(Received for publication, January 29, 1997)
From the Human Retrovirus Pathogenesis Group, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, Maryland 21702-1201
We identified a region in the human Ran GTPase-binding protein RanBP1 that shares similarities to the nuclear export signal of the inhibitor of the cAMP-dependent protein kinase. Mutational analysis confirmed that this region is responsible for the cytoplasmic accumulation of RanBP1 and can functionally replace the nuclear export signal of Rev of human immunodeficiency virus type 1. We showed that RanBP1 interferes with Rev-mediated expression of human immunodeficiency virus type 1, whereas the RanBP1 with inactivated nuclear export signal abrogates Rev function. Expression of a Rev-independent molecular clone, which is regulated via the constitutive transport element (CTE) of the simian retrovirus type 1, is not affected. These findings indicate that Rev and RanBP1 compete for the same nuclear export pathway, whereas Rev- and the CTE-mediated pathways are distinct. The inhibition of Rev function is independent of the ability of RanBP1 to associate with Ran and therefore, it is not likely a result of interference with Ran function. These data suggest that RanBP1 interacts with Rev at the putative nuclear receptor and, hence, shares a step in posttranscriptional pathway with Rev.
One of the best studied examples of regulation at the
posttranscriptional level is that of the Rev-responsive element
(RRE)1-containing mRNAs of HIV-1 (for
reviews, see Refs. 1-5). The Rev protein binds to the RRE and promotes
the export of the RRE-containing mRNA to the cytoplasm and their
expression. Rev contains an RNA-binding domain and a nuclear export
signal (NES). Mutations in the NES are transdominant (TD) and lead to
inhibition of Rev's nucleocytoplasmic export resulting in
inhibition of HIV expression. Nucleoporin-like proteins hRip/Rab have
been shown to interact with the NES of Rev and are believed to be
involved in the nuclear export of Rev (6, 7). Similar NES elements have
been identified in the inhibitor of cAMP-dependent protein
kinase (PKI) (8) and the 5 S RNA-binding protein TFIIIA (9) and have
been shown to functionally replace the NES of HIV-1 Rev. We identified
a related NES region in the Ran GTPase-binding protein 1 of human
(hRanBP1) (10, 11) and mouse RanBP1/HTF9-A (mRanBP1) (12) origin. They
belong to a family of proteins that bind to the GTP-bound Ran that
includes known and putative nucleoporins as well as proteins of unknown function (13-19). Ran GTPase is required for active transport of macromolecules through the nuclear pore (for recent reviews, see Refs.
20 and 21). Here, we show that the NES signals of RanBP1 and Rev
functionally cross-interfere, indicating a competition for a common
export pathway. NES()RanBP1 mutants completely abrogate Rev-mediated
regulation of HIV-1 without affecting the regulation mediated by the
posttranscriptional control element (CTE) of simian retrovirus type 1. Taken together, our data suggest that RanBP1 and Rev share a nuclear
export pathway, which is distinct from the CTE-mediated pathway.
HIV-1 molecular clone
pNL4-3 (22), the rev clone pNL4-3fB (23, 24),
the Rev-independent pNL43 Rev(
)R(
).S containing the CTE of SRV-1
(25), the rev expression plasmid pBsrev (26), the TD Rev expression
vectors pRevBL (27) and pRevM10BL (28) have been described. To generate
hybrid Rev-NES proteins, the PstI-BstEII fragment
of pBsrev was replaced by synthetic DNA encoding the heterologous NES
as shown on Fig. 1, substituting amino acids 72-88 of Rev. The RanBP1
gene was cloned by polymerase chain reaction (PCR) using cDNA from
human Jurkat cells. Mutations in the NES and RBD domain of RanBP1 were
introduced by PCR. The RanBP1 expression plasmids were constructed by
insertion of the PCR fragments into the
BssHII-BamHI sites of pB37R (29). The
GFP-containing plasmids have an insertion of the mutant sg25GFP
gene2 into the BssHII site
generating the N-terminal GFP-RanBP1 fusion proteins consisting of GFP
followed by a linker (Ala-Pro-Ala) and RanBP1. All recombinant genes
were sequenced on both strands. HLtat is a HeLa-derived cell line that
constitutively produces Tat protein (31). 293 is a human embryonic
kidney cell line. Cells were transfected by the calcium phosphate
coprecipitation technique, and total protein was extracted as described
(25). pRSVluc (32) or pL3luc (33) luciferase expression plasmids were
included in the transfection mixtures, and the luciferase activity was
measured as described (33). GFP fluorescence was measured in cell
lysates using CytoFluor fluorimeter (28).
