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Journal of Virology, May 2000, p. 4853-4859, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cytoplasmic Trafficking of the Canine Parvovirus
Capsid and Its Role in Infection and Nuclear Transport
Maija
Vihinen-Ranta,
Wen
Yuan, and
Colin R.
Parrish*
James A. Baker Institute for Animal Health,
College of Veterinary Medicine, Cornell University, Ithaca, New
York 14853
Received 20 October 1999/Accepted 14 February 2000
 |
ABSTRACT |
To begin a successful infection, viruses must first cross the host
cell plasma membrane, either by direct fusion with the membrane or by
receptor-mediated endocytosis. After release into the cytoplasm those
viruses that replicate in the nucleus must target their genome to that
location. We examined the role of cytoplasmic transport of the canine
parvovirus (CPV) capsid in productive infection by microinjecting two
antibodies that recognize the intact CPV capsid into the cytoplasm of
cells and also by using intracellular expression of variable domains of
a neutralizing antibody fused to green fluorescence protein. The two
antibodies tested and the expressed scFv all efficiently blocked virus
infection, probably by binding to virus particles while they were in
the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with
an infectious plasmid clone of CPV. Cytoplasmically injected full and
empty capsids were able to move through the cytosol towards the nuclear
membrane in a process that could be blocked by nocodazole treatment of
the cells. Nuclear transport of the capsids was slow, with significant
amounts being found in the nucleus only 3 to 6 h after injection.
| |
INTRODUCTION |
The infection of cells by viruses is
a multistep process that requires that the virus pass through a series
of barriers so as to deliver the genome into the appropriate region of
the cell for replication. The early steps of virus entry into cells
involve attachment to the cell surface, followed by penetration of the virion or its components into the cytoplasm either by direct fusion with the plasma membrane or after uptake into the endocytic pathway. During uptake in endosomes the release of enveloped virus nucleocapsids to the cytoplasm is in many cases associated with structural changes of
viral proteins triggered by exposure to an acidic pH or binding to
cellular receptors. Fusion of the viral envelope releases the viral
genome and other components into the cytoplasm (reviewed in reference
19, 30, and 32 to
34).
Less is known about the mechanisms by which nonenveloped viruses enter
a host cell, although many of those viruses use interactions between
the hydrophobic portions of the outer capsid proteins or of
lipid-conjugated proteins to allow the particle to penetrate the
cellular membranes (33, 34). Nonenveloped viruses may enter
the cell through either pH-dependent pathways (e.g., picornaviruses, adenoviruses, reoviruses, and parvoviruses [6, 20, 36, 39, 40,
44, 45, 51]) or pH-independent pathways (papovaviruses or
poliovirus [27, 47]), and some may also directly
penetrate the plasma membrane (rotaviruses [25]).
Nuclear replicating viruses must pass through the cytoplasm to access
the nuclear pore or the nucleus (28, 55). Although it has
been assumed that this was a largely passive process, it is now clear
that the intracytosolic movements of adenovirus and herpes simplex
virus 1 capsids use the active transportation system of the cell,
mediated by the microtubule cytoskeleton, to move through the cytoplasm
to the vicinity of the nuclear pore (49, 50). Polyomavirus
virions are endocytosed via small monopinocytotic vesicles derived from
the plasma membrane, and then it is suggested that they enter the
nuclei via direct fusion of the vesicles with the nuclear membrane
(21). The slow caveola-mediated entry of simian virus 40 (SV40) particles into cells appears to be followed by targeting of
virions to the endoplasmic reticulum, where the viruses induce the
formation of interconnected tubular smooth membrane structures (2,
9, 27). However, mechanisms by which the virion or viral genome
is translocated from the endoplasmic reticulum to the nucleus for
replication are not known. It appears that the SV40 capsids enter the
cytoplasm of cells during infection, as infection can be blocked by
intracytoplasmic injection of antibodies against VP1 or VP3
(38). Microinjected SV40 particles in the cytoplasm of cells
were imported into the nucleus within 1 h, and they infected the
cell, indicating that trafficking through the cytoplasm is part of the
infectious pathway (10).
