THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 35, Issue of August 30, pp. 32036 –32045, 2002
Printed in U.S.A.
Functional Neutralization of HIV-1 Vif Protein by Intracellular
Immunization Inhibits Reverse Transcription and Viral Replication*
Received for publication, February 26, 2002, and in revised form, May 23, 2002
Published, JBC Papers in Press, May 30, 2002, DOI 10.1074/jbc.M201906200
Joao Goncalves,a,b Frederico Silva,a,c Acilino Freitas-Vieira,a,d Mariana Santa-Marta,a,c
Rui Malhó,e Xiaoyu Yang,f,g,h Dana Gabuzda,f,h,i and Carlos Barbas III j
From the aURIA-Centro de Patogénese Molecular, Faculdade de Farmácia, University of Lisbon, 1649-019 Portugal,
the eDepartment of Plant Biology, Faculdade de Ciencias Lisboa, University of Lisbon, 1600 Portugal, the fDepartment of
Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, Massachusetts 02115, the Departments of
g
Pathology and iNeurology, Harvard Medical School, Boston, Massachusetts 02115, and the jSkaggs Institute for
Chemical Biology and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
Human immunodeficiency virus type 1 (HIV-1)-encoded Vif protein is important for viral replication and
infectivity. Vif is a cytoplasmic protein that acts during
virus assembly by an unknown mechanism, enhancing
viral infectivity. The action of Vif in producer cells is
essential for the completion of proviral DNA synthesis
following virus entry. Therefore, Vif is considered to be
an important alternative therapeutic target for inhibition of viral infectivity at the level of viral assembly and
reverse transcription. To gain insight into this process,
we developed a Vif-specific single-chain antibody and
expressed it intracellularly in the cytoplasm. This intrabody efficiently bound Vif protein and neutralized its
infectivity-enhancing function. Intrabody-expressing
cells were shown to be highly refractory to challenge
with different strains of HIV-1 and HIV-1-infected cells.
Inhibition of Vif by intrabody expression in the donor
cell produced viral particles that do not complete reverse transcription in the recipient cell. The anti-Vif
scFv was shown to be specific for Vif protein because its
function was observed only in nonpermissive cells (H9,
CEM, and U38). Moreover, transduction of peripheral
blood mononuclear cells with an HIV-derived retroviral
vector expressing Vif intrabody was shown to confer
resistance to laboratory-adapted and primary HIV
strains. This study provides biochemical evidence for
the role of Vif in the HIV-1 lifecycle and validates Vif as
a target for the control of HIV-1 infection.
Human immunodeficiency virus type 1 (HIV-1)1 is a complex
* This work was supported by Fundação para a Ciência e Tecnologia
Grant POCTI/33096/MGI/2000, by funds from the Comissão Nacional
de Luta Contra a SIDA, and by National Institutes of Health Grant AI
37470 (to C. F. B.). The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
b
To whom correspondence should be addressed: Faculdade de
Farmácia de Lisboa. URIA-Centro de Patógenese Molecular. Av. Das
Forças Armadas, 1649-019 Lisboa, Portugal. Tel.: 351-21-7946489; Fax:
351-21-7934212; E-mail: joao.goncalves@ff.ul.pt.
c
Supported by a Bolsa de Investigação Científica from Fundação para
a Ciência e Tecnologia.
d
Recipient of a doctoral fellowship from Fundação para a Ciência e
Tecnologia.
h
Supported by National Institutes of Health Grant AI 36186 and an
Elizabeth Glaser Scientist Award from the Pediatric AIDS Foundation.
1
The abbreviations used are: HIV-1, human immunodeficiency virus,
type 1; HIV-2, human immunodeficiency virus, type 2; VL, light chain
variable region; VH, heavy chain variable region; scFv, single-chain
antibody fragment; ELISA, enzyme-linked imunosorbent assay; PBMC,
retrovirus that contains a number of accessory genes not present in other retroviruses. One of the critical determinants of
HIV-1 infectivity in vivo is the Vif protein. Vif (virion infectivity
factor) is essential for the establishment of productive infection
of HIV-1 in peripheral blood lymphocytes and macrophages in
vitro and for pathogenesis in animal models of AIDS (1–7). In
cell culture, vif-defective HIV-1 is able to replicate in some
T-lymphoblastoid cell lines termed permissive (CEM-SS,
SupT1, C8166, and Jurkat), whereas Vif is required in other
cell lines, such as H9, U38, or MT-2, termed nonpermissive (2,
4, 6, 8, 9). Vif acts during late steps of the viral life cycle to
increase the infectivity of HIV-1 virus particles as much as
100 –1000-fold. Vif plays a critical role in the cell-free transmission of HIV-1 and also appears to be important for cell-tocell virus transmission (4 – 6). Previous studies have shown
that Vif acts to enhance viral infectivity during the production
of virus particles, most likely by affecting virus assembly or
maturation (10 –14).
Vif is a phosphorylated 23-kDa protein in multimer form
that is abundantly expressed in the cytoplasm of infected cells
(15–18, 22–25). Vif has been shown to interact or co-purify with
membranes (15, 21), intermediate filaments (22), HIV-1 Gag
(23, 11), and, most recently, viral RNA (16, 19, 26). It was also
proposed that Vif counteracts the tyrosine kinase Hck as an
inhibitor of HIV-1 replication (27). Virus particles produced in
the absence of a functional Vif protein can bind and penetrate
susceptible cells but are defective in their ability to synthesize
proviral DNA, most likely indirectly as a result of an effect on
a component of the virus core (6, 24, 25, 28). Virus uncoating,
reverse transcription, transport of the preintegration complex
to the nucleus, and subsequent integration of the viral DNA
into the host genome are necessary steps that must occur for
infection to be established (30).
Intrabodies are single-chain variable region antibody fragments expressed and confined intracellularly, where they can
bind to viral proteins and other targets (29, 31, 33–37). The
early events of the viral life cycle are potential therapeutic
targets that could be inhibited using anti-HIV-1 Vif intrabodybased strategies and may therefore represent a new therapeutic approach to inhibit HIV-1 reverse transcription. Therefore,
strategies that prevent or limit expression of Vif are expected to
be beneficial in the treatment of HIV-1 disease (29, 39).
peripheral blood mononuclear cells; PHA, phytohemagglutinin; HRP,
horseradish peroxidase; FITC, fluorescein isothiocyanate; HA, hemagglutinin; CAT, chloramphenicol acetyltransferase; PBS, phosphatebuffered saline; MOI, multiplicity of infection; PLAP, placental alkaline
phosphatase.
32036
This paper is available on line at http://www.jbc.org
Neutralization of Vif Protein by Intracellular Immunization
Here we have investigated the potential benefit of intracellular neutralization of Vif activity (36, 37, 40). We developed a
Vif-specific single-chain antibody from immunized rabbits that
was expressed intracellularly and confined to the cytoplasm.
