Nuclear localization of Vpr is crucial for the efficient replication of HIV-1 in primary CD4+ T...

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Virology 327 (20

Nuclear localization of Vpr is crucial for the efficient replication

of HIV-1 in primary CD4+ T cells

Sayuki Iijimaa,b, Yuko Nitahara-Kasaharaa, Kiyonori Kimataa, Wen Zhong Zhuanga,

Masakazu Kamataa, Maya Isogaic, Masanao Miwab, Yasuko Tsunetsugu-Yokotac, Yoko Aidaa,*

aRetrovirus Research Unit, RIKEN, Wako, Saitama 351-0198, JapanbInstitute of Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

cDepartment of Immunology, National Institute of Infectious Disease, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

Received 22 March 2004; accepted 17 June 2004

Available online 8 August 2004

Abstract

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr appears to make a substantial contribution to the replication of

HIV-1 in established T cell lines when HIV-1 is present at very low multiplicities of infection. However, the role of Vpr in viral replication

in primary CD4+ T cells remains to be clarified. In this study, we generated a panel of viruses that encoded mutant forms of Vpr that lacked

either the ability to accumulate in the nucleus and induce G2 arrest or the ability to induce apoptosis, which has been shown to occur

independently of G2 arrest of the cell cycle. We demonstrate here that the nuclear localization of Vpr and consequent G2 arrest but not the

induction of apoptosis by Vpr are important for viral replication in primary CD4+ T cells at both high and low multiplicities of infection.

Viruses that encoded mutant forms of Vpr that failed to be imported into the nucleus in the presence of cytoplasmic extracts from primary

CD4+ T cells in an in vitro nuclear import assay replicated at drastically reduced rates. Thus, Vpr might be a key regulator of the viral

nuclear import process during infection in primary CD4+ T cells. By contrast, a mutant form of Vpr that exhibited diffuse cytosolic staining

exclusively in an immunofluorescence assay of HeLa cells and was not imported into nucleus by the cytosol from HeLa cells was

effectively imported into the nucleus by cytosol from primary CD4+ T cells. This Vpr mutant virus replicated well in primary CD4+ T cells,

indicating that cellular factors in primary CD4+ T cells are indispensable for the accumulation of Vpr in the nucleus and, thus, for viral

replication. Our results suggest that the nuclear import of Vpr might be a good target in efforts to block the early stages of replication of

HIV-1.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Vpr; Nuclear localization; HIV-1; Primary CD4+ T cell; G2 arrest; Apoptosis

Introduction

The accessory gene vpr of human immunodeficiency

virus type 1 (HIV-1) encodes a 15-kDa nuclear protein of 96

amino acids that is strongly conserved in primate lentivi-

ruses, all of which include a Vpr-like protein (Emerman,

0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2004.06.024

* Corresponding author. Mailing address: Retrovirus Research Unit,

RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Fax: +81 48 462

4399.

E-mail address: [email protected] (Y. Aida).

1996; Planelles et al., 1996). Thus, it is likely that Vpr plays

an important role in the life cycle of such viruses. Vpr has

many biological functions, being involved in incorporation

of virions (Cohen et al., 1990a; Connor et al., 1995;

Mahalingam et al., 1995a; Schuler et al., 1999; Tung et al.,

1997); nuclear localization (Gallay et al., 1996; Heinzinger

et al., 1994; Jenkins et al., 1998; Kamata and Aida, 2000;

Lu et al., 1993; Popov et al., 1998a; Vodicka et al., 1998);

induction of cell cycle arrest at the G2/M phase (He et al.,

1995; Jowett et al., 1995; Re et al., 1995) or at the G1 phase

(Nishizawa et al., 1999, 2000a, 2000b); weak activation of

04) 249–261

S. Iijima et al. / Virology 327 (2004) 249–261250

several viral promoters, including those of HIV-1 (Cohen et

al., 1990b); induction of the terminal differentiation of

certain types of cells (Le Rouzic et al., 2002); and

association with a variety of several cellular factors

(Agostini et al., 1996; Bouhamdan et al., 1996; Mahalingam

et al., 1998; Refaeli et al., 1995; Sawaya et al., 1998; Wang

et al., 1995; Withers-Ward et al., 1997; Zhao et al., 1994).

The various biological activities of Vpr appear to be

correlated with specific structural features of the protein

(Di Marzio et al., 1995; Mahalingam et al., 1995b, 1995c,

1995d, 1997; Piller et al., 1996; Wang et al., 1996; Yao et

al., 1995; Zhao et al., 1996). Analysis of the three-

dimensional structure of Vpr by nuclear magnetic resonance

(NMR) revealed three well-defined a-helices, namely,

amino acids 17–33, 38–50, and 56–77, which are sur-

rounded by flexible amino- and carboxy-terminal domains

(Morellet et al., 2003). Mutational analysis has suggested

the importance of the first of the three a-helical domains in

nuclear localization and in the expression, stability, and

incorporation into virions of Vpr (Mahalingam et al., 1995a,

1995c; Yao et al., 1995). The second domain also appears to

be critical for the incorporation of Vpr into the virions and

for the oligomerization feature of Vpr (Singh et al., 2000),

while the third domain contains a determinant that is

involved in nuclear localization and, in particular, in the

translocation to the nucleus of the preintegration complex

(PIC) in nondividing cells (Kamata and Aida, 2000; Nie et

al., 1998). The carboxy-terminal 20-aa arginine-rich tail,

which contains a cryptic nuclear localization signal (NLS),

makes a major contribution to the nuclear localization of

Vpr (Lu et al., 1993; Mahalingam et al., 1997; Yao et al.,

1995; Zhao et al., 1996, 1998). Truncation of amino acid

substitutions in this region results in failure to induce cell

cycle arrest (Di Marzio et al., 1995; Mahalingam et al.,

1997; Zhao et al., 1998). Thus, it appears that the carboxy-

terminal region of Vpr is also required for the induction of

cell cycle arrest.

