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Journal of Virology, August 2000, p. 7179-7186, Vol. 74, No. 15
0022-538X/00/$04.00+0
Two Putative 
-Helical Domains of Human
Immunodeficiency Virus Type 1 Vpr Mediate Nuclear Localization by
at Least Two Mechanisms
Masakazu
Kamata and
Yoko
Aida*
RIKEN Tsukuba Institute, Tsukuba, Ibaraki
305-0074, Japan
Received 24 February 2000/Accepted 8 May 2000
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ABSTRACT |
To identify the domains of Vpr that are involved nuclear
localization, we transfected HeLa cells with a panel of expression vectors that encode mutant Vpr protein with deletions or substitutions within putative domains. Immunofluorescence staining of transfected cells revealed that wild-type Vpr was localized predominantly in the
nucleus and the nuclear envelope and certainly in the cytoplasm. Introduction of substitutions or deletions within 
H1 or H2
resulted, by contrast, in diffuse expression over the entire cell. In
addition, double mutations within both of these -helical domains led
to the complete absence of Vpr from nuclei. Next, we prepared HeLa cells that express chimeric proteins which consist of the H1 and
H2 domains fused individually with green fluorescent protein (GFP)
and a Flag tag and extracted them with digitonin and Triton X-100 prior
to fixation. Flag-H1-GFP was detected in the nucleus but not in the
cytoplasm, while Flag-H2-GFP was retained predominantly in the
nucleus and in a small amount in the cytoplasm. The immunostaining patterns were almost eliminated by substitutions in each chimeric protein. Thus, it appeared that the two -helical domains might be
involved in nuclear import by binding to certain cellular factors. Taken together, our data suggest that the two putative -helical domains mediate the nuclear localization of Vpr by at least two mechanisms.
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TEXT |
The mechanisms of nuclear import of
the preintegration complex (PIC) are necessary for infection by human
immunodeficiency virus (HIV) of nondividing cells, such as quiescent T
lymphocytes, terminally differentiated dendritic macrophages, and
microglial cells (5, 38). At least three HIV type 1 (HIV-1)
proteins, namely, the matrix protein (MA) (4), integrase
(IN) (13), and viral protein R (Vpr) (29, 36),
have been identified as possible mediators of the nuclear import of the
PIC. MA and IN contain classical, simian virus (SV40)-like, nuclear
localization signal (NLS) sequences (5, 14), and their roles
appear to be mediated by importin /
heterodimers (1).
By contrast to MA and IN, Vpr contains two discrete nuclear targeting
signals that use two different import pathways, both of which are
distinct from the NLS- and M9-dependent pathways (17). Vpr
interacts with importin (29, 36), but this interaction
is not blocked by a peptide that corresponds to the NLS of the SV40
large T antigen, a result that suggests that the site at which Vpr
binds to importin is not the NLS cargo site (14, 29).
Likewise, Vpr interacts with several nuclear pore proteins that contain
FXFG repeats, such as Nsp1p, Nup1p, and Pom121 (9, 28, 36).
However, the NLS of Vpr has not been fully characterized and the
nuclear import pathway that involves Vpr has also not been defined.
In addition to its role in nuclear transport, Vpr has a number of other
functions, which include the induction of cell cycle arrest at the
G2/M phase (16, 18, 30) or at the G1
phase (25b, 28) both positive and negative regulation of
apoptosis (2, 7, 12, 33; Nishizawa, M., M. Kamata,
T. Myojin, Y. Nakai, and Y. Aida, submitted for publication), weak
activation of several viral promoters that include those of HIV-1
(6), and induction of the terminal differentiation of
certain types of cell (20). The various biological
activities of Vpr have been correlated with specific structural
features of the protein (8, 21-25, 37, 39, 40).
Computer-assisted secondary-structural analysis of Vpr predicts the
presence of two -helical segments between residues 17 and 34 and
between residues 46 and 74 (22, 24, 34). Mutational analysis
has suggested the importance of the first of these two -helical
domains in nuclear localization and also in the expression, stability,
and incorporation into virions of Vpr (8, 22, 24, 39). A
peptide derived from the first -helical domain has been reported to
mediate nuclear transport of peptide-conjugated bovine serum albumin in
permeabilized cells (19). Moreover, the second of these
domains appears to contain a determinant that is involved in
translocation to the nucleus of the PIC in nondividing cells
(25). By contrast, recent work has indicated that a
20-amino-acid (aa) arginine-rich region in the carboxy-terminal tail of
the protein (residues 77 to 96) might be necessary for its nuclear
localization (17, 39, 41). Thus, the domain(s) of Vpr that
might be important for nuclear localization remains controversial.
