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Journal of Virology, August 1999, p. 7087-7092, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Interaction of Human Immunodeficiency Virus-Derived
Vectors with Wild-Type Virus in Transduced Cells
Anatoly A.
Bukovsky,
Jin-Ping
Song, and
Luigi
Naldini*
Cell Genesys, Foster City, California 94404
Received 12 October 1998/Accepted 15 April 1999
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ABSTRACT |
The interaction of human immunodeficiency virus (HIV)-derived
vectors with wild-type virus was analyzed in transduced cells. Vector
transcripts upregulated by infection had no measurable effect on HIV
type 1 (HIV-1) expression but competed efficiently for
encapsidation, inhibiting the infectivity and spread of HIV-1 in culture and leading to mobilization and recombination of
the vector. These effects were abrogated with a
self-inactivating vector.
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TEXT |
Gene transfer vectors based on human
immunodeficiency virus type 1 (HIV-1) have shown significant potential
for gene therapy (1, 12, 20, 21, 28). However, several
safety concerns must be addressed before their clinical testing
(8, 14, 19, 26, 30). Among them are the possible adverse
interactions with wild-type virus in recipients who are or become
infected by HIV-1. cis-acting sequences in the vector are
responsive to the regulatory proteins of HIV-1
(19; Fig. 1) and could
affect HIV-1 replication and lead to the mobilization and recombination of the vector in infected cells. To address experimentally these issues
we infected lymphocytes previously transduced by HIV-derived vectors and monitored HIV-1 replication and vector expression. Control
cells were transduced by a murine leukemia virus (MLV) vector or an HIV
vector with a deletion in the long terminal repeat (LTR) (
U3) that
prevents transcription and activation by HIV-1 infection
(31).
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FIG. 1.
Schematic drawing of the types of vectors used in this
work. The transduced proviral forms are shown. All vectors shown carry
an internal expression cassette for EGFP driven by the PGK promoter
(PGKp). The following viral cis-acting sequences
are labeled and shown as dark boxes if derived from HIV-1 or as
crosshatched boxes if derived from MLV: the LTRs with the constituent
U3, R, and U5 regions, the leader sequence containing the major splice
donor site (SD) and the packaging signal ( ), the portion of the
gag gene included for optimal packaging efficiency crossed
out to indicate that the reading frame is closed by mutationsthe
env-derived splice acceptor sites (SA), and the RRE for the
HIV vectors. The deletion of enhancer (enh) or enhancer and promoter
sequences (enh/pro) from the U3 region of the LTR is indicated for
the MLV and the self-inactivating U3 HIV vectors, respectively. The
types of vector transcripts discussed in the text are indicated by
dotted arrows.
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Replication of HIV-1 is inhibited in cells transduced by
HIV-derived vectors.
Human SupT1 lymphoid cells were transduced by
vectors expressing the enhanced green fluorescent protein (EGFP) from
an internal phosphoglycerokinase (PGK) promoter (Fig. 1). Vectors were
produced by transient transfection of 293T cells with matched transfer and gag pol packaging constructs, derived either from HIV-1
or MLV, and a third plasmid expressing the vesicular stomatitis virus (VSV) envelope (21, 30). The U3 HIV vector was produced
by a transfer construct in which the 3' LTR sequence from base pair 
418 to 18 relative to the beginning of R was deleted. The
transduction of this vector replicates the deletion in the upstream LTR
(self-inactivation [31]). Vector titers were
determined in HeLa cells and used at similar multiplicities of
infection (ranging from 5 to 20 in different experiments). Pools of
EGFP-positive transductants and control nontransduced cells were
challenged with two different amounts of HIV-1 (Fig.
2A). Virus stock was produced by the
transfection of 293T cells with the proviral plasmid R8, a
lymphocytotropic hybrid of the HXB2D and NL43 isolates, which expresses
all HIV reading frames (9). No significant difference in HIV
replication was observed in nontransduced cells and in cells transduced
by MLV or U3 HIV vector. However, in HIV-transduced cultures, the peak of p24 antigen accumulation in the supernatant was delayed in an
infection dose-dependent manner, and the cells remained viable much
longer after infection.
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FIG. 2.
