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Gene Therapy (2005) 12, 1353–1359
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Gene Therapy Progress and Prospects:
Viral trafficking during infection
EM Campbell and TJ Hope
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL, USA
The intracellular steps involved in viral infection, namely
cytoplasmic trafficking and nuclear import, are critical events
in the viral life cycle that have lagged behind other areas of
viral research. This review examines recent advances in our
understanding of these steps for viruses commonly employed as viral gene delivery vectors. Steps governing the
cytoplasmic trafficking and nuclear import of Herpes Simplex
virus, Human Immunodeficiency virus and Adenovirus are
reviewed in this article.
Gene Therapy (2005) 12, 1353–1359. doi:10.1038/
sj.gt.3302585
Keywords: virus; infection; trafficking; HIV; HSV; adenovirus; cytoskeleton; nuclear import
In brief
Progress
Prospects
Cytoplasmic trafficking requires active transport due
to properties of the cytoplasm that greatly impede
diffusion.
Most viruses overcome this barrier by engaging the
transport machinery of the host cell microtubule
cytoskeleton.
The microtubule motor complex dynein is involved
in the cytoplasmic transport of the viruses discussed.
Nuclear import of viral genomes is an active process
critical to infection or transduction of host cells.
Identification of the viral components that mediate
engagement and trafficking along microtubules will
likely lead to more efficient vector systems.
Some viruses may activate signal transduction pathways that augment their cytoplasmic trafficking, and
identification and utilization of these pathways could
aid in vector design.
Elucidation of the mechanisms by which viruses
facilitate the nuclear import of their genomes may
generally enhance transduction and possibly allow
for directed specific transduction of desired cell
populations.
Introduction
Viral trafficking in the cytoplasm of target cells has for
years been an area that lagged behind other areas of viral
research. Studies involving viral fusion, gene regulation
and viral production have predominated due to their
relative amenability to classical biochemical and cell
biology approaches. Models of viral infection have
therefore concentrated on these events, often leaving
readers with the impression that events between fusion
and the virus reaching its replicative compartment, most
frequently the nucleus, occur passively. However, it is
now obvious that most virions utilize active transport to
facilitate their nuclear localization.
One fact often overlooked is that the environment of
the cytoplasm is not something the size of a virion can
diffuse through. Cytoskeletal structures and organelles
present in the cytoplasm in fact create an environment
Correspondence: Professor TJ Hope, Department of Microbiology and
Immunology, University of Illinois at Chicago, 835 S. Wolcott Rm E-703,
Chicago, IL 60612, USA
that greatly hinders the diffusion of molecules and
molecular complexes greater than 500 kDa. For example,
when the density of the cytoplasm is taken into account,
it has been estimated that it would take 231 years for a
herpes virus to diffuse 10 mm in the axonal cytoplasm.1
The difficulties presented by limitations to diffusion in
the cytoplasm have forced viruses to evolve methods to
overcome this problem. It is becoming increasingly clear
that the solution to this problem for most viruses is to
utilize the cellular transport systems of the target cell
cytoskeleton to promote their cytoplasmic trafficking.
This review will discuss recent advances in our understanding of how different virions facilitate their cytoplasmic transport and nuclear translocation during
infection, with specific emphasis on those viruses most
utilized as gene delivery vectors.
The previously mentioned gap between research
examining viral trafficking during infection, and other
areas of viral research, has been bridged in two ways.
First, the use of pharmacological agents that disrupt
cytoskeletal structures and dynamics have repeatedly
demonstrated the importance of cytoskeletal-mediated
Viral trafficking during infection
EM Campbell and TJ Hope
1354
transport during infection for a large number of viruses
(reviewed in Campell and Hope2 and Dohner and
Sodeik1). These studies, however, must always be
cautiously interpreted because of the fact that cytoskeletal disruption has pleiotropic effects on cell function.
Therefore, experiments using such inhibitors must be
viewed as a useful starting point used to design other,
more definitive experiments. With that caveat, however,
such agents continue to provide insights into trafficking
processes that are otherwise very difficult to study.
The second major advance allowing the examination of
viral trafficking has been the use of fluorescent microscopy to visualize the behavior of individual fluorescently
labeled virions in target cells. The ability to monitor the
behavior of individual virions or groups of virions in
target cells combined with selective fluorescent labeling
of target cell components has been an invaluable tool in
revealing how viruses interact with target cell components
and compartments during infection. Again, as will be
discussed in more detail below, these experiments have
also indicated an intimate association of virions with the
host cell cytoskeleton during infection.
