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Journal of Virology, January 2001, p. 809-820, Vol. 75, No. 2
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.2.809-820.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effects of Homology Length in the Repeat Region on
Minus-Strand DNA Transfer and Retroviral Replication
Que
Dang1,2 and
Wei-Shau
Hu2,*
Department of Microbiology and Immunology,
School of Medicine, West Virginia University, Morgantown, West
Virginia 26506,1 and HIV Drug Resistance
Program, National Cancer Institute, Frederick Cancer Research and
Development Center, Frederick, Maryland 217022
Received 13 July 2000/Accepted 25 October 2000
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ABSTRACT |
Homology between the two repeat (R) regions in the retroviral
genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the
3' R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of
replication, viral titers from the vector with a full-length downstream
R were compared with viral titers generated from the other four vectors
with reduced R lengths. Viral titers generated from vectors with R
lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region
were significantly lower. Because expression and packaging of the RNA
were similar among all the vectors, the differences in the viral titers
most likely reflected the impact of the homology lengths on the
efficiency of minus-strand DNA transfer. The molecular nature of
minus-strand DNA transfer was characterized in 63 proviruses. Precise
R-to-R transfer was observed in most proviruses generated from vectors
with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were
predominantly used to generate proviruses from vectors with 3- or 6-nt
homology. Reverse transcription using RNA transcribed from an upstream
promoter, termed read-in RNA transcripts, resulted in most of the
aberrant transfers. These data demonstrate that minus-strand DNA
transfer is homology driven and a minimum homology length is required
for accurate and efficient minus-strand DNA transfer.
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INTRODUCTION |
Retroviruses are RNA viruses that
replicate through a DNA intermediate (49). Most retroviral
particles contain viral RNA; upon infection of the target cells, viral
RNA is copied into DNA by the viral enzyme reverse transcriptase (RT)
(2, 27, 51) and then integrates into the target cell
genome to form a provirus (49). Host cell RNA polymerase
II transcribes the provirus to generate viral RNA transcripts; the
full-length viral RNA is packaged into viral particles to serve as
genetic material for the next round of viral infection
(7).
Retroviruses have evolved to adapt to the dual phase of the life cycle.
Two of the adapted features are the genome structure and the mechanism
by which the viral DNA is synthesized. The proviral genome contains two
long terminal repeats (LTRs), one at each end of the viral DNA
sequences (7, 50). The LTR is composed of three sections,
unique 3' (U3), repeat (R), and unique 5' (U5) regions; each plays
important roles during the viral life cycle (7). The U3
region contains the promoter from which viral RNA transcripts are
expressed (7, 36). R and U5 are both important in the
process of reverse transcription of the viral RNA into DNA, U5 for the
initiation and R for the extension of viral DNA synthesis (7, 26,
48). The viral RNA transcript, which includes sequences from the
upstream R to the end of downstream R, is generated by the host cell
RNA polymerase II and is shorter than the provirus (7, 36,
50). It is advantageous for the viral DNA to contain an active
promoter at the 5' end of the sequences to ensure active RNA
transcription; however, the upstream U3 is missing from the viral RNA,
which is the template for viral DNA synthesis. Therefore, retroviruses
undergo two strand transfer steps to first regenerate the LTR by
joining the U3 and R-U5 sequences and then to duplicate the LTR and
place it at both ends of the viral DNA.
Reverse transcription is initiated near the 5' end of the viral RNA by
using a tRNA primer that has hybridized to the primer binding site
(PBS) adjacent to the U5 sequence (7, 11). Because viral
RNA is plus-sense, the first strand of DNA synthesized is referred to
as minus-strand DNA. Minus-strand DNA synthesis copies U5 and R, in a
step termed minus-strand DNA transfer, and reverse transcription
switches to using the 3' viral RNA as a template and continues DNA
synthesis (7, 11, 48). This step joins together the U3, R,
and U5 sequences. Minus-strand DNA synthesis continues to copy the RNA
template, including the U3 and adjacent polypurine tract (PPT)
sequences. Once minus-strand DNA synthesis passes the PPT, RT makes a
specific cut in the RNA strand at the 3' end of the PPT and uses the
RNA as a primer for plus-strand DNA synthesis (5), which
copies U3, R, U5, and the first 18 nucleotides (nt) of the tRNA
sequences (7). This plus-strand DNA is then transferred to
the 5' end of the viral DNA to duplicate the LTR. Once DNA synthesis is
completed, the resulting viral DNA has two LTRs, one on each end of the
viral genome (11).
Minus-strand DNA transfer joins U3 with R-U5 to regenerate the LTR
during DNA synthesis. Although it is a critical step of reverse
transcription, the exact mechanism of minus-strand DNA transfer remains
unclear. The current hypothesis is that minus-strand DNA synthesis
copies U5 and R to form minus-strand strong-stop DNA and that the RNase
H function of RT degrades the RNA template in the DNA-RNA hybrid
(5, 47, 52). The single-stranded minus-strand strong-stop
DNA contains an R region that is complementary to the R region at the
3' viral RNA. Using this complementarity, the nascent DNA can hybridize
to the 3' viral RNA, and the reverse transcription complex can continue
copying the U3 sequences (7).
This hypothesis predicts that minus-strand DNA transfer depends on the
hybridization of the two R regions; thus, the RNase H activity of RT
and the homology in the R region are critical for minus-strand DNA
transfer. It has been shown that abolishing the RNase H activity of RT
prevents minus-strand DNA transfer (5, 47), which supports
this hypothesis. However, little is known about the requirement of R
homology during minus-strand DNA transfer.
The R regions of different retroviruses vary in size, from 15 nt in
mouse mammary tumor virus (MMTV) to 247 nt in human T-cell leukemia
virus type 2 (HTLV-2) (33, 42). Premature minus-strand DNA
transfer has been observed in murine leukemia virus (MLV), spleen
necrosis virus (SNV), and human immunodeficiency virus type 1 (HIV-1)
at low frequencies, with R regions of 69, 82, and 97 nt, respectively
(24, 25, 28, 37, 55). These events indicated that partial
R sequences are able to mediate strand transfer in these viruses. The
effect of R homology length in HIV-1 was examined by truncation of the
3' R; delayed viral replication kinetics were observed when the 3' R
was shortened to 30 or 15 nt (3). The effect of removing R
homology was examined by using a chimeric vector containing R regions
from MLV and SNV that did not have significant homology
(55). The chimeric vector replicated at a reduced titer
compared with its counterpart containing two highly homologous R
sequences; molecular analyses indicated that most of the minus-strand
DNA transfer events were mediated by short stretches of homologous
sequences. Taken together, these data indicated that most retroviruses
probably could use a shorter R sequence to mediate minus-strand DNA
transfer. However, the effect of R homology length on the efficiency of
minus-strand DNA transfer is unclear.
In this report, we explored the impact of R homology length on the
efficiency and accuracy of minus-strand DNA transfer. Using MLV-derived
vectors and a system that allowed only one round of viral replication,
we examined the ability of 3, 6, 12, 24, and 69 nt of R homology to
mediate minus-strand DNA transfer. The molecular nature of the strand
transfer and transfer junctions was also analyzed.
