Function of Dynein and Dynactin in Herpes Simplex Virus Capsid Transport

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Perl Molson - 28 Nov 2004 09:46 GMT

After fusion of the viral envelope with the plasma membrane, herpes
simplex virus type 1 (HSV1) capsids are transported along microtubules
(MTs) from the cell periphery to the nucleus. The motor ATPase
cytoplasmic dynein and its multisubunit cofactor dynactin mediate most
transport processes directed toward the minus-ends of MTs.
Immunofluorescence microscopy experiments demonstrated that HSV1
capsids colocalized with cytoplasmic dynein and dynactin. We blocked
the function of dynein by overexpressing the dynactin subunit
dynamitin, which leads to the disruption of the dynactin complex. We
then infected such cells with HSV1 and measured the efficiency of
particle binding, virus entry, capsid transport to the nucleus, and
the expression of immediate-early viral genes. High concentrations of
dynamitin and dynamitin-GFP reduced the number of viral capsids
transported to the nucleus. Moreover, viral protein synthesis was
inhibited, whereas virus binding to the plasma membrane, its
internalization, and the organization of the MT network were not
affected. We concluded that incoming HSV1 capsids are propelled along
MTs by dynein and that dynein and dynactin are required for efficient
viral capsid transport to the nucleus.
INTRODUCTION

To initiate a successful infection, animal viruses bind to the cell
surface, penetrate into the cytosol, and target their genome to the
sites of viral transcription and replication. For many viruses this is
the host nucleus (Whittaker et al., 2000). Particular neurotropic
viruses that enter at the presynaptic plasma membrane, such as herpes
simplex viruses, are transported over long distances because the site
of entry is far away from the nucleus. Herpes simplex virus type 1
(HSV1) is a human pathogen that initially replicates in epithelial
cells of the oral cavity. Amplified virus enters neurons and is
transported to the neuronal nuclei located in the trigeminal ganglion
(reviewed in Enquist et al., 1998). After lytic infection of some
neurons, a latent infection is established (Wagner and Bloom, 1997).

We have calculated that it would take 231 years for a herpes virus
capsid to diffuse by 10 mm in the axonal cytoplasm (Sodeik, 2000).
High concentrations of protein, the cytoskeleton, and organelles cause
molecular crowding in the cytoplasm, which effectively restricts free
diffusion of molecules larger than 500 kDa (Luby-Phelps, 2000). Thus,
virions and subviral particles are transported by active processes.
Besides hijacking vesicular transport during endocytosis and
secretion, viruses also exploit the host's cytoskeleton directly for
their itinerary (Sodeik, 2000; Ploubidou and Way, 2001).

HSV1 virions consist of four structural components: DNA, capsid,
tegument, and envelope (Steven and Spear, 1997; Zhou et al., 2000).
The icosahedral capsid with a diameter of 125 nm surrounds the
double-stranded viral DNA of 152 kb. The tegument, the hallmark of all
herpes viruses, is an amorphous layer of ~20 proteins. It is localized
between the capsid and the viral envelope that contains ~12 membrane
proteins.

For cell entry the envelope of HSV1 fuses with the plasma membrane.
Different molecules such as heparan sulfate proteoglycans, members of
the tumor necrosis receptor family (HVEM), and the immunoglobulin
family (nectins) serve as receptors for the HSV1 viral glycoproteins
gB, gC, and most importantly gD (reviewed in Spear et al., 2000). The
fusion of the viral envelope with the plasma membrane is mediated by
the viral glycoproteins gB, gD, gH, and gL (Spear et al., 2000).

All tegument proteins and the capsid with the DNA are released into
the cytosol. In epithelial cells and in axons of cultured neuronal
cells, incoming cytosolic capsids are transported along microtubules
(MTs) to the nucleus (Kristensson et al., 1986; Topp et al., 1994;
Topp et al., 1996; Sodeik et al., 1997). Electron microscopy and
careful quantification demonstrated that ~70% of cytosolic capsids
bind to nuclear pores and that concomitantly these capsids have lost
their electron-dense core (Sodeik et al., 1997). Using an in vitro
uncoating assay, Ojala et al. (2000) demonstrated that capsid binding
to the nucleus requires importin-beta and that the release of the
viral DNA is triggered by the interaction with the nuclear pore.
Transcription, viral replication, and capsid assembly take place in
the nucleus (for reviews see Steven and Spear, 1997; Roizman and
Knipe, 2001).

