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J Biol Chem, Vol. 273, Issue 49, 32568-32575, December 4, 1998
From the John Innes Centre, Norwich Research Park, Colney,
Norwich NR4 7UH, United Kingdom
Initiation of DNA plus-strand synthesis in most
reverse-transcribing elements requires primer generation by reverse
transcriptase-associated RNase H at one or more template polypurine
tracts (PPTs). We have exploited infectious clones of the plant
pararetrovirus cauliflower mosaic virus carrying redundant ectopic
plus-strand priming elements to study priming in vivo.
Ectopic priming generated an additional discontinuity in progeny virion
DNA during infection of plants. We found that altering the length of
the 13-base pair PPT by ±25% significantly reduced priming
efficiency. A short pyrimidine tract 5' to the PPT, highly conserved
among diverse reverse-transcribing elements, was shown to play an
important role in PPT recognition in vivo. The predominant
DNA plus-strand 5' end remained 3 nucleotides from the PPT 3' end in
mutant primers that were longer or shorter than the wild-type primer.
Use of an ectopic redundant primer to study
replication-dependent priming was validated by
demonstrating that it could rescue infectivity following destruction of
the wild-type priming elements. We propose a model for plant
pararetroviral plus-strand priming in which pyrimidines enhance
PPT recognition during polymerase-dependent RNase H
cleavages, and suggest that fidelity of primer maturation during
polymerase-independent cleavages involves PPT length measurement and 3'
end recognition by RNase H.
Retro-elements (REs),1
the propagation of which involves copying genomic RNA into DNA mediated
by reverse transcriptase (RT), are found in organisms representing the
major groups of eukaryotes. REs include animal retroviruses, animal and
plant pararetroviruses, and retrotransposable elements (1).
Although REs are diverse, most share several common features. Important
among these is the pol gene product specifying multiple
enzyme activities associated with reverse transcriptase (RT) involved
in RE DNA synthesis and processing (2, 3).
REs have a different origin of replication for synthesis of each strand
of the double-stranded (ds)DNA phase. First, reverse transcription of
the genomic RNA into minus-strand DNA is initiated in most REs by a
host tRNA (4). During minus-strand synthesis, RT-associated RNase H
activity degrades the RNA template (5, 6). This degradation appears to
occur in at least two stages. Processive DNA minus-strand synthesis is
accompanied by a polymerase-dependent, non-processive RNase
H degradation of the template leaving RNA fragments. These fragments
are probably further degraded by subsequent polymerase-independent
RNase H cleavages (7-9). The RNA primer for DNA plus-strand synthesis
is generated during these steps by RNase H cleavage at a specific site
called the polypurine tract (PPT), 13-18 nucleotides long. For REs
producing an integrating linear double-stranded DNA with long terminal
repeats (LTRs), precision of plus-strand initiation is essential for RE
viability as one LTR border is defined by this step. Therefore,
understanding the role of the PPT is important, particularly in
retroviruses where plus-strand initiation is a possible target for
therapeutic control.
Several studies of PPT function have involved analysis of products made
by RT added to RNA primer/DNA template combinations in vitro
(10-13) but it is not clear how such reactions relate to plus-strand
priming in vivo. Mutagenesis of the LTR-associated PPT could
render an RE non-functional in vivo limiting usefulness of
this approach. Members of the lentivirus and spumavirus groups of
retroviruses, and the yeast retrotransposon Ty1, have a second central
PPT colinear with the pol gene (14-16), which could be more
amenable to analysis in vivo. Studies have shown that
mutations in the pol-associated PPT, leaving the colinear
pol amino acid sequence unchanged, impaired priming and
retroviral replication in transfected cells (17, 18) but did not affect
Ty1 transposition in yeast (16). Charneau et al. (18) also
inserted a 29-bp linker containing the PPT sequence into a new site in
the HIV-1 genome, where it primed DNA plus-strand synthesis. With this
approach, the primer could be moved potentially to a new genome site
for analysis without interference from colinear functions.
Plant pararetroviruses, such as cauliflower mosaic virus (CaMV),
offer great potential to study plus-strand priming because clones of
their circular double-stranded DNA genome can be infectious to plants,
and, some members of the group have multiple PPT-associated priming
sites (19). Different plant pararetroviruses have 1-3 PPTs close to
virion DNA discontinuities, which are structurally similar to the HIV-1
pol-associated DNA discontinuity (Fig. 1). CaMV has two
PPTs; one is colinear with a non-essential gene and can be deleted
without loss of viral infectivity (20, 21). Priming at the CaMV PPTs
in vivo is less that 100% efficient (22, 23), but
additional priming seems to occur at other PPT-like sequences (24) and
by hairpin fold-back (22, 25), although it is not known if these extra
events contribute to viral replication. CaMV PPTs have functional
polarity (26), and we have recently shown that plus-strand priming can
be initiated in vivo by insertion of a PPT at an ectopic
genome site (27). These features have enabled us to investigate the
pararetroviral plus-strand priming mechanism in vivo.