HIV-1 replication is inhibited in lymphocytes transduced
by HIV-derived vectors. The inhibitory activity is mediated by the
vector LTR. (A) SupT1 cells transduced by HIV-derived ( ), U3
HIV-derived
( ), and
MLV-derived
( ) vectors
encoding EGFP or nontransduced cells ( ) were infected with the
indicated p24 equivalent of HIV-1 and scored for the accumulation of
p24 antigen in the culture supernatant (upper panels) and cell
viability (lower panels). (B) SupT1 cells were transfected (grey
markers, with each marker representing an individual clone) or
transduced () with HIV-derived vector encoding the neomycin
resistance gene, selected in G418-containing medium, and infected with
HIV-1 as above; control cells are also shown (). HIV-1 replication
was inhibited in all the cultures containing the vector, independent of
the mode of delivery. (C) Primary CD4-positive lymphocytes were
transduced with HIV-derived () and U3 HIV-derived
( ) vectors
encoding EGFP and selected for transgene expression or were not
transduced and mock sorted () and infected as described above. The
averages of duplicate determinations are shown for a representative
experiment of three performed.
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Cells infected by HIV-1 may become resistant to subsequent challenge
with the virus due to interference with viral entry by
downmodulation
of the CD4 receptor from the cell surface (
17)
or possibly
at later steps preceding viral gene expression (
29).
However, it is unlikely that any one of these mechanisms was
responsible
for the inhibition observed in transduced cells, as the
vector
does not transfer viral genes and was packaged by a construct
with all HIV gene products known to induce CD4 downmodulation
deleted
(
30). Moreover, direct immunostaining showed no difference
in CD4 levels between parental and transduced cells (data not
shown).
To rule out the possibility that the process of infection
by vector
particles establishes the interference, we introduced
an HIV vector
expressing the neomycin resistance gene into SupT1
cells by
electroporation of the corresponding plasmid and cloned
the
transfectants in a selective medium. HIV-1 replication was
significantly inhibited both in the transfectant clones and in
a pooled
population of transduced cells obtained as described
above (Fig.
2B).
These results indicate that the inhibition of
HIV-1 replication in
transduced cells is independent of the mode
of vector delivery and of
transgene type and is mediated by functional
HIV LTRs in the
vector.
The inhibition of HIV-1 replication and improved cell survival were
also observed in primary lymphocytes (Fig.
2C). CD4-positive
lymphocytes were obtained from healthy blood donors by Ficoll
gradient
centrifugation and negative immunoselection of CD8-positive
cells,
stimulated with anti-CD3- and anti-CD28-coated Dynal beads
for 1 day,
transduced with EGFP vectors as described above, selected
by
fluorescence-activated cell sorting (FACS), and cultured in
AIM-V
medium (Gibco) with 100 U of interleukin 2 (Chiron) per
ml for virus
challenge. HIV-1 replication was inhibited only in
cells transduced by
the HIV vector with intact
LTRs.
The initial steps of HIV-1 infection are not inhibited in
transduced cells.
To clarify the mechanism of inhibition we
analyzed the steps of HIV-1 replication in cells transduced by
different types of vectors. We used virions transiently complemented by
HIV-1 gp120 (pEnvHXB plasmid [25]) or VSV G
glycoprotein (VSV.G) in a single-round infection assay (10).
VSV.G was used to increase the efficiency of infection (3,
5). The viral genome had a deletion in the env gene
and contained the bacterial chloramphenicol acetyltransferase (CAT)
gene in place of the nef gene (HXBH10envCAT plasmid
[25]). All infected cells had similar levels of CAT
activity 3 days after infection (Fig. 3).
CAT activity was not detected in cells exposed to virus produced
without an envelope, and it was dramatically reduced in cells
infected by a virus carrying a defective integrase (25).
These data indicate that the inhibitory activity of the vector on
HIV-1 replication is exerted at a postintegration step.
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FIG. 3.
Lymphocytes transduced by HIV-derived vectors are
permissive to all stages of HIV-1 infection, leading to the integration
and expression of the early genes. SupT1 cells, transduced as indicated
on the left, were infected by HIV-1 virions containing the CAT gene in
place of nef and pseudotyped by the HIV-1 or VSV envelope.
Duplicate cultures infected by the types of virions shown on top were
extracted and assayed for CAT activity after the indicated dilution.
All the cultures infected by the same viral pseudotype had similar
levels of CAT activity, indicating that the integration and expression
of the early genes were not affected by the vector. The VSV pseudotype
was much more infectious than the HIV pseudotype, as shown by the
higher dilution of extract yielding similar CAT activity. No activity
was detected in cells exposed to virions produced without envelopes.
