining the molecular basis of caveolar
internalisation.
In addition to the recent data indicating that SV40
enters cells via caveolae, it is of significance that many
coxsackieviruses and echoviruses bind to the GPI-linked
receptor, decay-accelerating factor (DAF or CD55), as
GPI receptors are known to cluster in caveolae.47–49 HIV
also utilises a GPI-anchored PM receptor in a number of
CD4-negative human cells,50 whilst Semliki Forest virus51
binds to galactosyl ceramide, another molecule expected
to cluster in, and enter cells via, caveolae.
Under some conditions caveolae subsequently fuse
with endosomes.8,39,52 For example, the HIV gp120
Rev. Med. Virol. 7: 199–209 (1997)
N. E. BISHOP
molecules bound to a GPI-anchored form of CD4,
internalise at a very slow rate into caveolae, and are
subsequently found within endosomes.53 Following
internalisation, GPI-linked CD4 recycles through a
primaquine-insensitive compartment, in contrast to the
recycling of the transmembrane form of CD4 which
recycles through a primaquine-sensitive vesicular compartment. Thus, whilst these two forms of the CD4
receptor recycle from distinct populations of early endosomes, their subsequent co-localisation indicates that the
two endocytic pathways later converge. Therefore, at
least in certain circumstances, molecules entering cells via
caveolae enter acidic endosomal compartments. Viruses
entering cells via caveolae, would thereby encounter a
low pH environment, and those that are acid-sensitive
would be expected to gain access to the cytoplasm at
that time.
Although some caveolae fuse with late endosomes, the
fate of markers may differ depending on whether ligands
are bound for endocytosis or transcytosis. For example,
although both native and modified albumins can be
internalised by caveolae, depending on the cell type,
only modified albumins are directed to endosomes and
lysosomes, whilst native albumins are transcytosed.32
Caveolar vesicles internalised during okadaic acid treatment of cells accumulate in the perinuclear region,
showing that this route may be capable of delivery of
ligands/viruses to different cellular locations than the
clathrin-coated endocytic route. Further details on the
functions and modes of action of caveolae will be
facilitated by recent reports of the isolation of purified
caveolae from cells.27
ENDOCYTOSIS VIA MACROPINOSOMES
It has been known for many years now that some cells
undergo a dramatic stimulation of membrane ruffling and
fluid-phase endocytosis following exposure to growth
factors or phorbol esters. The endocytic vesicles stimulated by such treatment are large cytoplasmic vacuoles up
to several ìm in diameter (typically 0·5–2·5 ìm) which
can be seen by light microscopy, and are termed macropinosomes (see Figure 3). The increased fluid-phase
endocytosis induced by such reagents is associated with
vigorous surface ruffling, although surface ruffling is not
in itself sufficient for stimulated endocytosis.54 A ruffle
is formed by a band of actin polymerisation directed
outwards near the PM and, unlike clathrin-dependent
endocytosis, macropinocytosis is therefore typically
inhibited by cytochalasin D and colchicine, suggesting a
role for microtubules and microfilaments in entry and
cycling via this pathway.55–57 The requirement for filaments, however, may vary between cell lines.10,58 Significantly, macropinocytosis has recently been shown to be
exploited by a bacterial pathogen to invade epithelial
cells and macrophages.59
Evidence also implicates macropinosomes in the entry
of EBV and West Nile virus (WNV) into cells. EBV infects
two cell types: B lymphocytes and epithelial cells, and
? 1997 John Wiley & Sons, Ltd.