Virus, Plants, and Kits--
CaMV isolates used were Cabb B-JI
and Aust (21), and CM4-184 (20) maintained as infectious clones.
Infection studies were with turnip plants (Brassica
rapa-rapifera, cv. "Just Right"). Celite abrasive was supplied
by Celite Corp. pGEM cloning vectors were from Promega. Mutagenesis was
performed using the QuikChange procedure (Stratagene). Primer extension
was performed with the T7 sequencing kit supplied by Amersham Pharmacia Biotech.
Infection of Plants--
Turnip seedlings were grown in a
greenhouse (minimum day length, 16 h) at 20 °C. Plants were
mechanically inoculated using Celite abrasive with sap from infected
plants, or cloned viral DNA constructs (1-4 µg/plant) digested with
a restriction enzyme to liberate viral DNA from the cloning vector.
Symptoms were scored when consistent on a minimum of 4 plants.
Construction of Mutant Viral Genomes--
Molecular biological
manipulations were based upon those described by Sambrook et
al. (28). Artificial plus-strand primers, (+)ps elements, were
made by annealing pairs of complementary, 5'-phosphorylated
oligonucleotides (Table I, oligonucleotides 1-28) generating terminal
NheI sites. The annealed oligonucleotides were cloned into
the unique NheI site introduced into infectious CaMV 35 S
promoter deletion mutants M4 and M10 (29) as described by Noad et
al. (27). Insert orientation was checked by PCR.
Authentic CaMV (+)ps elements were inactivated by mutagenesis as
follows. For the (+)ps1 element colinear with gene V, the clone MP12
(27) was digested with PstI and the fragment (3430-5386) containing the cloning vector (pGEM5) inserted at the unique
SalI site (4836), and the site to be mutated (~4210), was
self-ligated and cloned. Mutagenesis was performed using the QuikChange
procedure with mutagenic oligonucleotides (Table I, oligonucleotides 29 and 30). The mutation was checked by sequencing, and an NaeI
fragment (pGEM5 site to CaMV site 3910) containing the change was
isolated and used to replace the wild-type fragment in the full-length CaMV clone pBJI5Sal, generating clone PB1. The (+)ps2 priming element
colinear with gene II was mutated by exploiting the adjacent unique
XhoI site to design an oligonucleotide containing this site
and the mutations (Table I, oligonucleotide 31). A fragment was
amplified between this and an upstream site (740-761), and an
NsiI-XhoI fragment (1042-1644) was isolated from
the PCR product. This fragment was used to replace a similar fragment
in a BstEII (126)-XhoI subclone of the CaMV
genome (producing clone BB5) and this, in turn, replaced the wild-type
fragment generating the full-length clone BB8. The (+)ps2 sequence was
also deleted using a strategy similar to that for BB8 but employing an
oligonucleotide with a 33-bp in-frame deletion (Table I,
oligonucleotide 32) relative to the wild-type CaMV sequence, generating
clone BB12. Clone BB9, containing both mutant (+)ps elements, was made
by replacing the BstEII/XhoI-cut fragment from
BB8 with that from BB5 producing clone BB9. A mutant genome containing
the modified (+)ps1 and the deleted (+)ps2 was created by replacing the
SpeI(110)-XhoI fragment of PB1 with that from
BB12 producing clone BB21. Construct PM1 comprised the deleted gene II
region of CaMV isolate CM4-184, the mutated (+)ps1 element, and a
linker in the 35 S promoter. PM1 was made by ligating the 3.0-kb
StuI fragment from clone MP12 (27) to the complementary
7.6-kb StuI fragment of clone PB1. Construct GG5 was a
modification of PM1 in which an artificial (+)ps element (Table I,
oligonucleotides 1-2) was introduced in forward orientation into the
NheI linker in the 35 S promoter. All mutations introduced
into the viral genome were checked by sequencing before infection and
after recovery from plants if constructs were infectious.
Analysis of Virion DNA Discontinuities--
Priming of DNA
plus-strand synthesis was determined by detecting discontinuities in
CaMV virion DNA purified as described by Covey et al. (30).
Denaturing gel electrophoresis of CaMV virion DNA and detection by
blot-hybridization was as described by Noad et al. (27).
