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Journal of Virology, July 1999, p. 6171-6176, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Contributions of Viral Splice Sites and
cis-Regulatory Elements to Lentivirus Vector
Function
Yan
Cui,
Tomoo
Iwakuma, and
Lung-Ji
Chang*
Department of Molecular Genetics and
Microbiology, Gene Therapy Center, and University of Florida Brain
Institute, College of Medicine, University of Florida, Gainesville,
Florida 32610-0266
Received 28 October 1998/Accepted 9 April 1999
 |
ABSTRACT |
The mobile transgene constructs of most human immunodeficiency
virus (HIV)-based lentivirus vectors currently in use contain viral
long terminal repeats, a 5' untranslated region, gag
sequences, and env sequences that include the
Rev-responsive element (RRE). In this study, we examined the
possibility of deleting HIV splice sites and gag and
env sequences from an HIV type 1 recombinant vector
established in our laboratory as part of our ongoing efforts to improve
this vector system. Mutations in the major splice donor site (SD)
markedly reduced viral RNA expression but had little effect on vector
titer. Deletion of gag or env sequences,
excluding RRE, led to a moderate reduction in vector titer.
Interestingly, deletion of RRE slightly reduced viral RNA expression
but markedly impaired vector function. Combined deletions of RRE,
gag (except for the first 40 nucleotides), env,
and the SD mutation resulted in a twofold increase in cytoplasmic viral
RNA expression and a recovery of vector efficiency to ~50% of the
wild-type level. This increase in cytoplasmic RNA levels is likely to
be due, at least in part, to effects of the TE671 host cells, a human
rhabdomyosarcoma cell line used for vector production in our system, on
the cytoplasmic distribution of spliced and unspliced viral RNA. These
results show that optimal lentivirus vector function can be maintained in the absence of multiple essential viral elements.
| |
TEXT |
Lentiviruses have advantages over
traditional Moloney murine leukemia virus-derived vectors in transient
and long-term transduction of various types of human cells (11,
23, 30, 31). To establish a safe lentivirus vector system, it is
necessary to delete essential viral sequences without compromising
vector functions. The human immunodeficiency virus (HIV) 5'
untranslated region (UTR) contains several essential regulatory
elements, including the primer binding site for initiation of reverse
transcription and a structure of four stem-loops as part of the
packaging signal (
) for genome packaging (13, 15, 28).
Previous studies demonstrated that deletions in this region markedly
reduced packaging efficiency and HIV replication (12, 25).
Besides the four stem-loops in the 5' UTR, other sequences have been
identified throughout the HIV genome as additional packaging signals.
These include 5' gag, pol (13), 3'
env (13, 24), TAR (14, 17, 29), and
the 5' poly(A) hairpin motif in U5 (18, 39). Deletion of any
of these elements from the vector system may diminish vector function.
Another factor affecting vector function is the level of cytoplasmic
viral RNA expression controlled by viral regulatory proteins and
genetic elements (reviewed in references 19 and 20). This regulation is mediated by Rev, which
counteracts the nuclear retention of viral RNA controlled by splice
sites and cis-repressive sequences (CRS) (or inhibitory
sequences [INS]) to allow nuclear export of Rev-responsive element
(RRE)-containing RNAs (3, 7-9, 32, 35). Some of these
CRS/INS elements have been mapped to reside within the splice donor
site (SD) (6, 21, 36), p17 of gag (5, 37,
38), pol (16, 27, 33), and env
(32). Deletion of these elements may affect viral RNA expression and thus vector function.
We have recently established an HIV type 1-based lentivirus vector
system, HP/TV, which produces high vector titers, more than
106 infectious particles per ml upon transfection of human
TE671 cells with three plasmids (pTV, pHP, and pHEFVSV-G), without
detectable replication-competent virus (11). To further
improve the safety and payload of this vector system, we attempted to
delete more of the essential viral elements in pTV. Through mutation
and deletion analyses, the contributions of these genetic elements to
viral RNA expression and vector function were elucidated.
Mutations of the 5' splice site down-regulate cytoplasmic RNA
expression but have only moderate effects on vector titer.