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MATERIALS AND METHODS |
Plasmid construction and definition.
Retroviral vectors were
constructed using standard cloning techniques (40). All
primer and linker sequences are available upon request. In this report,
the p in the vector name (e.g., pMMQD3) refers to the plasmid, whereas
the vector name without the p (e.g., MMQD3) refers to virus derived
from this plasmid.
Briefly, pLXSN (32) was digested with
HindIII (located downstream of the simian virus 40 [SV40] promoter), treated with Klenow fragment of the DNA polymerase,
digested with XbaI (located in the downstream U3 region),
and then treated with calf intestinal alkaline phosphatase to produce a
4.6-kb DNA fragment containing the SV40 promoter and the plasmid
backbone. Plasmid pAR2 (55) was digested with
SalI, located upstream of the hygromycin phosphotransferase B gene (hygro) (12), treated with Klenow
fragment, and then digested with XbaI, located in the
downstream U3 region. This procedure generated a 1.4-kb DNA fragment
containing hygro. Ligation of the hygro DNA
fragment to the digested pLXSN backbone generated pMSM2, an MLV-based
vector plasmid containing the SV40 promoter and hygro. Next,
pMSM2 was digested with ClaI plus NdeI and
treated with calf intestinal alkaline phosphatase to excise the
downstream LTR. To construct pMSM3, a linker containing the MLV PPT,
the attachment site (att), and a BglII site was
ligated to the digested pMSM2. A 101-bp linker containing the SNV R
region (82 nt) was inserted into the unique BglII
restriction site in pMSM3 to generate pMSM4. Various linkers generated
from annealed synthesized oligonucleotides were inserted into the
BglII and MluI sites of pMSM4 to generate pMSM6,
pMSM12, pMSM24, and pMSM69. These linkers contained different lengths
of MLV R sequences; each linker also contained a unique restriction
enzyme site marker. These restriction enzyme sites were
Bst1107, HindIII, EcoRI, and
ClaI, for pMSM6, pMSM12, pMSM24, and pMSM69, respectively.
It was later found that SNV R sequences do not provide an adequate
signal for the cleavage of RNA transcription in murine cells. To
introduce a functional polyadenylation signal for these constructs, a
0.4-kb DNA fragment containing the SV40 polyadenylation signal was
inserted into these plasmids. The SV40 DNA fragment was isolated from
pJD220svhygro (8), which had been digested with
ClaI and Bst1107, followed by treatment with Klenow fragment, and was inserted into plasmids digested with SalI and treated with Klenow fragment. This insertion
generated pMMQD3, pMMQD6, pMMQD12, pMMQD24, and pMMQD69 from pMSM4,
pMSM6, pMSM12, pMSM24, and pMSM69, respectively.
Cells, transfections, and virus propagations.
D17 and PG13
cells were obtained from the American Type Culture Collection. D17 is a
dog osteosarcoma cell line that is permissive to infection by MLV
(38). PG13 is a murine cell line that expresses MLV
gag-pol and gibbon ape leukemia virus env
(31). Both cells were grown in Dulbecco's modified
Eagle's medium supplemented with 6% (D17) or 10% (PG13) calf serum
(HyClone Laboratories, Inc.). Penicillin (50 U/ml, Gibco) and
streptomycin (50 µg/ml, Gibco) were also added to the medium. Cells
were maintained at 37°C with 5% CO2. Hygromycin
(Calbiochem) selection was performed at 120 µg/ml for D17 cells and
240 µg/ml for PG13 cells.
Viral vectors were transfected into PG13 cells by the calcium phosphate
precipitation method (
40). Transfected PG13 cells
were
pooled, expanded, and plated at a density of 5 × 10
6
cells per 100-mm-diameter dish. Fresh medium was added to the
cells
24 h before the virus was harvested. Virus-containing cell
culture
medium was centrifuged at 6,000 ×
g for 10 min to
pellet
cellular debris. The supernatant was then aliquoted for use in
the infections, RT assay, and isolation of cell-free virion
RNA.
Tenfold serial dilutions of each virus were used to infect D17 cells.
Viral infections were performed in the presence of Polybrene
(50 µg/ml) for 4 h at 37°C with 5% CO
2. The infected
cells were
subjected to hygromycin selection, and viral titers were
determined
by the number of hygromycin-resistant D17 cell colonies.
Individual
D17 cell clones containing proviruses were isolated for
analysis
of minus-strand DNA transfer
junctions.
RNA isolation and analysis.
PG13 cells were plated at a
density of 5 × 106 cells per 100-mm-diameter dish.
Cellular RNA was isolated 48 h later using Trizol (Gibco/BRL) as
specified by the manufacturer. The integrity of the cellular RNA was
examined by gel electrophoresis and the inspection of the rRNA bands.
To obtain cell-free virion RNA, viruses were harvested as described
above and concentrated by centrifugation at 25,000 rpm
for 90 min in a
Beckman SW28 rotor. Viral pellets were resuspended
in diethyl
pyrocarbonate-treated water. This preparation was divided
into two
aliquots, one for the RT assay (described below) and
the other for the
RNA analysis. To monitor for any RNA loss during
the isolation
procedure, an aliquot of SNV (CG4) (
10) was added
as an
internal control to the viral preparation used for RNA analysis.
Sodium
dodecyl sulfate (0.1%, final concentration) and tRNA (200
µg/ml,
final concentration) were added to lyse the mixture of
viruses.
Phenol-chloroform extractions were performed and the
viral RNA was
precipitated with ethanol by standard methods. Viral
RNA was
resuspended in diethyl pyrocarbonate-treated water. Alternatively,
cell-free virion RNA was isolated using the QIAamp viral RNA kit
(Qiagen).
RNase protection assay.
RNase protection assay analyses were
performed on the cellular and cell-free virion RNA as specified by the
manufacturer (Ambion); these results were quantified using the
ImageQuant program on a PhosphorImager (Molecular Dynamics).
RT assay.
Viruses were harvested and concentrated as
described above. RT assays were performed using standard procedures
(2, 13, 51). Briefly, an aliquot of each virus was added
to a reaction mixture containing 50 mM Tris (pH 8.0), 0.6 mM
MnCl2, 60 mM NaCl, 0.5% IgePal CA-630 (Sigma), 1 U of
RNasin (Boehringer Mannheim Biochemicals) per µl, 0.05 µg of
oligo(dT)/µl, 0.1 µg of poly(A)/µl, 80 µM dTTP, 10 mM
dithiothreitol, and 10 µCi of [3H]dTTP (72 Ci/mmol;
ICN). The reaction mixture was incubated at 37°C for 1 h and
then precipitated with 1 ml of 10% ice-cold trichloroacetic acid for
1 h on ice. The viral mixture was filtered through a 0.45-µm-pore-size GN6 Metricel membrane (Gelman Sciences) and washed
twice with 10% trichloroacetic acid. Radioactivity incorporated into
the newly synthesized DNA by RT was measured using a scintillation counter.