CAT expression was dependent on the integration of the HIV genome, as
shown by the comparison between cells infected by virus carrying
wild-type or mutant defective integrase (D116A). The migration of the
different acetylated forms of chloramphenicol is indicated on the
right. A representative experiment of three performed is shown.
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We then analyzed vector expression in the transduced SupT1 cells. As
expected, Northern analysis showed that HIV infection
stimulates vector
transcription from the LTR and leads to the
accumulation of both
unspliced and spliced transcripts (Fig.
4).
FACS analysis showed that some of
these transcripts contribute
to EGFP expression, as the superinfection
of transduced cells
results in the appearance of a distinct population
of cells with
increased expression of EGFP (Fig.
5). These cells represented
the infected
fraction as shown by secondary staining with phycoerythrin
(PE)-conjugated anti-p24 antibodies (KC57-RD1; Coulter). In contrast,
EGFP expression was unaffected by infection of cells transduced
by the
U3 HIV vector.
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FIG. 4.
Northern analysis of lymphocytes transduced by the EGFP
vectors indicated on the bottom and infected by the p24 equivalent of
HIV-1 virions indicated on the top, probed for expression of the EGFP
gene. The expected positions of transcripts initiated from the LTR or
the internal PGK promoter are indicated. HIV-1 infection increases
transcription from the vector LTR. The transcripts accumulate partly
unspliced and partly spliced into acceptor sites downstream from the
RRE or in the PGK promoter sequence, thus contributing to transgene
expression. Note the smaller size of the PGK-driven transcript and the
lack of HIV-induced transcripts in cells transduced by the
self-inactivating U3 HIV vector. All lanes contained similar amounts
of RNA by ethidium bromide staining (data not shown).
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FIG. 5.
FACS analysis of SupT1 cells transduced by HIV-derived
or U3 HIV-derived vector expressing EGFP and infected by HIV-1
virions pseudotyped with (+) or without () the VSV envelope,
after immunostaining for p24. All HIV-infected cells in the culture
transduced by vector with intact LTRs overexpress EGFP, while transgene
expression was unaffected in cells transduced by the U3 vector. Note
that all the cells shown were transduced by EGFP vectors and so are
fluorescent; nontransduced cells, either infected or not with HIV-1,
mapped to the left of the FACS diagram (data not shown).
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Vector transcripts initiated from the LTR contain the transactivation
response element (TAR) and the Rev response element
(RRE), binding
sites for the Tat and Rev proteins of HIV (
17).
Kim et al.
(
13) and Corbeau and Wong-Staal (
6) reported a
decreased expression of transfected HIV-1 proviral plasmid in
cells
transfected (
13) or transduced with HIV vectors
(
6).
Corbeau and Wong-Staal also showed a decreased
expression of a
Tat- or Rev-dependent reporter gene cotransfected with
a Tat or
Rev plasmid in HIV vector-transduced cells (
6).
These results
indicated a decoy effect of the vector RNA as previously
observed
for TAR and RRE (
4,
15,
16,
27). However, probing
an
actual infection, we did not find significant differences in
expression
of early (see CAT expression described above) or late genes
of
HIV-1 in cells transduced by HIV vectors. The expression of the
late
gene product p24 was similar in cells transduced by vector
with
wild-type or
U3 LTR by immunostaining (Fig.
5) and immunocapture
in
the supernatant (data not shown). Thus, the decoy activity
of vector
transcripts on the Tat and Rev proteins of the infecting
virus was too
small an effect to be noticeable in a single-cycle
infection and
consequently to explain the observed inhibition
of HIV-1
replication.
Vector transcripts compete for encapsidation and reduce transfer of
the viral genome.
We then rescued HIV-1 particles from the
infected transduced cells to measure the encapsidation and transfer of
the viral and vector genomes. SupT1 cells transduced with EGFP vectors
were infected by virions generated by 293T cells transfected with the plasmid pR8.7, an NL43-derived construct with all four accessory genes
deleted, and a plasmid encoding VSV.G to make the first round of
infection more efficient. The newly released virions were collected and
assayed for RNA content and infectivity. RNA was purified from
p24-matched viral pellets and analyzed by RNase protection
(HybSpeed RPA kit; Ambion) (Fig. 6). The
templates for the riboprobes were prepared by using the
Lig'nScribe kit (Ambion). A 556-bp fragment (nucleotides 677 to 1233 of HIV-1 proviral DNA) was amplified from the R8 plasmid by PCR with
primers 1s (5'-GCTCTCTCGACGCAGGACTCGG-3') and 2as
(5'-GTGATATGGCCTGATGTACCAT-3'), and a 566-bp fragment was
amplified from pHR2 vector plasmid with primers 1s and 3as
(5'-GTGCTACTCCTAATGGTTCAA-3'). The fragments were ligated to
an adapter containing the T7 promoter and used as templates for a
second PCR with primer 1s and a T7 primer provided with the kit.