Efficiency of plus-strand priming at the ectopic site (+)ps3 was
determined semiquantitatively by scanning autoradiograms following
denaturing Southern blot analysis. Scanned lanes were analyzed using
ImageQuant software to integrate peak areas. Precise determination of
DNA plus-strand 5' ends was by primer extension using appropriate
primers upstream of the (+)ps2 and (+)ps3 elements, respectively.
Primer extension was performed on 1-2 µg of virion DNA using the
method described by Medberry et al. (31).
The Polypurine Tract and 5'-Pyrimidines Are Both Required for
Plus-strand Priming in Vivo--
We have adopted a new descriptor for
plant pararetroviral plus-strand priming sequences, (+)ps, since the
term PPT is an incomplete definition and previous abbreviations in
plant pararetroviruses referred to virion DNA gaps rather than priming
elements themselves. The two authentic CaMV (+)ps elements are
co-linear with viral genes (Fig. 1). The
CaMV reverse transcriptase gene (gene V) contains (+)ps1, and the aphid
transmission protein gene (gene II) contains (+)ps2. Although gene II
is not essential for viral replication and can be deleted, our
studies2 of (+)ps2 have shown
that mutagenesis can interfere with co-linear functions affecting CaMV
pathogenicity. To circumvent this problem, we exploited our discovery
that a redundant 31-bp copy of (+)ps2 inserted into the CaMV 35 S
promoter (Figs. 1A and
2A) efficiently primes DNA
plus-strands during infection of plants with no detected effect on
viral pathogenicity (27). Our assay for functionality of the inserted
primer is to measure generation of a new discontinuity in progeny CaMV
DNA. Following denaturing gel electrophoresis, virion DNA of the M4
vector, lacking the inserted priming element (Fig. 2A),
resolves as three major single-stranded fragments (Fig. 2B)
comprising the genome-length product of minus-strand priming (
The relative activity of mutant (+)ps elements was then tested at the
ectopic site (Fig. 3). This was quantified by comparing the intensity
of the 3.1-kb band (
Further mutants were constructed to investigate the effects of small
deletions and insertions on primer efficiency and primer fidelity
(described below). We confirmed involvement of the pyrimidines by
removing 2 of the 5 T nucleotides in a construct lacking upstream purines that interfere with priming, (Fig.
4, construct (+)ps3:b) and
observed a reduction in the level of priming efficiency to 75% that of
the full-length element. In an attempt to increase priming efficiency,
we added 3 extra T nucleotides (construct (+)ps3:a), but this produced
priming with an efficiency indistinguishable from the wild-type
sequence (Fig. 4). Removal of 3 of the 7 G nucleotides (construct
(+)ps3:d) significantly reduced priming efficiency to 11%. A slight
reduction in efficiency was also found with construct (+)ps3:c
containing 2 extra G nucleotides (Fig. 3). These experiments define the
limits of the cis-acting elements required for CaMV
plus-strand priming in vivo. Thus, maximal priming activity
requires 5 5' pyrimidines and about 13 nucleotides in the PPT. Loss of
more than 3 purines virtually abolishes priming, as does loss of all of
the pyrimidines. Increasing the length of the PPT also reduced priming
efficiency.
Factors Affecting Plus-strand Priming Fidelity in Vivo--
The 5'
ends of the authentic CaMV plus-strands have been mapped previously to
fixed positions (32), as is the case with other REs. Therefore, we
wanted to determine (i) whether priming fidelity was maintained at the
ectopic site and (ii) the effects of mutations on priming precision
in vivo. We first checked the plus-strand 5' end at the
authentic (+)ps2 site in gene II, by primer extension. The 5' end was
found to be at a G residue 3 nucleotides upstream of the 3' end of the
PPT portion of the (+)ps2 site (Fig.
5A). This is 1 nucleotide
upstream of that previously reported by Richards et al.
(32). In addition to the predominant 5' end, lesser secondary ends were
observed 1 or 2 nucleotides to the 5' side of the major end.
Interestingly, a gene II variant (d2) we have obtained following
mutagenesis,2 with 2 of the PPT G nucleotides substituted
for T nucleotides, showed essentially the same priming fidelity
as the wild-type sequence (Fig. 5, A and
D).
We then determined priming fidelity at the (+)ps3 ectopic site and
compared it with that at the authentic site. Priming by (+)ps3:o gave
the same predominant 5' end as was found at the wild-type (+)ps2
priming site (Fig. 5, compare B with
A). In different primer extension experiments,
the degree of heterogeneity around the major 5' end sometimes varied.
