Yale University School of Medicine, Department of Cell Biology, New Haven, Connecticut 06520-8002
Herpes simplex virus 1 fuses with the plasma membrane of a host cell, and the incoming capsids are efficiently and rapidly transported across the cytosol to the nuclear pore complexes, where the viral DNA genomes are released into the nucleoplasm. Using biochemical assays, immunofluorescence, and immunoelectron microscopy in the presence and absence of microtubule depolymerizing agents, it was shown that the cytosolic capsid transport in Vero cells was mediated by microtubules. Antibody labeling revealed the attachment of dynein, a minus end-directed, microtubule-dependent motor, to the viral capsids. We propose that the incoming capsids bind to microtubules and use dynein to propel them from the cell periphery to the nucleus.
The entry of animal viruses into their host cells involves adsorption to cell surface receptors, penetration into the cytosol, and uncoating of the viral genome. For viruses that replicate in the nucleus, entry also
entails extensive movement of viruses and viral capsids
through the cytoplasm. Transport over long distances is
particularly critical for viruses that infect neurons in which the site of entry can be located far from the cell body and
the nucleus.
Since movement of virus-sized particles through the cytosol is not likely to occur by free diffusion due to high viscosity and steric obstacles (Bray, 1992 In this study we have focused on the intracytosolic transport of incoming HSV-1 capsids in Vero cells. HSV-1 has
three structural components: capsid, tegument, and envelope. The capsid consists of the viral DNA of 152 kbp and
a proteinaceous shell comprising six different proteins; it
has a diameter of 125 nm, and the proteins are arranged in
an icosahedron formed by 12 pentons and 150 hexons (Booy et al., 1991 The infectious cycle begins with virus binding to heparan sulfate receptors on the cell surface followed by glycoprotein-mediated fusion of the envelope with the plasma
membrane (for review see Spear, 1993 We analyzed the transport of incoming HSV-1 capsids
to the nucleus in fibroblasts and found that it occurs rapidly and efficiently by an MT-mediated mechanism. The
incoming capsids shed most of the associated tegument
proteins and bind dynein, an MT-dependent, minus end-
directed motor in the peripheral cytosol, whereafter they
are transported along MT toward the cell center. The viral capsids thus make use of the machinery responsible for
retrograde organelle transport in animal cells.
Cells and Antibodies
BHK-21 cells were grown in Glasgow's MEM with 5% FCS and 10% tryptose phosphate broth, and Vero cells were grown in MEM with 7.5% FCS
and nonessential amino acids. Both media contained 100 U/ml penicillin,
100 µg/ml streptomycin, and 2 mM glutamine. All tissue-culture reagents
were obtained from GIBCO BRL (Gaithersburg, MD). Both cell lines
were maintained as adherent cultures in a 5% CO2 humid incubator at
37°C and passaged twice a week. We used rabbit polyclonal antibodies
against VP-5 (NC-1), VP-19c (NC-2), DNA-containing capsids (anti-HC),
empty capsids (anti-LC; all provided by Roselyn Eisenberg and Gary Cohen, University of Pennsylvania, Philadelphia), and a mouse mAb 8F5 against
VP5 (provided by William Newcomb and Jay Brown, University of Virginia, Charlottesville). All rabbit polyclonal antibodies to viral proteins
were preadsorbed against paraformaldehyde (PFA)-fixed, Triton X-100
(TX-100)-permeabilized Vero cells. They were used at the highest possible concentration, giving no signal in noninfected cells. An mAb against
ICP4, an early herpes protein, was obtained from Advanced Biotechnologies, Inc. (Columbia, MD). We used a mouse monoclonal anti-tubulin
(1A2; provided by Dr. Thomas Kreis, University of Geneva, Switzerland; Kreis, 1987 Virological Techniques
Preparation of Stock Virus.
HSV-1, (strain F), designated passage 1, was
obtained from American Type Culture Collection (Rockville, MD).
BHK-21 cells grown to confluency at 37°C for 3 d were infected with an 8 ml/175 cm2 flask of MEM containing 0.2% (wt/vol) BSA and <0.01
plaque-forming units (PFU) per cell. After 2 h, 25 ml per flask of normal
medium was added to the cells, and after 3 d, the medium containing secreted extracellular virus was collected. All the following steps were performed at 4°C. Cells and debris were removed by spinning at 2,000 rpm for
10 min, and the virus was collected by centrifugation from the medium using a JA-14 rotor (Beckman Instruments, Inc.. Palo Alto, CA) at 12,000 rpm for 90 min. The resulting pellets were resuspended by gentle shaking
overnight in MNT buffer (30 mM MES, 100 mM NaCl, 20 mM Tris, pH
7.4). After waterbath sonification (4°C; three times for 30 s each), the suspension derived from 30 flasks was layered on top of one linear 10-40%
(wt/vol) sucrose gradient in MNT buffer and centrifuged in a Beckman
SW28 at 15,000 rpm for 45 min. The virus-containing band in the middle
of the gradient was detected by light scattering, collected (~3 ml per gradient), aliquoted, snap frozen in liquid nitrogen, and stored at Plaque Assay.
Virus was diluted in 10-fold steps into MEM with 0.2%
BSA and incubated in 24-well dishes with just confluent Vero cells for 1-2 h
at 37°C in a humidified 5% CO2 incubator. Unbound virus was removed
by three washes in MEM-BSA, and the cells were further incubated in
normal medium for 30 h. Plaques were counted using a dissecting microscope after fixation in absolute methanol ( Preparation of [3H]Thymidine-labeled virus.
BHK cells grown in an 850cm2 roller bottle to ~70% confluency were infected with a multiplicity of
infection (MOI) of 0.1 PFU per cell in MEM-BSA (0.2% wt/vol) containing 20 mM Hepes, pH 7.4. At 2 h postinfection (PI), the inoculum was removed and normal growth medium containing 5 mCi [methyl-3H]thymidine (Amersham Lifescience, Amersham, UK) was added. After 2 d, the
cells and the medium were collected and transferred to 4°C, the cells were
pelleted by a low speed spin, and the resulting supernatant was spun in a
Beckman SW40Ti at 13,700 rpm for 2 h. The pellet was resuspended by
gentle shaking overnight in MNT buffer, layered onto a linear 10-40%
(wt/vol) sucrose gradient in MNT buffer, and spun in a Beckman SW40Ti
at 15,800 rpm for 1 h. The gradient was fractionated into 1-ml fractions,
and the radioactivity in each fraction was determined by scintillation counting. The peak fractions were pooled, aliquoted, snap frozen, and
stored at Infection and Drug Treatments.
Cells were inoculated with virus for 2 h
at 4°C with constant rocking in RPMI medium containing 0.2% (wt/vol)
BSA, 20 mM Hepes, pH 6.8, to allow virus binding. Viral infection was initiated by shifting the cells to growth medium and 37°C in a humidified 5%
CO2 incubator. In those experiments analyzing incoming viral particles,
0.5 mM cycloheximide was added to the medium to prevent synthesis of
new viral proteins and progeny virus. In some experiments, the cells were
incubated for 1 h before infection in normal medium containing 2 µM nocodazole, 10 µM vinblastine, 2 µM colchicine, 20 µM taxol, or 0.5 µM cytochalasin D and were maintained in these drugs during the infection. Nocodazole was stored as a stock at 20 mM, vinblastine at 10 mM, colchicine
at 20 mM, taxol at 20 mM, and cytochalasin D at 10 mM, all in DMSO and
at Electron Microscopy
Vero cells grown on culture dishes or glass coverslips (for flat embedding)
for 1 d to just confluency were infected with HSV-1 at an MOI of 500 PFU
per cell (or 150 PFU/cell for the nocodazole experiment) in the presence
of cycloheximide. The virus was dialyzed before infection against RPMI
containing 0.2% BSA for 2 h (75 kD cut off) to remove sucrose. The infected cells were analyzed by three different protocols.