To identify in further detail the functional domains of Vpr that are
involved in nuclear localization, we used immunofluorescent staining
and the powerful technique of confocal microscopy, which allows
three-dimensional imaging of the localization of protein in cells. In
another study, we have identified the localization of Vpr during the
24-h period after transfection, namely, during the time when Vpr is
expressed at a detectable level but is minimally effective in inducing
G2 arrest (M. Nishizawa, M. Kamata, T. Myojin, Y. Nakai,
and Y. Aida, submitted). Using a panel of expression vectors that
encode mutated forms of Vpr, we first demonstrated that both
-helical domains, but not the carboxy-terminal arginine-rich tail,
contribute to the subcellular localization of Vpr. We then constructed
expression plasmids that encode chimeric proteins that consist of the
putative -helical domains of Vpr fused to green fluorescent protein
(GFP) and a Flag tag. The results we obtained with these chimeric
proteins indicate that the two -helical domains both mediate nuclear
transport of Vpr and that they operate by at least two mechanisms.
Identification of domains that are involved in the nuclear
localization function of Vpr.
To identify the domain(s) involved
in nuclear localization of Vpr, we used a series of plasmids that
encode variants of Vpr with deletions in different regions of the
protein (26), as shown schematically in Fig.
1A.
In designing these mutants, we targeted five putative structural regions on the basis of the amino
acid sequence (8, 22-24, 37, 39): (i) the amino-terminal domain from aa 1 to aa 16; (ii) the first amphipathic -helical region, extending from aa 17 to aa 34 (H1); (iii) the second amphipathic -helical region, extending from aa 46 to aa 74 (H2); (iv) the leucine zipper-like domain from aa 60 to aa 81; and (v) the
arginine-rich carboxy-terminal domain from aa 77 to aa 96.
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FIG. 1.
Subcellular localization of wild-type and mutant forms
of Vpr with specific deletions. (A) Plasmids that encode mutant forms
of Vpr and were generated by PCR from HIV-1NL43. The
predicted first -helical domain (H1) is located near the amino
terminus, and a second -helical domain (H2) is located near the
carboxyl terminus. A leucine zipper-like domain and an arginine-rich
region are also indicated. The Flag tag is in gray, and the Vpr
portions are in black. (B) Laser sectioning analysis of the
localization of immunostained wild-type Vpr. HeLa cells were
transfected with pME18Neo, which encodes Flag-tagged wild-type Vpr. At
24 h after transfection, cells were subjected to
immunofluorescence staining with Flag-specific MAb M2 and
Cy3-conjugated antibodies against mouse IgG. Serial laser sections
through a representative cell that expressed wild-type Vpr protein were
obtained by confocal laser scanning microscopy at 0.6-µm intervals.
Photomicrographs are numbered from 1, which corresponds to the nuclear
periphery, through 12, which corresponds to the region through which
the cell was adsorbed to the slide. The staining of the cytoplasm
becomes more obvious toward the center of the cell in sections 8 through 10. (C) Photomicrographs of HeLa cells expressing wild-type
and mutant forms of Vpr. HeLa cells were transfected with
pME18Neo, which encodes Flag-tagged wild-type Vpr (a and l); C81 (b);
C74 (c); C59 (d); N17 (e); N35 (f);  17-34 (g); 46-74 (h);
60-81 (i); 17-74 (j); or control vector pME18Neo-Flag (k). At
24 h after transfection, cells were subjected to
immunofluorescence staining with either Flag-specific MAb M2 (a to k)
or normal mouse IgG (l) and Cy3-conjugated antibodies against mouse
IgG. Cells were then analyzed by confocal laser scanning microscopy on
a focal plane near the center of each nucleus. Bar, 20 µm.