The distribution of ends shown in Fig. 5D has been assessed
from two or more primer extension experiments. None of the mutations
studied altered the position of the major 5' end relative to the 3' end
of the PPT. However, the relative abundance and positions of the
secondary 5' ends varied in some mutants. For instance, (+)ps3:t showed
a slight bias of minor ends to the 5' side and (+)ps3:x to the 3' side of the major 5' end (Fig. 5, B and D). Mutations
affecting the 5' pyrimidines had no significant effect on the major 5'
end. The most significant effect on plus-strand priming fidelity was observed in construct (+)ps3:c in which 2 additional G residues had
been inserted into the PPT. This construct produced two major 5' ends
with the predominant end located 3 nucleotides from the PPT 3' end, as
before. However, the second major end was displaced 2 nucleotides
to the 5' side, which places this end at the same relative position
from the 5' end of the PPT as in the wild-type sequence (Fig.
5D). These experiments show that DNA plus-strand 5' end
positioning by the ectopic priming element is essentially the same as
at the authentic site. Additionally, the start site appears to be
controlled by the length of the PPT portion of the (+)ps element with a
primary fixing point at the PPT 3' end.
Priming from Tandemly Arranged (+)ps3 Elements--
During
assembly of one (+)ps3 construct into CaMV vector M10, we isolated a
clone with three tandem (+)ps3:o elements, each in forward orientation
(Fig. 6A). This construct was
infectious and relatively stable, although a recombinant with a single
(+)ps3:o element was cloned from PCR reactions on progeny virion DNA.
Primer extension of the (+)ps3:3o virions showed a correctly positioned DNA 5' end at each of the three priming sites (Fig. 6B).
However, the discontinuities resulting from priming at the upstream
elements would only be detectable by primer extension if priming at the respective downstream element(s) had not occurred (Fig. 6C,
i-iii). Priming at all three tandem sites in any one virion
DNA molecule (Fig. 6C, iv) would not be
detectable by primer extension, so we cannot exclude more than one
priming event per molecule. An additional 5' end was mapped to a
non-authentic purine-rich sequence in the most distal element (Fig.
6B) that we had not seen in other primer extension
experiments. This experiment indicates that the presence of adjacent
primers does not affect primer fidelity, although there may have been
some competition for selection and generation of additional ends due to
the complexity of the sequence environment.
Pararetrovirus Infectivity Rescued by an Ectopic Plus-strand
Primer--
The experiments above demonstrated the relative efficiency
of variant (+)ps elements when they were redundant to viral
replication. Although primer redundancy is a normal feature of CaMV, we
wanted to be sure that priming at the ectopic site could be fully
functional in replication. To achieve this, we resolved to destroy
authentic CaMV plus-strand priming and to rescue infectivity with a
single ectopic priming element. The CaMV (+)ps1 element is colinear
with the RT gene; therefore, destruction of this primer must not
disrupt RT. We introduced single nucleotide changes converting purines to pyrimidines and, vice versa, without changing the RT amino acid
sequence, producing construct PB1, which was infectious (Fig. 7, A and B).
Progeny PB1 virion DNA isolated from plants had only one discontinuity
in each strand producing predominantly genome-length single-stranded
DNA fragments (8 kb) on denaturation together with a predicted fragment
of 6.4 kb arising from defective second strand transfer (Fig. 7,
C and D). Next, the (+)ps2 element was disrupted
with third nucleotide changes, in construct BB8. This was also
infectious with characteristics suggesting priming from only one (+)ps
site (Fig. 7, C and D). To test the effectiveness of both sets of mutations, we produced construct BB9, which should have
been completely disabled in plus-strand priming. In the first infectivity test, we inoculated 15 turnip seedlings with BB9 DNA and
none showed symptoms. In a second round of BB9 inoculations, 1 plant
from 15 showed symptoms. Analysis of the virion DNA from this plant
indicated that priming had occurred only at the mutated (+)ps2 site but
that the mutated sequence had been
retained.3
Because of the residual infectivity exhibited by BB9, we made two
further constructs in which the (+)ps2 element was deleted. BB21 had a
33-bp in-frame deletion removing (+)ps2, which, together with the
(+)ps1 mutations, completely abolished infectivity. We also constructed
a hybrid CaMV, PM1, comprising the gene II region from CaMV isolate
CM4-184 with its 421-bp deletion removing (+)ps2, the inactivated
(+)ps1 element, and a linker in the 35 S promoter capable of receiving
a new (+)ps element. PM1 was not infectious but infectivity was rescued
by insertion of a (+)ps:o element (Table I) into the linker generating
construct GG5 (Fig. 7). This clearly demonstrates that CaMV infectivity
can be made dependent upon an artificial plus-strand primer in an
ectopic genomic location and validates the functional assay of mutants
at the redundant ectopic site.