Cytoskeletal transport machinery is utilitized
by viruses
Utilization of cellular transport machinery has been a
dominant theme in the examination of how viruses facilitate
their intracellular trafficking. It is therefore important to
briefly describe these systems, and we would also refer the
reader to other, more comprehensive reviews.3,4
The maintenance of eukaryotic cellular organization is
developed and maintained by the transport machinery of
the cytoskeleton, which includes actin microfilaments,
microtubules and intermediate filaments. Intermediate
filaments are thought to primarily provide mechanical
stability to cells and will therefore not be discussed
further. The actin and microtubule cytoskeletons are
formed from polymerization of monomeric subunits to
form dynamic, three-dimensional structures in cells that
provide the primary mechanisms of intracellular transport of molecular complexes greater than 500 kDa
(reviewed in Dohner and Sodeik1). Both the microtubule
and actin cytoskeleton form polarized filaments to which
subunits are added to a growing, plus end in a complexly
regulated process.5 These filaments then serve as the
platforms that engage ATP-driven molecular motors to
transport cargo throughout the cell (Figure 1). Transport
along actin microfilamants is driven by myosin motors,
with some myosins transporting cargo towards the plus
end of filaments and others transporting cargo in the
opposite direction. Another method of actin-based
motility utilized by cells as well as intracellular pathogens, is the utilization of actin nucleating proteins to
generate ‘actin comets’ that essentially propel cargo on
the tip of a branched, rapidly polymerizing scaffold.5
While actin-based transport of cargo is thought to
occur most frequently at the cell periphery and is,
relatively speaking, used for short-ranged transport of
cargo, longer-ranged transport is provided by the
microtubule cytoskeleton (Figure 1). In addition to the
polarity of individual filaments, microtubules also
possess an organized, functional polarity in most cells
whereby minus ends collectively end in a perinuclear
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Figure 1 Cytoskeletal transport. The actin and microtubule cytoskeleton are
responsible for trafficking numerous endogenous cargos, as well as viruses
and intracellular bacteria, throughout the cell. In general, myosin motors
facilitate short-range traffic of cargos in the cell periphery, whereas the
dynein- and kinesin-microtubule-based motors transport cargos longer
distances throughout the cell. Grey colored dots adjacent to cytoskeletal
filaments represent myosin motors transporting cargo along microfilaments
and dynein and kinesin motors transporting cargo along microtubules.
area termed the microtubule-organizing center (MTOC)
and plus ends extend into the cell periphery. Directional
transport along microtubules is achieved by ATP-driven
molecular motor complexes. In general, kinesins drive
cargo towards the plus ends of microtubules extending
to the cell periphery while dynein motor complexes
drive cargo towards the minus ends located near the
nucleus. Less is known about what determines the cargo
specificity. However, a known critical player in the
transport function of microtubules is dynactin (reviewed
by Schroer6), which is a multiprotein dynein cofactor
required for dynein function under many conditions.
There is evidence that dynactin facilitates the interaction
between cargo, particularly membrane bound cargo, and
dynein.6 Disruption of the dynein complex with a
dominant-negative protein known as dynamitin
abolishes dynein-mediated traffic. However, the finding
that dynactin disruption also abolishes the plus- and
minus-end transport of some cargos that undergo
bidirectional transport7 has led to the notion that
dynactin may act to coordinate the net transport of
bidirectionally transported cargo.3
Herpes simplex virus (HSV) trafficking and
nuclear import
HSV was one of the first viruses observed to associate
with microtubules during infection, and these earlier
Viral trafficking during infection
EM Campbell and TJ Hope
findings have been reviewed elsewhere.1,2 It is known
that HSV utilizes microtubules to facilitate their translocation to a perinuclear region in the cell (Figure 2).