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We transfected HeLa cells with expression vectors that encode wild-type
or mutant Vpr with an amino-terminal Flag tag by electroporation
as
described previously (
25a). Twenty-four hours after
transfection,
cells growing on coverslips were fixed for 15 min at
4°C in phosphate-buffered
saline (PBS) that contained 1%
formaldehyde and then permeabilized
for 5 min in PBS that contained
0.2% Triton X-100. The coverslips
were then incubated either with
monoclonal antibody (MAb) M2,
which recognizes the Flag tag (Sigma), or
with normal mouse immunoglobulin
G (IgG) and then with Cy3-conjugated
goat antibodies against mouse
IgG (Jackson ImmunoResearch). Cells were
then mounted on glass
slides in PBS that contained 50% glycerol, 0.1%
p-phenylene diamine,
and 0.02% sodium azide. Cells were
examined with a confocal laser
scanning microscope (LSM 10 or LSM 510;
Carl
Zeiss).
Serial sections through positively immunostained cells showed that
wild-type Vpr was localized predominantly in the nucleus
and nuclear
envelope and a certain amount was present in the cytoplasm
of
transfected cells (Fig.
1B). In addition, a small number of
discrete
dots that represented the products of immunostaining
were present in
the nuclei of most cells. The C74 and C81 proteins
with deletion of 22 and 15 aa at the carboxyl terminus gave patterns
of immunofluorescence
that were not significantly different from
that observed with wild-type
Vpr (Fig.
1C). The N17 mutant, which
lacked 16 aa at the amino terminus
of Vpr, showed a slightly increased
ability to reach the interior of
nuclei (Fig.
1C). Similarly,
the 17-74 mutant protein gave exclusively
strong nuclear staining
that was distinct from the nuclear staining
pattern obtained with
wild-type Vpr, which was more diffuse. These
results indicated
that 16 aa in the amino-terminal region and 22 aa in
the carboxy-terminal
region of Vpr are not essential for the nuclear
localization of
Vpr. By contrast, Vpr with a deletion of the
H1
domain (
17-34)
and the amino-terminally truncated Vpr that lacks the
34 aa that
include the
H1 domain (N35) had completely lost the
specific
capacity of wild-type Vpr for perinuclear localization. The
aberrant
localization was associated with punctate cytoplasmic
staining,
indicating that the first
-helical domain was a major
determinant
of the perinuclear localization of Vpr (Fig.
1C). Vpr with
a deletion
of the
H2 domain or of the leucine zipper-like domain
(
46-74
or
60-81, respectively) and a carboxyl-terminally
truncated Vpr
that lacks 37 aa that include part of the
H2 domain
(C59) were
barely detectable after immunofluorescence staining (Fig.
1C).
These mutant proteins were diffusely distributed throughout cells,
including their nuclei. By contrast, C74, which lacks part of
the
leucine zipper-like domain, was associated with a nuclear
localization
pattern similar to that of wild-type Vpr. Disruption
of the second
-helical domain, but not that of the leucine zipper-like
domain, had
a dramatic effect on subcellular localization and
on the level of
expression or stability of Vpr. The specificity
of the
immunostaining was demonstrated by the absence of signals
in cells
transfected with the control vector (Fig.
1C) and in
mock-transfected
cells (data not shown) and by the absence of
staining in cells
transfected with the plasmid that encodes wild-type
Vpr that were
immunostained with normal mouse IgG (Fig.
1C). Collectively,
our
results indicate that the putative
-helical domains that
extend from
aa 17 to aa 34 and from aa 46 to aa 74 influence the
localization of
Vpr.
Effects on the localization of Vpr of mutations within the two
-helical domains.
To confirm the roles of the two -helical
domains in the nuclear localization of Vpr, we constructed plasmids
that encode Vpr with site-directed mutations (Fig.
2A). To disrupt helical structures, four
bulky nonpolar leucine residues at positions 20, 22, 23, and 26 within
H1 of wild-type Vpr were replaced with smaller alanine residues
(21, 22) by ExSite PCR-based site-directed mutagenesis
(Stratagene). As primer and template DNAs, we used 5'-CCGAGGAAGCCAAGAGTGAAGCTGTTAGA-3',
5'-CCGCCTCGGCTGTCCATTCATTGTATGGCTCC-3', and pSK-Fvpr
(26), which encoded wild-type Vpr and a Flag tag with the
amino acid sequence
NH2-Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. An
XhoI-NotI fragment which includes the
site-mutated vpr gene and the Flag sequence of pSK-FLA
was then excised and subcloned into the expression vector pME18Neo
(35). The resultant plasmid encodes a protein designated
LA. Next, isoleucine at position 60 and leucine at position 67 within H2 of wild-type Vpr were changed to proline residues to yield
I60P and L67P, respectively, as described previously (26).