NON-CLATHRIN COATED ENDOCYTOSIS
Figure 3. Endocytosis via large, heterogenously sized macropinosomes. The macropinocytic pathway commences when ruffles form at
the PM due to actin polymerisation. The ruffles can be of many shapes
and sizes, ultimately fuse, and this leads to the formation of large
macropinosomes within the cytoplasm. The fate of these large
endocytic vesicles appears to vary with cell type. Markers may recycle
to the PM without interaction with vesicles from other endocytic
pathways. Alternatively they may decrease in size, acidify and
eventually fuse with lysosomes. Viruses depending on acidic conditions to enter cells may thus be able to infect cells via this pathway,
at least in some cell types.
whilst EM studies show the virus entering lymphoblastoid cells by direct fusion at the PM, entry into normal B
cells occurs by endocytosis into very large, non-coated
vesicles,60,61 a process acutely sensitive to the inhibitory
effects of chloroquine and chlorpromazine.62 EBV thus
enters B lymphocytes via a mechanism morphologically
resembling macropinocytosis. The apparently disparate
routes of EBV entry into epithelial cells and B cells may
have significance for the subsequent fate of nucleocapsids
in each cell type and on the route and mechanisms
responsible for intracellular transport to the nucleus.60
Individual WNV particles, and aggregates of viral
particles enter the macrophage-like cell line P388D1 via
two different mechanisms.63 Uptake of single virions is
mediated rapidly by clathrin-coated pits, whilst aggregates of five or more particles are endocytosed slowly by
extensions of the PM into large non-coated vacuoles of
0·2–0·5 ìm diameter. Specific inhibitors of macropinocytosis have recently been characterised and could now
be used to determine more directly whether this pathway
mediates productive virus-cell entry, as outlined below.
Amiloride and the stilbene compound SITS, (inhibitors
of Na + /H + exchange), block stimulated fluid-phasemarker uptake by macropinosomes in a variety of cells,
without affecting uptake of markers via clathrin coated
pits. These data confirm that the macropinocytic process
is independent of the clathrin-mediated pathway, imply
that macropinocytosis is extremely sensitive to acidification of the cytosol,3,62 and provide a means to study
ligands entering cells via this endocytic pathway.
Macropinosomes are large in relation to other endocytic vesicles and provide an efficient route for the non? 1997 John Wiley & Sons, Ltd.
203
selective endocytosis of solutes, receptors and ligands.
Presumably viruses are also taken up by macropinosomes; a possibility which can be formally investigated.
While macropinosomes are not typically known to concentrate receptors,64 there are exceptions. For example,
epidermal growth factor (EGF) receptors concentrate on
ruffles following ligand binding.65 Moreover, receptors
which concentrate in clathrin-coated vesicles are almost
certainly present, to some extent, on macropinosomal
membranes, and molecules entering these pathways may
reach different intracellular destinations.10 The intracellular fate of macropinosomes, however, does appear to
differ depending on the cell type. In macrophages, they
migrate to the centre of the cell, decrease in size, and
rapidly acidify, forming an endosome-like organelle
which merges with lysosomes.64,66 In A431 cells, on the
other hand, macropinosomes show little interaction with
endosome-like compartments, do not acidify, and they
form a distinct vesicle population which often recycles to
the PM.10 Conceivably, the fate of macropinosomes may
also depend on which receptors are stimulated during
their formation. Nonetheless, macropinosomes are
dynamic structures which can exhibit vesiculo-tubular
morphology and are generally capable of fusing with
each other.10
Macropinosomes, therefore, like coated vesicles, can
become acidified during passage through the cytoplasm.
In such situations, acid-sensitive virus particles typically
gaining cytosol entry from coated pits, could presumably
gain access in a similar manner from within acidified
macropinosome-derived vesicles.
MICROPINOCYTOSIS
Recent results confirm the operation of a further constitutive clathrin-independent endocytic pathway operates
(see Figure 4), separate from that mediated by caveolae or
macropinosomes, which can be up-regulated in compensation for a loss of clathrin-dependent endocytosis.67,68
In HeLa cells where clathrin-mediated endocytosis is
defective due to a defect in the dynamin gene, clathrinindependent endocytosis is induced and, subsequently,
fluid-phase uptake of horseradish peroxidase occurs at
wild-type levels.68 The fluid-phase pathway induced in
these cells is mediated by neither caveolae, nor macropinosomes, as cytochalasin D does not inhibit marker
uptake. In addition, amiloride, which has been shown to
selectively inhibit macropinocytosis in these cells, did not
affect marker uptake.68 The non-coated vesicles (micropinosomes) induced under these conditions are 270–
100 nm in diameter, slightly smaller than clathrin-coated
vesicles, and slightly larger than caveolae.