Disrupting microtubules using pharmacological inhibitors prevents this accumulation of HSV in the perinuclear
region.8,9 More recent investigations have focused on
determining the molecular mechanisms by which HSV
interacts with the microtubule transport machinery. As
would be expected, given the minus-end directional
transport HSV undergoes to reach the nucleus, numerous
reports indicate that HSV interacts with dynein to
facilitate its cytoplasmic transport.10,11 Using electron
microscopy and immunogold antibodies, Sodeik et al8
demonstrated that dynein is present in the protein
complex linking HSV virions to microtubules. Dohner
et al10 have shown that HSV virions colocalize with the
intermediate chain of the dynein protein complex as well
as with the p150-glued protein of the dynactin complex,
which is thought to link the dynactin complex to the
dynein complex. Moreover, overexpression of dynamitin,
to induce the disruption of the dynactin complex and
inhibit dynein-mediated transport,6 prevents the nuclear
accumulation of HSV virions in the nucleus and
expression of early HSV gene products following
infection.10 Taken together, these reports suggest dynein-mediated transport of HSV virions is a critical
component of HSV intracellular trafficking.
Other investigations have attempted to identify the
HSV proteins responsible for interacting with the
microtubule transport machinery. One HSV protein that
is a viable candidate for providing the interaction
between the incoming virion and the microtubule
cytoskeleton is the VP22 tegument protein. The tegument
is a virion compartment located between the capsid
1355
Figure 2 Microtubule transport of HIV and HSV. Both HIV (left panel) and HSV (right panel) have been observed to traffic in a microtubule-dependent
fashion. Both viruses accumulate at the MTOC during infection and have been observed to tightly associate with microtubules during infection by
fluorescent and electron microscopy. Reprinted with permission from McDonald et al21 and Sodeik et al.8
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Viral trafficking during infection
EM Campbell and TJ Hope
1356
and envelope and may participate in postfusion events
during HSV infection. Interestingly, VP22–GFP fusion
proteins colocalize with microtubules and also affect
microtubule dynamics, causing microtubule reorganization and stabilization when expressed in Vero cells
(reviewed in Campbell and Hope2 and Dohner and
Sodeik1). The ability of VP22 to bind microtubules has
been mapped to a C-terminal domain and a central
stretch of approximately 100 amino acids.12 VP22 may
also play a role in facilitating the trafficking past the actin
cortex, given the finding that VP22 also interacts with the
actin motor myosin IIA.13 These facts make VP22 one
of the most promising candidates for the mechanism
by which incoming HSV virions associate with microtubules during infection.
The herpes UL34 protein has also received attention as
a protein that may be responsible for the interaction
between HSV virions and microtubules during infection.
UL34 interacts directly with the intermediate chain of
dynein in yeast-two-hybrid models and glutathione Stransferase (GST) pull-down assays, and demonstrates
perinuclear localization that is inhibited by nocadozole, a
drug that disrupts the microtubule network.1,2 This
notion is also supported by the fact that UL34 directly
interacts with the viral capsid protein. However, UL34’s
candidacy as a protein responsible for affecting virion
trafficking during infection has recently been brought
into question by the finding that extracellular virions do
not contain detectable amounts of UL3414 and the fact
that deletion of UL34 does not prevent infection or
nuclear accumulation of HSV virions.
Another protein recently proposed to mediate HSV
retrograde transport during infection is the VP26
protein.11 Douglas et al11 demonstrated that VP26 binds
the dynein light chains RP3 and Tctex1 in yeasttwo-hybrid systems and in vitro binding assays. They
also found that microinjected HSV capsids lacking
VP26 failed to accumulate around the nucleus as well
as their VP26-negative counterparts.11 These observations make VP26 another attractive candidate to be
the viral determinant that is responsible for interaction between HSV and the microtubule cytoskeleton
during infection.
Following cytoplasmic trafficking to the MTOC, HSV
virions bind the nuclear pore complex (NPC) and release
their DNA genome into the nucleus (reviewed by
Whittaker15) (Figure 3). This process, reconstituted in
vitro, has been shown to be inhibited with antibodies to
the NPC complex or against importin b. In this sytem,
HSV binding of the NPC was dependent on both
importin b and Ran. It has also been found that
uncoating of the HSV capsid and subsequent release of
viral DNA requires both NPC binding and ATP.
Given that the genome of HSV is, in viral terms,
extremely large, a better understanding of the mechanisms by which HSV virions traffic within an infected cell
will likely lead to more specialized vectors in which the
genes not required for cellular transductions have been
removed. As will be discussed in the next section, the
removal of a few human immunodeficiency virus (HIV)
accessory genes not required for transduction from HIVbased vectors resulted in the production of higher titer
vectors from vector-producing cells. A similar if not
more dramatic benefit could easily be imagined for HSV
vectors as the HSV genes critical for gene transduction
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Figure 3 Nuclear import of adenovirus and HSV. Electron micrographs
showing the accumulation of (a) adenovirus and (b) HSV capsids at the
nuclear envelope. Reprinted with permission from Saphire et al16 and Ojala
et al.17
are identified and separated from the numerous other
genes in its expansive genome.