Both of these residues are located on the hydrophobic side of the helix
with an orientation favorable for dimerization through leucine zipper
interactions (31, 32). In addition, both amino acids are
strongly conserved in various isolates of HIV-1 (26), an
observation that suggests that they play important roles in the
activities of Vpr. Moreover, to generate double mutants with mutations
in H1 and H2 (LA/I60P and LA/L67P), we performed ExSite
PCR-based site-directed mutagenesis using pSK-FI60P or pSK-FL67P
(26) as templates and the primers described above. The
XhoI-NotI fragments were then subcloned into
pME18Neo. Twenty-four hours after transfection with each expression
plasmid, we examined the localization of Vpr by immunofluorescence
staining with Flag-specific MAb M2 and Cy3-conjugated antibodies
against mouse IgG (Fig. 2B).
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FIG. 2.
Subcellular localization of mutant forms of Vpr with
specific substitutions. (A) Replacement of isoleucine and leucine
residues within predicted -helical domains H1 and H2
(represented by shaded boxes) of Vpr. The positions of the substituted
amino acids are numbered, and amino acids are shown in the
single-letter code. Hatched boxes represent the Flag tag. (B)
Transfection of HeLa cells with pME18Neo, which encodes Flag-tagged
wild-type or mutant Vpr, or with control vector pME18Neo-Flag. At
24 h after transfection, cells were subjected to
immunofluorescence staining as described in the legend to FIG. 1 and
analyzed by confocal laser scanning microscopy with the focal plane
near the center of each nucleus. Bar, 20 µm.
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The isoleucine and leucine substitutions in
H2 (I60P and L67P)
resulted in complete loss of the highly specific wild-type
pattern of
perinuclear staining, with diffuse staining over the
entire cell.
Expression of
LA resulted in cytoplasmic staining
that was much more
widespread and diffuse than the perinuclear
foci of wild-type Vpr.
However, single substitutions in
H1 and
H2 still yielded intense
signals in the nucleus, suggesting that
both of the
-helical domains
act together to determine the subcellular
localization of Vpr. To
confirm this possibility, we examined
the intracellular localization of
double-mutant proteins with
mutations in both the
H1 and
H2
domains (
LA/I60P and
LA/L67P).
As expected, we observed
exclusively diffuse cytosolic staining
that was distinct from the
cytosolic staining of wild-type Vpr,
which was much less intense, and
we detected almost no nuclear
staining (Fig.
2B). A similar pattern was
observed when HeLa cells
were transfected with another double mutant,
17-34/L67P, with
the
H1 domain deleted and replacement of the
leucine at position
67 with proline (data not shown). Moreover, when we
examined the
participation of isoleucine at positions 74 and 81 in the
leucine
zipper-like domain in the subcellular localization of Vpr
(
26),
we found almost the same localization pattern as with
wild-type
Vpr (data not shown). These results indicate that both
-helical
domains were necessary for the nuclear distribution of
Vpr.
Both -helical regions of Vpr mediate nuclear uptake of chimeric
proteins that include the Flag tag and GFP.
We examined whether
the -helical regions of Vpr is sufficient for the nuclear
localization of proteins in transfected cells. We constructed plasmids
that express chimeric proteins that consist of the H1 or H2
domain, or of these domains with site-specific mutations, fused
individually at the carboxyl terminus to GFP to monitor the effects of
each -helical domain and fused at the amino terminus to the Flag tag
to facilitate immunofluorescence staining (Fig.
3A).
