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Journal of Virology, May 2001, p. 4922-4928, Vol. 75, No. 10
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4922-4928.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Transfer of Primer Binding Site-Mutated Simian Immunodeficiency
Virus Vectors by Genetically Engineered Artificial and Hybrid
tRNA-Like Primers
Anette Chemnitz
Hansen,1
Thomas
Grunwald,2
Anders Henrik
Lund,1,
Alexander
Schmitz,1
Mogens
Duch,1
Klaus
Überla,2 and
Finn
Skou
Pedersen1,3,*
Department of Molecular and Structural
Biology1 and Department of Medical
Microbiology and Immunology,3 Aarhus University,
DK-8000 Aarhus, Denmark, and Institute of Virology, Leipzig
University, D-04103 Leipzig, Germany2
Received 19 October 2000/Accepted 14 February 2001
 |
ABSTRACT |
Simian immunodeficiency viruses (SIV) harbor primer binding sites
(PBS) matching tRNA
or
tRNA
. To study determinants of primer usage
in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a
sequence (PBS-X2) with no match to any tRNA. By cotransfection of a
synthetic gene encoding a tRNAPro-like RNA with
a match to PBS-X2, the activity of this vector could be restored to a
transduction efficiency slightly lower than that of the wild-type
vector. A vector with a PBS matching tRNAPro
was functional at a level slightly below that of the wild-type vector,
but higher transduction efficiency could be obtained by cotransfection
of a gene for an engineered
tRNAPro-tRNA
hybrid with a match to PBS-Pro. The importance of tRNA backbone
identity was further analyzed by complementing the PBS-X2 vector with a
gene for a matching x2 primer with a
tRNA backbone, which led to three-
to fourfold-higher titers than those observed for the x2 primer with
the tRNAPro backbone. In summary, our results
demonstrate flexibility in PBS and primer usage for SIVmac239, with
PBS-primer complementarity being the major determinant, in analogy with
previous findings for murine leukemia viruses and human
immunodeficiency virus type 1.
| |
TEXT |
Retroviruses replicate through
reverse transcription of the viral RNA genome into a double-stranded
DNA form that is integrated into the genome of the host. The
virus-encoded reverse transcriptase (RT) enzyme mediates this process
through the use of a host-encoded tRNA. The 3'-terminal 18-nucleotide
(nt) segment of the tRNA primer molecule anneals to the RNA genome at a
complementary sequence known as the primer binding site (PBS). In order
to synthesize a double-stranded DNA copy, two template shifts are
involved. In plus-strand synthesis, the 3' 18-nt segment of the tRNA
primer is reverse transcribed into a DNA copy, which mediates the
second template shift by annealing to the minus-strand DNA copy of the PBS. Accordingly, the PBS sequence of the transduced provirus may
originate from a copy of the genomic PBS RNA sequence or from copying
of the 3' 18 nt of the tRNA primer.
Different tRNA species are used as primers among retroviruses; however,
for each group of viruses, the PBS is stably maintained during
replication. Murine leukemia viruses (MLV) and human T-cell leukemia
virus use a tRNAPro, the avian sarcoma-leukosis
virus (ASLV) group uses a tRNATrp, and a
tRNA is used as a primer for reverse transcription in mouse mammary tumor virus and in human and simian immunodeficiency viruses (HIV and SIV, respectively) (reviewed in
references 33 and 35).
Mechanisms of PBS maintenance during replication have been studied in a
variety of viruses. MLV mutated in the PBS to match the 3' end of other
tRNAs are able to use noncognate primers in multiple rounds of
replication (6, 30, 36). In contrast, PBS-mutated HIV-1
and ASLV may initially replicate, but in time revert to their cognate
tRNA during serial transfer of PBS-mutated viruses (7, 25, 47,
49). The selective reversion and maintenance of the cognate
primer in these viruses may be accomplished by non-PBS determinants.