Before the genome of the incoming virion can replicate, it must be
released from its capsid, and uncoating may occur at one or more of
several sites in the cell, ranging from the cell surface to the nuclear
matrix (19, 20, 22, 29). For viruses that replicate in the
nucleus, the genome and possibly some associated proteins must enter
that compartment. Most viruses utilize the nuclear import system of the
cell, including the nuclear pore complex, receptors, and import factors
(33, 55). The nuclear pore has an effective diameter of
about 26 nm when Xenopus oocytes are injected with coated
gold beads (13). Larger viruses appear to localize to the
nuclear pore and then to release their DNA for transport to the nucleus
associated with specific viral proteins (18, 26, 49). SV40
particles within the nuclear pore appeared to be about 21 to 24 nm in
diameter, while those seen within the nucleus had a diameter of 38.2 nm, suggesting that the capsid structure is distorted during nuclear
import (56).
Autonomous parvoviruses have a nonenveloped 25-nm-diameter capsid
that contains a single-stranded DNA genome. Canine
parvovirus (CPV) is a member of the feline parvovirus subgroup,
which includes several host range variants that infect carnivores. CPV
enters cells by receptor-mediated endocytosis into clathrin-coated
vesicles (42). CPV infection can be prevented by
lysosomotropic bases, indicating that infection involves an acidic
intracellular compartment (6), and temperature- and
microtubule-dependent transport steps are also required
(53). CPV capsids associated with cells appear not to be
degraded (54). However, the mechanisms of penetration of CPV
from the endosomes into the cytoplasm and nuclear targeting of the
virus or its genome prior to replication are still poorly understood.
In previous studies only a small proportion of CPV capsids injected
into the cytoplasm of cells were seen to enter the nucleus within 1 or
2 h (53, 54). CPV capsids added to cells are detected
only in endosomes for several hours (6, 42, 53, 54).
Adeno-associated virus (AAV) capsids are taken into cells in a
dynamin-dependent endocytic process (12), and labeled
capsids added to cells are seen in the nucleus (4, 5). Up to
50% of the input single-stranded DNA-AAV capsids may be filled in
after uptake into some cells (14, 15).
Here we examine the steps in CPV entry that occur between
internalization via receptor-mediated endocytosis and nuclear
replication of the virus. Both microinjection of monoclonal anti-capsid
antibodies and intracellular expression of a single-chain antibody
prevented CPV replication, showing that the capsid enters the cytosol
during productive infection. Full or empty CPV capsids microinjected into the cytoplasm of cells accumulated in a perinuclear area and were
mostly transported into the nucleus only after 3 to 6 h.
Depolymerization of microtubules by nocodazole treatment of cells
resulted in the capsids being found distributed throughout the
cytoplasm, and this treatment also inhibited nuclear transport.
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MATERIALS AND METHODS |
Viruses and cells.
CPV type 2 (CPV-d) was grown in NLFK
cells, and aliquots were stored frozen at 
70°C (43).
Empty and full CPV particles were purified and quantified as previously
described and stored in 10 mM Tris-HCl (pH 7.5) at 4°C
(1). NLFK cells were grown and maintained in a 1:1 mixture
of McCoy's 5A medium and Leibovitz L15 medium with 5% fetal bovine
serum (43). Virus-infected cells were detected by staining
for the newly produced viral NS1 protein using a monoclonal antibody
(MAb), obtained from Caroline Astell (57), conjugated to
Texas red (TxR).
Antibodies and intracellular expression of single-chain antibody
(scFv).
MAb 8 and MAb 14, which both recognize the intact CPV
capsid, were purified from hybridoma culture supernatant by
chromatography on protein G (54). Purified mouse
immunoglobulin G (IgG) was obtained from Sigma (St. Louis, Mo.).