The expression vectors included ATG
initiation codons for the Flag tag and for GFP, and the chimeric
proteins were detected by indirect immunofluorescence after reaction
with the Flag-specific MAb M2 and Cy3-conjugated antibodies against mouse IgG, to avoid confusion by green fluorescence derived from nonchimeric GFP, which might have been translated from the initiation codon of GFP. First, to generate a plasmid that encodes a Flag-Vpr-GFP fusion protein, the fragment containing vpr and Flag
sequences was amplified by PCR with the primers
5'-TAATCTCGAGATGGACTACAAGGACGAC-3' and
5'-TACCTCGAGATAGGATCTACTGGCTCC-3' with pSK-Fvpr
(26) as the template. The fragment obtained by PCR was
subcloned in the GFP expression vector pEGFP-N1 (Clontech Laboratories)
to yield pGFP-Fvpr. Next, plasmids pGFP-FH1 and pGFP-FH2 and
plasmids with site-specific mutations, such as pGFP-FLA/H1,
pGFP-FI60P/H2, and pGFP-FL67P/H2, were generated by PCR with the
following primers and templates: H1,
5'-GCGGATATCCGAATGGACACTAGAG-3' and
5'-ATCCCCGCGGAAAATGTCTAACAGC-3' with pSK-Fvpr as the
template; LA/H1, 5'-GCGGATATCCGAATGGACAGCCGA-3' and
5'-ATCCCCGCGGAAAATGTCTAACAGC-3' with pSK-FLA as the
template; H2, I60P/H2, and L67P/H2,
5'-GGCGGATATCCATCTATGAAAC-3' and 5'-CGCGGATCCCCAATTCTGAAA-3' with pSK-Fvpr, pSK-FI60P
(26), and pSK-FL67P (26) as the templates,
respectively. The H1 and LA/H1 fragments were prepared by
digestion with EcoRV and SacII, and the H2,
I60P/H2, and L67P/H2 fragments were prepared by digestion with
EcoRV and BamHI. Each fragment was subcloned into
pGFP-Fvpr that had been digested with same respective enzymes to
introduce the Flag tag at the amino terminus of each GFP fusion
protein. We also constructed expression vectors that encode a chimeric Flag-GFP fusion protein as a negative control and a chimeric Flag-SV40 NLS-GFP fusion protein as a positive control. The expression of each
chimeric protein was examined by radioimmunoprecipitation assay with
Flag-specific MAb M2. We observed bands of proteins with apparent
molecular masses consistent with the predicted sequences in our
analysis of HeLa cells that had been transfected with each of our
constructs (Fig. 3C). The molecular masses of Flag, GFP, and Vpr are 1, 27, and 15 kDa, respectively. Thus, the sizes of the various fusion
proteins were substantially below the limit of 40 to 60 kDa for passive
diffusion of proteins through the nuclear pore complex (15).
Therefore, we examined whether the fusion proteins were retained in the
nuclear compartment by binding to cellular factors after their entry
into the nucleus. To eliminate nonanchored chimeric proteins after
transfection of HeLa cells, we treated cells with digitonin at 40 µg/ml in PBS for 3 min on ice and washed them once with cold PBS and
then once with 0.025% Triton X-100 for 5 min on ice before fixation in
1% formaldehyde and permeabilization in 0.2% Triton X-100. The cells
on coverslips were then examined by immunofluorescence staining. To
confirm the validity of this assay, we used expression vectors that
encode chimeric Flag-GFP and Flag-SV40 NLS-GFP fusion proteins (Fig. 3D, a to d). The NLS in the large T antigen of SV40, as previously reported (1), is imported into the nucleus via the importin / pathway. As we had expected, in transiently transfected HeLa cells, the chimeric Flag-GFP fusion protein was distributed throughout the cells in the absence of prior treatment with digitonin and Triton
X-100 (Fig. 3D, a). Treatment with digitonin and Triton X-100
completely eliminated the fluorescence of Flag-GFP (Fig. 3D, b). By
contrast, the chimeric Flag-SV40 NLS-GFP fusion protein was localized
in the nucleus in both untreated and digitonin- and Triton
X-100-treated cells (Fig. 3D, c and d). Thus, it appears that treatment
with digitonin and Triton X-100 prior to fixation did not affect the
signal-mediated nuclear transport process.
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FIG. 3.
Nuclear import by predicted -helical domains
H1 and H2. (A) Construction of plasmids derived from pEGFP-N1
that encode wild-type Vpr, H1, H2, or a substitution mutant form
of Vpr fused at the carboxyl terminus to GFP and at the amino terminus
to the Flag tag. The helical regions of Vpr encoded by each plasmid are
in gray; the positions of the substituted amino acids are numbered, and
amino acids are indicated in the single-letter code. (B) Amino acid
sequences of H1, H2, and the NLS of the large T antigen of SV40.