Conventional Epon Embedding.
The cells were fixed with 1% glutaraldehyde (GA) in 200 mM cacodylate, pH 7.4, or with 2% GA in PBS containing 0.5 mM CaCl2 and 1 mM MgCl2 for 30 min at room temperature
(for MT analysis), treated with 1% OsO4 for 1 h (or 30 min for MT),
treated with 2% uranyl acetate in 50 mM maleate buffer, pH 5.2, for 1 h,
dehydrated using a graded ethanol series and propylenoxid, and pelleted
before embedding in Epon and cutting, or flat-embedded in Epon and cut parallel to the substrate (for MT).
Flat Embedding of Extracted, Immunolabeled Cells.
Cells were extracted
with 0.5% Triton X-100 in MT stabilizing buffer (MT buffer: 80 mM
Pipes-KOH, pH 6.8, 5 mM EDTA, 1 mM MgSO4, 10 µM taxol) for 2 min
at 37°C (or 4°C for the 0-min time point) before the addition of twofold
fixative (2% GA in MT buffer; Baas and Ahmad, 1992 Frozen Hydrated Cryosections.
The cells were transferred to 4°C and removed from the culture dish either by proteinase K treatment (25 µg/ml
for 2-3 min on ice) and fixed (4% paraformaldehyde and 0.1% GA in 250 mM Hepes, pH 7.4), or fixed first for 20 min and then collected by scraping. After pelleting, the cells were fixed further overnight. The pellets were infiltrated with 20% (wt/vol) polyvinyl pyrolidone and 2.0 M sucrose
in PBS and frozen in liquid nitrogen, and ultrathin sections were cut at
Quantitation.
To analyze the efficiency of capsid transport to the nucleus (see Fig. 5), 25 EM negatives of infected, flat-embedded cells were
taken at a magnification of 5,800 in a systematic random fashion for each
time point (Griffiths, 1993b
Quantitation of Virus Binding and Internalization
We assayed for virus binding and internalization as described previously
(Helenius et al., 1980 Light Microscopy
Vero cells grown on coverslips for 1-2 d to ~70% confluency were infected with HSV-1 at an MOI of 50 PFU per cell in the presence of cycloheximide. In one protocol, cells were fixed with 3% (wt/vol) paraformaldehyde for 20 min followed by quenching remaining fixative using 50 mM
NH4Cl for 10 min and permeabilization with 0.1% TX-100 for 4 min.
Here, all incubations were carried out in PBS, pH 7.4, at room temperature. In an alternative protocol, the cells were preextracted with 0.5% TX100 for 10 s at 37°C in an MT stabilizing buffer (80 mM Pipes, 2 mM
MgSO4, pH 6.8, 10 µM taxol), and then fixed in 100% methanol at Assay for Viral Protein Synthesis
Confluent Vero cells grown in 6-cm tissue-culture dishes were infected
with HSV-1 at an MOI of 0.5 PFU per cell for 30 min at 37°C and 5% CO2
in MEM containing 0.2% (wt/vol) BSA, and then incubated further in
normal growth medium. At various time points, they were transferred to
4°C, washed three times with ice cold PBS, scraped, pelleted, and resuspended in 0.3 ml per dish lysis buffer (MNT with 0.5% TX-100, 2 mM
DTT, 2 mM EDTA, and 10 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin). The nuclei were removed by centrifugation at 5,000 rpm for 5 min. A 50-µl aliquot of the resulting supernatant was used for
protein assays (bicinchoninic assay; Pierce Chemical Co., Rockford, IL), and 0.2 ml were solubilized in 5 × Laemmli sample buffer. Equal amounts
of protein were loaded onto a 7.5% SDS polyacrylamide gel for electrophoresis, and the separated proteins were blotted onto nitrocellulose
membranes using a semidry blotter. After blocking in 5% lowfat milk in
PBS with 0.1% Tween-20, the membranes were incubated with either
anti-NC-1 and calnexin or anti-ICP4 and calnexin, followed by HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Pierce Chemical
Co.). The secondary antibodies were visualized by enhanced chemiluminescence detection (Amersham Corp. or Pierce Chemical Co.) and Kodak
film (Eastman Kodak Co., Rochester, NY). The bands were quantified by
densitometry using a Visage 200 digital gel scanner (Bioimage, Ann Arbor, MI). The amount of viral protein (ICP4 or VP5) was normalized for
the amount of cellular protein (calnexin).
Early Events during HSV-1 Entry
Due to the large size of HSV-1 virions and capsids, early
virus cell interactions can be readily visualized by EM
(Campadelli-Fiume et al., 1988
After 1 h, the first capsids had reached the nucleus and
attached themselves to the nuclear pore complexes (Fig. 1,
g and h; see also Batterson et al., 1983 The rate and efficiency of cell surface binding and subsequent internalization was assayed quantitatively using
[3H]thymidine-labeled HSV-1. To monitor binding, cells
were incubated with labeled virus at an MOI of 10, 50, or
100 at 4°C, and the cell-associated radioactivity was measured at different times. As previously reported, a continuous increase in virus binding was observed up to 4 h with
no signs of saturation (Fig. 2 a; McClain and Fuller, 1994
To follow internalization biochemically, a protease protection assay was used (Helenius et al., 1980 Transport of Capsids from the Plasma Membrane
to the Nucleus
Indirect immunofluorescence microscopy was next used to
analyze the transport of individual capsids from the cell
periphery to the nucleus. The antibodies used were either
against VP-5 (anti-NC-1) or VP19c (anti-NC-2), the two
major capsid proteins, or against purified whole capsids
(anti-HC and anti-LC). All the antibodies gave similar results and had in common that they stained capsids that
were in the cytosol (Fig. 3, 1 h) but not capsids present in
surface-bound intact viruses (Fig. 3, 0 h). Presumably, this useful selectivity was caused by antigen accessibility; the
relevant capsid epitopes in the intact virus particles were
probably obscured by envelope and/or tegument components. In contrast, anti-capsid antibodies labeled viral
capsids on thawed cryosections (Sodeik, B., M.W. Ebersold, and A. Helenius, unpublished observations).
The capsids in the cytoplasm appeared as small, intensely labeled spots (Fig. 3, arrows, 1 h). Using immunoelectron microscopy on thawed ultrathin cryosections, we
confirmed that these spots represented single capsids (not
shown). All the structures labeled in the cytosol or at the
nuclear membrane by antibodies directed against the capsid or against VP5 represented single viral capsids (not
shown). The number of fluorescently labeled spots increased continuously during the first hour of warming. In
addition, their distribution within the cell changed with increasing time: from the cell periphery toward the nucleus
where they accumulated at the nuclear rim (Fig. 3, 2 h).