HIV trafficking and nuclear import
Following fusion of the human immunodeficiency virus
(HIV) envelope glycoprotein with CD4 and chemokine
coreceptors at the cell surface, the viral core must
traverse through the cytoplasm and induce the nuclear
import of the viral nucleoprotein complex, known as the
preintegration complex (PIC), and allow integration of
the viral genome into the host cell chromosome. The
characterization of these trafficking events, as well as the
viral proteins that facilitate them, remains an active area
of HIV research. The viral accessory protein Nef was one
of the first viral proteins identified that appears to play a
role in trafficking of virions in the cytoplasm of target
cells. Nef has been shown to be incorporated into virions
and increase virion infectivity (reviewed by Anderson
Viral trafficking during infection
EM Campbell and TJ Hope
and Hope18). Importantly, numerous studies have found
that Nef is no longer required to increase viral infectivity
when virions are pseudotyped with pH-dependent
envelopes, which are activated by the low pH of
lysosomes and therefore enter the cell through an
endocytic pathway. However, while the nature of the
envelope affects the ability of Nef to increase viral
infectivity, numerous studies agree that Nef does not
affect the fusogenicity of HIV virions.2,19,20 This suggests
that some property of these pH-dependent envelopes
independent of their target receptor and fusogenic
potential is responsible for their ability to complement
the infectivity-enhancing properties of Nef. Importantly,
these envelopes only fuse following the induction of
endocytosis and trafficking of a virion within an
endosome, some distance into the cell. A possible
solution is that Nef acts to allow the virus to bypass
the layer of cortical actin barrier present in cells. This
would be consistent with the above data, as well as
data demonstrating that Nef interacts with numerous
molecules associated with cytoskeletal rearrangement,
and our recent finding that disruption of the actin
cytoskeleton in target cells increases the infectivity of
Nef-deficient virions to wild-type levels.2
Our lab has also generated data demonstrating that
the PIC utilizes microtubule-based transport to achieve a
perinuclear translocation during infection21 (Figure 2).
Utilizing fluorescent labeling, HIV virions have been
observed to undergo bidirectional transport along
microtubules that result in the accumulation of labeled
virions in a perinuclear location. Moreover, viral reverse
transcription complexes (RTCs), identified by the incorporation of fluorescent nucleotides, were observed to be
associated with microtubules using correlative electron
microscopy (Figure 2). The accumulation of RTCs at the
MTOC appears to be dependent on the dynein motor
complex, as microinjection of anti-dynein antibodies,
known to disrupt dynein function, resulted in the
prevention of this perinuclear accumulation of RTCs,
and also, interestingly, resulted in these RTCs being
transported to the periphery of the cell.21 This not only
suggests that during infection, the RTC selectively
interacts with the minus-end dynein motor complexes,
but also that the RTC is capable of interacting with plusend kinesin motor complex, although this situation is
clearly not predominant in the presence of active dynein
motor function.
Another element of HIV infection that may affect viral
trafficking is the poorly described feature of viral
uncoating. Uncoating involves the sequential loss of
some viral structural proteins during infection (reviewed
by Dvorin and Malim22). This is supported by a study
examining PIC composition, which found that the p17MA
protein is a component of purified PICs, but that the
p24CA protein is not present. Immunofluorescent analysis
of RTCs that have incorporated fluorescent nucleotides
also showed that p17MA is present in most of these
complexes, while many contained no detectable p24CA.3
It is tempting to speculate that some level of viral
uncoating may, therefore, facilitate viral reverse
transcription and/or trafficking events during infection,
but currently there is only circumstantial evidence to
support this.
While many simple retroviruses require cell division
and breakdown of the nuclear envelope to gain access to
the host cell chromosome, lentiviral vectors such as HIV
show considerable more promise than their retroviral
counterparts because of their ability to transduce
quiescent, nondividing cells (reviewed by Bartosch and
Cosset23 and Cockrell and Kafri24). However, although
there is clear agreement that this is the case, the
mechanism that the viral PIC employs to traverse the
nuclear membrane of target cells remains clouded. This
confusion is due to the fact that a number of mechanisms
have been reported to be the primary determinant
allowing the PIC to achieve nuclear import. These have
been reviewed extensively elsewhere.25,26 Briefly, convincing arguments have been made that the nuclear
import activity of the PIC is mediated by the viral matrix,
Vpr, integrase proteins and a DNA element formed
during reverse transcription known as the central
DNA flap. There remains a lack of consensus as to
which, if any, of these elements are the primary
determinant governing nuclear import of the PIC, and
it may be that these elements act in a synergistic fashion
in many settings.