(C) Analysis by radiolabeling and immunoprecipitation of transfected
HeLa cells that express the indicated fusion proteins. One day after
transfection with the pEGFP-N1 plasmids indicated above the lanes, HeLa
cells were incubated with Redivue Pro-Mix (a mixture of
[35S]methionine and [35S]cysteine [1,000
Ci/mmol]; Amersham Pharmacia Biotech) at a concentration of 200 µCi/ml for 2 h. The cells were lysed at 4°C in lysis buffer
{0.15 M NaCl, 0.05 M Tris-HCl [pH 8.5], 5 mM
3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid
[CHAPS; Sigma]} with a cocktail of protease inhibitors (Boehringer
Mannheim Biochemicals) and centrifuged for 20 min at 17,000 × g and 4°C to remove cell debris. Supernatants were subjected
to immunoprecipitation with Flag-specific MAb M2 and protein
A-Sepharose 4FF (50% [vol/vol]; Amersham Pharmacia Biotech.). The
immunoprecipitates were dissolved in 0.05 M Tris-HCl (pH 6.8)-0.1 M
dithiothreitol-2% sodium dodecyl sulfate-10% glycerol-0.001%
bromphenol blue, heated at 100°C for 5 min, and analyzed by sodium
dodecyl sulfate-5 to 20% polyacrylamide gel electrophoresis with
subsequent fluorography. Arrowheads indicate the positions of chimeric
proteins. (D) Photomicrographs of transfected HeLa cells that express
the indicated fusion constructs. HeLa cells were transfected with
derivatives of pEGFP-N1 that encode Flag-Vpr-GFP (e and f),
Flag-H1-GFP (g and h), Flag-LA/H1-GFP (i and j),
Flag-H2-GFP (k and l), Flag-I60P/H2-GFP (m and n),
Flag-L67P/H2-GFP (o and p), Flag-SV40 NLS-GFP (c and d), or the
control protein Flag-GFP (a and b). At 24 h after transfection,
cells were treated (+) with digitonin at 40 µg/ml and then with
0.025% Triton X-100 (b, d, f, h, j, l, n, and p) or were not treated
( ) with these reagents (a, c, e, g, i, k, m, and o) and then they
were fixed in 1% formalin. The fusion proteins were detected by
indirect immunofluorescence staining with Flag-specific MAb M2 and
Cy3-conjugated antibodies against mouse IgG. Then they were examined by
confocal laser scanning microscopy at a focal plane near the center of
each nucleus. Bar, 20 µm.
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In the absence of initial treatment with digitonin and Triton X-100,
the pattern of immunofluorescence staining of the Flag-Vpr-GFP
fusion
protein was not significantly different from that of wild-type
Vpr, as
shown in Fig.
1B (Fig.
3D, e). The subcellular localization
was almost
completely retained even after the initial treatment
with digitonin and
Triton X-100 (Fig.
3D, f). The chimeric protein
with the first
-helical domain of Vpr, Flag-
H1-GFP, was targeted
to the nucleus,
with weak and diffuse cytoplasmic staining, in
cells that had not been
treated with digitonin and Triton X-100
(Fig.
3D, g). Treatment with
digitonin and Triton X-100 eliminated
much of the diffuse cytoplasmic
staining but not the nuclear staining
of Flag-
H1-GFP (Fig.
3D, h).
Thus, it appeared that the
H1 domain
might be anchored in the
nuclear compartment by binding to cellular
factors. After replacement
of the leucine residues at positions
20, 22, 23, and 26 within the
first
-helical domain with alanine
residues, the capacity for
nuclear localization of
H1 was attenuated
(Fig.
3D, i) and treatment
with digitonin and Triton X-100 almost
eliminated the fluorescence of
Flag-
LA/
H1-GFP (Fig.
3D, j).
Likewise, the nuclear localization
of the chimeric protein with
the
H2 domain, Flag-
H2-GFP, was more
clearly evident than that
of Flag-
H1-GFP in the absence of treatment
with digitonin and
Triton X-100 (Fig.
3D, k). After treatment with
digitonin and
Triton X-100, Flag-
H2-GFP was observed in the
cytoplasm, as well
as in the nucleus, as in the case of Flag-Vpr-GFP
(Fig.
3D, l),
suggesting that the
H2 domain remains in both the
nuclear and
cytosolic compartments as a result of binding to cellular
factors.
In the cases of Flag-I60P/
H2-GFP and Flag-L67P/
H2-GFP,
the capacity
for nuclear localization was almost totally absent (Fig.
3D, m
and o) and the immunostaining patterns were partially resistant
to the initial treatment with digitonin and Triton X-100 (Fig.
