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Journal of Virology, June 2001, p. 5677-5683, Vol. 75, No. 12
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5677-5683.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Adenovirus Type 5 E1B-55K Oncoprotein Actively
Shuttles in Virus-Infected Cells, Whereas Transport of E4orf6 Is
Mediated by a CRM1-Independent Mechanism
Tanja
Dosch,1
Florian
Horn,1
Grit
Schneider,1
Friedrich
Krätzer,1
Thomas
Dobner,2
Joachim
Hauber,1 and
Roland H.
Stauber1,*
Institute for Clinical and Molecular
Virology, University of Erlangen-Nürnberg, D-91054
Erlangen,1 and Institut für
Medizinische Mikrobiologie und Hygiene, Universität Regensburg,
D-93053 Regensburg,2 Germany
Received 16 January 2001/Accepted 20 March 2001
 |
ABSTRACT |
The E1B-55K and E4orf6 proteins of adenovirus type 5 are involved
in viral mRNA export. Here we demonstrate that adenovirus infection
does not inhibit the function of the E1B-55K nuclear export signal and
that E1B-55K also shuttles in infected cells. Even during virus
infection, E1B-55K was exported by the leptomycin B-sensitive CRM1
pathway, whereas E4orf6 transport appeared to be mediated by an
alternative mechanism. Our results strengthen the potential role of
E1B-55K as the "driving force" for adenoviral late mRNA export.
| |
TEXT |
Late in adenovirus infection, the
nuclear export of most cellular mRNAs is inhibited while viral mRNAs
accumulate in the cytoplasm and are efficiently expressed. There is
general agreement that human adenoviruses encode at least three early
gene products, E1B-55K, E4orf6, and E4orf3, which promote viral
replication by directly or indirectly modulating nucleocytoplasmic mRNA
export (for reviews see references 14 and
29). Mutant viruses lacking one or more of these proteins
are impaired in efficient transport of late viral transcripts and hence
in virus replication (3, 4, 14). E1B-55K and E4orf6 were
shown to interact with each other (15, 21, 26, 27), and
this complex appears to play a critical role in late viral mRNA export
(14). However, the detailed mechanism by which these
adenoviral proteins promote viral but appear to inhibit cellular
nucleocytoplasmic mRNA transport is still not well understood.
Previously, it was suggested that the shuttling of the E4orf6-E1B-55K
complex in uninfected cells is mediated predominantly by the E4orf6
protein (6). In contrast, we recently demonstrated that
E1B-55K alone is capable of nucleocytoplasmic shuttling via the CRM1
export pathway and contains a functional leucine-rich nuclear export
signal (NES) (12). However, the issue of whether E1B-55K
also shuttles in adenovirus-infected cells was not investigated. On the
other hand, several groups reported shuttling of E4orf6 using
heterokaryon assays (6, 8, 20, 23), and the postulated E4orf6 NES appeared to be important for virus viability
(34). In contrast, E4orf6 transport in heterokaryon assays
seemed to be independent of the postulated E4orf6 NES (20,
23) and the E4orf6 NES itself was reported to be inactive in a
heterologous system (12).
In order to understand these observations and to broaden knowledge of
the adenoviral nuclear mRNA export pathway, we addressed the
nucleocytoplasmic trafficking of E1B-55K and E4orf6 in virus-infected cells.
The NES export pathway is active in adenovirus-infected cells.
First, we investigated whether adenovirus infection interferes
specifically or nonspecifically with export, mediated by the E1B-55K
leucine-rich NES (12). Therefore, a hybrid protein
composed of glutathione S-transferase (GST) linked to green
fluorescent protein (GFP) and containing the E1B-55K NES (amino acids
83 to 93) (12) was microinjected into the nucleus of HeLa
cells infected with human adenovirus type 5 (H5wt300). The
recombinant GST-E1BNES-GFP export substrate was isolated from bacteria
under nondenaturing conditions, and injection was carried out as
described previously (25). To ensure infection of all
cells, an inoculum of 30 PFU/cell was used as described previously
(12). Following microinjection of GST-E1BNES-GFP (3 mg/ml)
into the nucleus of HeLa cells at different times postinfection (p.i.),
export was monitored directly by fluorescence microscopy using the
appropriate GFP filters as described previously (12).