It appears that Vpr is not absolutely required for viral

replication because deletion of Vpr is not lethal to viral

replication in vitro. However, it was recently reported that

Vpr is present in significant amounts in the serum of HIV-1-

infected patients and that it activates viral expression in

latently infected cell lines and resting peripheral blood

mononuclear cells (PBMC) (Levy et al., 1993, 1994). These

observations suggest that Vpr is very much involved in the

life cycle of HIV-1 and controls both the replication and

pathogenesis of this virus. Moreover, the fact that many

copies of Vpr are packaged in progeny virions suggests that

the protein might play a role early in the infectious process

(Cohen et al., 1990a). Experimental support for an early

functional role for Vpr comes from observations of a

contribution by Vpr to the nuclear import of proviral DNA

in nondividing cells, such as macrophages (Heinzinger et

al., 1994). In addition, Vpr synthesized de novo plays an

additional role in the replication of HIV-1 because Vpr that

is supplied in trans does not fully complement the

replication of Vpr-negative viruses in infected macrophages

(Connor et al., 1995). It is possible that Vpr might

upregulate the expression of viral genes.

By contrast to its well-documented role in nondividing

cells, the role of Vpr in the replication of HIV-1 in dividing

cells remains to be fully characterized. Vpr is not essential

for the replication of HIV-1 in vitro in proliferating T cells

and stimulated PBMC (Balliet et al., 1994; Cohen et al.,

1990a, 1990b; Connor et al., 1995; Hattori et al., 1990;

Ogawa et al., 1989; Rogel et al., 1995). However, Vpr

contributes substantially to the replication of HIV-1 in these

cells because viral replication is increased in dividing cells

when the multiplicity of infection is very low (Balliet et al.,

1994; Connor et al., 1995; Ogawa et al., 1989). Previous

evidence indicates that expression of the viral genome is

maximal during the G2 phase of the cell cycle and that Vpr

increases the production of virions by progression of the cell

cycle beyond the point when the LTR is most active (Goh et

al., 1998). These observations suggest a novel mechanism

for maximizing the production of virions in the face of the

rapid killing of infected target cells. Thus, the G2 arrest

induced by Vpr might enhance the production of HIV-1.

However, the role of Vpr in the replication of HIV-1 both in

dividing cells and in nondividing cells remains to be fully

clarified.

In this study, we attempted to confirm the roles of

various phenomena induced by Vpr in the replication of

HIV-1 in primary CD4+ T cells. CD4+ monocytes exist in

PBMC fraction (3–10%, depending on the donor), which

differentiate into macrophages during cultivation. Further-

more, macrophages can be infected with an X4-type HIV,

although the replication of this T-cell line-adapted HIV is

much less efficiently in macrophages compared to that in

activated T cells. Therefore, to characterize the biological

function of Vpr in T cells, we consider that it is crucial to

purify CD4+ T cells from PBMC. First, we developed a

panel of expression vectors that encoded Vpr proteins with

site-directed mutations in each of the putative domains,

namely, in the three a-helical domains and in the arginine-

rich carboxy-terminal domain, we produced mutants of Vpr

that lacked either the ability to localize to the nucleus and to

induce G2 arrest or the ability to induce apoptosis, which

occurs independently of G2 arrest (Nishizawa et al., 1999,

2000a, 2000b). Furthermore, to explore the roles, if any, of

Vpr-mediated G2 arrest, nuclear localization, and apoptosis

in viral replication, we monitored the replication of viruses

that encoded the various mutant forms of Vpr during

infection of primary CD4+ T cells by HIV-1. Our results

indicate that the nuclear localization of Vpr, as well as G2

arrest, is important for viral replication in primary CD4+ T

cells. We also found that Vpr mutant viruses with impaired

nuclear import of Vpr in vitro in the presence of

cytoplasmic extracts of primary CD4+ T cells exhibited

drastically reduced levels of viral replication, while mutants

that retained importability replicated well in primary CD4+

T cells.

S. Iijima et al. / Virology 327 (2004) 249–261 251

Results

Construction of plasmids and expression of substitution

mutants of Vpr

The Vpr protein does not include a canonical nuclear

localization signal (NLS). However, it appears to interact

with PIC and to play a critical role in nuclear transport of

the PIC (Bukrinsky and Haffar, 1998). Moreover, Vpr

interrupts the cell cycle at G2 by inhibiting the activation of

Cdc2 kinase, which is required for entry into the M phase

of the cell cycle (He et al., 1995; Jowett et al., 1995; Re et

al., 1995). Furthermore, a recent report indicates that Vpr

enhances virus production by delaying cells at the G2 phase

of the cell cycle, when the LTR is most active (Goh et al.,

1998). By contrast, the induction of apoptosis by Vpr

occurs independently of G2 arrest (Nishizawa et al., 1999,

2000a, 2000b). Interestingly, the various biological activ-

ities of Vpr are correlated with specific structural features

or domains of the protein as noted in the introduction and

shown in Fig. 1A.

Therefore, to identify the domains of Vpr that are

involved in nuclear localization, G2 arrest, and the apoptosis

that occurs independently of G2 arrest, we generated a panel

of expression vectors derived from pME18Neo that encoded

Vpr and variants with site-directed mutagenesis in each

putative domain of Vpr as follows. (i) We replaced His and

Ile at positions 45 and 46 within the second a-helical

domain by Trp and Ala, respectively (HI4546WA); (ii) we

replaced the bulky nonpolar Leu at position 67 within the

third a-helical domain by smaller Ala to introduce a

Fig. 1. Construction and expression of mutant forms of Vpr. (A) Plasmids

that included cDNAs for the mutant forms of Vpr were generated by site-

directed mutagenesis. The diagram shows schematic representations of the

variations of proteins and putative domains. (B) Western blotting of wild-

type and Vpr mutant proteins; cells were co-transfected with pME18Neo

that encoded Flag-tagged wild-type Vpr, L67A, R8788EA, L67P,

aLAL67P, HI4546WA, or the control plasmid pME18Neo-Flag, together

with pSV-h-galactosidase. Transfected cells were harvested and lysed 24 h

after transfection. Lysates with equal h-galactosidase activity were

subjected to Western blotting analysis with the Flag-specific MAb M2.

significant change of hydrophobicity (L67A); (iii) we

replaced Arg at positions 87 and 88 within the arginine-

rich domain by Glu and Ala (R8788EA); (iv) we replaced

Leu at position 67 within the third a-helical domain by Pro

(L67P) to disrupt helical structure (L67P); and (v) we

replaced four bulky nonpolar Leu at positions 20, 22, 23,

and 26 within the first a-helical domain and the Leu at

position 67 within the third a-helical domain by Ala and Pro

(aLAL67P) (Fig. 1A). We transfected HeLa cells with these

expression vectors, which included, for monitoring the

expression of wild-type and mutant proteins, cDNA for an

amino terminal Flag tag that encoded the sequence NH2-

Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (Fig. 1A). West-

ern blotting analysis with MAb M2, which recognizes the

Flag-tag, indicated that each mutant form of Vpr was

expressed at detectable levels in the corresponding trans-

fected cells 24 h after transfection (Fig. 1B). Interestingly,

the aLAL67P mutant exhibited migration slightly slower

than that of wild-type Vpr. This difference in electrophoretic

migration may have resulted from the altered conformation

of mutant Vpr proteins relative to the wild-type polypeptide

or changes in the hydrophobic face (aLA) of the first helical

domain (Mahalingam et al., 1997).