MTs are polar hollow protein cylinders of tubulin with a fast-growing
and -shrinking plus end usually located toward the cell periphery and
a minus-end mostly stabilized by attachment to the centrosome, the
major microtubule organizing center (MTOC; Nogales, 2000). Most if not
all minus-end-directed MT transport is mediated during interphase by
dynein motors, whereas kinesins transport cargo toward the opposite
direction (Vallee and Sheetz, 1996; Hirokawa, 1998). Cytoplasmic
dynein is a 20 S MT-activated ATPase consisting of two dynein heavy
chains (DHC), two intermediate chains (DIC), four light intermediate
chains (DLIC) and four different classes of light chains (DLC; Karki
and Holzbaur, 1999; King, 2000). Dynein is responsible for the
perinuclear localization of several organelles around the MTOC and
retrograde organelle transport in axons and is active during mitosis
(Vallee and Sheetz, 1996; Hirokawa, 1998).

In many cases dynein is assisted by a second 20 S protein complex,
called dynactin (Vallee and Sheetz, 1996; Karki and Holzbaur, 1999).
It consists of 2 copies of p150Glued, 4 molecules of dynamitin, p62,
~10 copies of Arp1 (actin-related-protein 1), possibly 1 conventional
actin, Arp11, and actin capping protein (p37 and p32), p27, p25, and
p24 (Holleran et al., 1998; Eckley et al., 1999). p150Glued can bind
directly to DIC and thus link dynein to dynactin (Karki and Holzbaur,
1995; Vaughan and Vallee, 1995). Dynamitin, at high concentrations
after transient transfection, dissociates the dynactin complex
(Echeverri et al., 1996; Eckley et al., 1999). Excess dynamitin
affects all tested dynein-mediated transports in vivo and in vitro:
e.g., spindle organization, chromosome transport, and the subcellular
localization of several membrane organelles (Echeverri et al., 1996;
Burkhardt et al., 1997; Presley et al., 1997; Valetti et al., 1999;
Sharp et al., 2000).

Quantitative immunoelectron microscopy showed that DHC colocalizes
with incoming herpes virus capsids (Sodeik et al., 1997). Here, we
demonstrate that incoming HSV1 capsids also colocalized with DIC and
the p150Glued subunit of dynactin. To test whether HSV1 capsids use
cytoplasmic dynein for their transport to the nucleus, we transiently
transfected cells with dynamitin, subsequently challenged them with
HSV1, and measured virus binding, internalization, capsid transport to
the nucleus, and immediate-early viral gene expression. High
concentrations of dynamitin and dynamitin-GFP clearly reduced the
number of viral capsids transported to the nucleus compared with
untransfected cells. Because fewer capsids reached the nucleus,
presumably fewer viral genomes were delivered to the nucleoplasm, and
the amount of viral protein synthesis was reduced. Overexpression of
dynamitin did not downregulate virus receptors at the plasma membrane,
because both virus binding and internalization were not reduced. We
propose that incoming HSV1 capsids are propelled by dynein along MTs
and that functional dynactin is required for their efficient
transport.

Bidirectional HSV1 Capsid Transport along Microtubules?

Many subcellular structures and progeny GFP-tagged alphaherpes viruses
(Smith et al., 2001; Willard, 2002) can move bidirectionally along
MTs. Interestingly, overexpression of dynamitin did not lead to a
random capsid distribution but to their MT-mediated accumulation in
the cell margins (Figures 7 and 9). This suggested that besides
dynein, HSV1 capsids might also use a plus-end-directed MT motor.
Plus-end-directed capsid motility could be involved in further
transport from the MTOC to the nucleus, as would be required in cells
where the MTOC is not directly neighboring the nucleus. MT-mediated,
plus-end-directed capsid transport could also be involved in apical
entry of polarized epithelial cells (Topp et al., 1996). Moreover,
during HSV1 egress from neurons, capsids are transported anterogradely
to the presynapse (Miranda-Saksena et al., 2000; Ohara et al., 2000).
Because of the uniform polarity of MTs in axons, this transport has to
be catalyzed by a plus-end-directed motor.

