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Journal of Virology, December 2000, p. 12000-12001, Vol. 74, No. 24
0022-538X/00/$04.00+0
LETTERS TO THE EDITOR
The Nuclear Export Signal within the Adenovirus E4orf6
Protein Contributes to Several Steps in the Viral Life Cycle
 |
LETTER |
The concept of a Rev-like nuclear export pathway influencing
adenovirus late gene expression has recently been challenged by a note
in the Journal of Virology entitled "Adenovirus Late Gene
Expression Does Not Require a Rev-Like Nuclear RNA Export Pathway"
(5). The authors summarize that they "have tested the role
of NES-mediated RNA export during adenovirus infection, and find
that it is not essential for the expression of adenovirus late
genes." For the reasons outlined below, this conclusion seems misleading and not fully justified by the data.
We found that the E4orf6 protein of adenovirus type 5 contains a
nuclear export signal (NES), allowing the protein to shuttle between
the nucleus and cytoplasm (2). More recently, we observed that a functional NES of E4orf6 is required for multiple functions during infection, such as virus production, DNA replication,
accumulation and transport of viral mRNA, and late expression of viral
proteins (6). Nucleo-cytoplasmic shuttling of E4orf6 was now
confirmed by Eileen Bridge and coworkers (5). However, the
authors did not observe a significant reduction in the late expression
of viral proteins when comparing wild-type E4orf6 with a mutant lacking the NES. They conclude that the export function of E4orf6 is not critical for late gene expression. However, the experimental approach leading to this conclusion requires critical evaluation. The authors transiently transfected expression plasmids for E4orf6 and mutants, followed by superinfection with E4-deficient adenovirus. They then
stained the cells with antibodies to E4orf6 and simultaneously with
antibodies against late proteins. The proportion among the E4orf6-positive cells that also expressed late proteins was scored and
compared for various E4orf6 mutants. This score did not significantly differ between wild-type E4orf6 and the NES mutant. The problem with
this approach is that it selects for those cells that express high
levels of E4orf6
at least high enough to allow detection by
immunofluorescence. The experiment does not score those cells that
express the protein at levels that are too low for immunodetection but
high enough to support virus replication. However, such a low level of
expression would be what resembles the physiological situation during
infection. E4orf6 is expressed by the virus at levels that barely allow
immunodetection at late times after infection and essentially preclude
detection at earlier times or at low multiplicities of infection
(references 1 and 4 and our unpublished observations). Therefore, Rabino et al. (5) have almost certainly observed the effects of E4orf6 and mutants at supraphysiological intracellular concentrations. Our unpublished observations show that indeed, strong overexpression of E4orf6 using
newly developed transfection lipids makes the NES function unnecessary
for complementation of virus growth. However, this does by no means
reflect the situation of an infected cell. In the experiments described
in our previous report (6), we expressed E4orf6 using
electroporation, which allows a relatively even, albeit weak expression
of transfected plasmids. Under these conditions, the NES strongly
contributed to virus growth. Instead of counting cells that stain
positive for late proteins, we have documented several hallmarks of the
viral life cycle. Our results consistently show that DNA replication,
virus production, and late protein synthesis (assayed by
immunoprecipitation of hexon to allow overall quantitation) as well as
RNA levels and distribution all depend to a large extent on the NES
within E4orf6 (6).
Second, Rabino et al. (5) report that leptomycin B (LMB)
fails to inhibit the synthesis of late viral proteins when added 12 h postinfection, and they conclude that adenovirus late gene expression does not require a Rev-like nuclear RNA export pathway. Again, this conclusion is not fully justified by the data. First, the
authors do not provide an unequivocal test for LMB function in their
assays. They report an inhibitory effect on cell growth, but this may
be related to the general toxicity of LMB and does not provide a proof
that nuclear export was abolished by LMB at this concentration. Indeed,
the concentration used by the authors (10 nM) was previously shown to
inhibit nuclear export of the Rev protein only partially but not
entirely (3). Furthermore, the authors do observe a
reduction of late protein synthesis when LMB was added shortly after
infection. Thus, the data provided by Rabino et al. (5)
merely suggest that a nuclear export function of E4orf6 may be of
greater importance during the first 12 h after infection than afterward.