Analysis of G2 arrest, apoptosis, and nuclear localization of

substitution mutants of Vpr

To determine the effects of substitutions within the

putative domains of Vpr on G2 arrest, we transfected HeLa

cells with pME18Neo that encoded wild-type Vpr, L67A,

R8788EA, HI4546WA, or the control pME18Neo-Flag.

Forty-eight hours after transfection, we stained cells with the

Flag-specific MAb M2 and then with Alexa 488-conjugated

goat antibodies against mouse IgG for detection of cells that

expressed Vpr. Then we stained cells with PI for analysis of

DNA content (Fig. 2). Flow cytometric analysis revealed

that, in the case of cells transfected with the expression

vector that encoded wild-type Vpr, there was a dramatic

increase in the proportion of cells in the G2 phase of the cell

cycle (approximately 21.5% and 68.3% were at the G1 and

the G2/M phase, respectively, and the G2/M:G1 ratio was

2.31) compared with cells transfected with the control

vector pME18Neo-Flag (approximately 68.5% and 8.4%

were at the G1 and G2 phase, respectively, and the G2/M:G1

ratio was 0.13). Similarly, approximately 69.9% and 65.8%

of HeLa cells that expressed the mutant proteins L67A and

HI4546WA, respectively, were arrested at the G2 phase. By

contrast, cells that expressed the mutant protein R8788EA

had a reduced ability to induce G2 arrest (approximately

46.2% of cells were in G2 phase) as compared to the wild-

type Vpr.

The Vpr protein can inhibit cell proliferation by arresting

the cell cycle at the G2 phase and then it can induce

apoptosis after G2 arrest (Stewart et al., 1997). On the other

hand, we previously found that expression of endogenous

Vpr appears also to cause apoptosis independently of its role

Fig. 2. The DNA content of HeLa cells that expressed Flag-tagged wild-

type Vpr and mutant forms of Vpr. HeLa cells were transfected with

pME18Neo that encoded Flag-tagged wild-type Vpr, HI4546WA, L67A, or

R8788EA, or the control plasmid pME18Neo-Flag. Then 48 h after

transfection, cells were harvested for analysis of DNA content and stained

with Flag-specific MAb M2 and propidium iodide (PI). Cells that were anti-

Flag positive were analyzed by flow cytometry. Arrowheads indicate peaks

of cells at the G1 and G2/M phase. The G2/M:G1 ratio is indicated in the

upper right of each graph.

Fig. 3. Analysis of apoptosis of HeLa cells that expressed the mutant forms

of Vpr. HeLa cells were transfected with pME18Neo that encoded Flag-

tagged wild-type Vpr, HI4546WA, L67A, or R8788EA, or the control

plasmid pME18Neo-Flag. Then 24 h after transfection, cells were harvested

for analysis of apoptosis and stained with Flag-specific MAb M2 and PE-

conjugated antibodies against caspase-3. Cells that were stained with the

Flag-specific MAb were analyzed by flow cytometry. Actinomycin D

(Act.D), which is a potent inducer of apoptosis, was used as a positive

control. The percentage of caspase-3-positive cells is indicated. Each

column represents results from samples in two independent experiments.


in G2 arrest. Therefore, we examined the effects of

substitutions within the putative domains of Vpr on this

type of apoptosis. Within 24 h after transfection, we stained

cells with the Flag-specific Mab M2 and Alexa488-

conjugated goat antibodies against mouse IgG in combina-

tion with PE-conjugated rabbit antibodies against active

caspase-3, which plays a crucial role in the induction of

apoptosis. Then we analyzed the cells by flow cytometry. As

shown in Fig. 3, wild-type Vpr induced about 30–40% of

the apoptosis observed after treatment of cells with actino-

mycin D, a potent inducer of apoptosis. The R8788EA

mutant, which had exhibited a markedly reduced ability to

induce G2 arrest, retained the ability to induce apoptosis and

was actually twice as effective as wild-type Vpr. Similarly,

the HI4546WA mutant exhibited apparent caspase-3 activ-

ity. By contrast, in case of HeLa cells that had been

transiently transfected with the L67A expression vector,

only a small population of cells underwent apoptosis,

despite the strong ability of the encoded mutant Vpr to

induce G2 arrest. Thus, apoptosis did not depend solely on

the extent of G2 arrest induced by Vpr.

Next, we examined the subcellular localization of

substitution mutants of Vpr. Within 48 h after transfection,

we fixed HeLa cells in formaldehyde, labeled them with the

Flag-specific MAb M2, and analyzed them in an immuno-

fluorescence assay (Fig. 4). Vpr was localized predomi-

nantly in the nucleus and the nuclear envelope, with lesser

amounts in the cytoplasm of transfected cells. The

R8788EA mutant form of Vpr was localized similarly to

wild-type Vpr. The L67A mutant had lost the specific

capacity of wild-type Vpr for perinuclear localization and

was localized rather homogeneously in the nuclei. By

contrast, in HeLa cells that had been transiently transfected

with the vector that expressed the HI4546WA mutant, we

observed exclusively diffuse cytosolic staining, which was

distinct from the aforementioned cytosolic staining wild-

type Vpr, and there was almost no nuclear staining. In

addition, no signal was observed in transfected cells that

expressed wild-type Vpr and had been stained with pre-

immune serum.

Our results are summarized in Table 1. The HI4546WA

mutant with substitutions within the second a-helical

domain retained the capacity for inducing G2 arrest and

apoptosis but lost the ability to accumulate in the nucleus,

suggesting that the second a-helical domain might influence

the localization of Vpr. The R8788EA mutant, which

corresponded to Vpr with a disrupted carboxy-terminal

domain, was still capable of accumulating in the nucleus

and of inducing apoptosis, but its ability to arrest the cell

cycle at G2 phase was impaired. It appears likely that

specific basic residues in the carboxy-terminal domain

might not be essential for nuclear localization and apoptosis.

The L67A mutant, in which the Leu residue in the third a-

helical domain of Vpr had been was changed to Ala,

retained the ability to induce G2 arrest and accumulate in the

nucleus, but its ability to induce apoptosis was reduced.