If capsids were indeed able to travel along MTs in both directions, to
the minus and plus ends, specific signals must regulate which motor
the capsid is supposed to use during the different steps of the viral
life cycle. Thus, the direction of capsid motility must be tightly
controlled. The main transport direction during virus entry must be to
MT minus-ends to ensure net movement to the cell center and the
nucleus. However, if minus-end-directed, dynein-mediated transport was
inhibited by overexpressing dynamitin, these putative
plus-end-directed motors might have taken over and transported capsids
to the cell margins.
http://www.molbiolcell.org/cgi/content/full/13/8/2795#B56

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beatadje@email.com - 28 Nov 2004 10:11 GMT

http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=44236&action=stream&blobty
pe=pdf

Proc. Nati. Acad. Sci. USA
Vol. 91, pp. 6529-6533, July 1994
Microbiology
Axonal transport of herpes simplex virions to epidermal cells:
Evidence for a specialized mode of virus transport and assembly
MARK E. T. PENFOLD*t, PATRICIA ARMATIt, AND ANTHONY L. CUNNINGHAM*f
*Virology Department, Centre for Infectious Diseases and Microbiology,
Institute of Clinical Pathology and Medical Research, Westmead
Hospital, 2145, and
University of Sydney, 2001, New South Wales, Australia; and tSchool of
Biological Sciences, University of Sydney, 2001, New South Wales,
Australia
Communicated by Frank Fenner, January 31, 1994 (receivedfor review
August 2, 1993)
ABSTRACT To examinthe transmission of herpes splex
virus (HSV) from axon to epidermal cell, an in vitro m el
was constructed conis of human fetal dorsal root gnglia
cultured in the central chamber of a dual-chamber tisue
culture system separated from autologous dsn explants n an
exterior chamber by concentric steel cylinders adegto the
substratum through silicon grease and agarose. Axons grew
through the agarose viral barrier and trmated on
epidermal cells in the exterior chber. After ination of
HSV onto dorsal root g _, anterograde axonal tranport of
glycoprotein and nudeocapsid antigen was observed by confocal
miosopy to appear in exterior chamber axons within 12
h and in epidermal cells within 16 h, moving at 2-3 umm/h.
Although both enveloped and unenveloped nucleocapsds were
observed in the neuronal soma by t mi elecro microscopy,
only nucleocapsids were observed in the axons,
closely associated with microtubules. Nodule formation at the
surface of HSV-infeced axons, becoming more deWse at the
axon terius on e e al cells, and patches of axoemal
HSV glycoprotein D ex r were observed by s
(lmmuno)electron microscopy, probably reprnti virus
emerging from the axolemma. These finngs ly suggest
a s dmode of viral Iransport, assembly, and egress in
sensory neurons: microtubule-associated intermediate-fast anterogrde
aMonal trnport of unenveloped nuceoapids with
separate t sort of glycoproteins to the distal regions of the
axon and assembly prior to virus emergence at the axon
terminus.
Herpes simplex virus (HSV) initially infects epidermal cells
of the natural human host or animal models, followed by
entry into cutaneous sensory axonal twigs and retrograde
axonal transport to the neural soma and nucleus. This results
in acute ganglionitis and/or development ofneuronal latency.
Later intermittent viral reactivation, anterograde axonal
transport, and transmission back to epidermal cells in the
same or adjacent dermatomes may result in intermittent
asymptomatic shedding of virus in saliva or genital secretions
or in recurrent clinical lesions (1, 2). While most of these
stages have been carefully studied (3, 4), there is little data on
anterograde viral transport and transmission to epidermal
cells.
These latter stages are important for two reasons. (i) In
people infected with HSV, recurrent clinical lesions are much
less common than asymptomatic shedding (1, 2), but this ratio
is reversed during immunosuppression, reverting to normal
when immunosuppressive drugs are withdrawn (5). These
observations suggest that there is immune restriction of viral
replication in the epidermis in the immunocompetent host,
despite continuing axonal transport of HSV and asymptomatic
shedding. An in vitro system is needed to examine the
effects of immune factors, including cytokines, on HSV
transmission from axon to epidermal cell and subsequent
viral replication.
(ii) The transport of HSV-1 from neuronal soma via axons
to epidermal cells may be specialized. Ultrastructural observations
of HSV-1 replication and egress in cultured cell lines
showed that virus buds from the inner nuclear membrane into
the perinuclear cisterna. The mechanism of envelopment of
HSV and transport between the perinuclear cisterna and the
cell surface remains controversial. One hypothesis is that
enveloped virus is incorporated into vesicles and transported
to the extracellular environment via the endoplasmic reticulum
and Golgi apparatus (4, 6, 7). However, the presence of
unenveloped nucleocapsids within the cytoplasm and beneath
the cytoplasmic membrane has been interpreted to
represent a second "cytoplasmic envelopment" pathway of
viral egress where HSV is deenveloped at the outer nuclear
membrane, reenveloped by budding into cytoplasmic vesicles,