Export occurred efficiently early (Fig.
1A and B) and later (Fig. 1D, E, G, and
H) during infection. For each time point, about 30 cells were injected,
and the kinetics of export were similar in all cells assayed.
Comparable results were obtained in two independent experiments, and
export was also active in infected Vero cells (data not shown).
Microinjected GST-E4orf6NES-GFP was not exported in virus-infected HeLa
cells, as already shown for semipermissive uninfected Vero cells
(12) (data not shown). To verify infection, the cells were
stained with the monoclonal antibody B6-8, specific for the 72-kDa E2A protein (24), at early time points (Fig. 1C and F).
Staining with an antihexon antibody (Dako Diagnostics Ltd.) indicated
adenovirus late protein expression at 18 h p.i and hence active
late mRNA transport (Fig. 1I). Thus, the results strongly suggest that
adenovirus replication does not interfere with the E1B-55K NES-mediated
export pathway throughout infection.
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FIG. 1.
Adenovirus infection does not affect NES-mediated
export. Recombinant GST-E1BNES-GFP (3 mg/ml) was microinjected into the
nuclei of adenovirus-infected HeLa cells at different times p.i. (A, D,
and G). Export of the substrate was monitored by GFP fluorescence and
occurred with similar kinetics at early (A and B) or late times (D, E,
G, and H) of infection. Infection was verified by immunostaining
against the 72-kDa E2A protein (C and F) or the hexon protein (I).
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Adenovirus infection does not inhibit E1B-55K nucleocytoplasmic
transport.
Although the E1B-55K NES itself was active in
virus-infected cells, it was crucial to examine the nucleocytoplasmic
shuttling of the complete protein in the presence of other viral
proteins. Therefore, the E1B-55K-GFP hybrid (12) was
transiently expressed in H5wt300-infected HeLa cells (30 PFU/cell) using calcium phosphate-DNA precipitates (30),
and the cellular localization of E1B-55K-GFP was controlled at
different times p.i. E1B-55K-GFP localized predominantly to the
cytoplasm of infected cells, as reported for uninfected cells
(12) (Fig. 2A). At late
times p.i., E1B-55K-GFP became partially nuclear (Fig. 2C). This was
most likely caused by the accumulation of the E4orf6 protein, known to
direct E1B-55K to the nucleus in cotransfection experiments (12,
21, 27). Therefore, infection was performed using the
E4orf6-deficient virus mutant H5dl355 (10). In
the absence of E4orf6 E1B-55K-GFP was predominantly cytoplasmic even
at late times p.i. (Fig. 2E), indicative of active E1B-55K shuttling in
virus-infected cells. Productive infection was again verified by
anti-72-kDa-E2A staining (Fig. 2B), and the characteristic pattern of
viral factories indicated efficient adenovirus replication (Fig. 2D and
F). E1B-55K-GFP localization did not significantly change at later
times p.i. (20 and 24 h), although the cytopathic effect of
adenovirus infection resulted in cell rounding (data not shown).
Although E1B-55K has been reported to associate with the E4orf3 protein
in nuclear structures during H5dl355 infection
(15), the amount of expressed E4orf3 protein appeared to
be insufficient to target E1B-55K-GFP efficiently to the nucleus.
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FIG. 2.
E1B-55K-GFP shuttles in adenovirus-infected cells. HeLa
cells were transfected with the E1B-55K-GFP expression plasmid (3 µg
of plasmid DNA) and 10 h later infected with the indicated
adenoviruses (30 PFU/cell). The localization of E1B-55K-GFP was
monitored by fluorescence microscopy, and infection was verified by
immunostaining against the 72-kDa E2A protein (B, D, and F). In
H5wt300-infected cells, E1B-55K-GFP localized
predominantly to the cytoplasm at early times p.i. (A) and became
partially nuclear 16 h p.i. (C). Infection with the
E4orf6-deficient virus H5dl355 resulted in cytoplasmic
E1B-55K-GFP localization throughout infection (E), indicative of
active nuclear export. LMB treatment for 30 min caused nuclear
accumulation of E1B-55K-GFP, demonstrating that nuclear import was not
affected by adenovirus infection (G and H).