Western Blots and Immunoprecipitations
Western blots were performed with ECL kit (Amersham Corp.), using rabbit anti-GFP sera2 and anti-Ran monoclonal antibody (Transduction Laboratories). For native immunoprecipitation, HLtat cells transfected with GFP-RanBP1 expression plasmids were lysed in 10 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 2.5 mg/ml heparin by two freeze-thaw cycles, treated with 10 units/ml DNase I for 15 min at room temperature, cleared by centrifugation at 13,000 rpm for 10 min at 4 °C and gel-filtrated on Sephadex G50 against 0.2% Tween 20 in PBS (PBS-T). Rabbit anti-GFP or normal sera were captured to protein A-Sepharose in PBS for 1 h at 4 °C; the beads were then blocked in 10% milk, 1% casein, 1% gelatin, and 0.2% Tween 20 in PBS for 1 h at 4 °C. The beads were incubated with the cell extracts for 3-4 h at 4 °C, washed with PBS-T containing 1 mM EDTA, eluted with 1 M NaCl in PBS-T at room temperature, and analyzed on Western blots.
By data base searches for similarity to the nuclear export signals
of different PKI proteins, we identified a region in the human and
mouse Ran GTPase-binding protein 1 (RanBP1) lying outside the RBD.
Comparison of these regions with other NES, including that of Rev of
HIV-1 and the consensus NES of Rev, revealed conservation of a
functionally important pattern of hydrophobic residues (Fig. 1). To study the function of the identified NES-like
sequence, we generated hybrid proteins that have the Rev export signal
replaced with the NES-like elements of the human and mouse RanBP1 and, as control, with the previously described NES of PKI. We then tested
whether these proteins can activate gag expression from a
rev molecular clone of HIV-1 (Fig.
2). In the absence of Rev or in the presence of a
transdominant Rev (RevBL) Gag expression was barely detectable, whereas
in the presence of Rev high level of Gag was produced. Comparison to
wild type Rev shows that the Rev hybrid proteins containing the NES of
human or mouse RanBP1 had about 10% activity, whereas Rev containing
the PKI export signal showed 20% activity. Indirect immunofluorescence
analysis further revealed that the Rev-RanBP1 hybrid protein localized
in the nucleoli and translocated to the cytoplasm upon actinomycin D
treatment like Rev (not shown). Hence, the identified element from
RanBP1 can efficiently replace the activation/nuclear export signal of Rev, and the resulting hybrid protein has the characteristic
properties of Rev.
We next studied the role of the NES for the localization of the human
RanBP1. We generated a RanBP1 fusion with GFP and also introduced
mutations within the NES region (L183A, L186A, and V188A; see Fig. 1),
generating NES()RanBP1. The GFP-tagged wild type and mutant proteins
were studied upon transient transfection in living cells (Fig.
3). In HLtat cells, a HeLa-derived cell line, the wild
type GFP-hRanBP1 localizes to the cytoplasm and is excluded from the
nucleus. Confocal microscopy and immunogold electron microscopy
confirmed that the wild type protein is not detectable in the nucleus
(not shown). In contrast, the NES(
) mutant protein was found mostly
in the nucleus as well as in the cytoplasm. Thus, the NES(
) mutation
inactivates the nuclear export signal. Similarly, in human 293 cells,
the wild type GFP-hRanBP1 was found in the cytoplasm. Although the
localization of the NES(
)RanBP1 was shifted toward the nucleus, we
also detected many cells where the protein is predominantly
cytoplasmic. The difference in the effect of NES mutation in these two
cell types is unclear. After completion of our study, Richards et
al. (34) published similar data showing that cytoplasmic
localization of RanBP1 is determined by a NES. In their study, L186A or
V188A mutations resulted in nuclear accumulation of RanBP1 in hamster
BHK21 cells (34). Taken together, these data suggest that RanBP1 is a
nucleocytoplasmic shuttle protein.