Priming of DNA plus-strand synthesis is a critical step in RE
propagation. We report here the first detailed study of the cis-acting sequence requirements for this process during RE
viral infection of a whole organism. This was facilitated by the
ability to manipulate a redundant ectopic (+)ps element in infectious clones of the plant pararetrovirus CaMV. In particular, we were able to
study priming, independent of any requirement in replication or
pathogenicity. By measuring the percentage of virion DNA molecules containing a new discontinuity at the ectopic (+)ps3 location, we
estimated the approximate efficiency of priming at this site as about
85-90%. We have previously shown that priming at the authentic CaMV
plus-strand primers is less than 100% efficient, generating a
proportion of progeny DNA molecules lacking a discontinuity at each of
the two sites, respectively (23). Therefore, we would not expect
priming at the new site to be more efficient than at the authentic
primers so 85-90% probably approximates the efficiency of the
authentic primers as well.
The shortest artificial primer that gave maximal activity (construct p)
was 19 nucleotides long. We conclude that the complete element probably
encompasses 18 nucleotides assuming that the 5'-most A residue in this
construct is not a part of the primer. Further mutational analysis
should confirm this. Deleted variants of the artificial (+)ps3 element
which reduced the size of the 13 nucleotide PPT to 10 or fewer purines
significantly reduced the efficiency of priming. Priming was still
detectable when only the 7 G-box residues of the 13 nucleotide PPT were
retained, but deletion of the 7 G nucleotides leaving 5 purines
abolished priming. Priming efficiency was also diminished when the
length of the PPT was increased by 3 or more nucleotides. This suggests
a simple relationship between PPT length and priming efficiency, but
other factors must also be important because deletion of the first PPT purine caused a greater loss of priming efficiency than loss of the
first 2 purines (Fig. 3).
Unexpectedly, deletion of the 5 pyrimidines (T nucleotides) located
immediately 5' of the PPT (construct q) abolished detectable priming
(Fig. 3). Loss of the T nucleotides caused juxtaposition of an upstream
stretch of A nucleotides next to the PPT increasing the PPT length from
13 to 20 nucleotides. This change was most likely inhibitory to
priming, supporting our conclusion that the PPT has both a minimal and
maximal length for optimum efficiency. However, other mutants showed a
clear involvement of the 5' pyrimidines in primer recognition. Although
pyrimidine-rich sequences 5' to the PPT are highly conserved among REs,
in vitro experiments with retroviral PPTs have suggested
that the 5' pyrimidines are not necessary for priming (see
,e.g., Refs. 11 and 12). The differences between CaMV and
retroviruses could reflect a fundamental divergence in the priming
mechanism. However, due to the conservation of the 5' pyrimidines among
REs, we favor the explanation that some priming reactions in
vitro do not accurately reflect all of the priming steps in
vivo, as discussed by Klarmann et al. (13). One
explanation for this difference might reside in the design of
primer/template combinations used in in vitro studies. For example, use of a continuous RNA primer annealed to a DNA template (12)
allows assay of only the polymerase-independent RNase H cleavages and
excludes the initial round of polymerase-dependent RNase H
cleavages generating RNA fragments during reverse transcription (6).
Thus, we could rationalize our observation that the upstream pyrimidines play an enhancing role in primer recognition by proposing that they function in vivo during the
polymerase-dependent phase.
From our observations, we can propose a hypothetical model for
plus-strand priming in CaMV (Fig. 8). We
suggest that, during synthesis of the DNA ( Fidelity of priming at the artificial (+)ps3 site was the same as at
the authentic (+)ps2 site, although this was 1 nucleotide different
from that previously reported by Richards et al. (32). Since
the 5' pyrimidines had no apparent effect on priming accuracy, processing of the remnants of the pregenome template RNA to give the
precise cleavage for priming must only involve interaction between the
ribo-PPT and the active site of RNase H on a DNA template (Fig.
8A, iv-vii). Our results are consistent with a
model (Fig. 8B) in which there is an apparent preference for
interaction between the 3' portion of the PPT and RNase H since various
deletions of the PPT did not alter the position of the primary cleavage site 3 nucleotides upstream of the PPT 3' end (Fig. 4D).