After 4 h, virtually all the spots were localized at the nuclear membrane (Fig. 3, 4 h) where several hundred
capsids could be seen. Thus, incoming cytosolic capsids were transported efficiently from the periphery to the host
nucleus.
Capsids Bind to Microtubules
Double immunofluorescence microscopy using anti-capsid
and anti-tubulin antibodies showed that most of the
capsids that were not bound to the nucleus were associated with MT (Fig. 4, a and b). Moreover, in many cells,
capsids were seen to concentrate around the MT organizing center (MTOC) (Fig. 4 b). The close association of
capsids with MT was confirmed by EM (Fig. 4, c-e). Numerous cytoplasmic capsids were seen in close proximity
to cytoplasmic filaments with the expected 24-nm width
and MT-like morphology. Typically, the distance between
the capsid surface and the tubules was ~50 nm, suggesting
the presence of additional tethering components. The viral
DNA inside the MT-associated capsids could be seen as a
darkly stained material.
The tubules were unambiguously identified as MT by
antitubulin labeling of specimen that had been preextracted with TX-100 before fixation. After such extraction,
thicker sections could be viewed, allowing the analysis of
MT over longer distances. The MT-associated capsids generally contained the viral DNA (Fig. 4 f, arrows). At later
time points, most of the capsids were empty and typically
located at the nuclear pores and not at the nuclear envelope membrane (Fig. 4 g). Most of these capsids did not
contain DNA (Fig. 4, g and h). At 4 h postinfection, there
were occasionally empty capsids localized on MT in close
proximity to the nucleus (not shown). Whenever extracellular viruses were observed in these TX-100 extracted
samples, they were surrounded by very-electron dense material, which probably represented the viral glycoproteins and tegument proteins that were apparently not removed
during extraction (Fig. 4 h, curved arrow).
Quantitation of HSV-1 Entry and Capsid Transport
To quantify the kinetics of the cytosolic capsid transport,
we determined the subcellular localization of the incoming
virus by EM at various time points postinfection at an
MOI of 500 (Fig. 5). To compare this experiment with the
immunofluorescence microscopy, ultrathin sections were
cut parallel to the substrate through the basal region of the
cells (see Fig. 5 a). For each time point, 25 electron micrographs were taken in a systematic random fashion, and the
number and localization of cytosolic capsids was determined. Cytosolic capsids (Fig. 5 b) were localized at the
plasma membrane (Fig. 5 c), in the cytosol without obvious connection to any organelle (Fig. 5 d), at MT (Fig. 5 e),
or at the nucleus (Fig. 5 f).
After warming, the total number of cytosolic capsids increased rapidly, reaching a peak after 60 min (Fig. 5 b). At
later times the number of cytosolic capsids declined, suggesting that the capsids were ultimately disassembled. We
also scored whether the cytosolic capsids contained the
electron-dense viral DNA core or whether they appeared
empty (see Fig. 1). Up to 60 min after internalization, no
empty capsids were detected, whereas after 2 h 20% and
after 4 h 64% had lost their electron-dense DNA core (Fig. 5 b). Virions in endosomes peaked at 1 h and sharply
declined thereafter. They may have been degraded or recycled back to the cell surface, or capsids could have been
released by fusion into the cytosol.
At 15 min postinfection, almost 70% of all cytosolic
capsids were localized within 100 nm of the plasma membrane (see Figs. 1, c-f, and 5 c). All of the capsids close to
the plasma membrane contained the viral DNA core. At 1 h
postinfection, most of the cytosolic capsids (almost 80%)
had an intermediate location; they were neither close to
the plasma membrane or to the nucleus (Fig. 5, d and e). A
significant fraction (10% of all cytosolic capsids) colocalized with MT (see Figs. 4, c-e, and 5 e). Since the capsids (125 nm) are considerably larger than the section thickness (60-70 nm), the figure for colocalization with the MT
was most likely an underestimation. Of the cytosolic capsids, only one capsid (0.9% of all capsids at 4 h postinfection) was seen to colocalize with intermediate filaments
(not shown).
Cytosolic capsids arrived at the nucleus at 2 h postinfection, and at 4 h, the majority of cytosolic capsids (>60%)
was localized to the nucleus (Fig. 5 f). At the ealier time
points (up to 1 h), <4% of the cytosolic capsids were located at the nucleus. Of the nuclear capsids, 68% at 2 h
and 87% at 4 h were empty, suggesting that DNA release
occurred after arrival at the nucleus. The vast majority of
empty capsids were localized to the nucleus (Fig. 5 g). All
capsids at the nucleus were localized to the nuclear pore
complexes (see Fig. 1, g and h).
In summary, the electron microscopic experiment confirmed the immunofluorescence microscopy data. Cytosolic capsids were efficiently transported from the plasma
membrane via the cytosol to the nuclear pore complexes.
During transit, a significant fraction attached to MT. Empty
capsids appeared soon after their arrival at the nucleus,
and some of these were released into the cytosol. Ultimately, the capsids seemed to disassemble and disappear
because the overall number of cytosolic capsids decreased
late in infection. Interestingly, the appearance of cytosolic
capsids under the plasma membrane peaked before the
transport of virions into endosomes, strongly suggesting
that the early cytosolic capsids were indeed derived from
fusion of the virus at the plasma membrane rather than by fusion from endosomes.
Reduced Capsid Transport to the Nucleus
without Microtubules
To determine whether efficient capsid transport via the cytosol depended on an intact cytoskeleton, cells were infected at an MOI of 50 for 2 h in the presence of drugs that
affect MT or actin filaments. Immunofluorescence microscopy showed that neither taxol, a drug that prevents the
disassembly of MT (Wilson and Jordan, 1994
Since we were unable to quantify the localization of the
fluorescent spots representing cytosolic capsids reliably,
we infected Vero cells at an MOI of 150 in the presence or
absence of nocodazole and analyzed the embedded cell
pellets by quantitative EM. For each experimental condition, 50 electron negatives were taken in a systematic, random fashion and the localization of cytosolic capsids was
determined (Fig. 7, a and b). After 2 h of infection, 31% of
the cytosolic capsids had reached the nucleus, and only 25% were still in close proximity to the plasma membrane.
In contrast in the absence of MT, none had reached the
nucleus, and 58% were still in the region close to the
plasma membrane. After 4 h postinfection, 75% of all cytosolic capsids had reached the nucleus, 14% were at the
plasma membrane, and the remaining 11% were present
in the cytosol. In the presence of nocodazole, only 47% of
the cytosolic capsids had reached the nucleus, 18% were
still at the plasma membrane, and 36% were in the cytosol
distant from both the plasma membrane and the nucleus.
As nocodazole, vinblastine, and colchicine all affect the
network of MT (Wilson and Jordan, 1994 Viral Infection in the Absence of Microtubules
Whether nocodazole would have an effect on productive
infection was determined by monitoring the onset of viral
protein synthesis in the presence or absence of the drug.