The lentiviruses of the SIV group have attracted attention due to the
development of animal models for the study of AIDS (9) and
the development of lentivirus vectors for gene therapeutic purposes
(34, 43). Although SIVs and HIV-1 share common features, the SIVmac239 isolate used in this study differs from HIV-1 in lacking
an adenosine-rich loop situated in the U5 hairpin (3, 4),
which has been implicated in determining primer specificity in HIV-1
(19-24, 27, 46, 52). To study the determinants for primer
specificity in SIV, we have established a system in which the viral RNA
genome as well as the tRNA primer may be manipulated. By using
synthetic genes encoding tRNA-like molecules complementary to
PBS-mutated SIV vectors, it is demonstrated that the PBS is the primary
determinant for primer specificity in initiation of first-strand
synthesis and plus-strand transfer resulting in the formation of a
provirus. In this experimental setup, it is possible to study the
effect on the transduction titers exerted by sequences outside the
PBS-complementary 3' 18 nt of the tRNA-like primer, because no
endogenous competitor tRNA molecule is able to compete for binding to
the mutant PBS.
Design of PBS-mutated vectors and engineered tRNA-like
primers.
The PBS of a SIV-based green fluorescent protein (GFP)
vector (SIVGFP) was mutated, and constructs encoding putative tRNA-like primers were designed. SIVGFP-Pro contains a PBS-Pro for which the
corresponding tRNA proline is found in the cell. SIVGFP-X2, however,
harbors a PBS-X2 for which no matching endogenous tRNA exists (Fig.
1). In the case of SIVGFP-Pro, a PBS for
the tRNA proline was introduced into the PBS of SIVGFP by PCR-mediated site-directed mutagenesis (29) in which primer set A
(primers 1 and 2) (Table 1) and primer
set B (primers 3 and 4) generated two PCR products with SIVGFP as a
template. The two PCR products were combined in an overlap extension
reaction with primers 2 and 4. The resulting fragment was cloned into
SIVGFP. For SIVGFP-X2, the PBS was mutated at 11 nt positions that were
originally selected to give a minimal match to any known murine tRNA
(28) with primer set A (primers 5 and 2) and primer set B
(primers 6 and 4). The SIVGFP vector is an env deletion
mutant of SIVmac239, in which the nef gene has been replaced
by the enhanced GFP (EGFP) reporter gene (Fig. 1) (
envgfp in
reference 43).
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FIG. 1.
Vector structure. The SIVmac239-based vector SIVGFP is
shown at the top. The sequences of the introduced PBS mutations are
shown below. The mismatch marker mutation relative to the engineered
corresponding tRNA-like primer is shown in boldface.
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The constructs designed to encode the synthetic tRNA-like primer
molecules ptRNA
x2pro, ptRNA
x2lys-3,
and ptRNA
prolys-3 were constructed from
deoxyoligonucleotides and cloned as described
in reference
28. ptRNA
x2pro is identical to
ptRNA
x2 in reference
28, whereas
ptRNA
x2lys-3 and ptRNA
prolys-3 were constructed
by annealing the 22-nt elongation primer 7 to
the 127-nt primers 8 and
9, respectively. The sequences of primers
8 and 9 were based on that of
tRNA
(GenBank
accession no.
K01797)
(
41). The predicted structures of
ptRNA
x2pro and ptRNA
prolys-3 are
shown in Fig.
2A. The 3' 18-nt sequence
of ptRNA
x2pro matches that of PBS-X2, but
otherwise it resembles the sequence
of tRNA
Pro,
whereas ptRNA
prolys-3 matches PBS-Pro at the 3' end, but
resembles tRNA
.
The synthetic tRNA-like primers
were processed correctly with
regard to trimming of the tRNA precursor
at the 5' and 3' termini,
because the transcript sizes correspond to
the expected 75 and
76 nt for ptRNA
x2pro and
ptRNA
x2lys-3, respectively (Fig.
2B), as shown by Northern
hybridization after
transfection into human BOSC 23 packaging cells
(
37), as described
in reference
28. The
addition of the nonencoded CCA sequence
to the trimmed 3' end of the
synthetic tRNAs was verified by a
tRNA tagging assay (
31),
in which the presence of the CCA tail
was a designed prerequisite (Fig.
2C) for the synthesis of the
radioactively labeled product seen in Fig.
2D.