Cell lines and primary cells expressing the Vif intrabody were
shown to be highly refractory to challenge with the HIV-1 virus
or HIV-1-infected cells. Furthermore, replication of an HIV
virus constructed to express anti-Vif scFv in cis was highly
reduced after challenge with wild-type HIV-1. The formation of
completed reverse transcripts was reduced when cells were
infected with HIV-1 virus produced from intrabody-producing
cells. These results suggest that gene therapy approaches,
which deliver Vif intracellular antibodies, may represent a new
therapeutic strategy for inhibiting HIV reverse transcription.
EXPERIMENTAL PROCEDURES
Cells, Viruses, and Reagents
Cells—H9, U38, Jurkat, SupT1, and CEM cells were grown in RPMI
1640 medium containing 10% fetal bovine serum and antibiotics. All of
the cell cultures were maintained at 37 °C in 5% CO2. Human PBMC
from healthy donors were activated for 48 h with phytohemagglutinin
(PHA) and were then cultivated in RPMI 1640 medium supplemented
with 20% fetal calf serum and 50 units of interleukin-2/ml (Roche
Molecular Biochemicals). HeLa and 293T cells were grown in Dulbecco’s
modified minimal essential medium supplemented with 10% fetal calf
serum. Transduced cells were usually maintained in the presence of
puromycin (0.5 ␮g/ml). The tissue culture media and reagents were
from BioWhitaker.
Viruses—Plasmids coding for HIV-1NL4 –3, pHIV-1NL4 –3⌬vif, and
HIV-2ROD were obtained from the AIDS Research and Reference Reagent Program. HIV-1 primary strains Ac178 and strain Je524 isolated
from HIV-1-infected children were obtained from Prof. M. Helena
Lourenço (Faculdade de Farmácia de Lisboa, Lisboa, Portugal).
Plasmids—Plasmid pSVLvif expresses the vif gene of the HXB2
clone of HIV-1 under the control of the SV40 promoter (15). Plasmids for
the trans-complementation assay were described previously (15). Plasmid pComb3X is derived from pComb3H (33). The coding sequence for
anti-thyroglobulin scFv is cloned in plasmid pHEN obtained from Griffiths et al. (41). The envelope plasmid pMD.G, the packaging plasmid
pCMVR8.91, and the vector plasmid pRRL.SIN have been described
previously and were kindly provided by Didier Trono (42– 44). pHXBnPLAP-IRES-N⫹ was obtained from the AIDS Research and Reference
Reagent Program.
Antibodies—Rabbit polyclonal Vif was described previously (15). For
detection of Vif protein, rhodamine-conjugated sheep anti-rabbit immunoglobulin was used as a secondary antibody (Pierce). HRP-conjugated
anti-M13 phage antibody was obtained from Amersham Biosciences.
FITC-conjugated, HRP-conjugated, and nonconjugated anti-HA high
affinity antibody was purchased from Roche.
Proteins—Vif protein was derived from HIV-1HXB2 strain and purified as described (45, 46). HIV-1 protease was obtained from the AIDS
Research and Reference Reagent Program.
DNA Transfections
To produce large amounts of HIV-1 particles, 5 ⫻ 106 293T cells were
transfected by FuGENE (Roche) with 2 ␮g of wild-type HIV-1NL4 –3 or
mutant pHIV-1NL4 –3⌬vif. After 24 h Jurkat and H9 cells were cocultured during 48 h in the presence of 50 ␮g/ml of dextran. The cells
were maintained in RPMI medium plus 15% fetal calf serum. p24
antigen concentration was determined as recommended by the manufacturer (Innotest). For immunofluorescence and co-immunoprecipitation experiments, HeLa cells were co-transfected by the FuGENE reagent (Roche) according to the manufacturer’s protocol with 2 ␮g of
pSVLvif and 2-fold molar excess of plasmid encoding intrabodies or with
control plasmid, pcDNA3.1/Neo containing no insert. For co-immunoprecipitation, cell lysis was performed as described by Anderson (47).
For immunoprecipitation, magnetic protein A beads were used as described by the manufacturer (Miltenyi Biotec).
Replication Complementation Assay
A transient complementation assay was used to provide a quantitative measure of the ability of wild-type or mutant Vif proteins to
complement a single round of HIV-1 replication in trans. Briefly, H9
cells (107) were co-transfected by FuGENE (Roche), with 2 ␮g of either
pHXB⌬envCAT or pHXB⌬Avr⌬envCAT, 2 ␮g pSVIIIenv, and plasmids
32037
encoding scFv as described (49). The ability of the intrabody to inhibit
a single round of infection was measured by assaying for chloramphenicol acetyltransferase (CAT) activity in the transfected culture at 9 or 10
days after transfection. CAT assay was performed by ELISA (Roche).
Immunofluorescence Staining
HeLa cells transfected by FuGENE with 2 ␮g of plasmids were fixed
in phosphate-buffered saline (PBS) with 4% paraformaldehyde for 10
min at room temperature, permeabilized with PBS plus 0.1% Triton
X-100 for 5 min, and washed with PBS plus 2% fetal calf serum before
staining. The fixed cells were incubated with Vif antiserum (1:200) for
90 min at 37 °C, incubated with rhodamine-conjugated goat anti-rabbit
immunoglobulin antibody (1:200) for 20 min at 37 °C, washed, and
mounted on glass slides. For immunostaining of scFv, direct immunofluorescence was performed with FITC-conjugated anti-HA monoclonal
antibody (1:40). For double immunofluorescence staining of intrabodies
and Vif protein, FITC-conjugated anti-HA monoclonal antibody was
used in combination with Vif antiserum at similar concentrations. The
imaging setup consists of an Olympus IX-50 inverted microscope, Ludl
BioPoint filter wheels and a 12-bit V-scan cool CCD (Photonic Science).
Integrated control of filter wheel and image acquisition is achieved by
Image-Pro Plus 4.0 and Scope-Pro 3.1 (Media Cybernetics). The settings
for image acquisition (camera exposure time, filters, time interval, and
storing modes) were determined by custom-made macros. The images
were collected with Olympus 40⫻ or 100⫻ plan apo objectives (numerical apertures ⫽ 0.95 and 1.4, respectively).
Rabbit Immunization
A rabbit (New Zealand White) was treated with four subcutaneous
injections containing 50 ␮g of purified Vif protein in a 1-ml emulsion of
adjuvant (Ribi Immunochem Research, Hamilton, MT). The injections
were administered at 2–3-week intervals. Five days after the final
boost, spleen and bone marrow were harvested and used for total RNA
preparation (48, 50).