The variable domain sequences of MAb 8 light and heavy chain were
cloned and expressed as a single-chain antibody (scFv) in bacteria as
detailed by Yuan and Parrish (58), and that purified protein
bound and efficiently neutralized CPV capsids. That scFv was recloned
into the vector pEGFP-N (Clontech, Palo Alto, Calif.) so that it was
fused at its C terminus with the enhanced green fluorescent protein
(EGFP). Cells transfected with the plasmid expressing the scFv-EGFP
fusion protein or with pEGFP-N alone were inoculated 24 h later
with CPV and then incubated for a further 24 h. Cells were then
incubated with 3.7% (wt/vol) paraformaldehyde in phosphate-buffered
saline for 15 min, then with 0.1% Triton X-100 in phosphate-buffered
saline and 1% (wt/vol) bovine serum albumin. The transfected cells
were detected by EGFP expression, and the CPV-infected cells were
detected by staining with anti-NS1-TxR antibody. After being washed,
coverslips were mounted in Prolong medium (Molecular Probes, Eugene,
Oreg.) and examined by fluorescence microscopy. The proportions of
scFv-EGFP- or EGFP-expressing cells that became infected were compared
to the infection of the nontransfected cells in the same culture.
Cytoplasmic microinjection of antibodies.
Purified MAb 8 and
Mab 14 IgGs were dialyzed against 10 mM Tris-HCl-120 mM KCl (pH 7.4).
Cells at 7 × 103 per cm2 on Microgrid
coverslips (12-mm diameter, 175-µm grid size; Eppendorf, Hamburg,
Germany) were injected with 0.1 to 0.5 pl of antibodies at 5 mg/ml
using an Eppendorf 5246 Microinjector and Eppendorf 5171 Micromanipulator with Eppendorf capillaries. The cells were inoculated
with CPV either before the time of microinjection or up to 12 h
afterwards. The cells were then incubated for a total time after
inoculation of 24 h before paraformaldehyde fixation. Fluorescein
isothiocyante-labeled goat anti-mouse IgG was used to detect injected
cells, while virus infection was detected using TxR-conjugated
anti-NS1.
In control studies MAb 8 at 5 mg/ml was microinjected along with an
infectious plasmid clone of CPV (
43) at a concentration
of
200 µg/ml, and the cells were analyzed by staining for IgG
and viral
NS1. The colocalization of mouse IgG with viral capsids
was visualized
using an FITC-labeled goat anti-mouse IgG and rabbit
polyclonal
anticapsid antibody, followed by TxR-conjugated anti-rabbit
secondary
antibody. Cells were examined with a confocal fluorescence
microscope
(Bio-Rad Laboratories, Hercules, Calif.). The proportion
of cells
injected with MAb 8 and plasmid that became infected
was compared to
the proportion of control mouse IgG-injected cells
and also to the
noninjected cells in the same
culture.
Capsid microinjection and nocodazole treatment.
To further
examine the distribution and transport of capsids within the cell, we
injected full or empty CPV capsids at 2.5 mg/ml into the cytoplasm of
cells and then incubated the cells at 37°C for 0, 1, 3, 6, 12, or
24 h. The cells were then fixed and permeabilized as described
above and stained with MAb 8 (to detect the intact capsids) and with
TxR-anti-NS1, and the cells were then examined by confocal microscopy.
In some experiments the cells were incubated in medium containing 20 µM nocodazole for 1 h before microinjection; then, the drug was
maintained thereafter for 1, 6, or 12 h before fixation in 3.7%
paraformaldehyde. Some nocodazole-treated cells injected with capsids
were first incubated with drug for 6 h and then washed and
incubated in normal medium for an additional 6 h before fixation
and staining were carried out as described above. Control cells were
stained with antitubulin antibody (Amersham, Little Chalfont,
Buckinghamshire, United Kingdom) to confirm the nocodazole affect on
the microtubule structure.