Micropinosomal endocytosis is similarly observed on
uptake of univalent EGF into A431 cells. Ligand uptake
occurs via two distinct pathways in this cell type, neither
involving clathrin-coated pits.69 In the first pathway,
EGF is internalised into macropinosomes of 0·1–1·2 ìm
diameter, while in the second pathway, EGF clusters in,
and is subsequently internalised into micropinosomes, of
Rev. Med. Virol. 7: 199–209 (1997)
204
N. E. BISHOP
clathrin-independent endocytic pathway, along with
human rhinovirus type 2,24 while others bind to GPIlinked receptors, molecules known to cluster in caveolae
(see below). Semliki Forest virus entry into some cell
types has been reported via small non-coated vesicles,70
however their identity remains unknown. Thus, whilst
some compelling data indicate that viruses utilise endocytic mechanisms other than the clathrin-dependent
route (e.g. SV40; see below), further evidence remains
tantalisingly circumstantial. The opportunity now exists
to characterise these clathrin-independent endocytic
pathways and their role in viral replication.
Simian virus type 40
Figure 4. Schematic diagram of endocytosis via non-clathrin-coated
micropinosomes. This endocytic pathway originates at non-coated
vesicles at the PM, which bud into the cytoplasm to form micropinosomes with a diameter of 70–100 nm. Vesicles entering cells in this
manner become acidified and may fuse with early endosomes in
common with the clathrin-coated endocytic pathway (Figure 1). This
pathway may predominate in some cell types, or it can be upregulated
if the clathrin-coated endocytic pathway is blocked. It may be
advantageous for viruses depending on low pH, or endosomal
targeting, to be able to use both the non- and clathrin-coated vesicles
to enter cells.
2100 nm diameter. Interestingly, a role for these vesicles
in uptake of some picornaviruses has been suggested, but
not proven.24
A final example of micropinosomal uptake is that in
Hep2 cells, where tracer molecules can be visualised in
both coated vesicles (average 110 nm diameter, excluding
the coat) and non-coated vesicles, the latter being significantly smaller (95 nm diameter) than the former. Markers
internalised by micropinosomes in this cell line merged
with endosomes were subsequently found to be delivered
to acidic endosomes.16 These data indicate that the
clathrin-coated and micropinosomal entry pathways
merge, at least in some circumstances, so that acidsensitive viruses would be capable of uncoating under
these conditions.
NON-CLATHRIN-DEPENDENT VIRAL
ENDOCYTOSIS
It is clear that while clathrin-coated vesicles are involved
in the entry of many viruses, other endocytic mechanisms
also occur (see Table 2). EBV has been detected entering
cells via large, non-coated macropinosomal vesicles20 and
uptake has been reported to be sensitive to cytochalasins.21 Papovaviruses and SV40 are known to be internalised predominantly into non-coated vesicles which
are very small, tight-fitting22,23 and resemble caveolae
morphologically. In the picornavirus family, polioviruses
appear to enter cells via coated vesicles and by a
Rev. Med. Virol. 7: 199–209 (1997)
Two different mechanisms of endocytosis have been
demonstrated for SV40 cell entry. Uptake of individual
SV40 particles into CV-1 cells occurs predominantly via
smooth (60 nm) ‘monopinocytic vesicles’, resulting in
delivery to the nucleus, whereas uptake of clusters of
virions, by larger clathrin-coated vesicles, does not result
in nuclear transport.17,23 Most of the incoming virus
particles internalised by the former mechanism, remained
undegraded for many hours, and about one third of these
rapidly entered the ER, with greater numbers reaching
this region at later times post-infection.17 The pathway
followed by the majority of SV40 particles entering cells
was therefore not the classical coated-pit pathway, nor
did it seem to be the ‘classic fluid-phase pathway’ (i.e. not
via micropinosomes) as virus did not co-localise with
horseradish peroxidase.17
The ultrastructural similarities between caveolae, and
uncoated invaginations containing SV40 and its receptor,
MHC-I, prompted investigation on whether SV40
co-localises with caveolae. When such studies were
instigated, SV40 entry was indeed found to occur via
these vesicles. These studies made use of inhibitors of
caveolar function,27 and the co-localisation of virus with
caveolar markers, in conjuction with immunoelectron
microscopy.28 A model has therefore been proposed
where caveolar-association is necessary for the entry of
SV40 into the cell and for subsequent events leading to
delivery to the ER.28
Polyomavirus
As described for SV40, polyomaviruses gain entry into
mouse kidney cells and mouse embryo cells in two ways:
via ‘monopinocytic vesicles’ which lead to the nucleus
and productive infection, and via a degradative, nonproductive pathway terminating in lysosomes.22,71 The
monopinocytic vesicles described do not have a clathrin
coat and are smooth vesicles of 50–70 nm and may
therefore be caveolae or micropinosomes. This remains to
be experimentally determined.