To explore whether the localization of RanBP1 depends on its
interaction with Ran, we introduced mutations (K93A, R95A, G96A, and
G98A) in the RBD of the GFP-tagged RanBP1 and NES()RanBP1. These
changes affect amino acids that are well conserved between different
Ran-GTP-binding proteins (14). Coimmunoprecipitation assays under
nondenaturing conditions (Fig. 4) were used to confirm the inability of the RBD mutants to associate with Ran. Western immunoblots verified similar levels of Ran (Fig. 4A) and
GFP-RanBP hybrid proteins (Fig. 4B) in the different
lysates. While GFP-RanBP1 has an expected apparent molecular mass of
about 50 kDa, mutation of NES affected the electrophoretic mobility of
the protein. We also detected faster migrating proteins produced from
all expression vectors, that may represent N-terminal truncations
within GFP moiety (Fig. 4B). The same extracts were
immunoprecipitated with either preimmune serum (Fig. 4C) or
anti-GFP antiserum (Fig. 4D) and analyzed on Western
immunoblots using the anti-Ran antibody, which confirmed that only
wild type RanBP1 and the NES(
) mutant protein associate with Ran,
whereas the mutation of RBD abolished this interaction. Subcellular
localization studies (Fig. 3) in HLtat and in 293 cells showed that
RBD(
)RanBP1 localizes primarily to the cytoplasm, like the wild type
protein, demonstrating that this localization is not dependent on the
interaction with Ran. The RBD(
) mutation also did not affect the
nuclear accumulation of the NES(
)RanBP1 in HLtat cells. In 293 cell
line, the RBD(
) mutation led to a predominant nuclear accumulation of
NES(
)RanBP1 in all cells. These data indicate that the RBD and NES
determinants of RanBP1 may have different strength in different cell
types. Taken together, these experiments suggest that the ability to bind Ran does not contribute significantly to the shuttling properties of RanBP1.
The similarity of the nuclear export signals of RanBP1 and Rev suggests
that these proteins rely on a common nuclear export pathway. To test
this possibility, we studied the effect of Rev on the subcellular
localization of GFP-hRanBP1 in living cells (Fig. 5). We
found hRanBP1 to shift from the cytoplasmic to the nuclear localization
in the presence of Rev (Fig. 5, A-C). As a result,
fluorescence was found over the entire cell (Fig. 5A, a
typical cell is indicated by arrow) or was predominantly
nuclear (Fig. 5C). In contrast, the presence of TD Rev
protein M10BL had no effect on RanBP1 localization (Fig.
5D). Immunoblot analyses performed on extracts prepared from
the same cells showed comparable levels of Rev and RevM10BL proteins
(not shown), which excludes the possibility that the relative amount of
Rev or RevM10BL is responsible for this finding. In parallel
experiments, we showed that the localization of the NES()RanBP1 was
not affected by Rev or RevM10BL (Fig. 5, E and
F). Taken together, these data demonstrate that Rev
interferes with the nuclear export of hRanBP1 via its nuclear export
signal. Therefore, these findings suggest that Rev and RanBP1 compete
for a common NES-specific export pathway.