We
first mutated the consensus SD sequence in pTV by PCR site-specific
mutagenesis (primers are listed in Table
1). Mutations of SD from GGTG to GCAG
(SD1) or to GGGG (SD2) resulted in a moderate decrease (10 to 30%) in
vector titer (Fig. 1A). To determine the mutational effects of SD on pTV RNA expression, TE671 cells were transfected with 20 µg of pTV, 2 µg of pCEP4tat, 4 µg of pCMVrev, and 0.2 µg of pXGH5, which encodes human growth hormone, as a transfection control. Forty hours posttransfection, the cells were
lysed and cytoplasmic poly(A)+ RNA was harvested for
Northern analysis, as previously described (10). As shown in
Fig. 1B, wild-type pTV produced four different sizes of RNA: full
length, short intron spliced, a cytomegalovirus (CMV) promotor-driven
nlacZ transcript which comigrated with a spliced RNA
species, and large intron spliced. The SD mutations (SD1 and SD2), as
predicted, abrogated splicing. However, minute amounts of short
intron-spliced RNA, due to the use of a cryptic 5' splice site as
previously reported (36), were detected (Fig. 1B, lanes 3 and 4). It is noteworthy that mutations of SD reduced expression of
cytoplasmic full-length RNA by more than 70% compared with wild-type
vector, possibly due to decreased stability of the unspliced RNA or to
pretermination of transcription. It has been reported that the 5' SD
imposes a suppressive effect on activation of the polyadenylation site
in the 5' long terminal repeat (1, 2), and thus mutations of
SD may activate 5' polyadenylation. The decline in genomic RNA
expression (70%), however, did not correlate with the reduction in
vector titer (10 to 30%). This could be explained by the following two
possibilities. First, the amount of full-length RNA is always in excess
of the viral packaging requirement and thus is not a determining factor
of vector titer. Second, the elimination of spliced RNA by SD mutation abrogates its interference in genomic RNA packaging and thus results in
indirect enhancement of packaging of full-length RNA. In any event, our
results demonstrated that SD itself is not directly involved in HIV
packaging and can be mutated in the vector system.
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FIG. 1.
Analyses of SD mutations. (A) Schematic diagram of SD
mutations and relative vector titers. For virus production, plasmids
pTV, pHP, pHEF VSV-G, and pXGH were used to cotransfect TE671 cells.
Culture supernatants containing viral vectors were harvested at 48 h posttransfection and used to determine viral titer or to determine
transfection efficiency via radioimmunoassay measurement of human
growth hormone. The relative vector titer of each mutant was normalized
against pTV, which was arbitrarily set at 1.00. The titer of pTV was
consistently 2 × 105 to 10 × 105
CFU/ml. The relative titer represents an average of 4 to 7 independent
experiments ± standard error. (B) Northern analysis of
poly(A)+ cytoplasmic RNA of SD mutants (SD1 and SD2) in
transfected TE671 cells. The four major RNA spices are abbreviated as
follows: F, full-length unspliced RNA; ss, short intron-spliced RNA
(from 5' SD to SA in HIV env); CMV+s, CMV promoter-driven
nlacZ transcript plus a spliced RNA population with 5' SD
and a cryptic splice site in the CMV promoter; ls, long intron-spliced
RNA (from 5' SD to a cryptic SA in the nlacZ gene). A
lacZ probe was used for hybridization, as indicated.
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Most of gag and the entire env sequences,
except RRE, are dispensable for optimal lentivirus vector function.
gag and env sequences contain potential packaging
signals, splice sites, and CRS/INS elements. To examine their effects
on vector functions, a series of deletions in gag and
env were generated and tested. An array of deletions of 180, 361, 591, 824, and 1039 bp, in gag and env
(upstream of RRE) was made by PCR site-specific mutagenesis (Table 1;
Fig. 2A). Deletions of env 5'
to RRE and most of gag 3' to the first 255-nucleotide (nt)
coding sequence in pTV had no significant effects on vector titer (dl.1
to dl.4 [Fig. 2A]). Deletion of gag up to the first 40 nt
led to a 30% decrease in vector titer (dl.5 [Fig. 2A]). Northern
analyses showed that these mutants expressed cytoplasmic RNA at levels
similar to that of wild-type pTV (not shown). Therefore, deletions of 5' env and 3' gag did not significantly affect
RNA expression and vector titer. Deletion of gag 5' coding
sequences (dl.5) had a mild effect on vector titer (Fig. 2A), although
it did not affect viral RNA expression (Fig. 2C), suggesting a role for
the gag 5' 255 nt in RNA packaging. These results are
consistent with previous observations that the first 21 to 653 nt of
gag are important for RNA packaging (4, 13, 26, 28,
34).
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FIG. 2.
Analyses of 5' gag and env
deletions. (A) Schematic diagram of 5' gag and 5'
env mutants and relative vector titers. The relative titer
represents the mean of four to six experiments and is normalized to
that of pTV. (B) Schematic diagram of 3' env mutants and
relative vector titers. The relative titer represents an average of
four to nine experiments (except for env.dl.2) and is normalized
against pTV, which was set at 1.00. (C) Northern analyses of
gag and env deletion mutants. The full-length RNA
is denoted by asterisks.