Papillomavirus
While a fully permissive culture system for the replication
of human papillomaviruses has not been established, the
entry of empty virus-like particles (VLPs) has been
studied, and the receptor is known to be the á6 integrin
? 1997 John Wiley & Sons, Ltd.
Cell line
Endocytic vesicles
Methods of characterisation
Inhibitor sensitivity
References
Influenza virus
SV40
Canine kidney
CV-1 and HeLa
Non-clathrin-coated
Caveolae
Not tested
PMA; nystatin;
resistant to cytosol acidification
19
27,28
HIV
CD4-negative
Caveolae
Not tested
50
HIV
Transfected CHO
Caveolae
Primaquine resistant pathways
53
SFV
BHK-21
Caveolae
Not tested
51,70
WNV
EBV
P388D1
B lymphocytes
Macropinosomes
Macropinosomes
Morphological studies
Morphological similarities;
co-localisation with specific markers;
presence in purified caveolae;
slow uptake kinetics
Virus binds to receptors
expected to enter caveolae
Virus binds to receptor
expected to enter caveolae
Virus binds to receptor
expected to enter caveolae
Morphological similarities
Morphological similarities
62
21,60,61
Polyomavirus
Poliovirus
HRV2
Mouse kidney
HeLa
HeLa
Micropinosomes or caveolae
Non-clathrin-coated
Non-clathrin-coated
Morphological studies
Co-localisation of purified vesicles
Inhibitor studies
Coxsackieviruses
Various
Caveolae
Echoviruses
Various
Caveolae
Not tested
47–49
Enterovirus 70
HeLa
Caveolae
Not tested
49
Papillomavirus
CV-1 Caveolae
(or micropinosomes)
Morphological studies
Virus binds to receptor
expected to enter caveolae
Virus binds to receptor
expected to enter caveolae
Virus binds to receptor
expected to enter caveolae
Cytochalasin B
Not tested
Acutely sensitive to chloroquine,
chlorpromazine and cytochalasin B
Not tested
Balifomycin A1 resistant
Resistant to potassium
depletion and hypotonic shock
Not tested
22,71
25,26
24
73,74
72
205
Rev. Med. Virol. 7: 199–209 (1997)
Virus
NON-CLATHRIN COATED ENDOCYTOSIS
? 1997 John Wiley & Sons, Ltd.
Table 2. Non-clathrin-mediated mechanisms of virus uptake
206
subunit. Uptake of VLPs occurs via small, smooth endocytic vesicles with a 70–80 nm diameter and not by
clathrin-coated vesicles.72 It remains to be investigated
whether these vesicles represent caveolae or micropinosomes, although uptake in CV-1 cells is observed to be
inhibited by cytochalasin B, suggesting that caveolae
may be involved in this process.