RNA Isolation, cDNA Synthesis, Rabbit Antibody Library
Construction, and Sfi Cloning
Total RNA was prepared from rabbit bone marrow and spleen using
TRI reagent from the Molecular Research Center (Cincinnati, OH)
according the manufacturer’s protocol and was further purified by lithium chloride precipitation. First strand cDNA was synthesized using
the SUPERSCRIPT Preamplification System with oligo(dT) priming
(Invitrogen). The protocol and the oligonucleotide primers for the construction of chimeric rabbit antibody libraries, where rabbit VL and VH
sequences are combined with human C␬ and CH1 sequences, have been
described previously (32). Final PCR fragments encoding a library of
antibody fragments were Sfi-cut, purified, and cloned into pComb3X
vector. PComb3X contains a suppressor stop codon and sequences encoding peptide tags for purification (His6) and detection (HA) (50).
Expression and Purification of Fab Fragments
PCR fragments encoding Fab were generated by overlap extension
PCR and cloned into pComb3X vector. To express Fab, phagemid DNA
was transformed into the nonsuppressor Escherichia coli strain TOP
10F (50). Fab was purified from the concentrated supernatants of induced cultures by affinity chromatography. Purified Fab fragments
were analyzed by SDS-PAGE followed by Coomassie Blue staining and
Western blot with HRP-conjugated anti-HA monoclonal antibody. Protein concentration was determined by measuring the optical density at
280 nm by the classic Bradford method.
DNA Constructs
For the conversion of a Vif-specific Fab into a scFv, specific oligonucleotides primers were used to amplify VH and VL gene segments from
purified phagemid DNA isolated from J4, a Fab fragment specific for
the Vif protein. This Fab was isolated from an immunized rabbit by
using the phage display approach (50). The following primers were
used: VL, RSCVK1, 5⬘-GGGCCCAGGCGGCCGAGCTCGTGMTGACCCAGACTCCA-3⬘, and RKB9J0-B, 5⬘-GGAAGATCTAGAGGAACCACCTAGGATCTCCAGCTCGGTCCC-3⬘; and VH, RSCVH3, 5⬘-GGTGGTTCCTCTAGATCTTCCCAGTCGYTGGAGGAGTCCGGG-3⬘, and HSCG1234, 5⬘-CCTGGCCGGCCTGGCCACTAGTGACCGATGGGCCCTTGGTGGARGC-3⬘. The purified PCR products were assembled by another
PCR using the following primers: RSC-F, 5⬘-GAGGAGGAGGAGGAGGAGGCGGGGCCCAGGCGGCCGAGCTC-3⬘, and RSC-B, 5⬘-GAGGAGGAGGAGGAGGAGCCTGGCCGGCCTGGCCACTAGTG-3⬘. The resulting overlap PCR product encodes an scFv in which the N-terminal
32038
Neutralization of Vif Protein by Intracellular Immunization
VL region is linked with the VH region through a 7-amino acid peptide
linker (GGSSRSS) and a 18-amino acid peptide linker (SSGGGGSGGGGGGSSRSS), named scFv 4B and 4BL, respectively. The DNA fragment was gel-purified, digested with the restriction endonuclease SfiI,
and cloned into the appropriately cut phagemid vector pComb3X, a
variant of pComb3H. The binding activity of the expressed scFv was
confirmed, and the genes encoding the scFv were transferred to
pCDNA3.1/Neo (Invitrogen), pBabePuro vectors, and pRRL.SIN. A methionine initiation codon sequence was added into the 4BL and 4B scFv
by PCR amplification. The primers used for cloning in pCDNA3.1 were
scFv5, 5⬘-GGCATGGGGGCCCAGGCGGCCCAGCTC-3⬘, and scFv3, 5⬘GCCACCACCCTCCTAAGAAGC-3⬘. Similar primers were used for
cloning anti-Vif scFv in pBabePuro and pRRL.SIN: Vector/scFv5, 5⬘-CGCGGATCCGCGGGCATGGGGGCCCAGGCGGCCGAGCTC-3⬘, and
Vector/scFv3, 5⬘-ACGCGTCGACGTCGGATATCGCGGCCGCGGAAGCCACCACCCTCCTAAGAAGC-3⬘. Downstream of the sequence, we
maintained a sequence encoding the HA tag sequence (YPYDVPDYA)
followed by a stop codon. Control scFv anti-thyroglobulin was amplified
from pHEN2 and Sfi-cloned in pComb3X. A methionine was introduced
at the N terminus, and the DNA fragment with a HA-tag at the C
terminus was cloned into the appropriately digested vector DNAs. The
modified pcDNA3.1/Neo plasmid encoding 4BL, 4B, and anti-thyroglobulin was designated pI4BL, pI4B, and pThyr. Anti-Vif scFv I4BL
and anti-thyroglobulin was cloned in pHXBnPLAP-IRES-N⫹ by inserting the gene in place of PLAP-IRES-nef. The primers used for cloning
were scFv5/HIV, 5⬘-ATAAGAATGCGGCCGCTAAACTATATGGGGGCCCAGGCGGCCGAGCTC-3⬘, and scFv3/HIV, 5⬘-CCGCTCGAGCGGGCCACCACCCTCCTAAGAAGC-3⬘.
In Vitro Studies of Anti-Vif Antibody Fragments
The methods for bacterial expression of anti-Vif scFv were described
before (50). Briefly, scFv were cloned in pComb3X that allows the
expression in the supernatant and purification with a Ni2⫹-NTA column caused by the presence of a His6 tag at the C-terminal end. After
induction of scFv expression by growth in 1 mM isopropyl-␤-D-thiogalactopyranoside for 20 h at 30 °C, analysis of supernatants indicated
that both anti-Vif scFv were expressed. For assays of expression of
antibody fractions, 1 ml of culture supernatant was concentrated (Amicon 10) and separated by 12% PAGE. For purification of ScFv and Fab
proteins, 100 ml of bacterial supernatant were concentrated to 2 ml
(Centricon 30), and Ni2⫹ columns were used (CLONTECH). Relative
binding affinities of anti-Vif ScFv were determined via ELISA after
coating of the wells with 200 ng of purified recombinant HIV-1 Vif
protein. Purified anti-Vif scFv and Fab were diluted at various concentrations starting at 5 ␮g/ml and added to the wells for further incubation. After washing the wells with PBS, horseradish-conjugated anti-HA monoclonal antibody was applied for detection. Recognition of Vif
by anti-Vif scFv and Fab was next demonstrated by Western blot
analysis. Vif protein and HIV-1 protease (200 ng) were separated by
PAGE and transferred to nitrocellulose membranes. After blocking, the
proteins were probed with anti-Vif Fab and scFv antibody fragments
and then with horseradish-conjugated anti-HA monoclonal antibody as
a secondary antibody. The proteins were visualized using the ECL
system (Amersham Biosciences).