and released at the. cell surface by exocytosis (8, 9).
Some recent data suggest this pathway may be abortive (6,
10). However, due to the unique anatomy of sensory. neurons,
axonal transport of virions over distances of 10-100 cm
and egress to epidermal cells may involve modifications of
stages of these transport pathways.
Therefore, we developed a unique chamber system in
which the neural soma in the central chamber and the axonal
termini of individual human neurons plus autologous epidermal
cells in the exterior chamber are maintained in separate
environments. Previous two-chamber in vitro systems employed
rat neural tissue alone (11). Our in vitro model allowed
inoculation of sensory neurons in the central chamber and
observation of anterograde transport of HSV-1 in axons and
transmission from axon termini to epidermal cells in the
exterior chamber.
Proc. Nati. Acad. Sci. USA 91 (1994) 6533
the cytoplasm of the soma support the hypothesis that there
are marked differences in the pathways of transport, assembly,
and egress of HSV-1 in the soma and axons of human
neurons:
(i) Conventional assembly and transport of enveloped
virions within vesicles to the cytoplasmic membrane, similar
to that observed in cultured cell lines, may occur over short
distances within the neuronal soma by either or both of the
two hypothetical pathways (4, 6-10). This may result in
infection of neighboring neurons or support cells.
(ii) Separate anterograde transport of unenveloped nucleocapsids
and viral glycoprotein may occur within axons resulting
in assembly at a distal site. This transport is probably
microtubule-facilitated and may follow deenvelopment of
enveloped virions at the outer lamella of the nuclear membrane
prior to axonal transport of nucleocapsids, thus sharing
some steps with the "cytoplasmic envelopment" pathway (8,
9).
This hypothesis is further supported by our finding that
anterograde axonal transport of nucleocapsids and glycoproteins
was rapid whereas transport of enveloped virus through
the endoplasmic reticular would be relatively slow. Association
of nucleocapsids with axonal microtubules was also
strongly suggested by the relatively rapid velocity of transport
(2-4 mm/h or 50-100 mm/day), which is similar to the
well-defined intermediate to fast microtubule-associated axonal
transport of organelles, neurotransmitters, and proteins
(3-17 mm/h) (23, 24) and the proven microtubule-associated
retrograde transport of nucleocapsids in rat axons (17). Close
contact is highly likely between nucleocapsids that are 80-
100 nm in diameter and microtubules in distal axons that are
70-100 nm apart (see Fig. 3). Our hypothesis could explain
retention of the two pathways of viral transport, assembly
within infected cell lines (6-10), and differences in their
relative importance in different cell types.
Alternative interpretations of the marked predominance of
unenveloped nucleocapsids in distal axons in static TEM
images could be that deenveloped virions were transported
distally immediately after virus entry in the central chamber
or represent reinfection with progeny virions (10). However,
the kinetics of neuronal expression of HSV-1 glycoprotein
and capsid antigens, their anterograde axonal transport observed
by confocal microscopy, and the presence of only
unenveloped nucleocapsids in axons support the hypothesis
that this is indeed the main method of axonal transport.
Hence viral glycoproteins must be transported separately
from nucleocapsids, probably within the neuritic transport
vehicles commonly observed within axons, analogous to the
transport of endogenous neuronal proteins (23). This hypothesis
should be tested by future studies using transmission
immunoelectron microscopy.
Intermediate-to-fast translocation of organelles, neurotransmitters,
and proteins along axonal microtubules is associated
with the cellular proteins, kinesin and a cytoplasmic
dynein (MAP-IC), which produce movement in the anterograde
and retrograde directions, respectively (23, 24). Separate
interactions between different neuronal and viral tegument
or capsid proteins should, therefore, facilitate such
anterograde or retrograde transport of virions and could offer
important therapeutic targets.
Sparse nodules along HSV-infected axons (with concentrations
at the axon terminus) corresponded to focal patches
of axonal membrane containig viral glycoproteins. These
could be explained as viruses emerging by exocytosis or
other mechanisms. It is unlikely that nodules are adherent
virions released from other sites because of the kinetics of
appearance of nodules, their distribution (concentration at
the axon terminus), and the absence of patches of glycoprotein
staining on adjacent fibroblasts that also have receptors
for HSV. The mechanism for the marked increase in density
of viral nodules in terminal axons is unknown but probably is
determined by interactions with underlying epidermal cells.
We have not yet directly observed HSV emerging from the
axolemma facing the epidermal cell. Elucidation of the exact
mechanism ofHSV emergence from axons at all sites should
require further exhaustive observations of serial sections by
TEM.

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