Picornaviruses
The receptors for enterovirus type 7049 and at least six
different echovirus serotypes,47,48 is decay-accelerating
factor (DAF or CD55). In addition, whilst the major
receptor for coxsackievirus B strains is the 46kD ‘CAR’
protein,73 coxsackieviruses B1, B3, and B5 have the
additional capacity of binding to DAF in some susceptible cell lines.74,75 A GPI anchor attaches DAF to PM, and
DAF receptors are expected to co-localise with caveolae,
thereby mediating viral endocytosis. The role of caveolae
in the entry of these viruses was determined by
co-localisation studies using protein markers now known
to be found in caveolae, by the use of specific inhibitors
of caveolar function, and by the isolation of viral particles
in purified caveolar vesicles.
Polioviruses (PV) were originally thought to enter cells
by utilising the acidic conditions in clathrin-coated pits,
but recent work has cast doubt on the role of vesicle
acidification,25,76 and indeed of clathrin-coated pits,26 in
PV infection. Instead, mechanisms of uncoating depending on divalent cations, or low ionic strength, have been
proposed to play a critical role in PV disassembly.25,77
The presence of ion pumps in small, non-coated cytoplasmic vesicles and macropinosomal ruffles,9 and an
IP3-sensitive Ca2+ channel78 and ATP-dependent Ca2+
pump79 in caveolae, may therefore prove important in
this regard. Subcellular fractionation studies during PV
entry into HeLa cells find no correlation between the
major PV-containing endocytic vesicles and typical endosomal, lysosomal or PM markers26 further supporting a
role for an alternative endocytic route(s) in the entry of at
least some picornaviruses in some cell types.
N. E. BISHOP
of cell infection. As endocytosis via each of these
pathways may lead virus particles to acidic compartments, uncoating of acid-sensitive viruses may occur
regardless of the endocytic route followed. Macropinosomes,58,66 caveolae,38 and micropinosomes16 can be
acidified, and low-pH conditions are not uniquely
encountered by markers following the clathrin-mediated
route. Whether the ionic conditions in caveolae, macropinosomes and micropinosomes contribute to productive
virus uncoating is as yet unknown.
A specific route, on the other hand, may be essential
for some viruses, in that only one route may target
particles to the correct area within the cell, or simply
because the viral receptor preferentially localises in certain regions of the PM. The information discussed here
indicates that endocytic pathways may develop differing
ionic environments, be up-regulated or inhibited by
different conditions, and do not always deliver ligand to
the same region within the cell.
Many studies on viral entry into cells have not given
consideration to the existence of non-clathrin coated
endocytic routes, as the data confirming the role of these
pathways have previously been controversial, and few
techniques were available to pursue their function.
Further studies of the entry of some viruses may now
prove fruitful since more information on these pathways
is available. As with studies on the clathrin-coated
endocytic pathway, viruses will prove useful ligands in
the study of these pathways.
In light of the recent confirmation of entry of SV40
into cells via caveolae, and circumstantial evidence for
many viruses entering by caveolae and other distinct
non-clathrin-coated endocytic invaginations, these
aspects of viral replication clearly warrant further attention. At least five entry pathways can potentially be
exploited by viruses: (i) direct fusion at the PM, (ii)
endocytosis into clathrin-coated vesicles, (iii) endocytosis
into caveolae, and entry by (iv) macropinocytosis and (v)
micropinocytosis.
ACKNOWLEDGEMENTS
CONCLUDING REMARKS
The studies discussed here indicate that viruses enter into
cells, not merely by direct fusion at the cell surface or by
clathrin-dependent endocytosis, but also by additional
mechanisms of endocytosis. There are insufficient data in
many cases, at present, to determine which non-clathrincoated pathways are used by specific viruses, although it
is clear that SV40 replication requires clustering of virus
particles in PM caveolae. The main aim of this review,
was to demonstrate that this aspect is a growing field in
virus replication and to summarise the current knowledge
relevant to this area.
A single viral species may be able to utilise more
than one pathway, or only one endocytic pathway,
productively. Utilisation of more than one endocytic
route may be a desirable trait, ensuring maximal chance
Rev. Med. Virol. 7: 199–209 (1997)
I thank Dr Paul Luzio for his criticisms of the manuscript
and Dr Colin Watts for helpful comments on this topic.
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