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FIG. 2.
(A) Structure of the murine tRNAPro
(16) and tRNA (38) and
putative structures of synthetic tRNAs. Mutations relative to
tRNAPro and tRNA are boxed.
(B) Northern hybridization. Ten micrograms of total RNA from human BOSC
23 cells transfected with ptRNAx2lys-3 (lane 2) or
ptRNAx2pro (lane 3) was separated on an 8%
polyacrylamide-urea gel, blotted, and hybridized to an X2-specific
probe (primer 10). The DNA markers are shown in lane 1. (C) tRNA
tagging assay. Total RNA from BOSC 23 cells was incubated with a
biotinylated oligonucleotide complementary to the 3' 18 nt of
tRNAPro (31) or
tRNAx2lys-3 (primer 11), respectively. Following magnetic
separation, the annealed tRNA was extended in the presence of
[32P]dATP and separated on an 8%
polyacrylamide-urea gel. (D) tRNA tagging gel showing total RNA from
BOSC 23 cells incubated with the tRNAPro-specific
oligonucleotide (lane 2). In lane 3, total RNA from BOSC 23 cells
transfected with ptRNAx2lys-3 was incubated with primer 11. Lane 1 shows DNA size markers.
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A mismatch between vector PBS and primer was introduced by altering 1 nt in the PBS of SIVGFP-X2 and SIVGFP-Pro, yielding
SIVGFP-X2m and
SIVGFP-Prom, respectively (Fig.
1), to distinguish
the PBS sequences of
the primer and PBS origin during
replication.
Complementation of PBS mutations by designed tRNA-like
primers.
The effect of PBS mutations on the infectivity of SIVGFP
was determined by transient transfections of 293T cells
(293ts/A1609) (10) with the different mutants
of SIVGFP together with the vesicular stomatitis virus glycoprotein G
(VSV-G)-encoding pHIT-G plasmid (14) following
calcium phosphate coprecipitation (44). Table
2 shows the SIVGFP vector titers in the
supernatant of the transfected cells determined on 293A cells (Quantum
Biotechnologies, Laval, Canada). 293A cells, a subclone of 293 cells,
were used for titration since they show stronger adherence to plastic
dishes than 293T cells. The transfection efficiency was monitored by determination of capsid antigen levels in the supernatant of the transfected cells by employing an HIV-1 capsid enzyme-linked
immunosorbent assay kit (Innogenetics, Ghent, Belgium). The 293A target
cells were seeded in 24-well plates at a density of 5 × 104 cells per well. The following day, the medium
was removed and cells were incubated for 2 to 4 h with serial
dilutions of 200 µl (per well) of the supernatant from the
transfected cells. Two days after infection, the number of GFP-positive
cells was counted. The infectivity of the vectors was calculated by
dividing the vector titer by the amount of capsid antigen and is
expressed relative to the infectivity of wild-type SIVGFP.
Mutation of the PBS of SIVGFP to the X2 sequence reduced the titer by
at least 4 orders of magnitude with essentially no background
(Table
2). By the addition of a synthetic tRNA-like primer,
ptRNA
x2pro, matching PBS-X2, the infectivity of
SIVGFP-X2 was increased
by at least 4 orders of magnitude and was only
fivefold lower
than the infectivity of SIVGFP wild-type particles. The
PBS-mutated
vector could therefore be complemented by the synthetic
ptRNA-like
primer matching the 18 nt of the PBS-mutated
vector.
Direct sequence evidence for ptRNA
x2pro usage was
obtained from amplification with primers 12 and 13 and sequencing the
PBS region
of a mixed pool of cell lysates originating from
transductions
with the supernatant of SIVGFP-X2m transfected cells. In
the left
panel of Fig.
3, the PBS
sequence of the PBS-X2m origin is shown,
whereas the panel to the
right shows the PBS sequence, originating
from a copy of
ptRNA
x2pro, thus providing the evidence
for primer usage. According to the
mechanism of reverse transcription,
the PBS sequence of a transduced
provirus originates from copying the
genomic PBS RNA or the 3'
18 nt of the tRNA primer. Consequently, it
would be predicted
that 50% of the clones would contain the primer
sequence. It was
demonstrated that 5 subclones out of 13 analyzed
contained the
artificial tRNA sequence.