Vpr-mediated G2 arrest and nuclear localization, but not

apoptosis, are correlated with viral replication in primary

CD4+ T cells

To explore what roles, if any, are played by Vpr-mediated

G2 arrest, nuclear localization of Vpr, and Vpr-induced

apoptosis in viral replication, we examined a panel of

Fig. 4. Subcellular localization of mutant forms of Vpr. HeLa cells were transfected with pME18Neo that encoded Flag-tagged wild-type Vpr, HI4546WA,

L67A, or R8788EA, or with the control plasmid pME18Neo-Flag. Then 24 h after transfection, cells were subjected to immunofluorescence staining with Flag-

specific MAb M2 and Alexa488-conjugated goat antibodies against mouse IgG and analyzed by confocal laser scanning microscopy. Scale bar, 20 Am.


viruses that encoded Vpr and its mutant, derivatives such as

L67A, R8788EA, and HI4546WA. We compared the

replication of a virus coding for a wild-type Vpr and of

the a Vpr-negative ATG-mutant (DVpr) to that of viruses

that encoded substitution mutants of Vpr using activated

primary CD4+ T cells from three donors. At 3- to 4-day

intervals, we collected culture supernatants and evaluated

the kinetics of virus production as by quantitation of the p24

antigen by an ELISA. Typical kinetics of replication of the

various viruses in activated primary CD4+ T cells from one

donor (TM) are shown in Fig. 5. The virus that encoded

wild-type Vpr was apparently infectious at low (0.5 ng of

p24 antigen) and high (5 ng of p24 antigen) viral inputs, and

replication reached a maximum 21 days after infection and

then decreased. Some delay in the production of viruses was

observed in cultures after infection of cells with viruses that

encoded Vpr+ and DVpr. In the case of viruses that encoded

Table 1

Characterization of Vpr and mutants

Mutant or virus G2 arrest Apoptosis Localization

Vpr +++ +++ WTb

HI4546WA +++ ++ Cytoplasm

L67A +++ Fc Most at the nuclear envelope

R8788EA F +++ WT

DVprc NTd NT NT

a The viruses containing an amount of 0.5 ng (low) and 5 ng (high) of p24 antigb Subcellular pattern of Vpr expression detected by immunofluorescent staining:

nuclear envelope and a certain amount was present in the cytoplasm.c Effect on activity compared to that of wild-type Vpr, which was taken as 100%; +d NT, not tested.

HI4546WA and R8788EA, respectively, a form of Vpr that

is unable to localize in the nucleus and a form that has a

reduced ability to induce G2 arrest, the kinetics of HIV-1

replication were similar to those of the DVpr virus. The

growth of these mutated viruses was severely impaired at a

low viral input as compared to that at a high viral input. By

contrast, the L67A mutant, which retained its ability to

induce G2 arrest and to localize in the nucleus but had lost

its ability to induce apoptosis, showed an increased rate of

viral replication. This result was reproducible and was

observed also in primary CD4+ T cells from the two other

donors.

Our results suggest that the ability of the Vpr protein to

localize in the nucleus and its ability to induce G2 arrest, but

not its ability to induce apoptosis, are important for viral

replication in primary CD4+ T cells at both of high and low

viral inputs (Table 1).

Infectivitya

High Low

After 14 days After 21 days After 14 days After 21 days

+++ +++ +++ +++

+ ++ + +

+++ +++ +++ +++

+ ++ + +

+ +++ + +

en were used.

bwtQ indicated wild-type pattern with intense expression in the nucleus and

++++, N200%; +++, 100–80%; ++, 79–50%; +, 49–10%;F, 9–1%;�, b1%.

Fig. 5. Kinetics of p24 antigen production after infection with wild-type and

mutant forms of Vpr. Activated CD4+ T cells were infected with wild-type

virus or with viruses that encoded mutant forms of Vpr. Results are shown

for infections that corresponded to 5 ng (A) and 0.5 ng (B) p24 antigen.

Cells were maintained for 4 weeks, and virus production was monitored in

terms of levels of p24 in the culture supernatant, as determined by an

ELISA every at 3- to 4-day intervals.


Mutant viruses in which Vpr has lost its ability of nuclear

import are unable to proliferate in primary CD4+ T cells

In previous studies, Vpr appeared to contribute substan-

tially to the replication of HIV-1 in proliferating T cells.

Viral replication was enhanced in dividing cells when Vpr

was present under certain conditions, such as when the

multiplicity of infection was very low (Cohen et al., 1990b,

Yao et al., 1998). A previous report by Goh et al. (1998)

indicated that Vpr increases virus production by delaying

cells at the G2 phase of the cell cycle, when the LTR is most

active. In the present study, we found that the ability of Vpr

to accumulate in the nucleus, as well as its ability to induce

G2 arrest, might contribute to the efficient replication of

HIV-1 in primary CD4+ T cells, as shown in Table 1 and

Fig. 5. However, the extent to which Vpr contributes to the

nuclear import of proviral DNA during viral infection in

primary CD4+ T cells remains to be clarified.

To examine whether the nuclear import of Vpr is

promoted by a cytoplasmic extract from activated primary

CD4+ T cells, we performed an assay in vitro in which we

monitored the nuclear import of Vpr in digitonin-permea-

bilized HeLa cells (Fig. 6). As reported previously, the

region between residues 17 and 74 (N17C74), which is the

actual NLS of Vpr, appears to be indispensable for the

nuclear translocation of wild-type Vpr (Kamata and Aida,

2000). Indeed, using a chimeric protein that consisted of the

region between residues 17 and 74, designated N17C74,

fused at its amino-terminal end to GST and at its carboxy-

terminal end to GFP, we were able to confirm the subcellular

localization of the chimeric protein in a nuclear import assay

in vitro (Kamata et al., unpublished data). As shown in Figs.

6A and B, nuclear import of N17C74 was rescued upon

addition to the in vitro system of a cytoplasmic extract of

HeLa cells and of primary CD4+ T cells.