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RESULTS |
Anticapsid antibodies in the cytoplasm and the nucleus of cells
block CPV infection.
We microinjected two MAbs against assembled
capsids into cells prior to CPV inoculation. The antibodies were seen
throughout the cytoplasm and nucleus (Fig.
1A and B). In several independent experiments only between 0 and 0.35% of the MAb 8- or MAb 14-injected cells became infected and expressed NS1, while 32 to 45% of the noninjected cells in the same dishes became infected (Fig. 1C). The
effect of the injected MAb 8 or MAb 14 was specific for the virus
capsid and was not a nonspecific effect of microinjection on the cells,
as control IgG injected into the cells did not affect virus infection
(Fig. 1B and C).
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FIG. 1.
Microinjection of cells with of MAb 8 or MAb 14, but not
with control antibodies, resulted in the cells becoming resistant to
infection. A) NLFK cells were injected with MAb 8 and then inoculated
with CPV. The cells were dual stained for IgG (green) or for the viral
NS1 protein (red). B) NLFK cells were injected with a control IgG and
then inoculated with CPV. The cells were stained for IgG (green) or for
the viral NS1 protein (red). C) The percentage of MAb 8- or MAb
14-injected cells infected in the various experiments compared to
noninjected cells in the same dishes. Controls include NLFK cells
injected with control mouse IgG.
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|
To determine whether the block to infection was due to a specific
effect on the infecting capsid and not to a nonspecific
effect on some
later step in viral replication, we coinjected
cells with MAb 8 and the
infectious plasmid clone of CPV. In different
experiments NS1
expression was seen in between 18 and 33% of the
injected cells, not
significantly different from values seen for
plasmid microinjected
alone in the same experiment (Fig.
2;
results
not shown). It is likely that the injected cells that did not
show virus infection did not enter S phase during the time of
incubation, preventing virus DNA replication. In MAb 8-injected
and
CPV-infected cells, the IgG appeared as small labeled aggregations
which were either in the perinuclear cytoplasm or within the nucleus
(Fig.
2A). Immunofluorescence staining using anti-mouse IgG to
detect
MAb 8 and polyclonal anticapsid antibodies confirmed that
those spots
represented aggregated viral capsids (Fig.
2B). In
contrast, in
noninfected cells diffuse immunofluorescence of the
IgG was seen
throughout the cytoplasm and the nucleus (Fig.
1 and
2).
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FIG. 2.
CPV infection of cells microinjected simultaneously with
MAb 8 (5 mg/ml) and the infectious plasmid clone of CPV (200 µg/ml).
(A) MAb 8 detected using FITC-labeled goat anti-mouse IgG (green) and
viral infection visualized with TxR-labeled anti-NS1 antibody (red).
The insert shows another cell subjected to the same treatment. (B)
Capsids were present within the aggregates seen in the cells. Injected
MAb 8 was detected using FITC-labeled goat anti-mouse IgG (green),
while capsids were detected using rabbit polyclonal anti-capsid IgG and
TxR-conjugated anti-rabbit antibody (red). The insert shows another
cell subjected to the same treatment.
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MAb 8 expressed as an scFv fused to EGFP accumulated in both the
nucleus and the cytoplasm of the transfected NLFK cells.
Cells which
expressed the scFV-EGFP completely resisted infection
(0% infection),
while control cells expressing only EGFP showed
normal levels of
infection (20 to 30%) (Fig.
3).
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FIG. 3.
(A) The insert in the plasmid expressing the variable
domains of MAb 8 fused to EGFP. The light variable domain
(VL) was linked to the heavy variable domain
(VH) with three repeats of GlyGlyGlyGlySer sequence
(G4S)3 and then fused in frame with EGFP. (B)
Intracellular expression of the variable domain ScFv of MAb 8 and
susceptibility to infection with CPV. Cells transfected with a plasmid
expressing MAb 8 scFv fused to EGFP (scFv-EGFP) were inoculated with
CPV. Control cells (EGFP) were transfected with a plasmid expressing
EGFP alone. After a further 24 h of incubation, the cells were
fixed, examined for GFP expression (green), and stained with a
TxR-labeled anti-NS1 (red). Low- and high-magnification views of
typical fields are shown in each case (to the left and right,
respectively).