Identification of these proteins and nucleic acid
structures involved in nuclear import are important for
the design of recombinant HIV and other lentivirusbased gene transfer agents. For example, a vector
packaging system lacking the Nef, Vif and Vpr genes of
HIV has been shown to transduce growth arrested cells
and monocyte-derived macrophages as efficiently, or
better, than packaging sytems containing these genes
(reviewed by Bartosch and Cosset23 and Cockrell and
Kafri24), suggesting that, at least in the context of
lentiviral gene delivery vectors, Vpr is not required for
efficient transduction of nondividing cells. Although
vector systems without the central polypurine tract
(cPPT) that mediates the formation of the central DNA
flap are known to efficiently transduce cells, various
groups have shown that incorporation of the cPPT into
HIV-based vectors can improve transduction efficiency
of nondividing cells between two- and 10-fold.23,24
1357
Adenovirus trafficking and nuclear import
It has been known for quite some time that adenovirus
virions associate with microtubules. Adenoviruses are
nonenveloped viruses that induce their own endocytosis
by binding of its cellular receptor. Virions subsequently
escape from the endosomal compartment via a mechanism that requires lowering of endosomal pH. Owing
to space limitations, we confine our discussion to the
cytoplasmic trafficking of adenovirus, but refer the
reader to more comprehensive reviews examining
adenovirus endocytosis and endosomal escape.27,28
Following entry into the cytosol, numerous studies
have suggested that adenovirus utilizes microtubulebased transport to facilitate perinuclear accumulation.
Observations that adenovirus associates with microtubules during infection and in vitro are almost 30 years old
(reviewed in Campbell and Hope2). However, work in
recent years has extended these observations to generate
a much clearer picture of the events occurring during
adenovirus infection. The first of a recent flurry of
reports directly implicating microtubule-based transport
during adenoviral infection came from Suomalainen et al.
They found that Ad2 particles underwent bidirectional
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Viral trafficking during infection
EM Campbell and TJ Hope
1358
movement that resulted in a net displacement of virions
towards the MTOC with peak velocities in the micrometer per second range.29 This transport was inhibited
by microtubule depolymerization as well as by disrupting dynein function through dynamitin.29 Similar results
were reported by Leopold et al examining the cytoplasmic trafficking of Cy3-labeled Ad5 virions. They also saw
virions transported rapidly in curvilinear paths towards
the nucleus in a fashion that was inhibited by microtubule depolymerization and microinjection of functionblocking antibodies directed at dynein, but not by actin
depolymerization.30 However, this study found that Ad5
trafficking occurred almost exclusively in the minus-end
direction,30 different from the bidirectional motility of
Ad2 virions described by Suomalainen et al.29 Another
report confirmed the net minus-end trafficking of
adenovirus in enucleated cells.31 In an elegant series of
experiments in which cell nuclei were removed from
cells by centrifugation, Bailey et al31 demonstrated a
stable interaction between adenovirus and the MTOC.
This stable interaction, in addition to supporting the role
of microtubules during adenovirus infection, also suggests that nuclear factors may be required for inducing
the translocation of adenovirus from the MTOC to the
nucleus during infection. Microtubule-directed motion
has also been shown to occur during infection by adenoassociated virus.32 This study observed fluorescently
labeled virions to undergo directed motion in curvilinear
paths at speeds consistent with microtubule-based
transport. Moreover, these authors observed individual
virions traveling along identical curvilinear paths,
suggesting transport of multiple virions on a single
microtubule.32
The events facilitating microtubule-mediated nuclear
transport of adenovirus have also been investigated. It
has been reported that both the frequency and velocity
of minus-end trafficking events during infection is
increased by transient stimulation of the protein kinase
A and p38 MAP kinase pathways by adenovirus.33
Despite the implication of these pathways, the precise
mechanism by which this enhancement in trafficking
occurs remains poorly defined. A recent report by Kelkar
et al34 sought to determine the factors required for the
adenovirus/microtubule interaction. Using an in vitro
microtubule binding assay, these authors examined the
binding of adenovirus to polymerized microtubules in
the presence or absence of microtubule-associated
proteins (MAPs), including dynein. They found that
adenovirus bound microtubules strongly only in the
presence of MAPs, and that the ability of MAPs to induce
microtubule binding to adenovirus was greatly reduced
when the MAP fraction was depleted of dynein.34
Following arrival at the nuclear membrane, adenovirus deposits its genome into the host cell nucleus
(Figure 3). Studies examining the nuclear import of
adenovirus throught the NPC have focused on identifying the cellular proteins involved in this process
(reviewed in Greber and Fornerod35). It has been noted
that nuclear import of adenovirus DNA can be inhibited
by excess of other NLS-containing proteins, suggesting
that adenovirus utilizes the classical nuclear import
pathway to achieve nuclear localization of its genome.