Next, in addition to the HI4546WA mutant, which had

lost the ability to localize in the nucleus in HeLa cells, as

described above, we selected two mutant forms of Vpr,

namely, L67P and aLAL67P that localize in the cytoplasm

of HeLa cells that had been transfected with expression

vectors that encoded these mutants as shown Figs. 1B and

6C. Moreover, these two mutants retained weak ability to

induce G2 arrest and apoptosis (data not shown). First, we

constructed plasmids for the expression L67PN17C74,

aLAL67PN17C74, and HI4546WAN17C74 as chimeric pro-

teins in which Vpr sequences were fused to GFP and GST,

and then equal amounts of recombinant protein used in a

nuclear import assay in vitro (Figs. 6A and B). We also

compared the replication of viruses that encoded for

HI4546WA, L67P, and aLAL67P in stimulated primary

CD4+ T cells from three donors (Fig. 6E). Interestingly,

nuclear import of HI4546WAN17C74 was rescued by

cytoplasmic extracts of primary CD4+ T cells but not of

HeLa cells, and the virus that encoded HI4546WA

replicated efficiently in activated primary CD4+ T cells.

These results indicate that a cellular factor, present in

stimulated primary CD4+ T cells but not in HeLa cells, can

support the nuclear entry of Vpr and, also, viral replication.

Indeed, in Fig. 6D, immunofluorescence staining revealed

the HI4546WA mutant yielded intense signals in the nuclear

in human lymphoid Jurkat. By contrast, the L67PN17C74 and

aLAL67PN17C74 proteins were unable to enter nucleus even

in the presence of cytoplasmic extracts of HeLa cells or of

primary CD4+ T cells. Moreover, the viruses encoding these

mutant proteins were unable to proliferate in primary CD4+

T cells (Fig. 6E).

As summarized in Table 2, our results indicate that the

viral phenotype with respect to the nuclear import of Vpr

closely correlates with infection by HIV-1 in primary CD4+

T cells.

Discussion

The result of the present study of mutant forms of Vpr

leads to four major conclusions. First, the results suggest the

importance of Vpr in the replication of HIV-1 in primary

CD4+ T cells at both low and high viral inputs. At low viral

input, in particular, the growth of virus that encoded the

Vpr-negative ATG mutant (DVpr) was considerably

impaired as compared to that of the wild-type virus. This

result supports previous observations that viral replication is

enhanced in proliferating T cells and primary PBMC when

Vpr is present at very low viral input (Balliet et al., 1994;

Connor et al., 1995; Ogawa et al., 1989). At high viral input

(5 ng), we found that the replication of DVpr virus was

significantly less effective than that of the wild-type virus in

primary CD4+ T cells during infection for 14 days. Second,

our results show that the phenotype with respect to nuclear

import of Vpr is closely correlated with HIV-1 infection in

primary CD4+ T cells. Moreover, viruses that encoded

mutant forms of Vpr that were unable to enter the nucleus

upon addition of a cytoplasmic extract of activated primary

CD4+ T cells were also unable to proliferate in stimulated

Fig. 6. Nuclear import and kinetics of viral replication. (A) Coomassie blue-stained gel of GST- and GFP-tagged N17C74 and substitution mutants of Vpr. (B)

In vitro nuclear import assay of the mutant forms of Vpr. Scale bar, 20 Am. (C and D) Subcellular localization of mutant forms of Vpr in CD4+ T cells and HeLa

cells (C) and Jurkat cells (D). Cells were transfected with pME18Neo that encoded Flag-tagged wild-type Vpr, L67P, aLAL67P, HI4546WA. Then 24 h after

transfection, cells were subjected to immunofluorescence staining with Flag-specific MAb M2 and Alexa488-conjugated goat antibodies against mouse IgG

and analyzed by confocal laser scanning microscopy. Scale bar, 20 Am. (E) Kinetics of viral replication. Activated CD4+ T cells were infected with viruses that

encoded wild-type or mutant Vpr equivalent to 0.5 ng of p24 antigen. The production of viruses was monitored in terms of levels of p24 in the culture

supernatant determined by ELISA at 3- or 4-day intervals for 3 weeks.


primary CD4+ T cells. Thus, the nuclear import of Vpr

might be a good target in attempts to block early events in

the replication of HIV-1. Third, our data revealed that the

ability of Vpr to induce G2 arrest might be important for

viral replication in primary CD4+ T cells because replication

of the virus that encoded R8788EA, which had reduced

ability to induce G2 arrest, was clearly impaired. This

observation strongly supports the results of Cohen et al.

(1990b), Goh et al. (1998), and Yao et al. (1998), who

reported that Vpr appeared to make a substantial contribu-

tion to the replication of HIV-1 in proliferating T cells. They

found that viral replication was enhanced in dividing cells

when Vpr was present under certain conditions, such as

when the multiplicity of infection was very low. In

particular, Goh et al. (1998) indicated that the G2 arrest

induced by Vpr contributed to the enhanced replication of

HIV-1 in CD4+ T cells, suggesting that this phenomenon

might explain why Vpr is so strongly conserved in primate

lentiviruses. Fourth, our results demonstrate that any cellular

factors in stimulated primary CD4+ T cells are indispensable

for nuclear entry of Vpr into the nucleus and for viral

replication. Although the Vpr mutant HI4546WA exhibited

diffuse cytosolic staining exclusively in an immunofluor-

escence assay of HeLa cells, this mutant protein yielded

intense signals in the nucleus in Jurkat cells, its nuclear

import was effectively restored by cytoplasmic extract of

primary CD4+ T cells, and a virus that encoded this mutant

Vpr was able to infect primary CD4+ T cells. This

Table 2

Correlation between nuclear import of wild-type or mutant Vpr in vitro and infectivity

Virus Localization Nuclear import with extract from Infectivitya

HeLa cells Jurkat cells HeLa cells CD4+ T cells

Vpr Nucleus Nucleus ++b + +++

HI4546WA Cytoplasm Nucleus F ++ ++

L67Pc Nucleus b Cytoplasm NTd � � FaLAL67Pc Cytoplasm NT � � F

a The viruses containing an amount of 0.5 ng (low) of p24 antigen were used.b Extent of activity; �, negative; F, weakly positive; +, positive; ++, strongly positive.c The L67P and aLAL67P mutants exhibited week abilities to induce G2 arrest and apoptosis.d NT, not tested.


observation suggests that host proteins might play key roles

in the life cycle of HIV-1.

In the viral replication assay in primary CD4+ T cells,

the viruses that encoded the mutant forms of Vpr that had

lost the ability of nuclear import, namely, aLAL67P and

L67P exhibited significantly reduced rate of replication.