Cell extracts prepared after different periods of infection
were subjected to immunoblotting using antibodies against
an early HSV-1 protein, ICP4, and a late structural protein, VP5. The results in Fig. 7 c show that the synthesis of
both viral proteins was delayed and reduced in the absence of MT. Calnexin, an integral membrane protein of
the ER (Hammond and Helenius, 1994 When immunofluorescence microscopy using ICP4 was
performed, we found that, under control conditions, viral
protein synthesis commenced in many cells already at 2 h
postinfection. In contrast, in the presence of nocodazole,
only very few cells showed ICP4 labeling. At 4 h postinfection, all control cells were strongly labeled for ICP4,
whereas in the absence of MT, significantly less cells show
strong labeling for ICP4 (data not shown). However, at
later time points, all cells synthezised ICP4, in the presence and absence of MT.
Thus, depolymerization of MT delayed the onset of viral
protein synthesis in cultured fibroblasts, but did not prevent viral infection per se.
Dynein Colocalizes with Incoming Capsids
Our results strongly suggested that incoming herpes
capsids move along MT toward the nucleus. Therefore, we
ask whether they would use host MT-dependent motors
for their transport. Since the antibody against tubulin often obscured the morphology of putative tethering factors
located between the viral capsids and the MT (see Fig. 4),
we repeated these experiments without immunolabeling. Electron-dense material was usually attached to the vertices of the capsids that were associated with MT (Fig. 8).
The morphology of the appendages was variable, but, in
some cases (Fig. 8 d, arrow), they had the dimensions and
the shape of cytoplasmic dynein, a minus end-directed
MT-dependent motor. Dynein is a Y-shaped protein complex with a length of ~50 nm. It is connected to its cargo
via the stalk and to MT through two 14-nm globular domains (Gilbert and Sloboda, 1989
To test whether cytoplasmic dynein was, indeed, associated with the capsids, we used an affinity-purified antibody directed against the heavy chain of cytoplasmic dynein (Vaisberg et al., 1993
Table I.
Quantitation of Dynein Immunolabeling
; Luby-Phelps, 1994
),
viruses most likely exploit the cell's motile functions for
transport. The limited information available suggests that
viruses can make use of both microtubules (MT)1 and actin filaments. EM analysis has shown MT binding of certain viral capsids, such as adenovirus and reovirus in vivo
and in vitro (for review see Luftig, 1982
) as well as herpes
simplex virus 1 (HSV-1) in neurons (Lycke et al., 1984
, 1988;
Penfold et al., 1994
). Way and co-workers have described
actin filament-dependent intra- and intercellular transport
of vaccinia virus during egress from cells (Cudmore et al.,
1995
). Moreover, many viruses take advantage of the retrograde movement of endocytic organelles during entry.
They are internalized by receptor-mediated endocytosis and carried within coated vesicles and endosomes from
the cell periphery toward the perinuclear area of the cell
(see Greber et al., 1994
; Marsh and Helenius, 1989
).
; Cohen et al., 1980
; Heine et al., 1974
;
Spear and Roizman, 1972
). VP5 constitutes the major protein component of both hexons and pentons (Newcomb
et al., 1992
; Trus et al., 1992
). The tegument is a protein
layer between the envelope and the capsid and contains
~12 different viral polypeptides (Roizman and Furlong,
1974
; Roizman and Sears, 1996
). While the functions of
most of these remain to be identified, one is a protein kinase (LeMaster and Roizman, 1980
), one induces shut off
of host cell protein synthesis (Read and Frenkel, 1983
),
and some are known to be activators and modulators of viral gene expression (Batterson and Roizman, 1983
; Campbell et al., 1984
; McLean et al., 1990
). The viral envelope
contains at least 12 different membrane proteins, some of
which are involved in receptor binding and fusion.
). After release into
the cytosol and dissociation from some of the tegument
components, the capsids move through the cytosol to the
nucleus and bind to nuclear pores (Batterson et al., 1983
;
Lycke et al., 1988
), whereafter the genome is released into
the nucleus. Transcription, replication of viral DNA, and assembly of progeny capsids take place within the host nucleus (for review see Roizman and Sears, 1996
; Steven and
Spear, 1996
). The synthesis of early viral proteins peaks after 4-6 h of virus entry, and the first progeny viruses are
produced after 8 h (Honess and Roizman, 1973
).
Materials and Methods
), a rabbit antipeptide anti-calnexin (Hammond and Helenius,
1994
), and a rabbit polyclonal, affinity-purified antibody directed against a
bacterially expressed fragment of dynein heavy chain (provided by Dr.
Eugeni Vaisberg, University of Colorado at Boulder; Vaisberg et al.,
1993
). Fluorescently labeled secondary antibodies (Texas red- or fluorescein-conjugated goat anti-rabbit or goat anti-mouse) were obtained from
Jackson ImmunoResearch Laboratories (West Grove, PA), and rabbit
anti-mouse was obtained from Organon Teknika-Cappel (Durham, NC).
80°C. All
experiments described here were carried out with gradient-purified virus
from passage three.
20°C) and Giemsa staining.
Titers of 109-1010 PFU/ml and protein concentrations of 0.5-2 mg/ml were
obtained using this protocol.
80°C. This preparation had a titer of 1 × 107 PFU/ml and contained 0.1 mg/ml protein.
20°C.
). The cells were
fixed for 20 min at room temperature and rinsed with PBS, and remaining
fixative was inactivated by three rounds of freshly prepared 2 mg/ml sodium borohydride in PBS, pH 7.4, for 15 min. After blocking with 10%
FCS in PBS, the cells were incubated with primary antibody, with secondary rabbit anti-mouse antibody (if the primary was not a rabbit antibody), and with 9 nm protein A-gold (University of Utrecht, The Netherlands), each in 10% FCS in PBS for 30 min at room temperature with extensive washings with PBS in between. After a second fixation in 1% GA with
2 mg/ml tannic acid in 100 mM cacodylate buffer, pH 7.4, for 10 min, the
cells were contrasted using 2% OsO4 for 10 min and 2% uranyl acetate in
50 mM maleate buffer, pH 5.2, for 1 h. They were embedded in Epon and
ultrathin sections parallel to the substrate were prepared. All Epon sections (first and second protocols) were further contrasted using lead citrate
and uranyl acetate.
100° to
120°C and immunolabeled as previously described (Griffiths,
1993a
; Sodeik et al., 1992
).
). The negatives were analyzed under a dissecting
microscope, and the number and localization of cytosolic capsids was
determined. Capsids within 100 nm of the plasma membrane were scored
as being at the plasma membrane, and within 50 nm as being on MT and
at the nuclear pore complex. The remaining capsids were scored as being
apparently free in the cytosol. Virions present in endosomes were also
counted. To quantify the effect of nocodazole (see Fig. 7), 50 negatives of
conventionally embedded cells infected in the presence or absence of nocodazole (10 µM) were taken in a systematic random fashion. Cytosolic
capsids were scored as being at the plasma membrane, in the cytosol, or at
the nuclear pore complex. To quantify the dynein labeling, 25 micrographs of cryosections were taken at a magnification of 16,900. Since incoming herpes capsids were rare structures, the specimen was systematically translated in a defined direction, and a micrograph was taken every
time a cytosolic capsid came into view. These were printed (× 2.7) and the
area of each structure was outlined (virions, capsids, mitochondria, nuclei,
and cytoplasm). A lattice grid was placed over the prints, and the number
of points and gold particles falling into each structure was counted. The
formula G/P*d2 gives the labeling density (gold particles per µm2), where
G is the number of gold particles, P is the number of points of the lattice
grid over the structure, and d is the distance between points (Griffiths,
1993b
).