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Antibody injected after virus inoculation blocks infection.
The kinetics of the blockade of CPV infection by antibody was examined
by inoculating the cells with virus, injecting MAb 8 into the cytoplasm
at various times afterwards, and then determining the proportions of
infected cells (Fig. 4). A large increase
in the proportion of virus-infected cells was observed only after 8 h, and by 12 h there was little effect on the rate of the
infection (Fig. 4).
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FIG. 4.
Effect of the timing of MAb 8 injection on infection by
CPV. Cells incubated with CPV for 1 h at 0°C were warmed to
37°C and then injected with MAb 8 at various times between 1 and
12 h later. The percentage of cells that became infected after
injection at the various times after warming was determined by staining
for the injected MAb and for NS1 to indicate the virus infection. The
percentage of infection of noninjected cells in the same cultures was
used as a control.
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Virus in the cytoplasm is transported into the nucleus after a
period of delay.
Both full and empty particles were distributed
throughout the cytoplasm immediately after microinjection (Fig.
5). By 1 h of incubation at 37°C
the capsids were more concentrated around the nucleus. Capsids appeared
to be transported into the nucleus in two phases. Up to 3 h after
injection, only small amounts of capsids were seen inside the nucleus,
but by 6 h, 30 to 50% of the injected cells showed substantial
nuclear localization of the capsids (Fig. 5). Twenty-four hours after
injection of empty capsids, almost all of the capsids were seen inside
the nucleus (results not shown), and those cells did not show viral
infection detectable by staining for NS1. However, in cultures injected with full capsids many of the injected cells and cells surrounding them
showed NS1 expression at 24 h but not at 1, 3, or 6 h
(results not shown).
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FIG. 5.
Localization of CPV capsids after microinjection into
the cytoplasm of cells. (A) Differential interference contrast and
fluorescence confocal image of cells 2 h after injection, showing
the specificity of staining for the virus in the injected cells. (B)
Full or empty CPV capsids injected into the cytoplasm of the cells that
were either fixed immediately (0 h), or incubated for 1, 3, or 6 h
at 37°C prior to fixation. The cells were stained for the capsid with
MAb 8, followed by FITC-conjugated anti-mouse secondary antibody.
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To determine whether capsid transport through the cytosol towards the
nuclear area depended on an intact microtubule network,
we monitored
the localization of full and empty capsids between
1 and 6 h after
cytoplasmic injection in cells pretreated for
1 h with nocodazole.
Immunofluorescence staining of nocodazole-treated
cells for tubulin
showed extensive disruption of microtubules
(Fig.
6C). At 1 and 6 h full and empty
capsids were both distributed
randomly throughout the cytoplasm with
little perinuclear accumulation
or nuclear localization (Fig.
6A), and
none of the cells was infected
as seen by anti-NS1 antibody staining
(results not shown). In
some cases the capsid-injected cells treated
with nocodazole were
washed and incubated in normal medium for a
further 6 h, in which
case nuclear accumulation of capsids was
observed in between 10
and 15% of cells, indicating the reversible
nature of inhibition
(Fig.
6A). Empty or full capsids injected into
cells and incubated
in the presence of the drug for 12 h appeared
to be aggregated,
but they showed little nuclear localization (Fig.
6B). Some of
the cells injected with full capsids and incubated for
12 h with
nocodazole became infected and showed NS1 expression
(Fig.
6B),
but this was not seen for the empty capsid-injected cells.
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FIG. 6.