3D, n
and p). In addition, detection of Flag-
H1-GFP was more
sensitive to
treatment with Triton X-100 than that of Flag-
H2-GFP:
Flag-
H1-GFP, but not Flag-
H2-GFP, was almost completely
undetectable
in cells that had been treated with digitonin at 40 µg/ml and
0.05% Triton X-100 (data not shown). These observations
suggest
that maintenance of the two
-helical structures is important
for binding to cellular factors and, in addition, that the nuclear
targeting mediated by the
H1 and
H2 domains involves at least
two
mechanisms.
Our results suggest that the karyophilic properties of HIV-1 Vpr can be
attributed to the two putative
-helical domains located
between
residues 17 and 34 and between residues 46 and 74. (i)
Introduction of
mutations into the
H1 and
H2 domains of Vpr
partially impaired
the capacity for nuclear localization. (ii)
Double mutations within the
H1 and
H2 domains resulted in complete
loss of the nuclear and
nuclear-membrane localization of Vpr.
By contrast, the carboxyl
terminus of Vpr, which most closely
resembles a classical NLS, is
greatly involved in its nuclear
localization (
17,
39,
41).
However, contrary results have
also been reported: this region can be
deleted with no impairment
of nuclear localization (
8).
Moreover, a peptide derived from
this region did not function as an
active NLS (
19). Our results
clearly indicate that the
carboxy-terminal region of Vpr is not
absolutely required for nuclear
localization. The C81 mutant,
namely, Vpr with a deletion of 15 aa at
its carboxyl terminus,
gave an immunofluorescence staining pattern
similar to that of
wild-type Vpr. Furthermore, substitutions within the
H1 or
H2
domain of Vpr with an intact carboxy-terminal region
resulted
in diffuse expression throughout cells. We also showed that
the
H1 and
H2 domains independently targeted chimeric proteins
that
included GFP and a Flag tag to the nuclei of HeLa cells, and in
addition, each fusion protein was apparently retained in the nucleus
by
binding to cellular factors after entry into the nucleus. These
results
support previous suggestions that nuclear localization
of Vpr is a
signal-mediated process (
9,
17) and that Vpr
interacts with
importin
and with several nuclear pore proteins
that contain FXFG
repeats, such as Nsp1p, Nup1p, and Pom121 (
9,
28,
36). Thus,
the two
-helical domains might be involved
in nuclear import via
binding to various cellular
factors.
How does Vpr mediate nuclear targeting? It has been reported that Vpr
uses a signal-mediated import pathway that does not
involve NLS or M9
signals (
17,
29). Jenkins and colleagues
(
17)
showed that the nuclear import of Vpr in vitro proceeds
independently
of protein factors provided by a rabbit reticulocyte
lysate and also
independently of exogenously added nucleoside
triphosphates. These
characteristics of Vpr do not coincide with
those of NLS- or M9-binding
proteins (
1,
27), suggesting
that as yet unidentified
cellular factors are involved in the
import process. We did confirm
that maintenance of
-helical structure
is important for retention in
specific cellular compartments via
binding to cellular factors, and we
showed that the
H1 or
H2
domain might mediate nuclear
localization of Vpr by at least two
mechanisms. Further studies of the
nuclear import of Vpr should
yield new insights into the mechanism(s)
by which proteins move
from one side of the nuclear pore complex to the
other. It is
now essential to identify the receptor that is involved in
the
nuclear localization of
Vpr.
For entry of the PIC into the nucleus of a host cell, HIV has evolved
specific mechanisms that are independent of the breakdown
of the
nuclear membrane. These mechanisms involve mainly Vpr and,
to some
extent, MA (
10,
11). Such functional redundancy underscores
the importance of infection by HIV of nondividing cells, such
as
macrophages and dendritic cells, which are presumed to be among
the
first cells infected (
3). Therefore, further study is
required
to define clearly the function of the
H1 or
H2 domain in
the
infection of nondividing cells by HIV. Moreover, suppression of
the
nuclear import of the PIC is likely to have a profound effect
on the
ability of HIV to infect host cells. Thus, the
H1 and
H2 domains
of Vpr might be attractive targets for the development
of novel
anti-HIV
therapies.
| |
ACKNOWLEDGMENTS |
This study was supported in part by a grant from the Japan Health
Science Foundation, by grants for AIDS research from the Ministry and
Education, Science and Culture of Japan, and by a special grant for the
promotion of research from RIKEN.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: RIKEN Tsukuba
Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Phone: 81 298 36 3522. Fax: 81 298 36 9050. E-mail:
aida{at}rtc.riken.go.jp.
| |
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