The extent of such reductions was greater than observed

for the DVpr virus. The structure of Vpr is characterized

by three a-helices, which are folded around a hydrophobic

core that consists of Leu, Ile, Val, and aromatic residues,

and this structure might be related to the interactions of

Vpr with different targets (Morellet et al., 2003). Several

cellular proteins have been reported to associate with Vpr,

such as a 41-kDa cytosolic protein that forms a complex

with the glucocorticoid receptor protein (Refaeli et al.,

1995; Zhao et al., 1994), an unidentified 180-kDa protein

(Refaeli et al., 1995), Sp1 (Wang et al., 1995), TFIIB

(Agostini et al., 1996), uracil DNA glycosylase (Bouham-

dan et al., 1996), HHR23A (Withers-Ward et al., 1997), p53

(Sawaya et al., 1998), and a human 34-kDa homolog of

mov34 (Mahalingam et al., 1998). It was also suggested

recently that Vpr binds importin-a, which is known as a

classical NLS (Agostini et al., 2000; Popov et al., 1998a,

1998b), in addition to phenylalanine-glycine repeat (FG-

repeat) nucleoporins, which are components of the nuclear

pore complex (Fouchier et al., 1998; Le Rouzic et al., 2002;

Popov et al., 1998a; Vodicka et al., 1998). A critical step in

the nuclear transport process is the recognition of the NLS

by the importin a/h complex followed by the docking of the

NLS-containing importin a/h complex to nucleoporins that

are residents of nuclear pores. These results suggest that,

because mutations at positions 20, 22, 23, and 26 within the

first a-helical domain or at position 67 within the third a-

helical domain, aLAL67P and L67P might lose the ability

to interact with the cellular nuclear transport machinery.

This interference might be exerted through steric hindrance

of the Vpr domain involved in interaction with the cellular

factor nuclear transport machinery. By contrast, Zhao et al.

(1994) and Zhang et al. (2001) identified a cellular protein

of 180 kDa (VprBP) that interacted with Vpr specifically in

co-immunoprecipitation assays and they obtained experi-

mental evidence that recombinant VprBP blocks the nuclear

import of Vpr. Thus, a human cytoplasmic protein VprBP

might help retain Vpr in the cytoplasm and might induce

changes in cellular functions as well as in the life cycle of

HIV-1. It is possible that aLAL67P and L67P are able to

interact with factors that block the nuclear transport of these

mutants and do not interact with wild-type Vpr because the

viruses that encoded these mutant proteins replicated

significantly less efficiently than DVpr virus. Therefore,

our results suggest that cellular factors carrying key roles in

nuclear import of Vpr might be a good target in efforts to

block the early phase of the HIV-1 life cycle. By contrast,

HI4546WA was found almost exclusively in the cytoplasm

in our immunofluorescence assay of HeLa cells but

imported at a high level in the nucleus upon addition of

cytosol from primary CD4+ T cells, but not from HeLa cells,

and the virus that encoded HI4546WA successfully infected

primary CD4+ T cells. This observation in fact indicates that

the nuclear entry of Vpr may be dependent on cell-type-

specific factors. Further studies are required to define the

cellular factors that interact with Vpr and are involved in the

targeting of the PIC to the nucleus.

Future therapies will likely target viral proteins other

than the reverse transcriptase, protease, and integrase, or

select host proteins that have key roles in the HIV life

cycles. Indeed, small chemicals already exist that block Tat

transactivation (Lind et al., 2002) and Rev-dependent

export of viral transcripts from the nucleus to the cytoplasm

(Chao et al., 2000). Moreover, as a proof of principle,

dominant-negative mutant Tat, Rev, and Gag proteins have

been shown to block viral replication. The present study

suggests that it might be possible to exploit our Vpr mutants

as dominant-negative inhibitors of infection by HIV-1 and

that it might also be possible to target Vpr in the treatment

of AIDS.

Materials and methods

Construction of plasmids and infectious molecular clones

Generation of expression vectors, based on pME18Neo,

which encoded Flag-tagged wild-type Vpr; the substitution


mutants of Vpr, designated L67A, aLAL67P, and L67P; and

the control plasmid pME18Neo-Flag, was described pre-

viously (Kamata and Aida, 2000; Nishino et al., 1997;

Nishizawa et al., 1999, 2000a, 2000b). To generate the

substitution mutants designated R8788EA and HI4546WA,

we introduced site-specific mutations into pSK-Fvpr

(Tajima et al., 1998) by ExSite PCR-based site-directed

mutagenesis using a kit from Stratagene (La Jolla, CA) and

the following primers: 5V-AGGGCAGCAAGAAATG-

GAGCCAG-3Vand 5V-CTGTCGAGTAACGCCTATTC-

TGC-3Vfor R8788EA; and 5V-TATGAAACTTACGGGGA-TACTTGGG-3Vand 5V-CGCCCATTGTCCTAAGTTATGG-3Vfor HI4546WA. Each XhoI–NotI fragment, including

mutated vpr and Flag sequences, in pSK-Fvpr was excised

and subcloned into pME18Neo (Tajima et al., 1998).

To generate the infectious molecular clone HIV-1

pNL432 (Adachi et al., 1986), parent clones that encoded

for the Vpr-negative ATG-mutant (DVpr), and clones that

encoded substitution mutants of Vpr designated L67A,

HI4546WA, L67P, and aLA, we introduced site-specific

mutations into pUC19 included the NdeI–SalI (NdeI site at

5122 and SalI site at 5785) fragment of the infectious

molecular clone HIV-1 NF462 (Kawamura et al., 1994; a

kind gift from A. Adachi, Tokushima University, Japan) by

PCR with the following primers: 5V-GAACAAGCCCCAG-AAGACCAAGG-3V and 5V-ACCTGTCCTCTGTC-

AGTTTCCT-3Vfor DVpr; 5V-AAGCACTGTTTATCCATTT-CAGA-3V and 5V-GTTGCAGAATTCTTATTATGG-3VforL67A; 5V-TATGAAACTTACGGGGATACTTGGG-3V and

5V-CGCCCATTGTCCTAAGTTATGG-3V for HI4546WA;

5V-CAACCCCTGTTTATCCATTTC-3V and 5V-TTGCA-

GAATTCTTATTATGGCTTCC-3V for L67P; and 5V-CCGAGGAAGCCAAGAGTGAAGCTGTTAGA-3V and5V-CCGCCTCGGCTGTCCATTCATTGTATGGCTCC - 3Vfor aLA. Furthermore, to generate the pNL432 parent clone

that encoded for the substitution mutant aLAL67P, we

introduced a site-specific mutation into pUC19 that included

the construct for aLA, described above, by PCR with

primers 5V-CAACCCCTGTTTATCCATTTC-3Vand 5V-TTGCAGAATTCTTATTATGGCTTCC-3V. Each PflMI and

SalI fragment, including mutations, in pUC19 was excised

and subcloned into pSK that included the NdeI–SalI (NdeI

site at 5122 and SalI site at 5785) fragment of pNL432

(Adachi et al., 1986). To generate the pNL432 parent clone

that encoded the substitution mutant R8788EA, we intro-

duced site-specific mutations into the construct for aLA,

described above, with primers 5V-AGGGCAGCAA-

GAAATGGAGCCAG-3Vand 5V-CTGTCGAGTAACGCC-TATTCTGC-3V. The EcoRI–NdeI fragment, including

R8788EA, in pUC19 was excised and subcloned into pSK

which included the EcoRI–XhoI (EcoRI site at 5743 and

XhoI site at 8887) fragment of pNL432 (Adachi et al., 1986).