Given the finding that Rev interferes with the nuclear export of
RanBP1, we explored whether RanBP1 and its mutants affect Rev function
as measured by inhibition of Gag production of HIV-1. We cotransfected
the rev molecular clone of HIV-1 in the presence of Rev
expression vector and increasing amounts the wild type and
NES(
)RanBP1 expression plasmids (Fig. 6). The presence
of both wild type RanBP1 and NES(
)RanBP1 led to greatly reduced
levels of Gag production. While the presence of excess RanBP1 lowered
HIV expression about 10-fold (Fig. 6A), the presence of
NES(
)RanBP1 resulted in a dose-dependent complete inhibition (Fig. 6B) of Gag expression. Similar data were
obtained using the intact HIV-1 molecular clone NL4-3 (data not shown). The specificity of this inhibition was controlled for by cotransfection of a luciferase expression vector, driven by the HIV-1 or Rous sarcoma
virus long terminal repeat promoter, which showed no inhibition in the
presence of either wild type or mutant RanBP1 (Figs. 6, A
and B, dotted lines). To control for the levels of wild type and NES(
)RanBP1 expression, we measured GFP fluorescence in the lysates (Fig. 6E) and demonstrated that similar levels of
these proteins were expressed. Therefore, the distinct effects observed for the wild type and the NES(
)RanBP1 reflect intrinsic properties of
the two proteins. In summary, our results indicate that RanBP1 interferes with the posttranscriptional regulation by Rev, and vice
versa, Rev, via its own nuclear export signal, interferes with the
nuclear export of RanBP1. Therefore, the two proteins are likely to
compete for NES-specific components of the same export pathway. The
strong inhibitory effect of NES(
) RanBP1 on Rev function has been an
unexpected result, since such mutants are not predicted to associate
with and, hence, to compete for the putative NES receptors. This led us
to propose that NES(
)RanBP1 directly targets the NES-specific pathway
through interaction with the endogenous RanBP1, directly or through
interaction with another common factor. The presence of NES(
)RanBP1
in such a complex may interfere with its transport, resulting in the
observed inhibition of Rev activity. A similar mechanism has been
described for the inhibition of Rev function by TD Rev (28, 35). Our model would predict that the inhibitory activity of wild type and
NES(
)RanBP1 will specifically affect this NES-specific pathway, but
not other regulated nuclear export. Therefore, we tested whether the
presence of RanBP1 or its NES-mutant affect expression of the
Rev-independent HIV-1 molecular clone. This clone is Rev(
) and
RRE(
) and contains the CTE posttranscriptional control element of
SRV-1 replacing the Rev/RRE system (25, 29). As shown in Figs. 6C and
6D, the expression of the Rev-independent HIV-1 was not affected by
RanBP1 or the NES(-)RanBP1. These data further suggest that RanBP1 and
Rev share a specific nuclear export pathway, which is distinct from the
CTE-mediated export pathway.
We asked whether the inhibitory effect of RanBP1 depends on the ability
to associate with Ran and tested the effect of the RBD()RanBP1
proteins on the expression of the HIV-1 (Fig. 7). Saturating amounts of plasmids expressing RBD(
)RanBP1 or
RBD(
)NES(
)RanBP1 as well as RanBP1, NES(
)RanBP1, or GFP alone
were cotransfected with Rev-regulated
(Rev-dependent; Fig. 7, bottom) or
CTE-regulated (Rev-independent; Fig. 7, top)
molecular clones, and the Gag production was measured with antigen
capture assay. Measurements of GFP fluorescence in the lysates showed
comparable levels of expression for different mutant proteins (not
shown). RBD mutation had no significant effect on the inhibitory
activity of wild type and NES(
)RanBP1, suggesting that the
interaction of RanBP1 with Ran does not play a role in its specific
effect on Rev function. The NES(
)RBD(
)RanBP1 inhibited HIV
expression reproducibly slightly less than NES(
)RanBP1. However, this
difference (about 6-fold) is small as compared with 400-fold inhibition
obtained by NES(
)RanBP1 and may be attributed to an overall
structural effect of the RBD(
) mutation rather than the loss of the
interaction with Ran. Similar data were obtained from dose-dependent inhibition studies (data not shown). In
summary, these data indicate that the inhibition of Rev function by
RanBP1 is directly mediated through its nuclear export signal and is independent of RanBP1's interaction with Ran.
Ran GTPase has been implicated in nuclear export, although the
molecular mechanisms of such activity are not known. It is not clear
how the ability of RanBP1 to shuttle is related to its proposed
function as a participant of nuclear localization signal-mediated nuclear import. It should be noted that removal of the region containing NES did not affect the ability of RanBP1 to enhance the
binding of Ran-GTP to -karyopherin in vitro (36) and
therefore NES is dispensable for this reported function. However, the
presence of NES may suggest a role of RanBP1 in the cytoplasmic
delivery of GTP-bound form of Ran. In addition, the ability of RanBP1
to interact with both Ran-GTP and the NES-specific components of a
nuclear export pathway makes it a candidate physical link between Ran
and nuclear export. The likely role of RanBP1 in this model is to
tether active, nuclear form of Ran to the putative export intermediates
that are involved in trafficking of other NES-containing substrates,
such as Rev of HIV-1.
We thank M. Dasso, G. N. Pavlakis, M. Neumann, E. I. Afonina, R. Stauber, and G. A. Gaitanaris for sharing materials and for discussions; V. I. Romanov for immunocytochemistry and E. Hudson for confocal microscopy; and J. Bear and G. Gragerova for technical assistance.