PCR and DNA sequencing.
Hygromycin-resistant D17 cell clones
were isolated. Each cell clone was expanded and then lysed
(16). The lysate was incubated at 60°C for 1 h and
then at 95°C for 10 min. Primers located at the 3' end of
hygro and in U5 were used to amplify a segment of proviral
DNA by PCR. The U5 primer was biotinylated and was used to isolate the
PCR product using Streptavidin magnetic beads (Dynal Inc.) as specified
by the manufacturer. After denaturation of the DNA, the biotinylated
single-stranded DNA was collected for direct sequencing using an
AutoRead kit (Pharmacia). Alternatively, PCR products were sequenced
directly using a BigDye termination cycle sequencing ready reaction kit
(PE Applied Biosystems).
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RESULTS |
Retroviral vectors used to study minus-strand DNA transfer during
reverse transcription.
A series of five vectors was utilized to
examine the effects of R homology length on minus-strand DNA transfer;
the vector structures are illustrated in Fig.
1. These vectors were derived from the
pLN series of plasmids (32) and contained
cis-acting elements from Moloney murine leukemia virus
(MoMLV) and/or Moloney murine sarcoma virus, a virus derived from
MoMLV. The cis-acting sequences from MoMLV and Moloney
murine sarcoma virus contain high homology; for simplicity, these
sequences are referred to as MLV sequences in this report. The five
vectors contain identical sequences except for the downstream R
regions. Each vector contains the SV40 promoter, hygro
(12), and MLV-derived cis-acting elements, such
as the upstream LTR, PBS, extended packaging signal (
+),
PPT, and att. The downstream LTR of these vectors was
replaced with the 3' att, a variable stretch of homology to
MLV R, SNV R, and the polyadenylation signal from SV40. The SNV R and
SV40 polyadenylation signals were added to replace the signal located in MLV R. SNV R was first inserted with the intention of using the
polyadenylation signal within these sequences. It was later found that
SNV R was not sufficient in providing the polyadenylation signal within
this sequence context in murine cells (data not shown). A DNA fragment
containing the SV40 polyadenylation signal was then inserted into these
plasmids. It had been shown previously that the function of the SV40
polyadenylation signal was not affected by SNV R when the two sequences
were present in tandem (18-20).
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FIG. 1.
Structures of the MLV-based vectors used to examine the
effects of different homology lengths in the R region on minus-strand
DNA transfer. R, MLV R; SV pro, SV40 promoter; hygro,
hygromycin phosphotransferase B gene; PPT, polypurine tract;
att, attachment site; r in black box, SNV R region; SV ter,
SV40 polyadenylation signal. Various vectors contained different
lengths of the downstream MLV R sequences as indicated.
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Different lengths of the MLV R sequences were inserted at the 3' end of
the viral genome within the vectors pMMQD3, pMMQD6,
pMMQD12, pMMQD24,
and pMMQD69, which contained 3, 6, 12, 24, and
69 nt of the downstream
R sequences, respectively. All of the
various lengths inserted at the
downstream R started with the
5' end of the R; for example, pMMQD3
contained the first 3 nt
of R. The viral RNAs generated from these
vectors would have complete
R and U5 regions at the upstream sequences,
and the downstream
sequences would contain 5' portions of R or the
entire R region.
During reverse transcription, RT used a tRNA primer
and copied
U5 and R to form minus-strand strong-stop DNA. The 3' end of
the
nascent DNA was complementary to the downstream R sequences in
the
viral RNA; the length of this complementarity varied from
3 to 69 nt.
If the length of R between the nascent DNA and RNA
was sufficient to
mediate minus-strand DNA transfer, than it was
expected that the virus
would replicate efficiently and most of
the minus-strand DNA transfer
would be from R to R (R-to-R transfer).
In contrast, if the length of R
complementarity was not sufficient
to mediate minus-strand DNA
transfer, then it was expected that
the virus would have reduced
replication efficiency and the minus-strand
DNA transfer events would
be aberrant in
nature.
MMQD69 viral RNA had two full-length MLV R regions, mimicking the
structure of the wild-type viral RNA, and thus served as
a positive
control for the viral replication and minus-strand
DNA transfer
efficiency. The full-length R should mediate efficient
and precise
R-to-R minus-strand DNA transfer. The RNA transcripts
from all the
other vectors would terminate using the polyadenylation
signal in SV40
sequences. To ensure that MMQD69 had the same 3'
sequences in its RNA
transcripts as the transcripts from the other
vectors, the AATAAA
sequences in the 3' MLV R were changed to
AAGGAA,
which had been previously demonstrated to abolish the
polyadenylation signal (
15,
44,
45). The change of 2 nt
in
these sequences was not expected to affect minus-strand DNA
transfer
for the following reasons. First, these 2 nt were located
far from the
transfer junction, 49 and 50 nt away from the 5'
end of R. Second,
vectors with two R regions containing few mismatches
in the internal
regions have been reported to replicate efficiently
(
32,
55).
The viral RNA of MMQD24 had 24 nt of homology in the R region; this was
approximately one-third the homology length in wild-type
MLV. The viral
RNA of MMQD12 had 12-nt homology in the R region;
this was close to the
shortest naturally occurring R in MMTV (15
nt) (
7,
33). It
had been previously observed that 6-nt homology
could be used to
mediate minus-strand DNA transfer, although with
unknown efficiency
(
55). The viral RNA of MMQD6 had 6-nt homology
between the
two R regions; this vector allowed us to test the
efficiency of
minus-strand DNA transfer mediated by 6-nt homology.
The viral RNA of
MMQD3 had 3-nt homology between the two R regions;
this vector was used
to examine whether homology shorter than
6 nt could be used to mediate
minus-strand DNA
transfer.
Experimental protocol used to examine the effect of R homology
length on minus-strand DNA transfer.
The protocol used in this
study is outlined in Fig. 2. Each
retroviral vector was separately transfected into PG13 helper cells and
the transfected cells were subjected to hygromycin selection. Hygromycin-resistant cells transfected with each vector were separately pooled; all of the cell pools used in these experiments contained at
least 600 colonies. From the viruses harvested from these transfected cell pools, one portion was used for the measurement of RT activity, a
second portion was used for the isolation of cell-free virion RNA, and
the third portion was used to infect D17 target cells. Infected D17
cells were subjected to hygromycin selection, and the number of
resistant colonies was used to determine the viral titer. Viral titers
were standardized to RT activity. In addition, hygromycin-resistant D17
target cell clones were isolated. Portions of the proviral genomes were
amplified by PCR and characterized by DNA sequencing to determine the
molecular nature of minus-strand DNA transfer.
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FIG. 2.
Experimental protocol used to study minus-strand DNA
transfer in one round of retroviral replication.
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Cellular RNA and cell-free virion RNA were isolated from the
transfected PG13 cells and the viruses harvested from these cells,
respectively. The integrity and concentration of the cellular
RNA were
determined by visualization of the rRNA bands in agarose
gels and by
spectrophotometer readings, respectively. To monitor
the recovery of
virion RNA isolation, an aliquot of wild-type
SNV was added to the
cell-free virions prior to the concentration
of the virus particles by
ultracentrifugation as an internal control.