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|
To further dissect the effects of
env sequences on vector
function, six more deletions (dl.1 to dl.6) in
env 3' to
RRE, which
contains two 3' splice acceptor sites (SA), were generated
by
Bal31 exonuclease digestion (Fig.
2B). Deletion of 3'
env SA resulted
in an overall ~40% reduction in vector
titer (Fig.
2B), and Northern
analyses showed reduction in full-length
genomic RNA and abrogation
of the short intron-spliced RNA species
(Fig.
2C, lanes 4 and
5). Therefore, the sequences of
env SA
appear to have moderate
effects on full-length RNA expression as well
as on vector
function.
Deletion of RRE markedly diminishes vector titer.
To determine
the effects of RRE on lentivirus vector function, precise RRE deletion
was made by PCR mutagenesis (Table 1; Fig.
3A). RRE deletion alone led to a marked
reduction in vector titer (>90%). Interestingly, Northern analyses
indicated that the RRE deletion had only moderate effects on vector RNA
expression in TE671 cells (Fig. 3B, lane 2 versus lane 1). This is
inconsistent with previous observations that removal of RRE from HIV
usually abolishes cytoplasmic expression of full-length viral RNA
(24, 34). It is possible that the recombinant vector
backbone might contribute to this phenotype. Alternatively, the
specific cell type (TE671) that we used for production of vectors might
be different from those used by others. To clarify these issues, three
different cell lines
TE671, HeLa, and 293were transfected with
different vector constructspTV, dl.RRE, or pHP-EFgp (a pHP mutant
helper construct with rev deletion [Fig. 3C])in the
presence or absence of a rev plasmid. Cytoplasmic and
nuclear RNAs were harvested and analyzed by Northern blotting. The
results showed that pHP-EFgp did not transport unspliced RNA into the
cytoplasm of transfected cells in the absence of rev (Fig.
3C, lane 2). When rev was present, full-length, unspliced
RNA species were detected in the cytoplasm of all three cell types,
suggesting that a similar Rev phenotype can be seen with a
less-truncated HIV vector construct (Fig. 3C, lane 4). Interestingly,
when RNA of the wild-type pTV construct was examined, differences in
RNA partition ratio were observed in different cell types (Fig. 3D). In
the presence of Rev, HeLa cells exhibited the highest ratio of
cytoplasmic unspliced RNA to spliced RNA (2.38), while 293 cells
exhibited the lowest (0.19) (Fig. 3D, lane 8). However, both HeLa and
293 cells showed a loss of full-length RNA in the cytoplasm when
rev was absent (Fig. 3D, lane 6). TE671 cells, in contrast,
did not exhibit an obvious rev effect when transfected with
either pTV or dl.RRE (TE671 [Fig. 3D], 0.58 versus 0.68 for pTV and
0.44 versus 0.44 for pTVdl.RRE). Therefore, the specific RNA partition
phenotype of the RRE mutant in TE671 is due to both the cell type and
the specific pTV construct that we used.
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FIG. 3.
Effects of RRE deletion on vector titer and RNA
expression in different human cell types. (A) Schematic diagram of RRE
deletion and relative vector titers. (B) Northern analysis of
cytoplasmic RNA of the RRE deletion mutant in TE671 cells. (C) Northern
analyses of full-length pHP-EFgp RNA in the presence or absence of Rev
in TE671, HeLa, and 293 cells. Cells were transfected with pHP-EFgp, a
pHP mutant helper construct with rev deletion, in the
presence (+) or absence ( ) of a rev plasmid. Cytoplasmic
(C) and nuclear (N) RNA was harvested and hybridized with an
env probe from nt 8133 to 8465 of pNL4-3. (D)
Northern analyses of nuclear and cytoplasmic RNA of pTV and pTVdl.RRE
in TE671, HeLa, and 293 cells, in the presence and absence of Rev. A
single HIV env probe was used; it hybridized to both
full-length (F) and spliced (ss) RNA. The ratios of full-length to
spliced RNA in the cytoplasm, which were determined by quantification
of RNA bands with a Fuji BAS1000 phosphorimager, are indicated.
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|
Despite the presence of similar amounts of cytoplasmic full-length RNA
in TE671 cells transfected with wild-type pTV and the
dl.RRE mutant,
the titer of the RRE mutant was less than 10% of
that of wild-type
pTV, suggesting an alternative effect of RRE
on vector function
independent of the known RRE effect on cytoplasmic
viral RNA
expression.