32P-labeled antisense riboprobes 1 and 2 (Fig. 6B) were
then generated with T7 RNA polymerase, purified, hybridized with virion
RNA, digested with RNase A/T1 mix (0.5/20 U), and analyzed by
electrophoresis and autoradiography. The protection patterns of vector
and virus RNA are easily distinguished by the difference in length of
the protected fragments as explained in the legend to Fig. 6. Only in
virions released by cells transduced with HIV-derived vector with
wild-type LTR the protection pattern of the vector RNA was superimposed
on that of the viral genomic RNA (Fig. 6A). As the virion samples were
adjusted to an equal content of p24 and had similar amounts of total
RNA determined by phosphorimager analysis, it is evident that the
vector RNA competes efficiently for encapsidation and reduces
significantly the amount of HIV-1 RNA in the particles (approximately
sixfold).
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FIG. 6.
RNase protection analysis of virions collected from
transduced cells showing the efficient encapsidation of the vector and
displacement of the viral genome. (A) Virions were collected from
HIV-1-infected SupT1 cells previously transduced by HIV-derived vector
with wild-type or U3-deleted LTR or not transduced, as indicated
on top. Vector particles were from 293T transfectants. RNA was
extracted from the indicated amounts of particles (by p24 content) and
analyzed for RNase protection of two probes derived from the
virus sequence (NL43 isolate [probe 1]) or the vector sequence (HXB2
isolate [probe 2]). Each probe encompasses the HIV-1 leader sequence
and the first portion of the gag gene and gives a distinct
protection pattern of vector versus virus RNA. (B) The vector sequence
differs from the virus genome for the truncation, frameshift (fs), and
additional mutations (mut) introduced in gag for vector
construction and for the indicated 2-bp mismatches between the sequence
of the two viral isolates (pale arrows). As these differences cause
fragmentation of the probe, the expected size in base pairs of the
protected fragments is shown in panel B, and the actual size of
the major protected bands is indicated to the right of the gels in
panel A. The protection pattern of the vector RNA is
superimposed on that of the viral genome only in virions released
by cells transduced by vector with the wild-type LTR. A decreased
encapsidation of the viral genome is concurrently observed.
The additional lanes in the gel to the left were loaded with
mock-purified pellets from noninfected (Not inf.) transduced SupT1 as
negative controls (ND, p24 not detectable), showing also the
digestion of unprotected probe, and with the untreated probe. One of
two similar experiments performed is shown. nt, nucleotide; SD, splice
donor site.
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Rescued virions were then tested for gene transfer by adding an equal
amount of p24 antigen to cultures of C8166, a T-lymphoblastoid
cell
line with high sensitivity to HIV infection. Four hours after
infection
unadsorbed virus was removed and the cells were further
cultured in the
presence of 5 µM ritonavir (Abbott Laboratories)
and the CD4
[Cys(Bzl)]
84-Fragment 81-92 (C2796; Sigma) to limit viral
spread and syncytium
formation. After 3 days, the infected cultures
were scored for
EGFP and Gag expression by FACS analysis (Fig.
7). The number
of Gag-positive cells per
nanogram of p24 antigen infected was
reduced approximately fourfold for
the virions released from cells
transduced by HIV vector with wild-type
LTR compared to the virions
released from all other cells.
Concurrently, a fraction of cells
infected by this virus expressed
EGFP, which indicated efficient
vector mobilization. No EGFP-positive
cells were found in the
culture infected by virus derived from cells
transduced with the
U3 HIV vector. Thus, only some of the virions
produced by transduced
cells propagate the virus, while others transfer
the vector. The
observed reduction in transfer of the viral genome
should be amplified
during each round of infection and significantly
decrease the
rate of HIV-1 spreading in the culture.
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FIG. 7.