(A) Intracellular localization of capsids injected into
cells in the presence of nocodazole. Full or empty capsids were
injected into cells in the presence of nocodazole and then incubated
for 1 h (1h ND) or for 6 h (6h ND). Other cells were injected
in the presence of nocodazole, incubated for 6 h, washed, and
incubated for a further 6 h in normal medium (6h ND + 6h
chase). (B) Cells injected with full or empty capsids in the presence
of nocodazole and then incubated for 12 h with the drug (12h ND).
The cells were then fixed and stained with MAb 8 or with TxR-labeled
anti-NS1 to distinguish those that were infected. (C) Cells that were
untreated (ND) or incubated for 1 h with 20 µM nocodazole (ND)
were fixed and then stained with an antibody against tubulin.
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DISCUSSION |
Intracellular antibodies show that capsids enter the cytoplasm
during infection.
Here we show that the intact parvovirus capsid
must enter the cytoplasm of the host cell during infectious entry, as
infection could be almost completely blocked by intracytoplasmic
injection of MAb 8 and 14, which only recognize assembled capsids, or
by expression of the MAb 8 variable domain fused to EGFP. This effect was virus specific and not due to an indirect effect of the
microinjection process, as injection of cells with nonspecific IgG did
not affect infection.
That the blocking effect was due to antibody binding to the capsid was
confirmed by injection of infectious plasmid DNA, where
the infection
was not blocked by either antiviral MAb 8 or 14.
In the MAb-injected
cells which became infected, the capsid-antibody
complexes were
frequently observed in the cytoplasm. Although
capsids normally
accumulate in the nuclei of parvovirus-infected
cells, neither the site
of assembly nor any process of assembled
capsid transport into the
nucleus or cytoplasm has been defined
(
16,
48). These data
suggest that the CPV capsids can assemble
in the cytoplasm, that they
can be transported there after assembly
in the nucleus, or that both
processes can
occur.
After CPV inoculation most cells were still protected from infection by
injection with MAb 8 up to 8 h later, indicating that
separation
of the viral DNA from the capsid occurred only slowly
after cellular
entry. The reasons for this delayed process are
not clear. Slow
trafficking of the capsids through the endosomal
pathway has also been
suggested by the finding that bafilomycin
A1 added to cells 90 min
after virus inoculation reduces infection
by about 70%
(
42). However, there may also be a slow transport
of the
capsid through the cytoplasm or into the nucleus, or DNA
release from
the capsid may require cellular factors associated
with S-phase.
Several attempts to inject antibodies into isoleucine-deprived
and
aphidicolin-treated cells resulted in few cells surviving
(results not
shown), and so we have not been able to test the
last hypothesis. The
slow kinetics of infection are similar to
those seen during the uptake
of SV40, where infection could be
blocked by injection of antibodies
against nucleoporin up to 8
but not 12 h after virus inoculation
(
56).
The mechanism(s) of intracellular neutralization is not known for
certain, but the antibodies would likely bind directly the
incoming
virus in the cytoplasm, interfering with capsid functions
by masking
structures required for interactions with cellular
factors or for
nuclear transport, or they may prevent essential
conformational changes
of the capsid required for infection or
DNA release. Where sufficient
virus was present the antibodies
clearly aggregated the capsids, as
seen in cells infected with
CPV plasmids (Fig.
3).
In previous studies we did not observe infection by the injected
capsids (
53), while that was sometimes seen in these studies
12 or 24 h after we injected full virus capsids. We believe that
this difference was due to small variations in the procedures
used. It
is not clear that capsid injection would recapitulate
normal virus
transport during infection; very large amounts of
capsids were injected
into the cytoplasm, and it is also likely
that capsids were deposited
onto the plasma membrane during the
injection
process.
Capsids are transported within the cytoplasm and enter the nucleus
slowly.