The amounts of vector RNA
in the transfected cells and released
virions were analyzed by RNase
protection
assay.
Effect of R homology length on viral replication.
Three
independent sets of transfection and infection experiments were
performed using different DNA sets for each experiment. Viral titers
were determined and were standardized to the RT activity. A summary of
the standardized viral titer is shown in Fig.
3. The positive control of the
experiment, MMQD69, had two full-length copies of MLV R; this vector
generated titers with a mean of 4.3 × 102 CFU/ml.
MMQD24 and MMQD12 generated titers with means of 3.3 × 102 and 2.7 × 102 CFU/ml, respectively.
The titers generated by these three vectors were not significantly
different (P = 0.46 for MMQD69 and MMQD24, P = 0.27 for MMQD69 and MMQD12, and P = 0.559 for
MMQD24 and MMQD12; two-sample t test). This similarity in
the viral titers indicated that the ability of the virus to replicate
was not significantly affected by reducing the R homology from 69 nt to
24 or 12 nt, approximately one-third or one-sixth of the length of
wild-type R.
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FIG. 3.
Standardized viral titer. The number of
hygromycin-resistant colonies was used to calculate the viral titer.
The RT activity of each viral sample used for infection was measured
and utilized to standardize the viral titer. Error bars represent the
standard errors of the means from three independent experiments.
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In contrast, MMQD6 generated titers with a mean of 0.34 × 10
2 CFU/ml; these titers were approximately 13-fold lower
than those
generated by MMQD69. The differences between the titers of
MMQD6
and those from the three vectors with longer R homology
were statistically
significant (
P < 0.03,
P < 0.02, and
P < 0.02 when MMQD6 was compared
with
MMQD69, MMQD24, and MMQD12, respectively; two-sample
t
test).
MMQD3 generated titers with an average of 0.29 × 10
2 CFU/ml. These titers were also significantly different
from those
of the first three vectors (
P < 0.03,
P < 0.01, and
P < 0.02 when
MMQD3 was compared
with MMQD69, MMQD24, and MMQD12, respectively;
two-sample
t test). The titers generated by MMQD6 and MMQD3 were
not
significantly different from each other (
P = 0.58;
two-sample
t test).
These data indicated that a minimum length of R homology was needed for
the retrovirus to mediate minus-strand DNA transfer
to allow efficient
viral replication. When the viral genome had
more than 12 nt of
homology in R, the viral titers remained similar
to those from virus
containing 69 nt of homology. In contrast,
when the R homology length
was reduced to 6 or 3 nt, the viral
titers dropped significantly. The
alteration in the viral titers
among these constructs most likely
reflected the efficiencies
of minus-strand DNA transfer during reverse
transcription. However,
we could not rule out the possibility that the
differences among
the viral titers in these constructs were caused by
other factors,
such as the amounts of RNA in the transfected cells or
the viral
RNA packaged in the virions. To address these possibilities,
we
examined the amount of cellular RNA and cell-free virion
RNA.
Similar expression and packaging of vector RNA among all
constructs.
To examine the level of viral RNA expression in cells
transfected with various constructs, total cellular RNAs were isolated from transfected cell pools. Equal amounts of RNA isolated from each
cell pool were used in RNase protection assays to determine the amount
of virus-specific RNA. The strategy and probes used in these analyses
are shown in Fig. 4. Two types of RNA
transcripts were expected to be generated from these constructs, one
from the U3 promoter and the other from the SV40 promoter (Fig. 4A). Only the U3-derived transcripts contained + and were
expected to be packaged efficiently for viral replication; therefore,
the amounts of U3-derived transcripts were measured. U3-derived RNA
transcripts should also contain SV40 promoter sequences, which were
lacking in SV40 promoter-derived RNA transcripts. A 202-nt RNA probe
containing the 3' end of the SV40 promoter and the 5' end of the
neomycin phosphotransferase gene (neo) (21) was
generated by in vitro transcription using a DNA fragment amplified from
pLXSN (32) as a template. Viral transcripts from all
constructs driven by the U3 promoter should contain the SV40 promoter
sequences but not neo (Fig. 4A). Therefore, it is expected
that the SV40 promoter sequences but not the neo sequences
should be protected during the RNase digestion and generate a 103-nt
protected fragment. A representative RNase protection assay of the
cellular RNA from transfected cell pools is shown in Fig.
5A. The levels of U3-derived viral RNA
expression in cells transfected with different constructs were similar
(Fig. 5A and data not shown).
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FIG. 4.
Strategy and probes used to detect vector RNA levels in
transfected cells and cell-free virions by RNase protection assays. All
abbreviations are the same as in Fig. 1. (A) Detection of vector RNA.
Two types of transcripts were expected to be generated from the vector:
those derived from the SV40 promoter and those derived from the
upstream U3. The SV-neo probe, generated from a DNA fragment
amplified from pLXSN, was used to detect U3-derived transcripts. This
probe contained sequences from both the SV40 promoter and the neomycin
phosphotransferase gene (neo). Only the SV40 promoter
sequences were protected in the RNase protection assay. (B) Detection
of internal control RNA. The probe was generated from an amplified DNA
fragment, using pRD136 as the template, and contained a portion of
adenovirus tripartite leader sequence (AV tl) and a portion of SNV
gag. Only the gag portion of the probe was
protected in the RNase protection assay.
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FIG. 5.
RNase protection assay analyses of the expression and
packaging of vector RNAs. Lanes: M, RNA molecular weight markers; P,
full-length probe; 3, 6, 12, 24, and 69, samples from cells or viral
samples derived from vectors pMMQD3, pMMQD6, pMMQD12,
pMMQD24, and pMMQD69, respectively. The intensities of the
bands were quantified by PhosphorImager analysis (ImageQuant program).
(A) Vector RNA expression in cells transfected with different vectors.
(B) Vector RNA from cell-free virions harvested from the
vector-transfected cells. (C) SNV cell-free virion RNA used as an
internal control to monitor the yield of virion RNA isolation. The
probe shown in Fig. 4A was used to detect vector RNA shown here in
panels A and B, whereas the probe shown in Fig. 4B was used to detect
internal control RNA shown here in panel C.
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To quantify the amount of RNA in the cell-free virions released by the
transfected cells, viruses were harvested from these
transfected cells.
Prior to isolation of the viral RNA, each virus
sample was mixed with
an aliquot of SNV, which served as an internal
control to monitor the
loss of the viral RNAs during the isolation
procedure and RNase
protection assay. The amount of cell-free
virion RNA from each sample
was measured by RNase protection assay
with the same
SV40-
neo probe described for the cellular RNA analyses.
The
amount of the SNV RNA (internal control) in each sample was
measured by
RNase protection assay using a 224-nt RNA probe generated
from pRD136
(
30), which partially hybridized to SNV
gag
(Fig.
4B) to produce a 174-nt fragment. SNV and MLV are distantly
related
viruses; they do not contain significant homology in
gag at the
nucleotide sequence level. Representative RNase
protection assay
analyses of vector viral RNA and internal control SNV
RNA are
shown in Fig.