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FIG. 3.
Genetic evidence for tRNAx2pro usage. PCR
products spanning the PBS region from lysates prepared from a mixed
pool of transduced cells were cloned and sequenced. In the left and
right panels are shown the PBS sequences resulting from a copy of the
vector SIVGFP-X2m and primer tRNAx2pro, respectively. The
mismatch positions are indicated by arrows.
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|
Effect of using primers with a tRNA-like
backbone.
A comparison of the infectivity of SIVGFP-Pro in the
presence or absence, respectively, of a hybrid primer,
tRNAprolys-3, which had an acceptor stem matching PBS-Pro
but otherwise resembled the tRNA molecule
(Fig. 2A), showed that cotransfection of ptRNAprolys-3
increased the infectivity fourfold (Table 2). This suggests that
sequences or structures in the backbone of the cognate
tRNA compared to the endogenous
tRNAPro are important for optimal initiation of
reverse transcription or packaging. In these experiments, accurate
quantification of the expression levels is difficult due to abundant
expression of endogenous tRNAs closely homologous to the transfected
tRNAs. However, primer selectivity by the retroviral machinery during primer packaging, placement, and utilization may be the determining factor rather than abundance of the tRNA. To compare the effects exerted by the tRNA sequences outside the PBS complementary region, ptRNAx2lys-3 was designed, containing a
tRNA backbone, but matching the PBS-X2 (Fig. 2A),
and used in transient transfections. The infectivity of the SIVGFP-X2
obtained by cotransfection with ptRNAx2lys-3 resulted in
three- to fourfold-higher transduction titers relative to
cotransfection with ptRNAx2pro (Table
3).
While normalization of transfection efficiency by p27CA antigen levels
within each experiment gave consistent results, normalization
of
transfection efficiency between different experiments was not
found to
be very useful. Although CA antigen levels differed largely
between
different experiments (compare Tables
2 and
3), vector
titers did not
seem to increase in a linear way with increasing
p27CA antigen levels.
Therefore, CA antigen levels might not be
the limiting factor for
vector titer, or increased toxicities
at higher transfection rates
might lead to stronger inhibition
of formation of infectious vector
particles than protein expression.
To confirm that the vector titer
depends on the amount and type
of tRNA, two independent titration
experiments were performed
with cotransfection of increasing
amounts of ptRNA
x2pro and
ptRNA
x2lys-3, respectively, in cotransfections with
SIVGFP-X2 (Fig.
4A). For
amounts of tRNA
expression plasmid up to 1 µg, the transduction
titer increases for a
given amount of vector DNA. Increasing the
amount of ptRNA above the
saturating level seems to have an inhibitory
effect on transduction
efficiency. Throughout the entire range
of DNA concentrations, the
cotransfection of ptRNA
x2lys-3 results in higher
transduction titers than those of ptRNA
x2pro,
with the exception of the experiment using 2.5 µg of
ptRNA
x2pro. Such a preference for
ptRNA
x2lys-3 was not observed in an MLV system using
PBS-mutated Akv-based
vectors in which the addition of
tRNA
x2pro results in slightly higher titers than
those used for addition
of the ptRNA
x2lys-3 (Fig.
4B).
Construction of MLV-based vectors and the titer assay
was described
previously in reference
28. Thus, it seems that
SIV shows
a weak preference for synthetic tRNA-like primers with
a
tRNA
backbone.
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FIG. 4.
Titration of engineered tRNA-like DNAs differing in
their tRNA backbone compositions. Dose-response curves of the effects
of increasing amounts of engineered tRNA-like primer DNA
ptRNAx2pro and ptRNAx2lys-3 on the infectivity
of an SIV-based vector (A) and the titer of an MLV-based vector (B) are
shown. (A) Engineered tRNA-like primers were cotransfected in the
indicated amounts with SIVGFP-X2 and pHITG into 293T cells. The
infectivity (green fluorescence-forming units [GFU] per nanogram of
p27CA) in the supernatant of the transfected cells was determined on
293A cells. (B) Engineered tRNA-like primers were cotransfected in the
indicated amounts with the MLV-based vector pPBS-X2 (28)
into the packaging cell line BOSC 23. Vector titers in the supernatant
of the transfected cells were determined on NIH 3T3 cells. Black and
grey bars represent cotransfections with
ptRNAx2pro and ptRNAx2lys-3,
respectively. Replica experiments are shown for each amount of ptRNA
added.