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|
To ensure that the cytoplasmic localization of E1B-55K-GFP was not
merely caused by blocking nuclear import, the cells were
treated with
the NES export inhibitor leptomycin B (LMB) (10 nM
final concentration
for 30 min) (
12). If nuclear import was
inhibited by
adenovirus infection the cytoplasmic localization
of E1B-55K-GFP
should not change. However, LMB treatment resulted
in complete nuclear
accumulation of E1B-55K-GFP in all cells,
demonstrating that nuclear
import was not affected by H5
wt300
or H5
dl355
infection (Fig.
2G and H). At least 30 transfected
or infected
cells were studied, and similar results were obtained
in two
independent
experiments.
E1B-55K and E4orf6 shuttle in heterokaryon assays.
To address
whether E1B-55K is capable of shuttling not only when expressed in
trans but also when produced during adenovirus infection, we
performed heterokaryon assays. HeLa cells were infected with
H5wt300 at 30 PFU/cell, washed, and 8 h later seeded
with uninfected mouse NIH 3T3 cells at a ratio of 1:3. Cells were
cultured and fused 8 h later using polyethylene glycol (PEG 1500;
Roche Diagnostica GmbH) as described previously (33).
Briefly, cells were washed with phosphate-buffered saline, and 2 ml of
PEG was added for 2 min. Subsequently, the cells were extensively
washed with phosphate-buffered saline and further incubated at 37°C. Cycloheximide (50 µg/ml) was added 30 min prior to fusion and was
present throughout the experiments to prevent new protein synthesis. At
various time points, the cells were fixed and stained with the
anti-E1B-55K monoclonal antibody 2A6 (28) (Fig.
3). To discriminate between infected
human donor and uninfected mouse acceptor nuclei, staining was
performed with Hoechst 33258 (Molecular Probes) (1 µg/ml) for 15 min.
As already presented in various reports (1, 7, 17),
Hoechst dye produces a more punctate staining of mouse nuclei (Fig. 3)
than of HeLa nuclei. Figure 3A and B illustrate that virus-produced
E1B-55K was exported from the donor and imported into the mouse
acceptor nuclei as early as 30 min after fusion, indicative of active
nucleocytoplasmic shuttling. Staining of heterokaryons with the
E4orf6-specific monoclonal antibody RSA3 (19) also
detected E4orf6 in the acceptor nuclei (Fig. 3E and F), as reported for
uninfected cells (6, 8, 20). In contrast to the efficient
export of E1B-55K (Fig. 3A), the cells had to be incubated for at least
1 h to detect E4orf6 in the acceptor nuclei. As a control,
incubation of the fused cells at 4°C resulted in no detectable
accumulation of E1B-55K or E4orf6 (data not shown). Expression of late
viral proteins was verified by staining with a monoclonal antihexon
antibody, indicative of active late mRNA transport and translation at
16 h p.i. (data not shown).
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FIG. 3.
E1B-55K but not E4orf6 shuttles via the CRM1 export
pathway during virus infection. HeLa cells infected with
H5wt300 were fused with uninfected mouse NIH 3T3 cells
using PEG and fixed at the indicated times postfusion. Alternatively,
cells were incubated with LMB (10 nM final concentration) for 30 min
prior to fusion, and LMB was present after fusion. Staining was
performed with monoclonal antibodies against E1B-55K or E4orf6. E1B-55K
(A and B) (30 min postfusion) as well as E4orf6 (G and H) (1 h
postfusion) were detectable in the acceptor nuclei (arrows). Whereas
E1B-55K export was blocked by LMB treatment (D and E) (1 h postfusion),
E4orf6 was still detectable in the acceptor nuclei (J and K) (1 h
postfusion). Human donor and mouse acceptor nuclei were discriminated
by Hoechst staining (B, E, H, and K). To control for heterokaryon
formation, phase-contrast images of the respective areas are shown (C,
F, I, and L).