5B and C, respectively. These data showed that
the
cell pools transfected with different vectors produced similar
amounts of cell-free virion RNA (Fig.
5B and C and data not
shown).
These experiments demonstrated that cells transfected with different
vectors had similar RNA expression and that the vector
RNAs were
packaged and released at similar levels. Therefore,
the differences in
the viral titers generated by these constructs
were not caused by RNA
expression or packaging but most likely
reflected the impact of the R
homology lengths on minus-strand
DNA
transfer.
Characterization of the molecular nature of minus-strand DNA
transfer events using different lengths of R homology.
To study
the molecular nature of minus-strand DNA transfer events, individual
cell clones containing proviruses were isolated from five independent
infection experiments and from separate cell culture dishes. These cell
clones were generated with low multiplicities of infection (<0.001)
such that most of the cells should only contain one provirus
(probability of double infection, <0.00001). DNA lysates were
generated from these cell clones and used to amplify a portion of the
proviral structures. If minus-strand DNA transfer occurred from the R
of the minus-strand DNA to the R of the 3' viral RNA (R-to-R transfer),
the resulting viral DNA should contain hygro, PPT,
att, R, and U5 at the 3' portion of the genome (Fig.
6A). Using primers near the 3' end of
hygro and in U5, the PCR-amplified DNA fragment should be
0.45 kb in length (Fig. 6A). These primers should also be able to
amplify proviral genomes that did not use R-to-R transfer. Reverse
transcription of all of these vectors should be initiated from the PBS
and copy U5 and then R prior to strand transfer. Therefore, regardless of the acceptor template used for minus-strand DNA transfer, all of
these proviruses should have U5 sequences at the 3' end of the viral
DNA. The infected cell clones were selected based on the
hygromycin-resistant phenotypes; consequently, the proviruses from
these cell clones should contain hygro. Therefore,
proviruses using aberrant minus-strand DNA transfer during viral
replication should also have hygro and U5 sequences to allow
amplification of the viral genome. However, the size of the DNA
fragments amplified by PCR should not be 0.45 kb but should vary
depending on the locations of the donor and acceptor templates.
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FIG. 6.
Strategy used to analyze minus-strand DNA transfer
junctions. (A) Expected minus-strand DNA transfer and predicted PCR
product size. Thick lines, viral RNA; dotted lines with arrows, nascent
DNA with arrows pointing toward the direction of DNA synthesis; open
arrows, PCR primers; all other abbreviations are the same as in Fig. 1.
(B) Representative agarose gel showing the products of PCR
amplification from cell clones infected with various vectors. Lanes: M,
molecular size markers in kilobases; 3, 6, 12, 24, and 69, lysates used
for the PCR that were derived from cell clones infected with MMQD3,
MMQD6, MMQD12, MMQD24, and MMQD69, respectively.
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A representative agarose gel containing PCR-amplified DNA products from
different cell clones is shown in Fig.
6B. Most of
the PCRs using
lysates infected with vectors having at least 12-nt
homology (MMQD12,
MMQD24, MMQD69) gave rise to the expected 0.45-kb
DNA fragment (Fig.
6B
and data not shown); 37 of 40 proviruses
had the 0.45-kb fragment,
whereas the other three proviruses generated
DNA fragments of other
sizes. In contrast, PCR analyses from proviruses
generated by vectors
with 3- or 6-nt homology produced very different
results. Only 3 of 12 MMQD6 proviruses had the 0.45-kb DNA fragments,
whereas the other 9 proviruses produced DNA fragments of other
sizes (Fig.
6B and data not
shown). Of the 11 proviruses generated
from MMQD3, none had the 0.45-kb
DNA fragment, and all had DNA
fragments of different sizes (Fig.
6B and
data not
shown).
The molecular nature of minus-strand DNA transfer from these proviruses
was characterized by directly sequencing the PCR products.
These data
are summarized in Table
1. Of the 40 proviruses that
produced the 0.45-kb DNA fragments, 37 were produced by
vectors
with at least 12-nt homology and 3 were from MMQD6 (Table
1,
column 2); all 40 of these proviruses were generated by R-to-R
minus-strand DNA transfer. Moreover, all of the 40 transfer junctions
were precise; we did not observe misincorporation, deletion, or
addition of sequences. Sequencing analyses were also performed
on
the PCR DNA fragments generated from the 23 proviruses yielding
different sizes that varied from 0.43 to 2.0 kb. Two transfer
patterns
emerged from these analyses based on the usage of donor
and
acceptor templates: misaligned minus-strand strong-stop DNA
transfer and minus-strand DNA transfer using "read-in" RNA
transcripts
(see below).
The first pattern, misaligned minus-strand strong-stop DNA transfer,
was observed only in one provirus generated from MMQD3;
a summary of
the transfer pattern is shown in Fig.
7.
The PCR
fragment from this provirus was 0.51 kb and contained most of
the 3' end of
hygro, a portion of PBS, a portion of
+, the entire R, and U5. From these sequences, we
deduced that
the DNA was generated by two successive transfer events:
one from
R to
+ during minus-strand DNA transfer, and
the other from PBS to
hygro (Fig.
7A). Sequence alignment
indicated that there was a 5-nt
homology between the end of R and the
region of
+ that served as an acceptor template; these 5 nt were probably
used to mediate minus-strand DNA transfer (Fig.
7B).
Sequence
alignment also showed a 9-nt homology between the PBS and the
3'
hygro that bridged the two sequences, indicating that it
was
used to mediate the second strand transfer event (Fig.
7B). Figure
7C illustrates the possible events that generated this provirus.
During
reverse transcription, minus-strand DNA transfer used the
5-nt homology
to transfer to the
+ region; RT proceeded to copy 105 nt
of the
+ sequence and 11 nt of the PBS. RT then used the
9-nt homology
to switch the template to the 3'
hygro and
continued the DNA synthesis.
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FIG. 7.
Misaligned minus-strand DNA transfer using strong-stop
DNA. PBS, primer binding site; +, extended packaging
signal. (A) Illustration of the structure of an amplified PCR fragment
from MMQD3 provirus. (B) Determination of transfer junctions by DNA
sequence analyses and sequence alignment. Bold letters represent
provirus sequences, whereas regular letters represent vector sequences.
Open boxes indicate homology used for transfer. (C) Proposed strand
transfer events used to generate the provirus.
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|
Minus-strand DNA transfer from read-in RNA transcripts.
DNA
sequencing analyses of 22 proviruses with aberrant PCR fragment sizes
indicated that all of them contained portions of U3 sequences or the
entire U3 region. Generally, viral transcripts are directed by upstream
U3 sequences. These U3-derived RNA transcripts should begin with R,
contain sequences between the two LTRs and then the downstream U3, and
end with the downstream R prior to the addition of poly(A). Because the
U3 sequences from the downstream LTR were deleted in these vectors, U3
sequences were not expected to be present in the viral transcript (Fig.