Fig. 5.
Time course of HSV-1 entry. Vero cells infected at an
MOI of 500 in the presence of CH were fixed at various times
postinfection with 2% glutaraldehyde in PBS and embedded in
Epon for EM analysis. (a) Schematic cross-section perpendicular
to the substrate through an adherent Vero cell. For quantitation,
25 random electron micrographs obtained from sections cut parallel to the substrate through the basal part of the cells were
taken in a systematic fashion for each time point. "Basal" is defined as being within 2 µm distance from the substrate (30 sections of ~50-70 nm). (b) The total numbers of cytosolic capsids
(full and empty), cytosolic empty capsids, and virions in endosomes were determined for each time point. The number of cytosolic capsids increased rapidly until 1 h postinfection. At later
time points, less cytosolic capsids were detected, suggesting that
they were ultimately disassembled. In addition, the subcellular localization of each capsid was determined and the numbers were expressed as a percentage of the total (c-g). (c) At the earliest time point of 15 min, the majority of cytosolic capsids was localized close to the plasma membrane, from where they disappeared
rapidly later. (d) Several capsids could not be localized to a particular organelle. At 1 h postinfection, there was the highest
amount of those capsids, which were apparently free in the cytosol. (e) A significant portion of all cytosolic capsids, namely 10%
at 1 h postinfection, colocalized transiently with microtubules. (f)
Capsids began to appear at the nuclear pores at 2 h postinfection, and at 4 h, 60% of all cytosolic capsid had reached the nucleus. (g) The appearance of empty cytosolic capsids coincided with the arrival at the nucleus. Only at 4 h postinfection, there was a very
small portion of empty, cytosolic capsids. N, nucleus; PM, plasma
membrane.
[View Larger Version of this Image (21K GIF file)]
Fig. 7.
Quantitation of capsid transport and viral protein synthesis in the absence of MTs. Vero cells were infected at an MOI
of 170 in the presence of CH and in the absence or presence of
nocodazole to depolymerize microtubules. They were fixed at 2 (a) or 4 (b) h postinfection with 1% GA and collected by scraping
and pelleting, and Epon sections were prepared. For quantitation, 50 random electron micrographs were obtained in a systematic fashion for each time point and both treatments. The subcellular localization of each cytosolic capsid was determined. (a) At
2 h postinfection in the presence of microtubules, the majority of
cytosolic capsids was localized in the cytosol, and a significant
fraction had reached the nucleus. In the absence of microtubules,
the majority of capsids remained at the plasma membrane (PM),
some were in the cytosol, and none had reached the nucleus. (b)
At 4 h, >70% of the capsids had reached the nucleus. In the presence of nocodazole, a much smaller fraction was transported to
the nucleus, and more capsids remained at the plasma membrane
and in the cytosol. (c) Immunoblot. Vero cells were infected at an
MOI of 0.5 (without CH!) in the absence or presence of 5 µM nocodazole for various times. The cells were harvested and lysed in
0.5% TX-100 in buffer, the nuclei were pelleted, and the supernatants were analyzed by immunoblotting. To monitor viral protein
synthesis, antibodies to ICP4, an early viral protein, and to VP5, a
late viral protein, were used. Calnexin, a membrane-bound ER
protein, was used as a cellular marker protein. Equal amounts of
protein were loaded per lane, and the amount of ICP4 or VP5
was normalized with the amount of calnexin. ICP4 synthesis commences at 2 h PI, whereas VP5 is first detectable at 5 h PI. The
amount of ICP4 is maximal after 6 h PI. In contrast, VP5 synthesis increases during the whole time course of the experiment. At
all time points, lower amounts of both, ICP4 and VP5, are synthesized in the absence of microtubules.
[View Larger Version of this Image (12K GIF file)]
). To quantify binding, 6-well dishes containing Vero
cells grown for 2 d to just confluency were set on a metal plate on an ice
box and washed three times with ice cold RPMI-BSA, and [3H]thymidinelabeled virus was added (1 ml per dish). After incubation on ice for various amounts of time and intensive washings, the cells were scraped into 1 ml
RPMI-BSA and pelleted, and cell-associated radioactivity was determined
by scintillation counting. The data are expressed as mean values from duplicates. To assay for virus internalization, [3H]thymidine-labeled virus
was bound to cells at 4°C for 2 h. The cells were washed to remove unbound virus, and then shifted to normal medium at 37°C and 5% CO2 for
various lengths of time. The cells were transferred back to ice, washed,
and stored at 4°C until the last time point. All the following treatments
were performed at 4°C. The cells were washed with cold, protein-free PBS
and incubated with 1 ml per well proteinase K (2 mg/ml in PBS; Boehringer Mannheim GmbH, Mannheim, Germany). After 1 h, 1 ml of PBS
containing 1.25 mM PMSF and 3% (wt/vol) BSA was added to stop further proteolysis. The cells were collected, the wells were rinsed with wash
buffer (PBS containing 0.2% BSA), and the cells along with the wash
buffer were spun at 1,500 rpm for 10 min. The pellet was resuspended and
pelleted again; scintillation liquid was added to the resuspended pellet (0.5 ml) and counted in a liquid scintillation counter. The amount of internalized virus was expressed as cell-associated [3H]thymidine counts (mean
value from triplicates). Control cells, which were not warmed up after virus binding, showed that the proteinase K treatment removed 90-95% of
cell-bound virus.
20°C
for 4 min. Before immunolabeling at room temperature, the cells from
both protocols were washed again in PBS, transferred to 10% normal goat
serum for 30 min, and then labeled with the primary antibody in 10% goat
serum for 20 min. The coverslips were rinsed three times for 5 min and incubated with fluorescently labeled secondary antibodies in 10% goat serum for 20 min. For double-labeling experiments, the two primary and the
two secondary antibodies were applied simultaneously. After extensive
washing and one short wash in water, the coverslips were mounted in
Mowiol containing 2.5% (wt/vol) 1,4-diazabicyclo-[2.2.2]octane on glass
slides and examined with an Axiophot fluorescence microscope (Carl
Zeiss, Inc., Thornwood, NY) or a confocal fluorescence microscope (Bio
Rad Laboratories, Hercules, CA). Image processing was performed using
Adobe Photoshop (Adobe Systems, Inc., Mountain View, CA).
Results
; Fuller et al., 1989
; Lycke
et al., 1988
). We followed the incoming capsids at different
stages of entry in Vero cells (Fig. 1). The extracellular, surface-bound virus particles had the familiar features of a
herpes virus: a membrane envelope with spikes, a capsid
with an electron-dense DNA core, and a tegument layer
between the capsid and the envelope (Fig. 1, a and b). Images of fusion between the viral envelope and the plasma
membrane were readily obtained (Fig. 1, c and d). After
fusion, the capsids appeared to separate from the bulk of
the tegument since an electron-dense mass remained associated with the cytoplasmic surface of the plasma membrane (Fig. 1, e and f, arrowheads; see also CampadelliFiume et al., 1988; Fuller et al., 1989
).
Fig. 1.