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|
Shuttling of E1B-55K is mediated by active CRM1-mediated export,
whereas E4orf6 transport appears to be nonspecific.
To
discriminate between nonspecific transport and NES-mediated export, we
performed heterokaryon assays in the presence of LMB. LMB specifically
binds and inactivates the NES export receptor CRM1 (9,
13). Prior to fusions the cells were incubated with LMB (10 nM
final concentration) and cycloheximide (50 µg/ml) for 30 min, and the
drugs were present after fusion. One hour postfusion, the cells were
fixed and stained for E1B-55K or E4orf6. Interestingly, whereas E1B-55K
export was blocked by LMB treatment (Fig. 3C and D), E4orf6 was still
detectable in the acceptor nuclei (Fig. 3G and H). This experimental
approach allowed us to uncouple the transport of E1B-55K and E4orf6 and
to demonstrate that E1B-55K was actively exported via the CRM1 pathway
even in the course of an adenovirus infection. The smaller
(34-kDa) E4orf6 protein appeared to be transported by a
nonspecific mechanism, as already reported for other small proteins
(2, 5, 33). Ten different syncytia were examined for each
fusion, and similar results were obtained in two independent experiments.
After completion of this work, Rabino and colleagues reported a only
partial inhibition of E4orf6 shuttling by LMB in heterokaryon
assays
(
23). Although we did not observe this inhibition in
our
assay 1 h after fusion, this report supports our observations
on
E4orf6 export. Since LMB binds and inactivates CRM1 irreversibly
(
13), one would expect a complete inhibition of E4orf6
export
if transport is mediated by a CRM1-dependent NES. One could
speculate
that the prolonged LMB-PEG treatment (2.5 h, used by Rabino
et
al.) may have nonspecifically affected the cells in terms of the
cytotoxic side effects of LMB, explaining the partial inhibition.
On
the other hand, we found no rationale why E4orf6 transport
should be
affected by LMB, since the suggested NES seemed to be
inactive
(
12) and an alternative active leucine-rich NES has
not
yet been identified. To further clarify the LMB-sensitive
trafficking
of E4orf6, we expressed E4orf6 as a GFP hybrid, which
allows highly
sensitive detection in living cells (
12). In transient
transfections, E4orf6-GFP localized primarily in the nuclei of
HeLa
cells, although the protein was also detectable in the cytoplasm
(Fig.
4A). Assuming that E4orf6-GFP is
constantly shuttling via
the NES-mediated pathway, LMB treatment should
result in complete
nuclear accumulation by blocking export, as already
demonstrated
for a variety of other shuttle proteins (
11,
12,
16,
31,
32). However, even treatment with LMB (20 nM final
concentration)
for over 2 h did not change the steady-state
localization of E4orf6-GFP,
indicating that the localization of E4orf6
is LMB insensitive
and thus not mediated by a leucine-rich NES (Fig.
4B). To avoid
artifacts caused by overexpression, we studied especially
low-level-expressing
cells. In addition, E4orf6-GFP transport could not
be blocked
by LMB in cell fusion assays. Of note, a E4orf6-GFP hybrid
lacking
the postulated NES displayed a similar intracellular
localization
and transport behavior, and incubation of the transfected
cells
for 6 h at 39°C did not significantly change the
localization
of the E4orf6-GFP or the E1B-55K-GFP hybrid protein (data
not
shown). The mechanism by which relatively small proteins can be
transported between the nucleus and the cytoplasm in general and
its
biological significance for protein function awaits certainly
further
investigation.
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FIG. 4.
E4orf6-GFP does not respond to LMB treatment. HeLa cells
were transfected with the E4orf6-GFP expression plasmid (3 µg of
plasmid DNA) and 16 h later observed by fluorescence microscopy.
In living cells, E4orf6-GFP localized predominantly to the nucleus but
was also detectable in the cytoplasm (A). LMB treatment for 2 h
did not affect the intracellular distribution of E4orf6-GFP (B).
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|
In order to understand the adenoviral mRNA transport machinery it is
first essential to identify and characterize potential
adenoviral
shuttle proteins in the absence of other viral components.
Subsequently, transport has to be further analyzed in combination
with
other viral proteins, especially during viral replication.