The combination of SD, SA, RRE and gag-env CRS
mutations restores vector titer to 50% of the wild-type level.
The above results suggest that most of the HIV regulatory elements in
pTV, except for RRE, have only moderate effects on vector function. To
examine whether these regulatory elements could be deleted together, we
combined different mutations and analyzed their effects. The
combination of SD-SA mutation generates a vector with a near-wild-type
level of titer (dl.SD1/env.dl.6) (Fig.
4A), which apparently repaired the defect
of the env.dl.6 (SA) mutation (Fig. 2B). When RRE mutation was
included, the resulting dl.SD1/env.dl.6/RRE mutant exhibited a vector
titer close to zero (0.3% of wild-type level) (Fig. 4A).
Interestingly, further removal of gag and env sequences resulted in a mutant, dl.SD1/gag/env/RRE, which showed a
vector titer close to 50% of the wild-type level. This restored vector
function diminished to about 1% of the wild-type level when the SD
mutation was reverted to the wild type (dl.gag/env/RRE [Fig. 4A]),
again suggesting a negative effect of splice site (SD-SA) disparity.
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FIG. 4.
Analyses of combinations of RRE, SD, SA gag,
and env mutations. (A) Schematic diagram of combinations of
mutants and their relative vector titers. Relative vector titers are
averages of four to nine experiments and are normalized against pTV,
which was set at 1.00. (B) Northern analysis of cytoplasmic RNA of
different combinations of mutants, as indicated.
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|
Northern analyses demonstrated that when both SD and SA were deleted
(dl.SD1/env.dl.6) (Fig.
4B, lane 2), full-length viral
RNA expression
was reduced by about 50% but the vector titer was
maintained at 90%
of the wild-type level. Further deletion of
RRE from this mutant did
not reduce RNA expression to a great
extent (dl.SD1/env.dl.6/RRE) (Fig.
4B, lane 3), but vector function
was markedly diminished (~0.3% of
the wild-type level [Fig.
4A]).
This result corroborated the observed
effects of the pTV dl.RRE
mutant (Fig.
3). Surprisingly, when most of
the
gag (except for
the first 40 nt) and the entire
env CRS/INS, including RRE, were
deleted, a twofold increase
in full-length viral RNA expression,
compared to wild-type vector
(dl.SD1/gag/env/RRE) (Fig.
4B, lane
4), with an accompanying increase
in vector titer (~50% of the
wild-type level [Fig.
4A]), was
observed. When the mutated 5'
SD was reverted to generate
dl.gag/env/RRE, expression of full-length
viral RNA was again
suppressed to about 40% of the wild-type level
(Fig.
4B, lane 6) and
vector function was again abolished (~1%
of the wild-type level
[Fig.
4A]). Therefore, only when all of
the CRS/INS regulatory
elements, including SD, most of
gag, the
entire
env, and RRE, are deleted can optimal vector function be
restored.
In summary, we have demonstrated that deletion of essential HIV
regulatory elements, including splice sites,
gag,
env, and
RRE, from the HP/TV vector system is possible
without significant
loss of vector titer. Together with further
deletion of long terminal
repeat elements in pTV from a separate study
(
22), we have reduced
the HIV sequences in pTV to less than
550 bp. This has effectively
increased the lentivirus vector payload to
more than 9 kb, because
the HIV genome is approximately 9,700 nt. These
modifications
have effectively reduced the sequence homology between
pHP and
pTV and thus have greatly improved the safety of this vector
system.
Future modifications of remaining lentivirus sequences will
require
replacement of native viral essential elements with alternative
functional elements while necessary functions for efficient vector
production and gene transduction are
preserved.
| |
ACKNOWLEDGMENTS |
We thank Martin Stoltzfus and Maurice Swanson for critical comments
and Gang Guo and Edward Mason for technical assistance.
Lung-Ji Chang is a Markey Scholar of the Lucille P. Markey Charitable
Trust, and this work was supported by grants from the National Health
and Research Development Program (NHRDP) of Canada and the National
Institutes of Health (HL-59412) of the United States.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Rm. R1-252, ARB,
Department of Molecular Genetics and Microbiology, Gene Therapy Center, University of Florida, Gainesville, FL 32610-0266. Phone: (352) 392-3315. Fax: (352) 392-3133. E-mail:
lchang{at}college.med.ufl.edu.
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Journal of Virology, July 1999, p. 6171-6176, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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