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Previous Article | Next Article 
J Virol, June 1998, p. 4989-4996, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Human Cytomegalovirus oriLyt
Sequence Requirements
Yuao
Zhu,1,2
Lili
Huang,1,2 and
David G.
Anders1,2,*
The David Axelrod Institute, Wadsworth Center
for Laboratories and Research, New York State Department of
Health,1 and
Department of Biomedical
Sciences, State University of New York at Albany School of Public
Health,2 Albany, New York 12201-2002
Received 2 January 1998/Accepted 18 March 1998
 |
ABSTRACT |
The mechanisms of action and regulation of the human
cytomegalovirus (HCMV) lytic-phase DNA replicator, oriLyt,
which spans more than 2 kbp in a structurally complex region near the
middle of the unique long region (UL), are not understood.
Because oriLyt is thought to be essential for promoting
initiation of lytic DNA synthesis and may participate in regulating the
switch between lytic and latent phases, we undertook a mutational study
to better define its sequence requirements. Kanr gene
cassette insertions located an oriLyt core region between nucleotides (nt) 91751 and 93299 that is necessary but not sufficient for replicator activity in transient assays. In contrast, insertions into auxiliary regions flanking either side of this core
also required
for significant replicator activityhad little effect. To search for
essential components within the core region, we made a series of
overlapping, roughly 200-bp deletions, and qualitatively and
quantitatively assessed the abilities of the resulting constructs to
mediate replication. All but one of these deletions produced a
significant (i.e., greater than twofold) loss of activity, arguing that
sequences across this entire region contribute to replicator function.
However, two particularly critical segments separated by a dispensable
region, here called essential regions I and II, were identified. Within
essential region I, which overlaps the previously identified early
transcript SRT, two adjacent but nonoverlapping, roughly 200-bp
deletions abolished detectable replication. No single element or motif
from the left half of essential region I was found to be essential.
Thus, essential region I probably promotes replication through the
cooperation of multiple elements. However, four small deletions in the
right half of essential region I, which included or lay adjacent to the
conserved 31-nt oligopyrimidine tract (referred to as the Y block),
abolished or virtually abolished oriLyt activity. Together,
these results identify candidate oriLyt sequences within
which molecular interactions essential for initiation of
oriLyt-mediated DNA synthesis are likely to occur.
| |
INTRODUCTION |
Human cytomegalovirus (HCMV)
infection is endemic throughout human populations (1).
The lytic phase of HCMV infection often poses life-threatening diseases
in immunocompromised individuals, including patients with AIDS,
recipients of organ transplants, and those with malignancies undergoing
chemotherapy (14). HCMV lytic-phase DNA replication is not
well understood, but the overall picture appears to resemble that of
herpes simplex virus type 1 (HSV-1). After entering permissive cells
and uncoating, the HCMV genome is transported to the nucleus and the
genomic termini become fused to form a circular molecule which serves
as the template for subsequent transcription and replication
(19). The HCMV genome is replicated by a mechanism that
produces concatemers, probably some form of a rolling circle (7,
23), although other possibilities have not been excluded (6,
21). The concatemers are subsequently cleaved to unit length and
packaged during virion assembly (29).
The only identified HCMV lytic-phase replicator, oriLyt, was
mapped near the center of the unique long (UL) segment by
using a transient assay (3, 5, 22). Moreover, this region
contains an origin of DNA synthesis (13). Previous deletion
analysis defined a region of more than 3.0 kbp, extending roughly from nucleotides (nt) 90500 to 93930, which contains sequences that contribute to HCMV oriLyt replicator activity in transient
assays (3). This oriLyt region contains a
strikingly elevated density of direct and inverted-repeat sequence
elements, as well as base composition biases and strand asymmetries
(3, 22). On the basis of differing replicator structures and
the absence of an origin binding protein homolog, the mechanism of HCMV
oriLyt activation appears distinct from that of HSV-1
oriS and oriL (4,
25, 27). Recently, we identified an essential oligopyrimidine
sequence that we called the Y block, which overlaps the heterogeneous
3' end of a 210- to 250-nt, nonpolyadenylated, early transcript (SRT), and we hypothesized that the Y block and SRT may cooperate to promote
initiation of oriLyt-mediated DNA synthesis (17).
At least three other transcripts initiate near or cross
oriLyt (16, 17). Whether any of these
transcription units contributes to replicator activation remains to be
determined.
Despite these previous observations, oriLyt sequence
requirements have not been determined. Identification of the essential components is a crucial first step toward understanding the mechanisms of oriLyt function. In this study, we first differentiated
an oriLyt core region that is surrounded by auxiliary
sequences by using insertion mutagenesis. We then dissected the core
region with a comprehensive series of deletions and identified two
essential segments. Further analysis identified the critical elements
in one of the essential segments and confirmed the essential role of
the Y block.
| |
MATERIALS AND METHODS |
Virus and cells.