Therefore,
this study was undertaken to investigate the shuttling
capability of
the E1B-55K and E4orf6 proteins in the context of
adenovirus infection.
We found that not only the previously identified
E1B-55K NES
(
12) but also the complete E1B-55K protein was actively
exported by the CRM1-dependent pathway in adenovirus-infected
cells.
E4orf6, on the other hand, appeared to be transported via
a nonspecific
mechanism. Clearly, E4orf6 mediates essential functions
during
adenovirus infection, and various mutations in the E4orf6
protein have
been shown to inactivate or alter these biological
activities
(
12,
18,
20,
34). However, so far the observed
effects
could not be directly linked to nucleocytoplasmic transport.
Our study
also underlines the necessity of carefully characterizing
a potential
active shuttle protein. Cell fusion assays certainly
provide important
information but additional experimental approaches
are required. A
shuttle protein exported by the CRM1-dependent
export pathway should
meet the following criteria. First, export
should be inhibited by LMB
treatment. Second, mutations of essential
leucine residues in the NES
should abolish nuclear export. Third,
the NES should be transferable to
a heterologous protein and export
should be assayed in the
absence of import (e.g., by microinjection
of the respective
NES-containing recombinant substrate into the
nucleus). So far, E1B-55K
but not E4orf6 meets all these requirements
even in adenovirus-infected
cells (reference
12 and this report),
suggesting that it is more likely
to be the potential "driving
force" for adenoviral late mRNA
transport.
Recently, Rabino and colleagues (
23) reported that
adenoviral late mRNA transport and protein expression were not
inhibited
by excessive LMB treatment. This is surprising, since we
observed
a general cytotoxic effect of LMB after 5 to 6 h of
treatment,
whereas Rabino et al. found the expression of late viral
proteins
almost unaffected by LMB treatment for 12 h. Since the
activity
of different LMB preparations appears to vary and LMB is also
a cytotoxic drug, we routinely test the LMB preparations prior
to use.
Treatment of cells transiently expressing the human T-cell
leukemia virus type 1 Rex-GFP or the E1B-55K-GFP protein for 30
min resulted in complete nuclear accumulation of the GFP hybrid
proteins by blocking nuclear export as reported elsewhere (
11,
12). Therefore, the influence of LMB treatment on adenovirus
late mRNA export may require reevaluation. On the other hand,
the
observation of Rabino and colleagues (
23) emphasizes the
fact that it is still unclear if and how adenovirus late mRNAs
are
actively export by adenoviral shuttle proteins and what the
corresponding
cis-acting RNA elements are. Certainly, the
mRNA
transport mechanism during adenovirus replication is less well
understood than that in complex retroviruses, in which a single
transport protein interacts specifically with a defined
cis-acting
RNA element (e.g., the Rev-RRE axis for human
immunodeficiency
virus type 1 [22]). Given the multifunctionality of
the E1B-55K
and E4orf6 proteins, there are also other essential steps
in the
adenoviral replication cycle and in the processes of cellular
transformation where shuttling may be essential. There is therefore
an
urgent need for a recombinant type 5 adenovirus expressing
a
NES-deficient E1B-55K and/or E4orf6 protein, to investigate
the impact
of NES-mediated shuttling on mRNA transport and the
adenovirus life
cycle.
| |
ACKNOWLEDGMENTS |
We thank Bernhard Fleckenstein for continuous support. Purified
adenovirus type 5 and LMB were kindly provided by Walter Dörfler and the Novartis Research Institute Vienna, respectively.
This work was supported by the Deutsche Forschungsgemeinschaft,
Johannes und Frieda Marohn-Stiftung, and the Wilhelm-Sander Stiftung.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Clinical and Molecular Virology, University of Erlangen-Nürnberg,
Schlossgarten 4, D-91054 Erlangen, Germany. Phone: (49)-9131-8522102.
Fax: (49)-9131-8522101. E-mail:
rdstaube{at}viro.med.uni-erlangen.de.
| |
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Journal of Virology, June 2001, p. 5677-5683, Vol. 75, No. 12
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5677-5683.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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