Moreover, the hsc70 protein is required for the nuclear
translocation of viral DNA, but not the structural hexon
protein. The role of the hsc70 protein in nuclear import is
Gene Therapy
not clear, however, and the authors speculate that hsc70
may play a role in uncoating of the virus following
endosomal escape. They also suggest the alternative that
VP22 may play a role conjugating an adenoviral NLS
sequence to the nuclear import machinery, a role that hsc
is thought to play in yeast. Another report has identified
the CAN/Nup214 NPC protein as the docking site of
Ad2 capsids, and found this association not to require
additional cellular factors. These authors also find that
the nuclear histone H1 protein is responsible for
facilitating the uncoating of the adenoviral capsid at
the NPC and subsequent translocation of the viral
genome to the nucleus.35
As with the previous viruses mentioned, identification
of the adenoviral gene products required for viral
trafficking may allow not only for increased vector
production and transduction efficiency, but may also
limit the undesired immune responses generated at
times by adenoviral vectors as the genes critical for
transduction are dissected from those dispensable genes
not required for gene transduction.
Summary
It is now clear that most viruses use components of the
cytoskeleton to move within cells. As viruses are
increasingly used as gene delivery vectors, an improved
understanding of viral trafficking in the cytoplasm can
only increase the efficiency of these vectors, as unnecessary viral gene products are separated from those
required for gene transduction, potentially leading to
increased vector production and transduction, and also
possibly limiting undesired immune reactions in the
host. Such optimization of the cell biology of gene
therapy vectors will help bring the promise of gene
therapy to reality.
Acknowledgements
We apologize to any authors whose work was not cited
or cited indirectly due to space limitations. We thank
Omar Perez, Marta Melar and Shivani K Maffi for critical
review and discussions which improved the manuscript.
References
1 Dohner K, Sodeik B. The role of the cytoskeleton during viral
infection. Curr Top Microbiol Immunol 2005; 285: 67–108.
2 Campbell EM, Hope TJ. Role of the cytoskeleton in nuclear
import. Adv Drug Deliv Rev 2003; 55: 761–771.
3 Welte MA. Bidirectional transport along microtubules. Curr Biol
2004; 14: R525–R537.
4 Karcher RL, Deacon SW, Gelfand VI. Motor-cargo interactions:
the key to transport specificity. Trends Cell Biol 2002; 12: 21–27.
5 Pollard TD, Borisy GG. Cellular motility driven by assembly and
disassembly of actin filaments. Cell 2003; 112: 453–465.
6 Schroer TA. Dynactin. Annu Rev Cell Dev Biol 2004; 20: 759–779.
7 Deacon SW et al. Dynactin is required for bidirectional organelle
transport. J Cell Biol 2003; 160: 297–301.
8 Sodeik B, Ebersold MW, Helenius A. Microtubule-mediated
transport of incoming herpes simplex virus 1 capsids to the
nucleus. J Cell Biol 1997; 136: 1007–1021.
Viral trafficking during infection
EM Campbell and TJ Hope
1359
9 Mabit H et al. Intact microtubules support adenovirus and
herpes simplex virus infections. J Virol 2002; 76: 9962–9971.
10 Dohner K et al. Function of dynein and dynactin in herpes
simplex virus capsid transport. Mol Biol Cell 2002; 13: 2795–2809.
11 Douglas MW et al. Herpes simplex virus type 1 capsid protein
VP26 interacts with dynein light chains RP3 and Tctex1 and
plays a role in retrograde cellular transport. J Biol Chem 2004; 279:
28522–28530.