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Our data demonstrate that complementarity between the PBS and a
matching synthetic tRNA-like primer is the primary determinant
for
selection and use of the tRNA species in initiation of reverse
transcription in SIV. However, we note that the transduction titers
are
lower than for the wild-type vector. An MLV Akv-based vector
carrying
PBS-X2 was greatly impaired in its ability to replicate
in single-round
transfections, but could be rescued by the addition
of the designed
complementary tRNA
x2pro, resulting in a complete
provirus (
28). Similarly, an HIV-1
provirus with a PBS
complementary to the yeast tRNA
Phe relied on the
transfection of a yeast tRNA
Phe, using a
single-round transfection system (
51). Thus, SIVmac239
seems to be similar to MLV, ASLV, and HIV-1, in which the PBS
and tRNA
complementarity is the primary determinant for selection
of the tRNA
primer for initiation of reverse transcription and
the ability to
proceed through a complete replication
cycle.
Northern analysis of the expression levels of the synthetic tRNAs
relative to a control U2 small nuclear RNA probe (
48)
has
shown that ptRNA
x2lys-3 is expressed at higher levels than
ptRNA
x2pro in the producer cells used (data not
shown). However, it is not
clear whether the level of tRNA abundance
per se has any effect
on transduction efficiency, because the amount of
synthetic tRNA
DNA added in the transfections seems to be saturating
(Fig.
4).
Secondary determinants such as non-PBS interactions may be
responsible
for the apparent preference for the
tRNA
backbone, as has been demonstrated for HIV-1.
Viral genomic RNA
sequences other than the A-rich loop may interact
with the primer
in SIVmac239, although interactions between
tRNA
Pro and the Moloney MLV RNA have been found
to be restricted to the
PBS (
13). In HIV-1, primer
placement on the viral genome and
primer packaging may depend on
destabilization of the secondary
structure of the tRNA and the viral
RNA template by the p55
gag precursor (
1,
5,
8,
12,
17,
18,
26,
32,
39,
40), which also seems to be the
case for avian leukosis virus,
but not for MLV (
15).
Primer and RT interaction may also be
necessary for initiation of
reverse transcription (
1,
2,
11,
42,
50), although RT may
recognize the overall L-shaped
tRNA rather than specific features of
its cognate tRNA primer
(
45). Thus, we cannot exclude that
one or several of the possible
non-PBS interactions may contribute to
the observed higher titers
in SIV for the tRNA
backbone in this study;
however, the primary determinant for primer
specificity is the
complementarity between the viral PBS and the tRNA
primer in
SIVmac239.
| |
ACKNOWLEDGMENTS |
The first two authors contributed equally to this work and should
both be considered the first author.
The technical assistance of Ane Kjeldsen and Katrin Bräutigam is
gratefully acknowledged.
A. Schmitz acknowledges support by the German Academic Exchange Service
(DAAD/HSPIII) Program. This work was supported by Bavarian Nordic
Research Institute A/S, the Karen Elise Jensen Foundation, the Danish
Cancer Society, the German Research Foundation (Ue45-3/1), and the
Danish Natural Science and Health Science Research Councils.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular and Structural Biology, Aarhus University, C.F. Moellers
Allé, Bldg. 130, DK-8000 Aarhus, Denmark. Phone: 45 89423188. Fax: 45 86196500. E-mail: fsp{at}mbio.aau.dk.
Present address: Division of Molecular Carcinogenesis, The
Netherlands Cancer Institute, NL-1066CX Amsterdam, The Netherlands.
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Journal of Virology, May 2001, p. 4922-4928, Vol. 75, No. 10
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4922-4928.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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