Retroviral Gene Transfer: Generation of Transduced H9
and Jurkat Cells
The amphotropic packaging cell line PhoenixAmpho was transfected
with pBabe Puro plasmids encoding the anti-Vif scFv inserts (51). For
control purposes pBabe Puro plasmids encoding scFv specific to thyroglobulin were also used (41). These packaging cell lines were used to
generate virus-containing medium for two rounds of infection of H9 and
Jurkat cells in the presence of 10 ␮g/ml Polybrene (Calbiochem). Two
days after the last infection, transduced H9 and Jurkat cells were
selected in puromycin (0.5 ␮g/ml) by limiting dilution cloning. After 15
days of selection, analysis of cells for anti-Vif expression and infectability was started. The untransduced parental H9 and Jurkat cell lines
were named H9-P and Jurkat-P, respectively, and the H9 and Jurkat
cells transduced to express anti-Vif intrabodies were named H9-I4BL,
H9-I4B, Jurkat-I4BL, and Jurkat-I4B. The cell lines transduced to
express the control intrabody anti-thyroglobulin were named H9-thyr
and Jurkat-thyro.
HIV-1 Infection of H9 and Jurkat Cells
Transduced and untransduced H9 and Jurkat cells were infected at
a multiplicity of infection of 0.01– 0.05 for 6 h at 37 °C, washed, and
then cultured for up to 30 days. To monitor infection, aliquots were
FIG. 1. Relative binding affinities of anti-Vif Fab antibody
fragments. Isolated anti-Vif Fabs in a total of 96 clones were expressed
and used for binding to 200 ng of Vif protein. Fab binding to antigen was
detected by high affinity HRP-conjugated anti-HA monoclonal antibody. A total of 16 clones with signals that were higher than background were isolated. Background optical density signal in all clones
are less than 0.2. Fab J4 was chosen for further studies, because its
signal was more intense in several independent experiments.
taken from the cultures at the indicated time points, and the p24 levels
were determined in an HIV-1 ELISA (Innotest).
PCR Analysis of HIV-1 Reverse Transcription
DNase-I-treated stocks of HIV-1NL4 –3 or mutant pHIV-1NL4 –3⌬vif
produced from parental Jurkat and H9 cells and cells expressing antiVif scFv were used to challenge 1 ⫻ 106 H9 cells and PBMC in a volume
of 1 ml. For infection, a stock of 2 ng of soluble p24 antigen was used.
The PCR procedure to analyze reverse transcription was described
previously (28, 52). The primers used in this assay (M667/M661-LTR/
gag, nucleotides 496 – 695) amplify the late DNA products in reverse
transcription. Similar reaction conditions were used, except only the
24 h time point was analyzed. The products of all PCR reactions were
electrophoresed through 1.5% agarose gels, transferred to Hybond N⫹
membranes (Amersham Biosciences), subjected to Southern hybridization by using random-primed radiolabeled DNA probes (Amersham
Biosciences), and visualized by autoradiography. The DNA used for
radiolabeling were obtained following PCR amplification of segments of
pHIV-1NL4 –3 or a human ␤-globin expression vector with the relevant
primer pairs.
HIV Vector Stock Preparation
The stocks were prepared as described previously by transient cotransfection of three plasmids into 293T cells: pRRL.SIN.I4BL, the
envelope plasmid pMD.G, and the packaging plasmid pCMV⌬R8.91
(42– 44). The p24 concentration was determined by antigen ELISA
(Innotest). Vector production and gene delivery were done in a biosafety
level 2 environment. The in vitro transduction experiments were done
in six-well plates (Nunc). Filtered vector-containing medium was added
24 h after seeding PBMC (2 ⫻ 105 cells/well). When applicable, transduction of H9 and Jurkat cells was also performed. The transduction
protocol was repeated two more times in the following 48 h. For the
transduction, 100 ng of p24 was used. The multiplicities of infection
were estimated by assuming that 1 ng of p24 corresponds to 1000 –5000
transducing units.
RESULTS
Selection of Vif-specific Antibody Fragments from a Phage
Display Chimeric Rabbit Fab Library—A chimeric rabbit/human Fab library was generated from cDNA derived from spleen
and bone marrow RNA of an immune rabbit that had a strong
immune response to HIV-1 Vif (50). The phagemid vector
pComb3X was used, and an antibody library of ⬇ 9 ⫻ 107
independent clones was constructed and selected. Several Fab
clones that bound to purified Vif protein were isolated and
studied. The relative binding affinities reported for each of the
Fab clones are summarized in Fig. 1. The analyzed clones
showed some sequence variation with conservation of sequence
within some complementarity determining regions (data not
shown). One clone, J4, bound strongly to Vif protein in ELISA
and was chosen for further characterization and expression.
Neutralization of Vif Protein by Intracellular Immunization
32039
FIG. 2. Expression and relative binding affinities of anti-Vif Fab and scFv. A, TOP 10 F bacteria expressing anti-Vif 4 BL scFv (lanes 1
and 2), anti-Vif 4B scFv (lanes 3 and 4), and Fab J4 (lanes 5 and 6) at 2 and 18 h, respectively, by induction with 0.5 M isopropyl-␤-Dthiogalactopyranoside at 25 °C. Antibody fragments were expressed in the supernatant and precipitated by trichloroacetic acid at indicated time
points. The proteins were immunoblotted by HRP-conjugated anti-HA monoclonal antibody. B, bacteria culture supernatant expressing Fab J4,
scFv 4BL, and scFv 4B were used for evaluating relative binding affinities to 200 ng of Vif protein, HIV-1 protease, and bovine serum albumin by
ELISA. The results were measured by optical density at 405 nm. The data represent the results of three independent experiments.
Anti-Vif scFv Gene Expression in E. coli and in Vitro Interaction with HIV-1 Vif—The antibody fragment J4, that binds
purified Vif protein was derived from a Fab phage display
library. To express it in the cytoplasm in a lower molecular
weight form, J4 Fab was converted to a single-chain fragment.
Anti-Vif scFv was constructed by PCR amplification of VL and
VH fragments and covalently linked with a short peptide linker
of 7 amino acids (4B, GGSSRSS) and a long peptide linker of 18
amino acids (4BL, SSGGGGSGGGGGGSSRSS). It was expected that the short linker peptide would result in a dimeric
scFv protein (33). To determine whether the binding efficiency
of recombinant scFv to the HIV-1 Vif would correlate with
those of the parental Fab clone, both scFv 4BL and 4B were
expressed in E. coli, and binding to HIV-1 Vif was assessed by
ELISA and Western blotting. Both 4B and 4BL anti-Vif scFv,
together with J4 Fab, were expressed in bacterial cell culture
supernatant and purified by Ni2⫹ affinity columns. As the data
in Fig. 2 demonstrate, 4B and 4BL scFv have different binding
patterns. Binding of anti-Vif scFv prepared with a long linker
(4BL) binds Vif protein in ELISA at similar levels to Fab J4. In
contrast, anti-Vif scFv prepared with a short linker (4B) demonstrates comparatively a very low affinity. In contrast, nearly
background signals indicate very low nonspecific binding of
scFv 4BL to control proteins HIV-1 protease and bovine serum
albumin, a profile similar to that of Fab J4.