12 Martin A, O’Hare P, McLauchlan J, Elliott G. Herpes simplex
virus tegument protein VP22 contains overlapping domains for
cytoplasmic localization, microtubule interaction, and chromatin
binding. J Virol 2002; 76: 4961–4970.
13 van Leeuwen H, Elliott G, O’Hare P. Evidence of a role for
nonmuscle myosin II in herpes simplex virus type 1 egress.
J Virol 2002; 76: 3471–3481.
14 Reynolds AE, Wills EG, Roller RJ, Ryckman BJ, Baines JD.
Ultrastructural localization of the herpes simplex virus type 1
UL31, UL34, and US3 proteins suggests specific roles in
primary envelopment and egress of nucleocapsids. J Virol 2002;
76: 8939–8952.
15 Whittaker GR. Virus nuclear import. Adv Drug Deliv Rev 2003;
55: 733–747.
16 Saphire AC et al. Nuclear import of adenovirus DNA in vitro
involves the nuclear protein import pathway and hsc70. J Biol
Chem 2000; 275: 4298–4304.
17 Ojala PM et al. Herpes simplex virus type 1 entry into host cells:
reconstitution of capsid binding and uncoating at the nuclear
pore complex in vitro. Mol Cell Biol 2000; 20: 4922–4931.
18 Anderson JL, Hope TJ. Recent Insights into HIV Accessory
Proteins. Curr Infect Dis Rep 2003; 5: 439–450.
19 Tobiume M, Lineberger JE, Lundquist CA, Miller MD, Aiken C.
Nef does not affect the efficiency of human immunodeficiency
virus type 1 fusion with target cells. J Virol 2003; 77: 10645–10650.
20 Cavrois M, Neidleman J, Yonemoto W, Fenard D, Greene WC.
HIV-1 virion fusion assay: uncoating not required and no effect
of Nef on fusion. Virology 2004; 328: 36–44.
21 McDonald D et al. Visualization of the intracellular behavior of
HIV in living cells. J Cell Biol 2002; 159: 441–452.
22 Dvorin JD, Malim MH. Intracellular trafficking of HIV-1 cores:
journey to the center of the cell. Curr Top Microbiol Immunol 2003;
281: 179–208.
23 Bartosch B, Cosset FL. Strategies for retargeted gene delivery
using vectors derived from lentiviruses. Curr Gene Ther 2004; 4:
427–443.
24 Cockrell AS, Kafri T. HIV-1 vectors: fulfillment of expectations,
further advancements, and still a way to go. Curr HIV Res 2003;
1: 419–439.
25 Piller SC, Caly L, Jans DA. Nuclear import of the pre-integration
complex (PIC): the Achilles heel of HIV? Curr Drug Targets 2003;
4: 409–429.
26 Bukrinsky M. A hard way to the nucleus. Mol Med 2004; 10: 1–5.
27 Medina-Kauwe LK. Endocytosis of adenovirus and adenovirus
capsid proteins. Adv Drug Deliv Rev 2003; 55: 1485–1496.
28 Meier O, Greber UF. Adenovirus endocytosis. J Gene Med 2003; 5:
451–462.
29 Suomalainen M et al. Microtubule-dependent plus- and minus
end-directed motilities are competing processes for nuclear
targeting of adenovirus. J Cell Biol 1999; 144: 657–672.
30 Leopold PL et al. Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis. Hum
Gene Ther 2000; 11: 151–165.
31 Bailey CJ, Crystal RG, Leopold PL. Association of adenovirus with the microtubule organizing center. J Virol 2003; 77:
13275–13287.
32 Seisenberger G et al. Real-time single-molecule imaging of the
infection pathway of an adeno-associated virus. Science 2001;
294: 1929–1932.
33 Suomalainen M, Nakano MY, Boucke K, Keller S, Greber UF.
Adenovirus-activated PKA and p38/MAPK pathways boost
microtubule-mediated nuclear targeting of virus. Embo J 2001;
20: 1310–1319.
34 Kelkar SA, Pfister KK, Crystal RG, Leopold PL. Cytoplasmic
dynein mediates adenovirus binding to microtubules. J Virol
2004; 78: 10122–10132.
35 Greber UF, Fornerod M. Nuclear import in viral infections. Curr
Top Microbiol Immunol 2005; 285: 109–138.
Gene Therapy