8A). However, we observed many proviruses
containing U3 sequences; furthermore, all of these proviruses had
precise U3-R junctions and most of them used sequences from U3 or the
plasmid backbone upstream of U3 as donors for strand transfer. This
result indicated that these proviruses were generated from RNA
transcripts containing the U3 sequences located in the upstream LTR.
Therefore, we propose that these proviruses were generated from RNA
transcripts expressed from promoters upstream of U3 and were termed
"read-in" RNA transcripts (Fig. 8A). We hypothesize that during DNA
transfection of the vector into the helper cells, vector DNA integrated
into the helper cell genome randomly, with some of the DNA integrating
close to other promoters. The transcript expressed by these promoters
could read into the vector sequences and generate RNAs containing all
or a portion of the U3 sequences, followed by R, U5, and the rest of
the vector sequences.
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FIG. 8.
Detection of read-in RNA transcripts by RNase protection
assay. All abbreviations and symbols are the same as those in Fig. 4
and 5. (A) Three types of RNA transcripts that can be detected by RNase
protection assay. (B) Strategy and probe used to detect read-in RNA
transcripts. The full-length probe contains viral sequences (open
boxes) corresponding to R, parts of U3 and U5, and a 30-nt nonviral
sequence (gray box). The expected protected fragments are shown below
the probe. (C) Results from a representative RNase protection assay
examining total cellular RNA from transfected cells. Three distinct
bands are observed, corresponding to the three different transcripts.
In this example, the amounts of the read-in and R-terminated
transcripts are 3 to 5% and 6 to 8% of the U3-derived RNA
transcripts, respectively. RNA quantification was performed using a
PhosphorImager and the ImageQuant program.
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|
To examine whether read-in transcripts could be detected in transfected
cells, RNase protection assays were performed with
cellular RNA from
transfected cells (Fig.
8A). Three types of
viral transcripts might be
present in the cellular RNA that could
be detected by the RNase
protection assay: the U3-derived transcript
and two types of
transcripts from the upstream promoters. One
type of transcript, an
R-terminated transcript, would read into
U3 and terminate at the
upstream R; these RNA transcripts would
lack the rest of the viral
cis-acting elements and were not expected
to be packaged and
contribute to provirus formation. The other
type of RNA transcript, a
read-in transcript, would read into
U3, read through the poly(A) signal
in R, and contain the rest
of the viral sequences. Read-in transcripts
were expected to be
the precursors of the proviruses with U3
sequences.
A 219-nt probe was generated to hybridize to portions of the upstream
LTR shared by all of the vectors (Fig.
8B). Covering
the junctions of
U3, R, and U5, this probe hybridized to the 3'
73 nt of U3, the entire
R (69 nt), and the 5' 47 nt of U5. In
addition, the last 30 nt of this
probe contained sequences that
did not hybridize to the viral genome
and should be degraded during
the assay. In an RNase protection assay,
the U3-derived transcript
would contain R and U5 sequences and should
protect 116 nt of
the probe (69 nt of R and 47 nt of U5). R-terminated
transcripts
derived from the upstream promoter would contain U3 and R
and
should protect 142 nt of the probe (73 nt of U3 and 69 nt of R).
The read-in transcript would contain U3, R, and U5 and thus should
protect 189 nt of the probe (73 nt of U3, 69 nt of R, and 47 nt
of U5).
For all of the vectors used in this study, most of the
sequences from
the downstream LTR, including the U3 and U5, were
deleted; therefore,
this assay would not be complicated by the
U3 or U5 sequence near the
3' end of the
transcripts.
A representative gel from an RNase protection assay is shown in Fig.
8C. In this and other analyses, three bands were detected
from each
cellular RNA sample, with sizes corresponding to the
U3-derived,
R-terminated, and read-in transcripts. In all samples,
the U3-derived
transcripts were the dominant RNA species; read-in
RNA transcripts and
R-terminated transcripts were approximately
3 to 8% and 6 to 11% of
the U3-derived transcripts,
respectively.
Minus-strand DNA transfer junctions generated from read-in RNA
transcripts.
In the read-in RNA transcripts, R regions were no
longer at the 5' end of the RNA. DNA sequencing analyses revealed that
during reverse transcription, DNA synthesis did not terminate at the end of the R but continued to copy the U3 sequences; in some cases, RT
copied the entire U3 and went into the plasmid backbone adjacent to the
U3 sequences. These U3 or plasmid backbone sequences, instead of R,
were used as donor sequences for minus-strand DNA transfer.
In most of these transfer reactions, various regions of the 3' end of
hygro or sequences downstream of
hygro were used
as
acceptors (Fig.
9A), probably because
the function of
hygro was
selected in these experiments. A
summary of the locations of the
donor and acceptor sites is shown in
Fig.
9B. Sequence alignments
indicated that 15 proviruses were
generated by a single transfer
event, using U3 or other upstream
sequences as donors and 3'
hygro as acceptors. An example of
this type of junction is shown in
Fig.
9C; in this portion of the
provirus structure, 3'
hygro sequences
were joined to U3
sequences. Sequence alignment showed that 5-nt
homology existed at the
junction between the U3 and
hygro sequences;
the
minus-strand DNA transfer event was presumably mediated by
these 5-nt
sequences.
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FIG. 9.
Minus-strand DNA transfer of DNA synthesized from
read-in RNA transcripts and locations of the donor and acceptor
templates used for transfer. (A) Proposed model for the generation of
proviruses containing U3 sequences by reverse transcription of read-in
RNA transcripts. Pro, upstream promoter; zigzag line, host-cell genome.
(B) Summary of locations of the donor and acceptor templates used for
minus-strand DNA transfer. Locations of donor templates are shown below
the vector structure, whereas locations of the acceptor templates are
shown above the vector structure. Numbers above or below the
arrows indicate the vector from which the provirus was
derived: 3, pMMQD3; 6, pMMQD6; 12, pMMQD12; and 69, pMMQD69. (C) Example of a provirus generated from MMQD6
using 5 nt of homology between the donor template (U3) and acceptor
template (3' hygro) to mediate minus-strand DNA transfer.
Bold letters represent the proviral sequences, whereas regular letters
represent vector sequences. The open box indicates homology used for
the transfer.
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Three proviruses were generated by more than one transfer event. Two of
these proviruses underwent two transfers to join U3
to 3'
hygro with all the sequences originating from the viral
vectors. In the third provirus, five transfer events were observed
to
join U3 and
hygro with the incorporation of sequences from
an endogenous MLV isolated from
Mus musculus musculus (clone
Mxv4)
(
53). This provirus is likely to be generated from
copackaging
of endogenous MLV followed by subsequent recombination
events.
The exact number of transfers could not be determined in four
proviruses because a small stretch of non-vector-derived sequences
was
identified between U3 and
hygro. Homology searches of these
sequences using the GenBank database identified one of the sequences
as
containing high homology to a submitted sequence from
Candida albicans (accession number
AL033501). It is highly probable
that
these stretches of non-vector-derived sequences were from
the host cell
genome adjacent to the integrated transfected DNA
and thus were
included in the RNA transcripts. During reverse
transcription, RT
copied the vector-derived sequences, continued
to copy some of the host
sequences, and then used these host sequences
to mediate minus-strand
DNA transfer. If this was the case, then
only one transfer event was
used to join the minus-strand DNA
to the 3'
hygro sequences.