The specificity of binding of anti-Vif scFv 4BL was next
investigated by immunoblotting and immunofluorescence (Fig.
3). Vif protein and HIV-1 protease were separated by SDSPAGE and probed with anti-Vif Fab J4 and recombinant antiVif scFv 4BL. In the Western blot the major band of 23 kDa
corresponding to HIV-1 Vif was recognized strongly by the scFv
4BL and Fab J4. The recombinant antibody fragment, as expected from the ELISA results, did not recognize HIV-1 protease. Immunofluorescence studies also show a specific recognition of Vif protein by anti-Vif Fab and scFv 4BL (Fig. 3). Thus,
in opposition to scFv 4B, Vif protein is specifically recognized in
vitro by scFv with a long linker.
Cellular Expression of Anti-Vif scFv Co-immunoprecipitates
Vif Protein and Co-localizes with HIV-1 Vif in the Cytoplasm—To express the antibody fragments 4BL and 4B in the
cytoplasm of human T-lymphoid cells, an initiation codon was
appended to the N terminus of the proteins, and an HA epitope
was maintained at the C terminus. Expression plasmids were
constructed in pCDNA3.1Neo (pI4BL and pI4B). Because 4B
scFv was unable to bind Vif protein in ELISA, we anticipated
that its expression in the cell would not effectively trap Vif in
FIG. 3. Detection of antigens by Western blot and immunofluorescence. A, 200 ng of purified HIV-1 protease (lane 1) and Vif protein
(lanes 2 and 3) were separated by PAGE and transferred to nitrocellulose membranes. For immunodetection, purified Fab J4 (lanes 1 and 2)
and scFv 4BL (lane 3) was used, and HRP-conjugated anti-HA monoclonal antibody was used as a secondary antibody. B, detection of Vif
protein expressed in HeLa cells transfected with pSVLvif. The cells
were fixed in 4% paraformaldehyde, washed in PBS, and immunostained with purified anti-Vif Fab J4 (left panel) and scFv 4BL (center
panel). Nontransfected HeLa cells were stained with scFv 4BL (right
panel). High affinity FITC-conjugated anti-HA antibody was used for
detection of immunocomplexes. The display settings in back image are
approximately three times higher than normal images to allow visualization of nontransfected cells; settings identical to the experiment
images produce a completely black image.
the cytoplasm. As a control for the activity of scFv, the antibody
fragment anti-thyroglobulin (41) was cloned in the same eukaryotic vector and expressed in the human T-lymphoid cells.
The efficacy of intrabody expression for binding HIV-1 Vif
protein in the cell was examined by co-immunoprecipitation
and fluorescence microscopy studies (Fig. 4). HeLa cells were
co-transfected with intrabodies and pSVLvif and then lysed
48 h after transfections. The intrabody fragment I4BL was
immunoprecipitated with an anti-HA high affinity monoclonal
antibody and protein A magnetic beads. Cell lysis and immunoprecipitation were carried out using mild detergent and salt
conditions to allow co-immunoprecipitation of binding proteins.
Upon immunoprecipitation of cells transfected with pSVLvif
and pI4B, no Vif protein was detected by Western blot, whereas
co-transfection of pSVLvif with pI4BL resulted in co-immunoprecipitation of Vif protein (Fig. 4). This result is consistent
with previous data in ELISA showing Vif binding. As a control,
anti-thyroglobulin scFv expressed in HeLa cells did not bind
Vif because this protein did not co-immunoprecipitate with the
antibody fragments (data not shown).
To evaluate the cellular localization of the antibody fragment
I4BL expressed together with the antigen Vif, intracellular
staining of co-transfected HeLa cells using an antibody specific
to the HA tag and a polyclonal serum against Vif was performed in HeLa cells (Fig. 4). 48 h after co-transfection with
pI4BL and pSVLvif, HeLa cells were fixed in paraformaldehyde
32040
Neutralization of Vif Protein by Intracellular Immunization
FIG. 4. Interactions of Vif and scFv 4BL. A, transfected HeLa cells expressing scFv 4BL were stained with high affinity FITC-conjugated
anti-HA antibody (green signal; panel 1) and Vif protein with anti-Vif polyclonal serum plus rhodamine-conjugated anti-rabbit antibody (red signal;
panel 2) as described under “Experimental Procedures.” A different localization and punctated pattern is observed for Vif and scFv 4BL when
transfected separately. B, transfected HeLa cells expressing Vif protein and scFv 4BL were stained with high affinity FITC-conjugated anti-HA
antibody (green signal; panels 1 and 4) and anti-Vif polyclonal serum plus rhodamine-conjugated anti-rabbit antibody (red signal; panels 2 and 5)
as described under “Experimental Procedures.” A punctate pattern is indicated with arrows, indicating possible immunocomplex aggregates of Vif
and scFv 4BL. In the majority of cells, the punctate pattern was consistently observed in the cytoplasm. Overlay of panels 1 and 2 and panels 4
and 5 results in a yellow signal at sites of co-localization (panels 3 and 6). Immunofluorescence microscopy was performed with the imaging setup
described under “Experimental Procedures” using the appropriate excitation and emission filters. C, transfected HeLa cells expressing Vif protein
(lane 1), Vif protein and scFv 4BL (lane 2), Vif protein and scFv 4B (lane 3), and Vif protein and scFv anti-thyroglobulin (lane 4) were lysed at 40 h
with a low detergent buffer. The cell lysates with scFv were immunoprecipitated with high affinity anti-HA antibody as described under
“Experimental Procedures.” Nonimmunoprecipitated lysate of Vif protein (lane 1) and immunoprecipitated fractions were separated by PAGE
(lanes 2– 4). Western blot was performed with anti-Vif polyclonal serum and HRP-conjugated anti-rabbit antibody. Co-immunoprecipitation of Vif
is observed with scFv 4BL but is absent when scFv 4B and scFv anti-thyroglobulin are expressed (lanes 3 and 4).
and observed by fluorescence microscopy. Microscopy showed a
predominant punctate pattern of co-localization of these two
proteins in the cytoplasm, with nucleoplasm exclusion (Fig. 4).
In addition, Vif was consistently detected in the nucleus, and
scFv was only observed in the cytoplasm in the majority of cells
evaluated. When Vif and scFv 4BL are expressed alone, its
pattern of localization is different from the co-localization
staining. In contrast, when anti-thyroglobulin scFv was expressed together with anti-Vif scFv, no obvious co-localization
was detected (data not shown). These results demonstrate that
Vif protein is specifically recognized in the cytoplasm by the
intracellular expression of anti-Vif scFv and that its interaction
co-localizes Vif and scFv 4BL throughout the cytoplasm.