Entry and uncoating of HSV-1. Ultrathin Epon sections of Vero cells infected with HSV-1 at an MOI of 500 in the presence of cycloheximide (CH). After 2 h of virus binding at 4°C, the cells were either fixed immediately (a and b), or warmed up for 30 (c-f) or 60 min (g and h). (a and b) Binding. The main morphological features of the intact virus bound to the plasma membrane (PM) are the viral
envelope (b, arrowhead) with the viral spikes (a, arrowhead), the viral capsid (arrow), and the electron-dense viral DNA genome within
the capsid. (c and d) Fusion. Upon warming up, the viral envelope fuses with the plasma membrane, and the capsid (arrow) and the tegument proteins are released into the cytosol. (e and f) Release of the capsid. Preparations, which display a prominent contrast of the tegument, show that the tegument (arrowheads) stays behind at the PM. (g and h) Binding to the nuclear pore. At later time points, the
capsids (arrows) have arrived at the nuclear envelope (NE), where they are exclusively located in close apposition to the nuclear pore
complexes. Occasionally, fibers emanating from the pores (arrowheads) are visible, to which the capsids seem to bind. Almost all of the
capsids at the nuclear pores appear empty and have lost the electron-dense DNA core. Bar, 100 nm.
[View Larger Version of this Image (91K GIF file)]
; Lyke et al., 1988).
They seemed to bind to cytsosolic fibers emerging from
the pores (see arrowheads in Fig. 1 h). Since most of the
capsids on the nuclear envelope lacked the electron-dense
central mass, DNA release had apparently occurred. At
later time points, some empty capsids were also seen free
in the cytosol without connection to the nucleus. Occasionally, 1 h postinfection and later, intact and partially degraded virions were also seen in endosomes and lysosomes, indicating that a fraction of the inoculum was taken
up by endocytosis.
;
Shieh et al., 1992
). At 2 h
the adsorption period used in
all subsequent experiments
~40% of the added virus was bound.
Fig. 2.
Efficiency and kinetics of HSV-1 binding and internalization. (a) Using [3H]thymidine-labeled virus at different multiplicities of infection (MOI), the amount of virus bound to cells at
4°C was quantified after various time points. There is no apparent
saturation of virus binding to Vero cells. After 2 h, ~40% of the
virus added bound. (b) Using [3H]thymidine-labeled virus at different MOIs, the amount of virus internalized into cells was quantified after various time points. At time 0 min (equals 2 h of virus
binding at 4°C), proteinase K treatment removes >95% of the virus bound to the plasma membrane. Upon warming up to 37°C
and viral fusion, the virus is protected within the cells from the
protease treatment. After 30 min, the maximal amount of virus,
~70% of the bound virus, is internalized. The half-time (t1/2) of
internalization is 8 min. Depolymerization of microtubules by nocodazole (ND) has no effect on the kinetics and the amount of internalization.
[View Larger Version of this Image (17K GIF file)]
). After a 2-h
binding period in the cold, the cells were warmed up to
37°C for different periods of time to initiate penetration.
Thereafter, they were chilled to 4°C and treated with proteinase K to detach remaining cell surface viruses. The fraction of virus protected from the protease increased rapidly
after warming (Fig. 2 b) with a half-time of internalization
of 8 min. The overall protease resistance reached a level of
70% within 30 min. The efficiency and rate of internalization was identical over a multiplicity range from 3 × 10
4-10
MOI. Treatment of the cells with nocodazole, a drug that
depolymerizes MT (Fig. 2 b, ND), or with cytochalasin D,
which depolymerizes actin filaments (not shown), had no
effect, either on the efficiency of binding and internalization or on the kinetics of internalization. Consistent with
previous reports using acid inactivation and plaque assays
to assess virus internalization (Huang and Wagner, 1964
), these results showed that HSV-1 penetration occurs rapidly and efficiently.
Fig. 3.
Subcellular distribution of incoming HSV-1 capsids. Conventional immunofluorescence microscopy of Vero cells infected
with HSV-1 at an MOI of 50 in the presence of CH. The cells were fixed in 3% PFA, permeabilized with 0.1% TX-100, and labeled with
anti-HC, an antibody generated against purified capsids. After 2 h of virus binding at 4°C (0 h), there is no signal. At 1 h PI, numerous cytoplasmic capsids (arrows) distributed within the entire cytoplasm are detectable. At 2 h PI, the capsids begin to accumulate at the nucleus. At 4 h PI, most of the capsids decorate the nuclei. Note that at 1 h, the focus was close to the coverslip, whereas at 2 and 4 h, it was
at the nuclear membrane.
[View Larger Version of this Image (74K GIF file)]
Fig. 4.
Incoming HSV-1 capsids colocalize with MTs. (a) Conventional immunofluorescence microscopy. Vero cells infected at an
MOI of 50 in the presence of CH were fixed in methanol at 2 h PI, and double labeled with anti-VP5 (NC-1; FITC anti-rabbit) and antitubulin antibodies (IA2; rhodamine anti-mouse). The FITC and the rhodamine signals were documented simultaneously using the FITC filter set. Almost all capsids (arrows) not localized to the nucleus (N) colocalize with microtubules. (b) Confocal immunofluorescence microscopy. Vero cells infected at an MOI of 50 in the presence of CH were fixed in methanol 2 h PI, and double labeled with
anti-capsid (HC, white) and anti-tubulin antibodies (IA2, gray). Almost all capsids not localized to the nucleus (N) colocalize with microtubules. Note that in some cells, the viral capsids accumulate at the microtubule-organizing center (MTOC). (c-e) Conventional EM. At
1 h postinfection, Vero cells infected at an MOI of 500 in the presence of CH were fixed with 2% glutaraldehyde in PBS. Epon sections
were cut parallel to the substrate. Numerous incoming viral capsids are localized to microtubules (MT) as identified by their typical morphology, localization, and their 24-nm diameter. (f-h) Immunoelectron microscopy. Vero cells infected at an MOI of 500 in the presence
of CH were extracted at 1 h (f) or 4 h PI (g and h) with 0.5% TX-100 in MT buffer before fixation. They were labeled with rabbit anti-
tubulin followed by protein A-9 nm gold, and Epon sections were cut parallel to the substrate. The cytoskeleton, nucleus, and nuclear
pore complexes (NPC), as well as cytoplasmic capsids, were preserved excellently, whereas all membranous organelles were extracted. The microtubules (MT) are easily identified, since they are heavily decorated with antibody and protein A-gold. In rather thick sections (f), the microtubules can be traced over long distances, and many capsids (filled arrows) are localized on them. These capsids still contain their electron-dense DNA core. At later time points (g and h), most of the capsids have lost their electron-dense core (h, open arrow) and are mostly located in close proximity to the nucleus. Very often the capsids are bound directly to the outer ring of the nuclear
pore complex (g, NPC). Virions bound to the plasma membrane (h, curved arrowhead) have lost the trilaminar appearance of their
membrane, but apparently a lot of glycoprotein and tegument remains attached to them, causing a distinct morphology easily distinguished from cytoplasmic capsids (g). Bar, 100 nm.