The donor and acceptor sequences of 24 transfer events were identified
during the analyses. Among these, 5 transfer events
were performed
without any observable homology and 19 transfer
events were mediated by
short homology. Of the 19 transfer events,
16 were mediated by homology
shorter than 6 nt, 4 were mediated
by 1-nt homology, 4 were mediated by
2-nt homology, 3 were mediated
by 3-nt homology, 3 were mediated by
4-nt homology, and 2 were
mediated by 5-nt homology. In addition, three
transfer events
were mediated by more than 11-nt homology (data not
shown).
| |
DISCUSSION |
The efficiency and accuracy of minus-strand DNA transfer directly
affect the ability of the virus to replicate and to preserve the genome
structure. In this report, we delineate the role of R homology length
in minus-strand DNA transfer.
Impact of R homology length on efficiency and accuracy of
minus-strand DNA transfer.
In these experiments, viral titers
measured the efficiency of minus-strand DNA transfer, whereas the
molecular characterization examined the accuracy of the transfer events
by quantifying the frequencies of R-to-R transfer events. Our data
indicated that removing more than 80% of the length of R in MLV, from
69 to 12 nt did not significantly affect the efficiency or accuracy of minus-strand DNA transfer. However, both the efficiency and accuracy of
minus-strand DNA transfer were significantly diminished when the length
of R was further reduced to 3 or 6 nt. Considering that most of the
proviruses produced by vectors with 3- or 6-nt homology were generated
from read-in RNA transcripts, the efficiencies of accurate minus-strand
strong-stop DNA transfer in these vectors should have been even lower
than the titers indicated. Taken together, these data indicate
that a minimum amount of sequence homology is needed to perform
efficient and accurate minus-strand DNA transfer.
In our MLV-based system, the vector with 12-nt R homology could
replicate with an efficiency close to that of the vector containing
two
full-length R regions. The effect of reducing the R homology
in HIV-1
has been previously studied (
3). Truncations of the
downstream R from 97 nt in wild type to 37, 30, and 15 nt were
generated; decreasing the downstream R to 30 or 15 nt reduced
viral
replication kinetics. Because multiple rounds of viral replications
were allowed and the amounts of capsid-p24 released were used
to plot
the replication kinetics, it was difficult to directly
correlate the
difference between replication kinetics and efficiency
of minus-strand
DNA transfer. In our system, only one round of
viral replication was
allowed and the viral titers were generated
and then used for
statistical analyses. Although there seemed
to be a slight decrease in
the viral titers when the downstream
R was reduced to 12 or 24 nt,
statistical analyses indicated that
the difference was not significant.
In addition, because the R
regions of HIV-1 and MLV are different, we
could not rule out
the possibility that the primary sequence or
secondary structure
of the R region may affect the requirement for the
homology
length.
In our experiment, the downstream R was manipulated, which may affect
other events prior to minus-strand DNA transfer. However,
it was shown
that deletion of the 3' R did not affect viral titer
when other
homologous sequences were supplied for minus-strand
DNA transfer
(
6). It was also shown that viral RNA synthesis
was not
affected by abolishing the polyadenylation signal in the
downstream R
(
45). These data indicated that it is unlikely
that the 3'
R sequences affect the initiation of reverse transcription
and
synthesis of R-U5 DNA. We have shown experimentally that truncation
of
3' R does not affect virus production or viral RNA packaging.
Therefore, the differences in titers that we measured are most
likely
to be solely attributed to the efficiency and accuracy
of the
minus-strand DNA
transfer.
Proposed mechanism for the requirement of R homology and the
function of R.
We propose that a minimum length of R homology is
needed to align the newly synthesized DNA and the 3' RNA in the correct manner during minus-strand DNA transfer; reduction of homology length
to a certain level can result in misalignment and possible dead-end
reverse transcription products.
Although similar viral titers were generated by MMQD6 and MMQD3,
accurate minus-strand DNA transfer was observed in MMQD6
(3 of 12) but
not in MMQD3 (0 of 11). These data agree with our
previous observation
(
55) that 6-nt homology could be used to
mediate
minus-strand DNA transfer. However, 6-nt homology was
not sufficient to
mediate minus-strand DNA transfer with the efficiency
and accuracy of
the wild-type
virus.
If 6-nt homology is not sufficient to mediate efficient minus-strand
DNA transfer, whereas 12-nt homology in R generates a
wild-type
phenotype, what about the lengths between 6 and 12 nt?
By probability,
a 6-nt homology occurs every 4,096 nt; a 10-kb
viral genome is likely
to contain other copies of the 6-nt sequence.
By this calculation, a
7-nt homology occurring once in 16,384
nt is likely to be unique to the
R sequences. However, additional
lengths would reduce the probability
of the minus-strand DNA to
misalign with sequences having slightly less
homology, such as
those with 5- or 6-nt homology. Therefore, it is
possible that
the length of homology can be slightly reduced but
probably not
much from 12 nt before the efficiency and accuracy of the
transfers
are compromised. Indeed, the shortest naturally occurring R
regions
are 15 nt (MMTV) and 21 nt (Rous sarcoma virus) (
7,
33,
46).
If the virus needs only 12 nt to mediate minus-strand DNA transfer, why
do most retroviruses have longer R regions? In these
experiments, we
only examined the role of homology length in reverse
transcription.
However, most of the R regions contain
cis-acting
elements
for other functions as well. For example, the R regions
in many viruses
contain the polyadenylation signal for RNA transcription
(
7). HIV-1 R also has the
trans-activator
response region (
14,
39), which allows the binding of Tat
and Tat-associated cellular
proteins for transcription activation
(
29,
36). The R regions
in HTLV-1 and HTLV-2 contain the
Rex responsive element (
41),
which allows the binding of
Rex protein to regulate the level
of full-length and singly spliced RNA
(
17,
36). MLV R appears
to be important in increasing
cytoplasmic levels of full-length
viral RNA (
54). SNV R
also serves as part of a translation enhancer
(
4).
Therefore, the ability of the R sequences to mediate minus-strand
DNA
transfer is most likely not the only factor that impacts on
the
evolution of the R lengths; other aspects of viral replication
also
have strong influences on R lengths as
well.
Minus-strand DNA transfer can use sequences other than R to mediate
the transfer.
During reverse transcription of wild-type
retroviruses, R sequences are used for minus-strand DNA transfer. It
had been proposed that R sequences evolved to facilitate template
switching and that non-R sequences could not mediate minus-strand DNA
transfer (1). However, in our experiments with the
proviruses that were generated from the read-in RNA transcripts, U3
sequences, plasmid backbone sequences, and possibly cellular sequences
were observed to mediate minus-strand DNA transfer. Furthermore, in a
separate set of experiments, we also found that nonviral sequences
could be used to efficiently mediate minus-strand DNA transfer
(6). Taken together, these data indicate that the
homology, not the sequence context of R, mediates minus-strand DNA transfer.