Taken together, these considerations and the currently available data
suggest that the NES of E4orf6 significantly contributes to several
steps in the viral life cycle. One of our goals is the construction of
a recombinant adenovirus with a mutated NES in the E4orf6 protein. This
is expected to further clarify the role of this export signal in the
physiological context of an adenovirus infection.
| |
REFERENCES |
| 1.
|
Cutt, J. R.,
T. Shenk, and P. Hearing.
1987.
Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells.
J Virol.
61:543-552[Abstract/Free Full Text].
|
| 2.
|
Dobbelstein, M.,
J. Roth,
W. T. Kimberly,
A. J. Levine, and T. Shenk.
1997.
Nuclear export of the E1B 55-kDa and E4 34-kDa adenoviral oncoproteins mediated by a Rev-like signal sequence.
EMBO J.
16:4276-4284[CrossRef][Medline].
|
| 3.
|
Fornerod, M.,
M. Ohno,
M. Yoshida, and I. W. Mattaj.
1997.
CRM1 is an export receptor for leucine-rich nuclear export signals.
Cell
90:1051-1060[CrossRef][Medline].
|
| 4.
|
König, C.,
J. Roth, and M. Dobbelstein.
1999.
Adenovirus type 5 E4orf3 protein relieves p53 inhibition by E1B-55-kilodalton protein.
J Virol.
73:2253-2262[Abstract/Free Full Text].
|
| 5.
|
Rabino, C.,
A. Aspegren,
K. Corbin-Lickfett, and E. Bridge.
2000.
Adenovirus late gene expression does not require a Rev-like nuclear RNA export pathway.
J Virol.
74:6684-6688[Abstract/Free Full Text].
|
| 6.
|
Weigel, S., and M. Dobbelstein.
2000.
The nuclear export signal within the e4orf6 protein of adenovirus type 5 supports virus replication and cytoplasmic accumulation of viral mRNA.
J Virol.
74:764-772[Abstract/Free Full Text].
|
| | | | |
Matthias Dobbelstein
Institut für Virologie
Universität Marburg
Robert Koch Str. 17
35037 Marburg, Germany
Phone: 49 6421 2864318
Fax: 49 6421 2868962
E-mail: dobbelst{at}mailer.uni-marburg.de
|
| |
AUTHOR'S REPLY |
The precise role of the 34-kDa nuclear export signal (NES) during
adenovirus infection is of great interest. Dobbelstein et al.
(1), proposed a provocative and interesting model for the export of adenovirus late mRNAs in which the 34-kDa NES would direct
viral mRNA to be exported via a Rev-like RNA export pathway. In our
experiments, viral late gene expression did not require either the
34-kDa NES or the Rev-like nuclear export pathway that is mediated by
exportin-1 and inhibited by the drug leptomycin B (2). In
contrast, complementation experiments performed by Weigel and
Dobbelstein suggested that the 34-kDa NES was important for virus
yield, late protein production, late mRNA levels, and importantly, for
the accumulation of viral DNA (3). Since subsequent steps in
the viral life cycle depend on efficient DNA replication, it is very
possible that the role of the 34-kDa NES in this complementation system
stemmed from its ability to promote the efficient onset of DNA
replication. Why then, did we fail to detect a similar defect in DNA
accumulation and late gene expression in our transfection/infection experiments? As we stated in our publication, we think the most likely
explanation for the differences in our results stems from the
multiplicity-dependent phenotype of E4 deletion mutants in viral DNA
replication (see reference 2 for this discussion). We do not dispute Weigel and Dobbelstein's observation that the 34-kDa
NES can play a role in the adenovirus life cycle (3), especially at low multiplicities of infection. While these results raise the interesting possibility that 34-kDa NES-mediated protein shuttling may promote viral DNA replication, neither of our groups has
compelling evidence to support a role for the 34-kDa Rev-like NES in
the export of adenovirus late mRNA.