[View Larger Version of this Image (135K GIF file)]
), nor cytochalasin D, a drug that causes depolymerization of actin
filaments (Cooper, 1987
), affected capsid transport to the nucleus (Fig. 6 a, upper panels). In contrast, capsid accumulation at the nuclear membrane was significantly reduced by colchicine, vinblastine, and nocodazole (Fig. 6 a,
lower panels). These drugs interact with tubulin by different molecular mechanisms (Wilson and Jordan, 1994
). Labeling with anti-tubulin confirmed that colchicine and nocodazole depolymerized MT, whereas vinblastine caused
tubulin paracrystal formation (not shown). In the presence
of these drugs, the majority of capsids remained scattered
throughout the cytosol. To test whether the effect of nocodazole was reversible, the cells were analyzed 2 h after
drug removal. At this time, the MT had repolymerized,
and the previously dispersed capsids were concentrated on
the nuclear rim (Fig. 6 b).
Fig. 6.
Reduced transport of capsids to the nucleus in the absence of MTs. Immunofluorescence microscopy of Vero cells infected
with HSV-1 for 2 h at an MOI of 50 in the presence of CH and drugs affecting the cytoskeleton. (a) Cytoskeletal drugs. Under control conditions, almost all capsids accumulate around the nucleus. If the cells were infected in the presence of nocodazole, colchicine, or vinblastine, respectively (lower panel), there is no accumulation of viral capsids at the nucleus; instead, the capsids are scattered throughout
the entire cytoplasm. In cells infected in the presence of taxol or cytochalasin D, the capsids accumulate at the nucleus (upper panel).
The cells were fixed in 3% PFA, permeabilized with 0.1% TX-100, and labeled with an antibody directed against purified capsids (HC).
(b) Reversibility. In the presence of nocodazole, the amount of viral capsids localized to the nucleus is drastically reduced, whereas, after a 2-h chase in nocodazole-free medium, the capsids accumulate around the cell nuclei. Cells were infected in the presence of nocodazole for 2 h, fixed immediately (left panels), or washed and further incubated in normal medium for an additional 2 h before fixation
(right panels). After extraction with 0.5% TX-100 in MT buffer and fixation in methanol, the cells were double labeled with anti-tubulin
(left panels) and anti-capsid (HC, right panels).
[View Larger Versions of these Images (76 + 86K GIF file)]
), we concluded
that MT are directly or indirectly involved in the rapid
transport of capsids from the cell periphery to the nucleus
in Vero cells. In cells devoid of functional MT, the arrival
of capsids at the nucleus was significantly delayed.
), was used as a
control and remained constant. Labeling with [35S]methionine demonstrated that nocodazole had no overall effect on protein synthesis in uninfected or HSV-1-infected
Vero cells (not shown).
; Vale, 1990
; Vallee et al.,
1988
).
Fig. 8.
Incoming HSV-1 capsids display appendices at their
vertices. At 1 h PI, Vero cells infected at an MOI of 500 in the
presence of CH were extracted with 0.5% TX-100 in MT buffer
for 2 min at 37°C and fixed in 1% GA in MT buffer. They were
labeled with anti-tubulin followed by protein A-9 nm gold (a) or
embedded directly (b-d), and Epon sections were cut parallel to
the substrate. All viral capsids display some appendages on their
outside (arrowheads). The appendages are visible at three, four,
five, or all six corners visible in a section, which represent the
pentons of a viral capsid. Some of the appendices have a morphology very similar to purified cytoplasmic dynein (large arrow
in d): a large globular stalk with two smaller globular head domains. The capsid size is ~100-110 nm; the dynein-like appendices are ~50 nm. In thick sections of golden color, the appendices
are often buried in cytoskeletal material (b and d), whereas in
very thin sections of grey color (a and c), they are very prominent. The appendices have all kinds of different morphology with
varying sizes and electron density, but they are always located at
the pentons.
[View Larger Version of this Image (172K GIF file)]
). Immunoelectron microscopy
with the anti-dynein antibody was performed on ultrathin
cryosections of in situ fixed cells 1 or 2 h after initiation of
virus entry. In addition to a low level of labeling of the cytoplasm, dynein was found to be localized on the surface
of endosomes (Fig. 9 h), mitochondria (Fig. 9 a), and the
Golgi apparatus (Fig. 9 a). Very few gold particles were seen over the nucleus and the mitochondrial matrix (Fig.
9, a, c, and d). The mitochondria, which do not contain dynein, served as a background control. Compared with the
mitochondrial matrix, there was a slightly higher labeling
in the nuclei, and the cytoplasm was labeled 2.3-fold
higher than background (Table I).
Fig. 9.
Anti-dynein antibodies label incoming viral HSV-1 capsids. Thawed cryosections of Vero cells infected for 1 (c) or 2 h (a, b, and
d-h) in the presence of CH at an MOI of 500 were labeled with rabbit antibodies followed by protein A-5 nm (a-d, and g) or 10 nm gold
(e, g, and h). About 5% of the incoming viral capsids (a-f, and h) are labeled by anti-dynein (arrows), an affinity-purified antibody made
against a recombinant fragment of dynein heavy. Labeled capsids are localized close to the plasma membrane (g, PM), at the nuclear
envelope (c and d), or within the cytoplasm without obvious connection to either one (b). Note that the capsids within the extracellular
virions (f) or within the endosomes (g and h, E) are not labeled. Anti-dynein also labels the membranes of mitochondria (a, M), endosomes (h, E), and the Golgi complex (a, G) and diffusely the entire cytoplasm, whereas the mitochondrial matrix (a, b, and d) or the nucleus (d, N) show no labeling for dynein. Bar, 100 nm.
[View Larger Version of this Image (168K GIF file)]
In cells containing incoming HSV-1 capsids, dynein
was, in addition, found associated with many of the capsids
(Fig. 9, a-f). Capsids directly underneath the plasma membrane (Fig. 9 g) and within the cytoplasm (Fig. 9 b), as
well as capsids in close proximity to the nucleus (Fig. 9, a,
c, and d), were labeled with anti-dynein. On average, 13%
of the cytoplasmic capsids were labeled (Table I). Capsids
of intact virions, either extracellular (Fig. 9 f) or within endosomes (Fig. 9, g and h), were not labeled. Only 1.5% of
all capsids present in virions had colloidal gold particles in
close proximity (Table I). Thus, the labeling density was eightfold higher on cytosolic capsids compared with capsids within virions, ruling out the possibility that the antidynein antibody showed cross-reactivity to capsid proteins.
Given that the labeling efficiency using this technique does
not exceed 10%, e.g., only 10% of the primary antibody
present on the sections is detected by protein A-gold
(Griffiths, 1993a), these results showed that incoming cytosolic viral capsids associated with cytoplasmic dynein.
HSV-1 causes a range of diseases from benign common cold
sores to life-threatening encephalitis. It infects epithelial
cells of mucocutaneous membranes, and, as other herpesviridae, it establishes latent infections in sensory ganglia (Whitley and Gnann, 1993
). It is thought to enter neurons
by fusion at the presynaptic membrane, after which the
capsid is transported along the axon to the nucleus (Hill
et al., 1972
; Lycke et al., 1988
; Roizman and Sears, 1989
;
Topp et al., 1994
). The efficient infection of neurons and
other terminally differentiated cells has made HSV-1 a
useful vehicle for gene delivery with potential for gene therapy in the human brain (Glorioso et al., 1995
; Karpati
et al., 1996
).