Increasing the length of the PPT by 2 nucleotides again produced the
major priming site 3 nucleotides upstream from the PPT 3' end,
suggesting a primary measuring recognition between RNase H and the PPT
3' end. However, the presence of a major secondary site 2 nucleotides upstream of this suggests alternative PPT length measurement from the
PPT 5' end (Fig. 8B, ii and iii). This
might also explain heterogeneity in priming. An alternative model for
primer recognition would involve changes in shape of the RNase H active
site to fit around primers of different lengths rather than variations
in the way the PPT fits into a fixed-shape active site (Fig.
8B, iv).
It is likely that other structural features are important in defining
the 5' end, as suggested for HIV-1 by Powell and Levin (12). Various
studies have shown that purine for pyrimidine substitutions in the PPT
affect priming fidelity (11, 16, 18, 33). This remains to be determined
for CaMV (+)ps elements, but, in two of our primer mutants in which 2 adjacent PPT G residues were substituted by 2 A and 2 T nucleotides,
respectively, no change in the predominant DNA 5' end was observed. The
presence of immediately adjacent, tandem (+)ps elements in one of our
constructs also showed no effect of primer proximity on priming
fidelity, although there appeared to be competition for primer
selection and possibly a novel 5' end generated in a purine-rich region in the last tandem primer. The precision of the plus-strand 5' ends in
pararetroviruses is intriguing since they do not integrate an
LTR-containing provirus with precise boundaries like retroviruses and
retrotransposons. This conserved precision suggests that the RT-associated enzyme activities responsible for generating plus-strand primers are inextricably linked to production of a precise start site
rather than a frayed end, suggesting that pararetroviral RTs evolved
from retroviral or retrotransposon RTs rather than the converse.
We also confirmed that the artificial (+)ps element functioned
accurately and efficiently independent of its sequence environment. It
also seems to function equally well in at least three other randomly
chosen insertion sites (data not shown). Full functionality was
confirmed by use of the ectopic primer to rescue infectivity following
mutational inactivation of the authentic (+)ps sites. Suppression or
removal of the two authentic CaMV (+)ps elements resulted in complete
abolition of viral infectivity, suggesting that any minor additional
priming sites, previously reported by Maule and Thomas (24), were not
sufficient to establish infectivity. These observations raise the
question as to the requirement for redundant plus-strand primers in
CaMV and other REs. It has been suggested that an additional (+)ps site
increases the rate of replication of HIV-1, although it may also be
involved in a novel replicative strategy (35). There is no evidence
that multiple (+)ps sites in plant pararetroviruses confer a
replicative advantage. Pathogenically, CaMV variants with one (+)ps are
only marginally less severe than those with two, although this
difference could be due to an effect on colinear elements. One
explanation for plus-strand primer redundancy in plant pararetroviruses
might lie in their inherent inefficiency. Two or more primers could confer a slight adaptive advantage over a single primer in marginal environments. Even so, the ability to manipulate pararetroviral plus-strand priming mechanisms in vivo opens up new
possibilities to understand the relationship between in
vitro and in vivo studies of RT and to engineer new
types of RT and RE tools.
Experiments were performed under Ministry of
Agriculture, Fisheries and Food license PHF 1491/982/34.
*
This work was supported by the Biotechnology and Biological
Sciences Research Council.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
RE, retro-element; RT, reverse transcriptase; PPT, polypurine tract; (+)ps, DNA
plus-strand priming sequence; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; LTR, long terminal repeat; HIV, human
immunodeficiency virus; CaMV, cauliflower mosaic virus.
2
N. Al-Kaff, D. Turner, and S. Covey, unpublished observations.
3
R. Noad, D. Turner, and S. Covey, unpublished observations.
Analysis of Polypurine Tract-associated DNA Plus-strand
Priming in Vivo Utilizing a Plant Pararetroviral Vector
Carrying Redundant Ectopic Priming Elements*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-strand, 8 kb), and two fragments resulting from plus-strand priming at (+)ps1 (
-strand, 5.4 kb) and (+)ps2 (
-strand, 2.6 kb).
A 3.8-kb fragment ('-strand) is a product of failed second strand
transfer during reverse transcription (Fig. 2, B and C). The
presence of an additional primer in the 35 S promoter, in vector M4Gf,
produces a new discontinuity in the -strand (Fig. 3). The efficiency of priming at the new
site was measured in denaturing gels by assessing the reduction in
amount of the 5.4-kb single-stranded DNA fragment and replacement by
two additional fragments of 3.1 and 2.3 kb. The amount of residual
-strand DNA in M4Gf shows that priming at the new site is less than
100% effective. By scanning gels, integrating the peak areas (see Fig.