The EcoRI–XhoI fragment in pSK, in which included

R8788EA, was subcloned into pNL432.

For constructions of GST-N17C74-GFP, GST-

L67PN17C74-GFP, and GST-aLAL67PN17C74-GFP, fragments

encoding each sequence were obtained by PCR with 5V-GCGGATATCCGAATGGACACTAGAG-3Vand 5V-CGCGGATCCCCAATTCTGAAA-3Vusing pSK-Fvpr,

pSK-FL67P, and pSK-FaLAL67P (Kamata and Aida,

2000; Nishizawa et al., 1999) as templates, respectively.

For constructions of GST-HI4546WAN17C74-GFP, the frag-

ment of interest was obtained by PCRwith 5V-TATGAAACT-TACGGGGATACTTGGG-3Vand 5V-CGCCCATTGTCCTA-AGTTATGG-3V using pSK-FHI4546WA as templates. The

fragment was then subcloned into pGEX-6P3 at the BamH1

and Not1 sites.

All constructs described above were verified by nucleo-

tide sequencing with a BigDye Terminator Cycle Sequenc-

ing Kit and a Genetic Analyzer (ABI PRISM 310; PF

Applied Biosystems, Norwalk, Conn.).

Cell culture and transfection

Human cervical HeLa cells, African green monkey

COS-1 cells, and human T-lymphoid cell line Jurkat were

maintained in RPMI 1640 medium supplemented with 2–

10% heat-inactivated fetal calf serum (FCS), penicillin

(100 Ag/ml), and streptomycin (100 Ag/ml). For Western

blotting, immunofluorescence staining, and examinations

of apoptosis and the cell cycle, HeLa cells (1 � 107) were

transfected with 20 Ag of expression vector by electro-

poration in a 0.4-cm diameter cuvette using a gene pulsar

(Bio-Rad, Richmond, CA) operated at 300 V and 975 AF.COS-1 cells were used for generation of viral stocks.

Western blotting

Twenty-four hours after transfection, the expression of

Vpr was examined by Western blotting as described

previously (Nishizawa et al., 2000b).

Immunofluorescence assay

Twenty-four hours after transfection, HeLa cells growing

on coverslips were fixed in 1% formaldehyde-phosphate-

buffered saline (PBS) for 1 h at 4 8C, permeabilized for 10

min in PBS that contained 0.2% Triton X-100, and washed

twice with PBS. The cells were incubated for 1 h at 4 8C with

Flag-specific MAb M2 (Eastman Kodak, Rochester, N.Y.) or

normal mouse immunoglobulin G (IgG). After washing with

PBS, the cells were incubated for 45 min at 4 8C with

Alexa488-conjugated goat against mouse IgG (Molecular

Probes, Eugene, OR). Coverslips were washed with PBS and

mounted on glass slides. Immunofluorescence was visual-

ized with a confocal laser scanning microscope (LSM510;

Carl Zeiss, Jena, Germany).

Analysis of the cell cycle

Forty-eight hours after transfection, cells were harvested,

fixed in 1% formaldehyde and 70% ethanol, and then


incubated for 1 h with the Flag-specific MAb M2 (Eastman

Kodak). After washing with PBS, cells were incubated for 45

min with Alexa488-conjugated goat against mouse IgG

(Molecular Probes). Then cells were washed again with PBS,

incubated in PBS that contained propidium iodide (PI; 50 Ag/ml), RNaseA (50 Ag/ml), and FCS (2%, v/v) for 15 min at 37

8C. The fluorescence of 10000 cells was analyzed on a

FACScan system (Becton-Dickinson, Mountain View, CA)

with Cell Quest software (Becton-Dickinson). Data are

presented after gating to eliminate cells in which Alexa did

not emit strong fluorescence. Relative numbers of cells in the

G2/M phase were calculated with ModFit LT software

(Verity Software House, Topsham, ME).

Analysis of apoptosis

To analyze apoptosis, we used flow cytometry and two-

color immunofluorescence staining caspase-3 activity was

evaluated as follows. Twenty-four hours after transfection

of cells with 20 Ag of Vpr expression and mutant

expression plasmids, HeLa cells were harvested, fixed in

1% formaldehyde and 70% ethanol, and then incubated for

1 h with the anti-Flag-specific MAb M2 (Eastman Kodak).

After washing with PBS, cells were incubated for 45 min at

4 8C with Alexa488-conjugated goat antibodies against

mouse IgG (Molecular Probes). After cells had been

washed again with PBS, they were incubated for 1 h at 4

8C with phycoerythrin (PE)-conjugated rabbit antibodies

against active caspase-3 (BD Biosciences, Franklin Lakes,

NJ). The fluorescence of 10000 cells was analyzed on the

FACScan system (Becton-Dickinson). Data are presented

after gating to eliminate cells in which Alexa did not emit

strong fluorescence. Relative numbers of positive cells

were calculated as percentages.