The biochemical and morphological assays used in our
study showed that HSV-1 entry into Vero cells is both
rapid and efficient. Over a broad range of multiplicities,
we found that ~70% of bound virus penetrated with a
half-time of ~8 min. More than 60% of the capsids delivered to the cytosol were transported to the nucleus within
4 h of uptake, and binding to fibers extending from the nuclear pore complexes seemed to occur. The ratio of particles to plaque-forming units for HSV-1 is between 10 and
50 (Frenkel et al., 1975; McLauchlan et al., 1992
; Smith,
1964
; Watson et al., 1963
). Thus, if cells were infected at an
MOI of 50, and 40% of these bound, the number of particles entering the cells was 140-700 according to the proteinase K assay. We determined that 60% of the internalized virus, namely 80-420 particles, uncoat their viral
genome within 4 h postinfection as measured by a DNase protection assay (Ojala, P., B. Sodeik, M.W. Ebersold, and
A. Helenius, manuscript in preparation).
A majority of the capsids remained associated with nuclear pores after releasing their DNA (see also Batterson
et al., 1983; Lycke et al., 1988
). The apparent transient stability of the empty capsids contrasts with adenovirus, another virus that delivers its DNA to the nucleus through
the nuclear pore complexes, for which no empty capsids
could be detected at the nuclear pore complexes (Greber
et al., 1993
). We cannot formally rule out that, even though
the herpes capsids appeared empty at the nuclear membrane, viral DNA release had not been completed yet.
Our results demonstrate that MT are involved in capsid transport from the cell surface to the nucleus. Since the distance from the plasma membrane to the nucleus in Vero cells is not as large as in neurons, MT-assisted transport was not essential for infection, but it accelerated both the transfer of the capsids to the nucleus and the onset of viral protein synthesis. Having first detached from most of the tegument proteins at the plasma membrane, the capsids moved in minus end direction along MT to the MTOC. From there, they were transferred to the nuclear pores by unknown mechanisms. At early times (1-2 h PI), the majority of the capsids associated with the nuclear membrane proximal to the MTOC, but later the distribution became more homogenous around the nucleus.
Nocodazole and colchicine, which both depolymerize
MT, and vinblastine, which causes MT paracrystal formation, reduced capsid accumulation at the nuclear envelope.
These drugs were also reported to reduce the amount of
infectious HSV-1 recovered from rat dorsal root ganglia
infected in vitro (Kristensson et al., 1986) and from the
trigeminal ganglia after inoculation of the murine cornea (Topp et al., 1994
). We think that it is very likely that
capsids derived from virions bound to the "apical" plasma
membrane in our system (see Fig. 5 a) reached the nucleus
independent of MT, since the distance between the apical plasma membrane and the nuclear membrane is only
~200-300 nm. Thus, the larger the distance between the
plasma membrane where the virion had bound and the nucleus, the more the transport to the nucleus depended on
MT. In contrast with the results obtained in neurons (Kristensson et al., 1986
), taxol, which stabilizes MT, did not reduce the efficiency of capsid transport, suggesting that the
dynamic turnover or "treadmilling" of MT was not required for capsid transport in Vero cells. Depolymerization of actin filaments by cytochalasin D had no effect either on HSV-1 internalization or transport to the nucleus.
The presence of dynein heavy chains in the material attached to the cytosolic capsids indicated that the MT-mediated transport was powered by cytoplasmic dynein. Cytoplasmic dynein is a multisubunit protein complex of 1,270 kD consisting of two heavy chains, two to three intermediate
chains, and a variable number of small subunits (Holzbaur
and Vallee, 1994; Schroer, 1994
). It has a length of ~40-50
nm and a shape reminiscent of the letter Y. The two head domains contain the MT binding and ATPase sites, while
the base interacts with the cargo (Gilbert and Sloboda,
1989
; Vale, 1990
; Vallee et al., 1988
). Dynein is responsible
for minus end-directed movement of chromosomes and
membrane organelles along MT (Cole and LippincottSchwartz, 1995; Mitchison, 1988
). Since the plus ends of
MT are localized in the periphery of Vero cells close to the
plasma membrane and the minus ends are anchored at the perinuclear MTOC, dynein seems a logical choice as a motor for the transport of incoming viral capsids to the nucleus. The same applies to the axons of neurons, where the
MT have their plus ends toward the synapse, and the minus end anchored at the MTOC in the cell body (Black
and Baas, 1989
). Although some kinesin-like MT minus
end-directed motors have been implicated to play a role in
mitosis and meiosis, none of them has been demonstrated
to be involved in interphase transport processes (Barton
and Goldstein, 1996
; Hirokawa, 1996
). Since the MT-mediated transport that is crucial for infection of neurons also
operates in cultured fibroblasts, its analysis will be greatly
facilitated.
It is believed that dynein attachment to vesicles and kinetochores requires another large multimeric complex,
called dynactin, which contains 10 different proteins (Holzbaur and Vallee, 1994; Schroer, 1994
; Vallee and Sheetz,
1996
). So far, receptor molecules for dynein and dynactin
have not been identified on any cargo structures, but several studies indicate that the activity of dynein and its
binding to cargo may be regulated by phosphorylation (Allen, 1995
; Dillman and Pfister, 1994
; Lin et al., 1994
;
Niclas et al., 1996
). Whether the binding of dynein to
HSV-1 capsids involves dynactin or other cellular components and whether it is regulated by phosphorylation remains unclear at this point. The viral protein(s) that serve
as dynein receptors also remain to be identified. Since the
polypeptide composition of the capsids and tegument is
known, some potential candidates can, however, be singled out. The capsid itself contains six different proteins. Of these, only VP5 is known to be located at the pentons
(Trus et al., 1992
), which were the sites of preferential attachment of proteinaceous material onto cytosolic capsids
shown here. In addition to the bona fide capsid polypeptides, components derived from the tegument are also
likely to be present. For example, VP1-2, a 270-kD protein, is particularly tightly associated with the capsids
(Roizman and Furlong, 1974
) and may constitute part of
the material seen attached to the pentons in our electron
micrographs. Taking advantage of the relative simplicity
of the capsids, of existing virus mutants, and of the complete genomic sequence of the virus, work is in progress to
identify and to characterize the dynein receptor in HSV-1
capsids.
Received for publication 4 October 1996 and in revised form 13 December 1996.
B. Sodeik thanks Drs. Gary Cohen and Roselyn Eisenberg for their generous hospitality in their labs and many helpful suggestions. Victor Stolc is acknowledged for his contribution to the immunofluorescence studies. We also thank Lisa Hartnell, Linda Iadarola, Philippe Male, and Paul Webster of the Center of Cell Imaging in the Cell Biology Department at Yale University for teaching many aspects of EM, and Henry Tan for his superb photographic work. We are grateful to Drs. Gary Whittaker, Jani Simons, Päivi Ojala, Philippe Pierre, and Urs Greber for many stimulating discussions and critical reading of the manuscript.CH, cycloheximide; GA, glutaraldehyde; HSV-1, herpes simplex virus 1; MOI, multiplicity of infection; MT, microtubule; MTOC, microtubule organizing center; PFA, paraformaldehyde; PFU, plaque-forming unit; PI, postinfection; TX-100, Triton X-100.