I4BL Inhibits Vif Function in a trans-complementation Assay—To provide a qualitative and quantitative measure of the
biological activity of the anti-Vif scFv, a transient complementation assay in nonpermissive cells was used to examine the
abilities of the anti-Vif scFv and control scFv to inhibit a single
round of HIV-1 replication in trans (27). In a previous study, we
showed that the expression of Vif in trans in this assay restores
the efficiency of a single round of replication of a vif-defective
virus to the wild-type level (15). The Vif expresser plasmid is
co-transfected with a vif-positive (pHXB⌬envCAT) or vif-negative env-defective (pHXB⌬Avr⌬envCAT) HXB2 provirus plasmid that expresses CAT and an HIV-1 envelope expresser
plasmid. The HIV-1 virus particles produced in this assay
result in only a single round of infection, because the packaged
viral genome is defective for Env production. The efficiency of a
single round of virus replication is quantitated by measuring
the level of CAT enzyme activity in the infected cultures after
9 –10 days, the minimum time for a detectable signal above
background. The ability of the anti-Vif scFv to inhibit a single
round of HIV-1 replication in trans was examined. In the absence of Vif, replication of the vif-negative virus was ⬃10-fold
lower than that of the vif-positive virus (Fig. 5). Coexpression of
I4BL scFv in H9 cells reduced trans-complementation to 15% of
the wild-type level, as shown in Fig. 5. In contrast, the expression of I4B scFv, which does not bind Vif, caused a much less
FIG. 5. Neutralization of Vif function in a trans-complementation assay. The values shown by the black and white bars represent
the percentages of replication complementation in Jurkat and H9 cells,
respectively, relative to the value obtained for the wild type. The cells
were co-transfected with a wild-type Vif protein, scFv expressor plasmids, either pHXB⌬envCAT or pHXB⌬Avr⌬envCAT, and pSVIIIenv as
described under “Experimental Procedures.” Replication complementation for each testing plasmid was determined by the level of chloramphenicol acetyltransferase activity in cells co-transfected with
pHXB⌬Avr⌬envCAT relative to that obtained with pHXB⌬envCAT.
Background levels obtained when pSVIIIenv was not co-transfected
were 8 ⫾ 2%. The results shown are the means ⫾ S.E. of three independent experiments.
significant reduction in trans-complementation to 85% of the
wild-type level. Expression of anti-thyroglobulin scFv caused
no significant reduction in trans-complementation. Thus, the
biological activities of the anti-Vif scFv correlated directly with
their Vif binding activities in ELISA and co-immunoprecipitation assays, indicating that I4BL scFv is likely to inhibit HIV-1.
H9 Cells Expressing Vif-specific Intrabody I4BL Are Protected from HIV-1 Infection—The experiments described above
examined the biological activity of anti-Vif scFv in a transient
assay under conditions in which most virus transmission occurs by cell-to-cell spread (15, 49). To investigate whether
anti-Vif scFv can inhibit Vif function during several rounds of
Neutralization of Vif Protein by Intracellular Immunization
32041
FIG. 6. Infection of permissive and nonpermissive cell lines expressing anti-Vif intrabody. A, the infectivity assays were started with
HIV-1NL4 –3 produced after transfection of 293T cell lines with full-length proviral DNA. Upper left panel, Jurkat and H9 cells infected with
HIV-1NL4 –3 and HIV-1NL4 –3⌬vif. Upper right panel, Jurkat and H9 cells expressing anti-thyroglobulin scFv infected with HIV-1NL4 –3 and
HIV-1NL4 –3⌬vif. Lower left panel, Jurkat and H9 cells expressing anti-Vif scFv I4BL infected with HIV-1NL4 –3 and HIV-1NL4 –3⌬vif. Lower right
panel, cell proliferation kinetics with WST-1 reagent. B, Western blot of cell lysates of Jurkat cells expressing anti-Vif scFv I4BL and
anti-thyroglobulin scFv (lanes 3 and 4) and cell lysates of H9 cells expressing anti-Vif scFv I4BL and anti-thyroglobulin scFv (lanes 5 and 6). The
lysates of Jurkat and H9 cells not expressing scFv were also used in Western blot as negative controls (lanes 1 and 2).
HIV-1 replication, permissive (Jurkat) and nonpermissive (H9)
cell lines expressing intrabodies were generated. For these
experiments recombinant murine leukemia virus (MLV)-derived retroviral vectors encoding anti-Vif intrabody I4BL and
control intrabody anti-thyroglobulin were used to transduce
the human T cell lines H9 and Jurkat. Transduced cells were
established through puromycin selection and limiting dilution
cloning. Retroviral transduced cell lines were analyzed for intrabody and anti-thyroglobulin expression by Western blot
(Fig. 6). Intrabodies were also detected by staining permeabilized transduced H9 and Jurkat cells with an anti-HA antibody
(data not shown). The resultant H9-I4BL and Jurkat-I4BL cell
lines showed homogeneous and stable expression of intrabody
after puromycin selection during at least 3 months in cell
culture. Subsequent studies on virus replication were performed in these intrabody-expressing cell lines.
Parental and transduced permissive and nonpermissive cell
lines were challenged with HIV-1 utilizing the highly cytopathic viral strain HIV-1NL4 –3. Infectivity assays were performed with an MOI of 0.05 or 0.1 to mimic natural infections
(35). Parental nontransduced H9 and Jurkat cell lines supported vigorous replication of HIV-1, as shown by the initial
increases in HIV-1 p24 antigen, which peaked at ⬃14 –20 days.
In contrast, low levels of HIV-1 p24 antigen were observed in
the supernatants of H9-I4BL and Jurkat-I4BL as well as parental cell lines infected with HIV-1NL4 –3⌬vif at early time
points after infection. On days 14 –20 post-infection, H9-I4BL
cells showed ⬃94 –98% inhibition of HIV-1 p24 antigen production compared with the parental H9 cells. Similar results were
obtained in H9 cells infected with HIV-1NL4 –3 ⌬Vif. In contrast,
at the same time points of viral replication, Jurkat-I4BL cells
showed similar levels of HIV-1 p24 antigen production compared with parental Jurkat cells. These results indicate that
HIV-1 replication and infectivity was dramatically reduced in
nonpermissive H9 cells by expression of a Vif-specific intrabody
and were consistent in multiple independent experiments. In
addition, expression of control anti-thyroglobulin scFv in the
same cells had no significant inhibitory effect on HIV-1 replication, indicating that anti-Vif scFv is specific for Vif inhibition
in other nonpermissive cell lines (Fig. 6).
Previous studies have shown that intracellular antibody expression has no obvious negative effects on cell viability or
proliferation. Nevertheless, we quantified cell proliferation and
cell viability of transduced H9-I4BL and Jurkat-I4BL cells
compared with parental cells. The assay consists of a colorimetric assay based on the cleavage of the tetrazolium salt
WST-1 by mitochondrial dehydrogenases in viable cells
(Roche). The kinetics of the metabolism of WST-1 reagent
showed that H9-I4BL and Jurkat-I4BL cells have similar proliferation curves compared with parental cell lines (Fig. 6). The
evaluated time points were the same as those used for p24
antigen detection. Therefore, expression of anti-Vif singlechain antibody stably expressed in the cell inhibits HIV-1 replication in multiple rounds of infection.