2B), and comparing amounts of -strand, we estimate that
the new priming site mediated by oligonucleotide (+)ps3:o (Table
I) is recognized in 85-90% of virion
DNA molecules.
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Fig. 1.
RE genome organization and plus-strand
priming elements. A, the circular double-stranded DNA
genome of the plant pararetrovirus CaMV showing the positions of the
discontinuities adjacent to the DNA minus-strand primer, ( 
)pbs, and
the two plus-strand primers (black circles),
(+)ps1 and (+)ps2. Open reading frames (arrows
I-VI) and the promoters (boxes) for the 19 S
mRNA and the genomic 35 S RNA are shown. B, genomic RNA
reverse transcription templates with terminal repeats (r)
showing relative positions of plus-strand primers (black
circles) in CaMV, HIV-1, and the yeast retrotransposon, Ty1.
C, structures of DNA discontinuities adjacent to the CaMV
(+)ps2 element, and the HIV-1 pol-associated central
PPT.
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Fig. 2.
Priming in vivo by an ectopic
(+)ps element. A, in the wild-type (wt) CaMV
genome, the 35 S promoter (P35S) and the C-terminal domain
of gene VI overlap. An infectious 35 S promoter deletion mutant (M4)
contains a unique NheI site into which the (+)ps3 element
was inserted in forward orientation producing the infectious CaMV
vector M4Gf. B, denaturing gel and blot-hybridization
analysis of CaMV virion DNA from plants infected with M4 and M4Gf.
Efficiency of priming at (+)ps3 was determined by scanning the lanes,
integrating the peak areas (shown in arbitrary units in
square brackets) and comparing the relative
abundance of the -strand bands with a band not altered by the
ectopic priming event. C, single-stranded DNA components of
M4 virion DNA and M4Gf variants. The -strand in M4 is divided into
two components (
,
) by the new priming event in M4Gf. The
defective (def.) form is produced by a failed second strand
transfer.
View larger version (74K):
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Fig. 3.
Ectopic priming in vivo by mutant
primers. A, sequences of the (+)ps3 variants inserted
into CaMV vector M4. Italicized nucleotides indicate the
NheI insertion site. The relative priming activities
(RA) were expressed as a percentage of that produced by the
full-length primer (o); nd, not detected.
B, denaturing gel and blot-hybridization analysis of CaMV
virion DNA from plants infected with M4Gf carrying variant (+)ps
elements. Priming activity was determined by comparing the relative
amounts of the and bands.
Oligonucleotides used in constructs
-strand) with that of the 2.6-kb band
(-strand) as described under "Experimental Procedures." Both of
the authentic CaMV (+)ps elements have a similar organization, although
the sequences differ slightly. In each case, the PPT is preceded
immediately by a pyrimidine-rich sequence then an A-rich region 5' of
this (see Fig. 7A). We first tested the involvement of these
conserved upstream elements in priming at the ectopic site. Removal of
the 7-nucleotide A-rich sequence in construct (+)ps3:p had no
significant effect on priming compared with (+)ps3:o. However, deletion
of the 5 5' pyrimidines, conserved among REs, in construct (+)ps3:q,
appeared to abolish priming (Fig. 3B). Deletion of 6 nucleotides, mostly purines, immediately upstream of the G-box in
construct (+)ps3:s, reduced the frequency of priming to about 13% of
the level of the full-length ectopic sequence, while deletion of the
G-block, construct (+)ps3:r, eliminated detectable priming. The
seemingly essential nature of the pyrimidines was unexpected since
there is evidence that they are not required for retrovirus plus-strand
priming (12). However, deletion of the pyrimidines and the upstream
purines, leaving the PPT intact (construct (+)ps3:u), allowed a level
of priming approximately one-fifth that of the full-length primer.
Further deletion of the 6 5' nucleotides, predominantly purines, within
the PPT in construct (+)ps3:v, also abolished priming. Substitution of
2 G-box G nucleotides for A nucleotides in construct (+)ps3:t, reduced priming efficiency to about 68%. A more significant reduction was seen
in construct (+)ps3:w and, to a lesser extent in (+)ps3:x in which the
PPT was shortened by 1 and 2 nucleotides, respectively (Fig. 2). These
experiments show that maximal priming activity requires both a PPT
sequence of optimal length together with upstream pyrimidines.
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Fig. 4.
Effects of pyrimidine and purine tract length
on ectopic priming at the (+)ps3 site. A, sequences
inserted into the ectopic site together with their relative priming
activities determined as described in the legend to Fig. 3.
B, denaturing gel blot analysis of virion DNA isolated from
plants infected by the different constructs.
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Fig. 5.