When we injected CPV full or empty capsids into normal
cells, they rapidly became localized to a perinuclear region. This suggests that capsid transport was controlled by microtubules, perhaps
in a manner similar to that reported for the transport of adenovirus
and herpesvirus capsids or their proteins (23, 49, 50). The
association between the microtubules and nuclear transport was also
shown when the cells were preincubated with nocodazole to cause
microtubule breakdown, where the capsids became distributed throughout
the cytoplasm (Fig. 6). The cytoskeleton is closely associated with the
nuclear pore complex (17). It is therefore likely that
capsid transport with the microtubular system could facilitate nuclear
transport and that capsids would not otherwise be efficiently
transported through the cytoplasm due to the high viscosity and other
steric obstacles present (31).
We showed in previous studies that during a 1- to 2-h period after
injection, most CPV capsids are found within the cytoplasm,
with only
small amounts being transported to the nucleus (
53,
54).
Here we confirm those findings but also show that by 6
h after
injection there was significant nuclear transport of capsids
in between
30 and 50% of the cells. Nuclear transport required
intact
microtubules, as it was blocked for up to 12 h by incubation
of
the cells with nocodazole, while further incubation of the
treated
cells in normal medium restored nuclear transport (Fig.
6). The 26-nm
diameter of the parvovirus particle is close to
the maximum size that
is reported to pass through the nuclear
pore, and it is not clear
whether any structural change in the
capsids occurs during the process.
However, MAb 8 used to detect
the virus recognizes only intact
particles, so the intranuclear
particles seen were largely intact. As
nuclear transport of the
capsids appears to require virus transport to
the vicinity of
the nuclear pore, the small amounts of capsid found
within the
nucleus immediately after microinjection may be those that
were
deposited in the vicinity of the nucleus by the injection (Fig.
5A).
Other changes in either the capsids or the cells which may facilitate
nuclear import of the capsids after 3 h include exposure
of the
apparent nuclear localization sequence in the VP1 N terminus
(
11,
52), removal of bound calcium ions from the capsid, modification
of the capsid by cellular enzymes, or binding of the virions to
cellular components. As no differences were seen between the transport
of injected full or empty capsids, the transport events examined
here
do not appear to be affected by the exposed N-terminal sequences
of VP2
or by the viral DNA in full
capsids.
The transport of infecting viral capsids or nucleocapsids within the
cytoplasm of cells has been found to be a tightly regulated
process in
most cases that have been examined (
7,
28,
35,
37,
55). The
processes often involve multiple cellular factors,
changes in the virus
components or in the cell, and a variety
of specific interactions
between viral and host cell components.
AAV capsids have been reported
to be transported into the nucleus
when added to cells in vivo (
4,
5), and some of the viral
DNA is converted to a double-stranded
form in receptive cells
(
14). The autonomous parvoviruses
appear to differ in that most
endocytosed capsids are retained within
endosomes for long periods
and neither the capsid nor the viral DNA
appears to reach the
nucleus at significant levels (
42,
46,
53,
54). However,
the infection process must be closely regulated and
depend on
specific cellular factors, as one or two amino acid sequence
substitutions
on the surface of autonomous parvovirus capsids can alter
the
infectivity for specific cells by over 10
5-fold, and
the block to infection appears to occur after virus
binding and
endocytosis but before successful DNA replication
in the nucleus
(
3,
8,
24,
41).
In our future studies we will examine the process of virus transport
through the cytoplasm in detail and the role of the microtubule
network, as well as the specific cellular and viral factors that
control nuclear
transport.
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ACKNOWLEDGMENTS |
Gail Sullivan and Wendy Weichert provided excellent technical assistance.
This work was supported by grants NR 64951 from the Academy of Finland
and AI28385 from the National Institutes of Health (to C.R.P.).
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FOOTNOTES |
*
Corresponding author. Mailing address: James A. Baker
Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. Phone: (607) 256-5649. Fax: (607) 256-5608. E-mail: crp3{at}cornell.edu.
Present address: Department of Biological and Environmental
Science, University of Jyväskylä, FIN-40351
Jyväskylä, Finland.
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Abstract
Introduction