Proviral DNA Synthesis Is Impaired for Virus Produced from
H9 Cells Expressing I4BL Intrabody—The synthesis of viral
DNA in H9 cells exposed to HIV-1NL4 –3 produced from H9I4BL and Jurkat-I4BL cells was analyzed by PCR, with prim-
32042
Neutralization of Vif Protein by Intracellular Immunization
FIG. 7. Proviral DNA synthesis is impaired for virus produced
from H9 cells expressing I4BL intrabody. The total cell lysates
were prepared from aliquots of 5 ⫻ 104 cells and were subjected to PCR
analysis with a primer pair specific for late products of reverse transcription. A, H9 cells were challenged with DNase-treated stocks of
HIV-1NL4 –3 derived from H9 and Jurkat parental cells (lanes 1) and
cells expressing anti-Vif scFv I4BL (lanes 2). HIV-1NL4 –3⌬vif was also
produced from H9 and Jurkat cell lines and used as a control (lanes 3).
B, PHA-stimulated PBMC were challenged with similar viral stocks as
in A. C, H9 cells infected as in A were subjected to PCR analysis with
control primers for ␤-globin. A dilution series of H9 and PBMC was also
subjected to PCR amplification with the same primer pairs as in A and
B (right hand panels). These reactions were performed within the
semiquantitative range. All of the reaction products were visualized by
Southern hybridization and then by autoradiography.
ers specific for products of reverse transcription, as described
previously (6, 28, 52). Reverse transcription of retroviral RNA
includes a series of steps, beginning with the synthesis of the
minus strand strong stop and continuing with the elongation of
this minus strand, with the final product constituting the completed double-stranded DNA (30). The M667/M661 primer
pairs flank the primer-binding site and specifically detect
HIV-1 DNA within completed or nearly completed reverse transcripts. To determine whether proviral DNA synthesis is impaired, Jurkat-I4BL and H9-I4BL were co-cultured with 293T
cells transfected with HIV-1NL4 –3, and virus was isolated 9
days later. H9 cells were incubated with the same amounts of
virus normalized by TCID50, and aliquots of the infected cultures were harvested and lysed. Total cellular lysates were
then subjected to PCR analysis. The results in Fig. 7 showed
that late reverse transcripts were significantly reduced in H9
cells infected with HIV-1NL4 –3 derived from H9 intrabody-expressing cells (H9-I4BL). Completed or nearly completed reverse transcripts were reduced to 5% of the viral infection
originated with HIV-1NL4 –3 produced in H9 cells. PCR analysis
was also performed in H9 chronically infected cells after 12
days, and the results were similar. These results show that
infection of H9 cells with virus derived from Jurkat-I4BL
showed similar infection levels compared with viruses produced in the absence of intrabody expression (Fig. 7). In contrast, we demonstrate that HIV-1NL4 –3 virus derived from intrabody-producing cells is defective for reverse transcription.
Inhibition of HIV-1 Viral Infectivity by Anti-Vif I4BL Is Specific for Nonpermissive Cells—Vif modulates HIV-1 infection in
cultured T-cell lines in a cell-dependent manner (2, 4, 6, 8, 9).
To evaluate the specificity of viral inhibition in different cell
lines, a recombinant HIV-1 expressing I4BL or anti-thyroglobulin scFv in cis was generated. This recombinant HIV-1 was
derived from pHXBnPLAP-IRES-N⫹ by replacing the human
PLAP gene by I4BL or anti-thyroglobulin scFv, generating
pHXB-I4BL and pHXB-thyro. In this experiment, 293T cells
were transfected, and a high titer supernatant of HXB-I4BL,
HXB-thyro and HXB2⌬vif was obtained. HIV-1 supernatants
normalized for the same TCID50 were used to infect permissive
(Jurkat and SupT1), semi-permissive (CEM), and nonpermissive cells (H9, U38, and PBMC), and their ability to replicate in
these cells was assessed by performing standard virus growth
curves. Three patterns of results were obtained (Fig. 8). All of
the nonpermissive cells tested in this experiment did not support the spread of HXB-I4BL. Similar results were obtained
with HXB2⌬vif. In contrast, permissive cell lines supported
replication of all viruses used in this experiment. In CEM cells,
replication of HXB-I4BL exhibited an intermediate behavior,
although in some experiments its replication was higher than
expected. As a control, HXB-thyro was able to replicate at
wild-type levels in all cells tested (Fig. 8). HXB-I4BL was also
co-cultivated with HIV-1NL4 –3 in nonpermissive cells, and infection was monitored by p24 antigen production. In this coculture experiment MOI of HIV-1NL4 –3 were similar and five
times higher than HXB-I4BL. Compared with infection of HIV1NL4 –3 alone in nonpermissive cells, p24 antigen production
was dramatically reduced from 94 to 97% at similar MOI and
from 87 to 95% with higher MOI (data not shown). Thus, the
HXB-I4BL can control replication of wild-type virus. These
results show that the specificity of Vif inhibition by the intrabody correlates with the cellular requirements for Vif function,
confirming the specificity of anti-Vif scFv.
Inhibition of HIV Replication by Anti-Vif scFv in Transduced
Human PBMC—To evaluate the potential of anti-Vif scFv for
clinical application, PHA-stimulated human PBMC were
transduced with a retroviral vector that expresses anti-Vif scFv
I4BL. Lentiviruses are attractive vectors for gene therapy because of their ability to integrate into nondividing cells (42, 43,
44). The retroviral vectors used in this study are derived from
the HIV SIN system described previously by Naldini et al. (42).
This delivery system has been shown to be highly effective in
transduction of primary cells and offers significant advantages
for its predicted biosafety (42). Intrabodies were cloned in the
self-inactivating vector pRRL.SIN and expressed under the
cytomegalovirus promoter. Replication-defective retroviral
particles were generated by transient co-transfection of 293T
human kidney cells with the combination of three plasmids as
described under “Experimental Procedures.” The p24 antigen
concentration was determined, and the amount corresponding
to 100 ng was used to transduce PBMC. The transduction
protocol consisted of the daily infection of stimulated human
PBMC with fresh 100 ng of viral p24 supernatants for 3 days,
leading to transduction efficiencies, as measured by anti-HA
immunofluorescence, ranging from 40 to 48% for three different experiments. The efficiency of transduction was significantly higher then previous experiments with MLV vectors,
which may reflect the higher capability of HIV-based vectors
for infecting human cells.