HIV-derived vectors are mobilized by HIV-1 infection of
transduced cells. C8166 lymphocytes were used to score the virions for
transfer of the HIV-1 gag gene, by immunostaining with
anti-p24 antibodies, and of the vector, by EGFP fluorescence. Virions
were collected from SupT1 cells transduced by the EGFP vectors
indicated on top and adjusted to a p24 content of 8 ng for infection.
Three days after infection C8166 cells were analyzed by FACS after
immunostaining for p24. Virions derived from cells transduced by vector
with the wild-type LTR transferred the viral genome less efficiently,
as indicated by the percent of p24-positive cells, but mobilized the
vector, as indicated by the appearance of EGFP-positive cells. No
vector mobilization was detectable from cells transduced by the
self-inactivating U3 vector. Immunostained, uninfected C8166 cells
are shown in the right panel. A representative experiment of three
performed is shown.
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Interestingly, a reduced capacity of the virions to transfer the viral
genome was not fully compensated by the transfer of
the vector. This
could be explained if the vector RNA cannot accomplish
all
preintegration steps as efficiently as the viral genome, for
example,
for lack of some
cis-acting elements. Also, it is expected
that some of the virions produced by the transduced cells are
heterodimeric and that template switching during reverse transcription
may generate recombinants between the vector and the virus
(
11).
Some of these recombinants will score negative both
for Gag and
EGFP expression. Of note, in Fig.
7 a distinct
population of bright
EGFP-positive, p24-negative cells is observed in
the culture infected
by virions derived from HIV-transduced cells. In
the experiment
shown in Fig.
5, all EGFP-bright cells are p24
positive, as expected
from the presence of the HIV-1 genome expressing
the Tat protein.
The unique population of Fig.
7 may therefore have
received a
recombinant genome coding for both EGFP and a functional Tat
protein
but unable to express Gag. Taken together, these results
indicate
that the encapsidation of vector RNA displaces the viral
genome
and leads to reduced particle infectivity, vector mobilization,
and recombination, all of which further reduce the delivery of
intact
viral genomes to target
cells.
Given the high degree of conservation among different HIV-1 strains
within the sequences involved in encapsidation, it is
expected that the
interactions described in this study will apply
to most primary
isolates of the virus. Therefore, in view of anti-HIV
therapy, it is
attractive to think that following vector-mediated
transduction, HIV-1
itself may deliver antiviral therapeutics
to physiologically
relevant targets in vivo through vector mobilization.
In this case,
however, adjustments in the design of the vector
may be required if the
therapeutics target functions also necessary
for vector production. On
the other hand, it is difficult to predict
whether the inhibitory
effect of HIV vectors described here will
be similarly significant in
vivo, where the transduced cells may
be highly
diluted.
An important aspect of this study is that no interaction of HIV-1 with
a self-inactivating vector was detected even under
permissive in vitro
conditions. These data provide direct experimental
evidence of the
increased biosafety achieved by this type of vector
(
18,
31). It is thus expected that the use of a self-inactivating
vector would greatly reduce the risks associated with recombination
between vector and viral genomes and allow a more stringent control
of
transgene expression. Of note, the risks associated with recombination
must be considered as a function of both the vector sequence and
the
transgene delivered. In the case of vectors derived from HIV-1
or HIV-2
(
2,
7,
24), the vector sequences are derived
from laboratory
strains of the viruses and have a limited potential
of increasing the
diversity or pathogenetic potential of the existing
viral clades. A
different case may apply to vectors derived from
lentiviruses of
nonhuman origin (
22,
23). In this case, the
consequences of
their possible recombination with HIV-1 and of
the introduction of
novel sequences in the viral pool infecting
humans should be carefully
considered.
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ACKNOWLEDGMENTS |
Anatoly A. Bukovsky and Jin-Ping Song contributed equally
to this work.
We are indebted to Didier Trono for suggestions, Tom Dull, Michael
Scott, Minh Nguyen, and Michael Kelly for help with some of the
experiments, and Heinrich Göttlinger for the HIV-CAT constructs.
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FOOTNOTES |
*
Corresponding author. Present address: Laboratory for
Gene Transfer and Therapy, IRCC Institute for Cancer Research,
University of Torino Medical School, S.P. 142, 10060 Candiolo (Torino),
Italy. Phone: 39-011-993.3226. Fax: 39-011-993.3225. E-mail:
lnaldini{at}ircc.unito.it.
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Journal of Virology, August 1999, p. 7087-7092, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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