In the accompanying letter to the editor, Matthias Dobbelstein raises a
number of points to question the validity of our experimental system.
First, he claims that by assaying late protein production in
transfected cells identified by microscopy, we have studied only that
fraction of the transfected cell population expressing levels of the
34-kDa NES that can be detected by immunofluorescence. However, Western
blotting experiments confirmed that equal levels of wild-type and the
NES 34-kDa mutant resulted in similar levels of late proteins in
extracts prepared from transfected/infected cultures and suggest that
the results we obtained by microscopy reflect the level of
complementation present in our cultures (2). Second, he
claims that the 34-kDa protein produced from our transfected expression
constructs was not at physiological concentrations since we used a
commercial lipid-based transfection system rather than electroporation.
We do observe a range in the intensity of 34-kDa protein staining in
cells from our transfection/infection experiments. Interestingly, the
cells that are most intensely stained for the 34-kDa protein are not
usually expressing viral late proteins. In cells which are both 34-kDa
protein positive and late protein positive and have therefore
complemented late gene expression, the level of 34-kDa protein is
similar to that seen after a 24-h infection with wild-type Ad5 (data
not shown). Although we have no reason to believe that a
"supraphysiological" concentration of the 34-kDa NES mutant protein
was required for complementation in our system, we do agree that it
will be critically important to determine the role of the 34-kDa NES
when the mutant gene is expressed from the adenovirus genome in its
proper context during infection. Finally, Dobbelstein raises the
question of whether the leptomycin B treatment we used inhibited
nuclear export. In our experiments, leptomycin B treatment reduced
34-kDa protein shuttling in cell fusion experiments by about threefold,
indicating that it could interfere with the export of a protein
containing a Rev-like NES (2). We also studied the effect of
leptomycin B on the shuttling of 34-kDa proteins produced in
transiently transfected cells fused to untransfected cells. These
experiments were complicated by the fact that we observed some
"shuttling" of transiently expressed 34-kDa proteins in our assay
that was not NES dependent. This may be due to the presence of 34-kDa
protein in the cytoplasm of some transfected cells overexpressing the protein prior to making the cell fusions. Nevertheless, when we compared shuttling of a 34-kDa protein lacking an intact nuclear retention signal (NRS) and a double mutant carrying lesions in both the
NES and NRS, shuttling was only inhibited by leptomycin B (threefold)
when the protein had an intact NES (data not shown). This indicates
that mutations in the 34-kDa NES abolished leptomycin B-sensitive
shuttling and shows that leptomycin B treatment inhibits 34-kDa
NES-mediated export.
| |
REFERENCES |
| 1.
|
Dobbelstein, M.,
J. Roth,
W. T. Kimberly,
A. J. Levine, and T. Shenk.
1997.
Nuclear export of the E1B 55-kDa and E4 34-kDa adenoviral oncoproteins mediated by a Rev-like signal sequence.
EMBO J.
16:4276-4284.
|
| 2.
|
Rabino, C.,
A. Aspegren,
K. Corbin-Lickfett, and E. Bridge.
2000.
Adenovirus late gene expression does not require a Rev-like nuclear RNA export pathway.
J. Virol.
74:6684-6688.
|
| 3.
|
Weigel, S., and M. Dobbelstein.
2000.
The nuclear export signal within the E4orf6 protein of adenovirus type 5 supports virus replication and cytoplasmic accumulation of viral mRNA.
J. Virol.
74:764-772.
|
| | | | |
Eileen Bridge
Department of Microbiology
Miami University
Oxford, Ohio 45056
Phone: 513-529-7264
Fax: 513-529-2431
E-mail: BridgeE{at}muohio.edu
|
Journal of Virology, December 2000, p. 12000-12001, Vol. 74, No. 24
0022-538X/00/$04.00+0
This article has been cited by other articles:
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Orlando, J. S., Ornelles, D. A.
(2002). E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein. J. Virol.
76: 1475-1487
[Abstract]
[Full Text]