PBMC were challenged with HIV-1NL4 –3 and two clinical
isolates of HIV-12 in the range of MOI 0.05– 0.1. Fig. 9 shows a
dramatic inhibition of HIV-1NL4 –3 production by anti-Vif I4BL
transduction. Similar values of inhibition are obtained for the
HIV-1 clinical isolates. In contrast, HIV-1 replication in PBMC
transduced with anti-Vif I4B and anti-thyroglobulin showed
less inhibition or no inhibition, respectively. These results
show the potential for anti-Vif scFv for inhibiting HIV-1 replication in primary cells by ex vivo transduction.
2
M. H. Lourenço, personal communication.
Neutralization of Vif Protein by Intracellular Immunization
32043
FIG. 8. Cell-specific inhibition of HIV-1 replication in presence of anti-Vif scFv. Replication of HIV-encoding anti-Vif scFv I4BL and
anti-thyroglobulin was assessed in permissive and nonpermissive cells. In the left panels, permissive and semi-permissive cells Jurkat, SupT1, and
CEM cells, were infected with HIV-I4BL (A) and HIV-thyro (B). In the right panels, nonpermissive cells H9, U38, and PHA-stimulated PBMC were
infected with HIV-I4BL (C) and HIV-thyro (D). Notice in the upper right panel that the lower values of HIV-1 p24 concentration were measured.
The PBMC data were obtained with cells from three different HIV-1 seronegative donors. The data are representative of two independent
experiments.
FIG. 9. Inhibition of HIV infection by transduction with an HIV-derived retrovector encoding anti-Vif scFv. Purified PBMC from
three different HIV-1/HIV-2 seronegative donors were PHA-stimulated and transduced with SIN18-I4BL using the protocol described under
“Experimental Procedures.” Left panel, nontransduced PHA-stimulated PBMC were infected with HIV-1NL4 –3 (MOI ⫽ 0.05– 0.1); two HIV-1 clinical
isolates (strain Ac178 and strain Je524, Prof. H. L., Faculdade de Farmácia de Lisboa), 1 ng of HIV-1 p24 antigen/106 PBMC. Right panel,
transduced PHA-stimulated PBMC was challenged with similar HIV strains as in the left panel. The data are representative of at least two sets
of independent experiments.
DISCUSSION
The action of Vif is essential for the completion of proviral
DNA synthesis after virus entry, most likely as a result of its
effect during virus production (6, 25, 28). ScFv-based strategies
directed against Vif may be an effective approach to inhibit
these two crucial steps of the viral replication cycle (36 –38).
The genetic approach described here abrogates viral infectivity
and reverse transcription by functional deletion of Vif, thereby
inhibiting HIV-1 replication prior to integration of viral DNA in
the host genome. We showed that anti-Vif scFv binds to Vif in
32044
Neutralization of Vif Protein by Intracellular Immunization
the cytoplasm. We also showed that scFv co-immunoprecipitates Vif, probably because of its high affinity. Anti-Vif scFv
expressed transiently, constitutively, or by retroviral transduction abrogated viral replication in primary human T-lymphocytes. Wild-type viral particles produced from nonpermissive
cells expressing anti-Vif scFv have a defect in infectivity at the
level of reverse transcription because of neutralization of Vif in
trans. These results confirm previous studies demonstrating
that Vif acts in the producer cell and affects virus assembly.
Consistent with this model, the expression of anti-Vif scFv in
target cells had no effect on the infectivity of wild-type HIV-1.
The transducing vector used in this study was derived from
HIV-1 (42, 43, 44), which can transduce dividing and nondividing human T-lymphocytes. Comparative studies using a vector
derived from MLV demonstrated that the HIV-derived vector is
more efficient at protecting human T-lymphocytes from HIV
infection (data not shown). The effectiveness of neutralization
was assessed by challenging intrabody-transduced PBMC with
an HIV-1 laboratory-adapted strain and HIV-1 primary isolates. The Vif epitope recognized by the 4BL intrabody may be
dominant, because it neutralizes both laboratory adapted and
primary HIV-1 strains. Effective intracellular immunization
must be designed for highly divergent proteins that share only
a few peptide motifs because HIV-1 rapidly evolves compensatory mutations in response to selective pressures. Extended in
vitro challenge with HIV-1 is necessary to determine whether
Vif will acquire escape mutations associated with resistance.
Vif is predominantly localized in the cytoplasm of infected
cells. A small fraction is localized in the nucleus, reflecting a
possible role of Vif in this compartment. Our results show that
scFv expressed in the cytoplasm co-localizes with Vif. Thus, the
result of our study predicts that the cytoplasm is where Vif
functions during HIV-1 replication.
The requirement for Vif differs among cell lines (2, 4, 6, 8, 9).
Recent studies suggest that Vif enhances infectivity by overcoming an inhibitory factor present in nonpermissive cells (53,
54). When anti-Vif scFv was cloned in HIV-1 in place of the nef
gene, viral replication occurred only in permissive cells,
whereas in nonpermissive cells replication was similar to vifnegative HIV-1. The activity of the anti-Vif intrabody was
shown to be cell-specific, because in cis and in trans it had
inhibitory effects only in nonpermissive cells. Anti-Vif scFv will
help to validate candidates for HIV-1 cell-specific inhibitors
that can be neutralized by Vif (20, 54).
The results presented here extend previous studies demonstrating that high affinity single-chain antibodies can be stably
expressed in the cytoplasm (31, 34, 36, 35, 37). Folding of the
scFv in the reducing environment of the cytoplasm can form
functional binding sites as demonstrated by co-immunoprecipitation. Furthermore, the toxicity of the scFv was low as assessed by cell viability in intrabody-expressing human T-lymphocytes. We constructed a single-chain antibody by ligating
VL with VH using a short and long linker. It was anticipated
that a short linker peptide would result in a dimer scFv with
two sites for antigen binding (33). Nevertheless, we observed a
lack of binding of the 4B antibody. The reason for this result
might be reduced stability or folding yield at the growth temperature. The short linker peptide may inhibit the coherent
structure of the framework regions, thereby altering the binding affinity.
A large panel of scFvs against Vif is being constructed to
map epitope-binding regions and putative functional sites.
Using a panel of anti-Vif scFvs directed to specific intracellular sites, it may be possible to gain novel insights into Vif
function. In addition, this strategy will facilitate the devel-
opment of rational combinatorial gene therapies that simultaneously target different functional motifs.
Acknowledgments—We thank Didier Trono for providing the
pRRL.SIN.I4BL, pMD.G, and pCMV⌬R8.91 plasmids, Greg Winter for
providing pHEN-Thyroglobulin, and Prof. Helena Lourenço (Faculdade
de Farmácia de Lisboa, Portugal) for providing HIV-1 primary strains.
HIV-2ROD, pHIV-1NL4 –3⌬vif, and HIV-1NL4 –3 and HIV-1 Protease were
obtained from the AIDS Research and Reference Reagent Program.
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