Short GC-rich sequences were used to mediate aberrant minus-strand
DNA transfers.
Previously, we proposed that GC-rich sequences were
used in minus-strand DNA transfer junctions. However, minus-strand
strong-stop DNA was predominantly the donor template and only two other
junctions had been analyzed (55). In this report, some of
the analyzed proviruses were generated from read-in RNA transcripts and
used sequences other than the strong-stop DNA for minus-strand DNA transfer. This result allowed us to examine the content of the homology
used for the transfer. We analyzed the homology sequences from 16 minus-strand DNA transfer events using 1 to 5 nt of homology; of the 43 nt that were utilized, 29 were C/G (14 C and 15 G) and 14 were A/T (6 A
and 8 T). These data parallel the observation that GC-rich sequences
were used to mediate deletions in MLV and SNV (22, 23, 34,
35).
We also compared R sequences from 10 different retroviruses (data not
shown) and found that in general, R sequences were not
GC-rich (ranging
from 43 to 64%). However, the first nucleotide
of the R region in all
10 viruses was G or C, and the second nucleotide
of the R in 8 of 10 viruses was G or C as well. Together, these
data suggest that GC-rich
sequences may be preferred at the junctions
of the template-switching
events during reverse
transcription.
Minus-strand DNA transfer events are precise and occur without
frequent nontemplate addition of sequences.
In this report, we
analyzed a total of 64 minus-strand DNA transfer junctions: 40 R-to-R
transfer junctions and 24 non-R transfer junctions. We found that all
of the transfer junctions were precise and we did not observe any
nontemplate G additions or misincorporation of bases in the transfer
junctions. In a separate experiment examining the sequence requirement
of minus-strand DNA transfer, we also characterized 31 minus-strand DNA
transfer junctions by DNA sequencing and found that all of the 31 transfer junctions were precise (6). Taken together, a
total of 95 minus-strand DNA transfer junctions have been analyzed and
all were precise.
It was previously reported that nontemplate addition of G occurred
approximately 10% of the time during MLV replication
(
25).
At the 10% frequency, we should have observed the G
addition in
approximately 9 transfer events among the 95 events that
were
analyzed; however, we did not observe any provirus with a G
addition.
This result led us to conclude that nontemplate G addition
does
not occur at a 10% frequency during minus-strand DNA transfer;
rather, minus-strand DNA transfers are precise
events.
Read-in RNA transcripts and reverse transcription products
generated from read-in RNA transcripts.
In these experiments, we
observed RNA transcripts generated from upstream promoters that were
termed read-in RNA transcripts; these are the first demonstrations of
read-in RNA transcripts and proviruses generated from these RNAs.
Several factors contributed strongly to the observation of read-in RNA
transcripts and the resulting proviruses. First, the U3 viral promoter
was most likely weakened by the promoter interference from the internal
SV40 promoter (9); this effect may have strengthened the
upstream promoter and increased the probability of generating read-in
RNA transcripts. Indeed, the weakened U3 promoter was reflected in the
relatively low viral titers generated by these vectors; the vectors
with at least 12-nt R homology consistently generated titers between 2 × 102 and 6 × 102 CFU/ml (Fig. 3
and data not shown) rather than the usual 103 to
104 CFU/ml from PG13-transfected pools (data not shown).
Second, removal of the R homology reduced the efficiency of R-to-R
transfer and enhanced our ability to observe proviruses that used other mechanisms for minus-strand DNA transfer. Third, the removal of the
downstream U3 allowed the observation of a large number of proviruses
that would have otherwise gone undetected. If read-in transcription
occurred in a wild-type provirus, both U3 sequences would be in the
RNA. During reverse transcription, minus-strand DNA can use the U3
sequences to mediate transfer; the resulting provirus would have the
same genotype as those that used the R sequences to mediate
minus-strand DNA transfer.
This observation raised the questions of whether read-in RNA
transcription occurs in nature and what would be the genetic
consequences of such an event. If read-in RNA transcription occurs
in
the infection of wild-type viruses, it is likely to be less
frequent
than the events observed in this experimental system
for the reasons
described above. In most cases, it would have
little evolutionary
impact because the proviruses generated from
these transcripts are
likely to have the same genotypes as those
that were generated from
U3-derived transcripts. However, it is
possible that in rare instances,
read-in RNA transcription can
serve as a mechanism to incorporate host
sequences in viral RNA.
Similar to the RNA read-through transcription
(
15,
44,
45),
these host sequences in viral RNA can be
incorporated into the
proviral genome through high- frequency
recombination events during
reverse transcription. In some of the
proviruses analyzed in this
set of experiments, nonvector sequences
were found in the strand
transfer junctions. One of these sequences had
high homology to
reported sequences in
C. albicans,
suggesting that they were probably
derived from the host genome. These
data suggest that read-in
RNA transcription is a possible mechanism for
oncogene
transduction.
In summary, our findings demonstrate that there is a requirement for a
minimum homology length for accurate and efficient
minus-strand DNA
transfer and retroviral replication. Although
the length required for
minus-strand DNA transfer is significantly
shorter than the R lengths
in most retroviruses, many retroviruses
have evolved to contain longer
R regions for other important functions
affecting the regulation of
viral replication. Many of the features
of minus-strand DNA transfer
observed in this study parallel other
template-switching events during
reverse transcription. For example,
we found that minus-strand DNA
transfer can be mediated by short
stretches of homology, specific
sequences are not required for
the transfer, and transfer junctions are
precise and occur without
addition of sequences. These features are
very similar to those
observed in deletions and nonhomologous
recombination (
22,
23,
34,
35,
56). Therefore, these data
support that minus-strand
DNA transfer occurs via a mechanism similar
to those used in other
template-switching events during reverse
transcription, which
was hypothesized in a recently proposed "dynamic
copy-choice model"
(
43).
| |
ACKNOWLEDGMENTS |
We thank Mithu Molla for the construction of many of the
precursor plasmids needed for these experiments and Vinay K. Pathak for
continuous intellectual input and discussions. We also thank Krista
Delviks, William Fu, Vineet KewalRamani, Vinay K. Pathak, Dexter Poon,
and Terence Rhodes for critical reading of the manuscript and Anne
Arthur for expert editorial help. We thank John Coffin for discussions,
suggestions, and coining the term "read-in RNA transcript." We also
thank Jacqueline Dudley for help with clarifying the length of MMTV R.
This work was partially supported by extramural grants from the
National Institutes of Health (R29 CA 58345-01 to W.-S.H.) and American
Cancer Society (RPG MBC-97322 to W.-S.H.) and intramural funding from
the HIV Drug Resistance Program, National Cancer Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: HIV Drug
Resistance Program, National Cancer Institute, FCRDC, Room 336, Building 535, Frederick, MD 21702. Phone: (301) 846-1250. Fax: (301)
846-6013. E-mail: whu{at}ncifcrf.gov.
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Journal of Virology, January 2001, p. 809-820, Vol. 75, No. 2
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.2.809-820.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