Analysis of the DNA plus-strand 5' ends in
authentic and ectopic priming sites by primer extension.
A-C, primer extension of CaMV wild-type and mutant d2
virion DNA at the authentic (+)ps2 discontinuity in gene II
(A) or variants at the ectopic (+)ps3 site (B and
C), as described in Figs. 3 and 4, with the respective
C-track sequences as markers (M). D, the positions of the
DNA 5' ends defined by selected (+)ps elements determined by primer
extension. The size of the arrow reflects the frequency of
priming at a particular nucleotide determined from at least two primer
extension experiments.
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Fig. 6.
Priming at tandem (+)ps3 elements inserted
into the 35 S promoter. A, the vector was M10, a 35 S
promoter deletion mutant in which the unique NheI site was
upstream of the insertion site in vector M4 (see Fig. 2). A clone
(M10Gf3) was isolated with three tandemly arranged (+)ps3 elements. A
cloned PCR product from plants infected with M10Gf3 also showed the
presence of a recombinant (rec.) with only one of the
inserted primers. B, primer extension of M10Gf3
(left lane of left panel)
showing three 5' ends in their correct locations (closed
pointers). An additional minor 5' end mapped to a different
purine-rich region (open pointer).
Right panel, sequence of the in vivo
recombinant showing precise excision of two of the (+)ps3 elements.
C, possible structures generated following replication of
M10Gf3: i, priming at all three sites, only that nearest the
primer would be detectable; ii-iv, detected by primer
extension in B; v, structure of the
recombinant.
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Fig. 7.
Destruction of authentic plus-strand primers
and rescue of infectivity with an ectopic primer. A,
sequences of wild-type and mutant (superscript m)
primer sequences (+)ps1 and (+)ps2 in which third nucleotide changes
did not alter the colinear amino acid sequences. The putative priming
elements are boxed together with the mutated nucleotides
(lowercase letters). The sequence deleted in
construct BB21 is shown ( 
) B, Structure of variant
CaMV genomic RNA reverse transcription templates and their infectivity
(inf.). Fully functional and destroyed primers are shown as
black or open-crossed circles,
respectively. C, denaturing gel electrophoresis of virion
DNA of infectious constructs BB21, PM1, and GG5 recovered from infected
plants. D, structures of variants including the defective
strand transfer form of PB1 (def.).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
)strand,
polymerase-dependent RNase H cleavages are directed toward
the 5' pyrimidines. It is possible that, at this stage, the PPT is
completely separated from adjacent sequences (Fig. 8A,
iii), leading to enhanced recognition on the subsequent round of polymerase-independent RNase H cleavages. The PPT is then
processed to leave a precise 3' end from which the DNA plus-strand originates. The primer is recognized by RT, which extends the DNA
plus-strand on the DNA minus-strand template. Completion of the
plus-strand occurs after one circuit of the circular template, where it
slightly displaces the DNA plus-strand 5' end. Further progression by
strand displacement is prevented by inefficient strand-displacing
activity of the CaMV RT. The RNA primer on the DNA 5' end "ages"
leaving one or a few oligoribonucleotides (34). Aspects of this model
could be tested by comparing in vivo functions with in
vitro reactions using purified CaMV RT.
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Fig. 8.
Suggested model of DNA plus-strand priming in
CaMV. A, i, part of the RNA template for
reverse transcription adjacent to a plus-strand priming site shows the
PPT and the 5'-pyrimidines (pyr) with the advancing DNA
minus-strand; ii and iii,
polymerase-dependent RNase H introduces initial cleavages
into the RNA template during DNA synthesis (ii) and, into
the pyrimidines (iii) to enhance recognition of the PPT;
iv, polymerase-independent RNase H cleavages generate the
precise primer 3' end; v and vi, selection and
initiation of DNA plus-strand synthesis (v) and DNA
extension and displacement of RNA fragments (vi);
vii, limited displacement of the DNA plus-strand 5' end and
loss of primer ribonucleotides produces the characteristic "gap"
structure. B, hypothetical processing of the ribo-PPT to
produce the correct plus-strand DNA 5' end as in A
(iv), above. i, affinity of the ribo-PPT 3' end
for the RNase H active site positions the cleavage 3 nucleotides from
the 3' end. ii and iii, an overlong PPT can fit
in alternative ways generating different cleavage sites. iv,
alternative cleavage sites or different structured ribo-PPTs could be
accommodated by changes in the RNase H active site.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed. Tel.: 44-1603-452571;
Fax: 44-1603-456844; E-mail: covey{at}bbsrc.ac.uk.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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