Generation of virus stocks

We used two methods for the generation of virus

stocks, as follows. We performed transfections using

FuGENE 6 reagent (Roche, Indianapolis, IN) initially

according to the manufacture’s instructions and then, in

later experiments, under optimized conditions. Then the

DNA was diluted 1:10 (w/v) with serum-free Dulbecco’s

modified Eagle medium (GIBCO Laboratories, Grand

Island, N.Y.). And FuGENE 6 reagent (Roche) was

diluted 1:2 with 25 mM HEPES and incubated at room

temperature for 5 min. The mixtures were combined and

incubated together for a minimum of 15 min at room

temperature. The complex DNA was added to a culture of

cells in fresh medium, then COS-1 cells were incubated at

37 8C in an atmosphere of 5% CO2 in air for 4 h. Then

the medium was replaced by fresh medium and culture

was continued for 72 h. We introduced DNA by electro-

poration using COS-1 cells that had been washed once

with PBS and then resuspended at a concentration of 1 �107 cells/ml in 1� K-PBS-buffered media [10� K-PBS

buffer contained 308 mM NaCl, 1.207 mM KCl, 81 mM

Na2HPO4, 146 mM KH2PO42] plus 30 Ag/Al plasmid

DNA. Cells were transfected in a cuvette using the gene

pulsar (Bio-Rad), which was operated at 260 V and 970

AF. Then cells were cultured in fresh medium at 37 8C in

an atmosphere of 5% CO2 in air. Seventy-two hours after

introduction of DNA, culture supernatants were harvested

and filtered (pore size, 0.45 Am).

Titers of virus stocks were measured on the basis of the

amount of p24 antigen in each culture supernatants by an

enzyme-linked immunosorbent assay (ELISA) using a

combination of two antibodies, namely a p24-specific

MAb (Nu24) and peroxidase-labeled MAb10B5 (Tsunet-

sugu-Yokota et al., 1995).

Preparation of CD4+ T cells

We isolated PBMC on a Ficoll (lymphoceparll; IBL,

Hamburg, Germany) gradient from a healthy HIV-1-

seronegative donor. After PBMC had been collected, they

were washed twice, and then monocytes were eliminated

using a magnetic cell-separation system and microbeads

coated with a CD14-specific MAb (MACS system;

Militenyi Biotec, Bergisch Gladback, Germany). After

removal of monocytes, CD4+ T cells were isolated from

CD14� PBMC on a T-cell column, as described pre-

viously (Tsunetsugu-Yokota et al., 2003). Purified CD4+ T

cells (approximately 95–98% purity) were stimulated with

CD3-specific and CD28-specific MAbs in the presence of

IL-2 (20 U/ml; Genzyme, Cambridge, MA) and were

cultured at 37 8C in a humidified atmosphere of 5% CO2

in air.

Kinetics of virus production

CD4+ T cells were exposed to diluted virus stocks that

contained 0.5 or 5 ng of p24 antigen for 2 h at 37 8C.They were then washed three times and the infected cells

(5 � 105/well) were seeded in a 48-well tissue-culture

plate (Corning, Acton, MA). Cells were maintained in

RPMI 1640 that contained 10% FCS and IL2 (20 U/ml;

Genzyme). Culture supernatants were harvested at 3- or 4-

day intervals and viral production was monitored by

sequential quantitation of p24 antigen in cell-free super-

natants with an HIV-1 p24gag ELISA kit (RETRO TEC;

ZeptoMetrix, NY).

Expression and purification of Vpr and its derivatives

cDNAs encoding GST-N17C74-GFP, GST-L67PN17C74-

GFP, and GST-HI4546WA N17C74-GFP were cloned into

pGEX-6P3 (Amersham Biosciences, Uppsala, Sweden) at

the BamH1 and Not1 sites. The proteins were expressed in

Escherichia coli strains NovaBlue (Novagen). The cells

were cultured in 500 ml of Super Broth medium that

contained ampicillin at 37 8C with constant shaking (190


rpm). When absorbance at 550 nm had reached 0.8, 0.1 mM

isopropyl-1-thio-h-d-galactopyranoside was added to the

medium at 16 8C. After overnight culture at 16 8C, cells werecollected by centrifugation, resuspended in sonication buffer

[50 mM Tris–HCl (pH 8.3), 500 mM NaCl, 1 mM DTT, 1

mM EDTA], and lysed by sonication. Each lysate was

centrifuged and GST-tagged proteins in the supernatant were

allowed to absorb to glutathione-Sepharose 4B (Amersham

Biosciences). The resin was washed with sonication buffer

and then proteins were eluted with 16 mM glutathione-

containing sonication buffer. The proteins were dialyzed

against transport buffer [20 mM HEPES-KOH (pH 7.3), 110

mM potassium acetate, 2 mM magnesium acetate, 5 mM

sodium acetate, 2 mM EGTA, 2 mM DTT] and then

concentrated in a Vivaspin (Sartorius AG, Goettingen,

Germany) centrifuge concentrator. The solution was applied

to a HiTrap Q FF column (Amersham Biosciences) and

eluted with gradient buffer [1 M NaCl in transport buffer].

Peak fractions containing individual proteins were pooled

and dialyzed against transport buffer. The purity of each

recombinant protein was checked by SDS-polyacrylamide

gel electrophoresis (SDS-PAGE) and preparations of pro-

teins were stored at �80 8C.

In vitro nuclear import assay

In vitro nuclear import assay was performed based on a

previously described method (Adam et al., 1990). Cyto-

plasmic extracts of HeLa cells and activated CD4+T cells

were prepared by lysis of cells in cold, hypotonic buffer

[10 mM Tris–HCl (pH 7.5), 2 mM MgCl2, 3 mM CaCl2,

0.3 M sucrose, 1 mM dithiothreitol, 1 Ag/ml leupeptin, 1

Ag/ml aprotinin, and 1 mM phenylmethylsulfonylfluoride].

HeLa cells were permeabilized by treatment with 35 Ag/ml

digitonin (Fluka, Buchs SG, Switzerland) in transport

buffer on ice for 5 min. Adjusted to 40 Al reaction solution

with transport buffer were contained 0.7 AM GST- and

GFP-fusion protein plus in the presence of cytosol extract.

Cells were incubated in this reaction mixture at 29 8C for

20 min and reactions were stopped by fixation of cells with

1% formaldehyde in PBS for 30 min on ice. Cells have

been analyzed by confocal laser-scanning microscopy

(Radiance 2100; Bio-Rad, Hemel Hempsted, UK).

Acknowledgments

We thank Dr. A. Adachi (Tokushima University,

Tokushima, Japan) for kindly providing HIV-1 pNL432

and pNF462. This study was supported in part by a grant

for AIDS Research from the Japan Health Sciences

Foundation; by a Health Sciences Research Grant from

the Ministry of Hearth, Labour and Welfare of Japan

(Research on HIV/AIDS 13110201 and 16150301), by a

grant from the Japanese Foundation for AIDS Prevention;

by a grant for AIDS Research from the Ministry and

Education, Science and Culture of Japan; and by a

President’s Special Research Grant from RIKEN.

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Nuclear localization of Vpr is crucial for the efficient replication of HIV-1 in primary CD4+ T...

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