Low- to moderate-passage-number human
foreskin fibroblasts obtained from local hospitals or from Clonetics
(San Diego, Calif.) were used for all experiments. HCMV AD169 (American
Type Culture Collection VR-538) was passaged at low multiplicity and
stored as frozen stocks for which titers were determined. HCMV
nucleotide sequence coordinates are from the published DNA sequence
data (GenBank accession no. X17403 [8]).
Recombinant plasmids.
pGEM-7Zf(
)-based plasmids pSP54 and
pSP50, both containing fully active HCMV oriLyt
(3), were the progenitors of all the mutant plasmids.
Kanr cassette insertions (1.15 kbp) were made after
linearizing pSP50 (or pSP54 for insertion into the EcoRI
site) by partial digestion with either RsaI (100 series),
AluI (200 series), HaeIII (300 and L series),
EcoRI (KanEco), HincII (Kan601), PmlI
(Kan500), or BssHII (Kan403). In the case of
BssHI, the ends were blunted by using T4 DNA polymerase. The
Kanr cassette was excised from pKK5 by treatment with
EcoRI or BamHI, the ends were blunted by
treatment with T4 DNA polymerase (except for insertion into the
EcoRI site), the blunted fragment was agarose gel purified,
and the purified fragment was ligated with the linearized pSP50.
Transformants were selected on kanamycin-containing plates, and
selected clones were characterized by restriction digestion and by DNA
sequencing to determine the site of insertion; insertion sites for the
plasmids discussed here are given in Fig. 1A. Corresponding Kanr cassette deletion constructs were made by excising the
inserted Kanr cassette with PstI, leaving a
linker fragment composed of the sequences between the Kanr
cassette EcoRI or BamHI sites used for insertion
and the interior Kanr cassette PstI sites.
Most of the large, overlapping deletions, including pYZ13, pYZ14,
pYZ15, pYZ16, pYZ17, pYZ18, and pYZ19, as well as the smaller deletions
pYZ1, pYZ3, pYZ3', pYZ4, pYZ5, pYZ6, pYZ7, pYZ8, pYZ9, pYZ10, pYZ11,
and pYZ12 were constructed by using a PCR-based overlap extension
method (15). The designed deletions were each introduced
into DNA fragments through two steps of PCR with a pair of central and
a pair of flanking primers and pSP54 as the template. Each engineered
DNA fragment was cloned into pGEM-7Zf(), sequenced, and then excised
with appropriate restriction enzymes and ligated into pSP54 in place of
the corresponding wild-type segment. Deletion plasmids, the respective
primers, the restriction sites used, and the deletion coordinates are
summarized in Table 1. Primer sequences
and coordinates are summarized in Table
2. Except for deletions in pYZ1, pYZ3,
pYZ3', pYZ4, pYZ5, and pYZ6, the PCR-generated deletions were designed
to introduce a unique PstI site in place of the deleted
sequence. Plasmid YZ15L was constructed by ligating the
PstI-XcmI fragment of pYZ15 into the corresponding sites of pYZ14, thus combining the deletions of pYZ15 and
pYZ14. By the same strategy, pYZ15R, pYZ15LR, pYZ18L, and pYZ18LL were
constructed by combining deletions of pYZ16 and pYZ15, of pYZ16 and
pYZ14, of pYZ18 and pYZ17, and of pYZ18 and pYZ16, respectively.
A version of pSP54 with the vector BstXI site eliminated by
T4 DNA polymerase treatment was selected and named
pSP54
BstXI(v). Plasmid YZ20 was constructed by cleaving
pSP54BstXI(v) at the unique NotI (nt 92,887)
and BstXI (nt 93,142) sites, blunting with T4 DNA
polymerase, and religating. The region between nt 93,101 and 93,240 of
the HCMV (AD169) genome spans a large dyad symmetry and is variably
reiterated (2). In pYZ20, this sequence is not reiterated
and thus does not contain a complete copy of the dyad. Another selected
clone containing the pYZ20 deletion, pYZ20+1r, carries a complete copy
of the reiterated segment. Plasmid YZ22 was made by restricting
pSP54BstXI(v) with BstXI and
BamHI (93,361), blunting with T4 DNA polymerase, and
religating.
Plasmids SP90 and SP72-24 were generated by bidirectional exonuclease
III digestion as described previously (5), with the Erase-a-base system (Promega, Madison, Wis.), after cutting pSP62 and
pSP61 (3) with NotI and SphI,
respectively. The extent of deletions was determined by sequencing.
Plasmids LH13, LH34, and LH50 were produced by excising the
Kanr cassette from pSP50Kan13, pSP50Kan34, and
pSP50Kan50, respectively, with PstI and religating.
The manipulated region of all mutant plasmids was sequenced to confirm
the intended mutations and to ensure the integrity of the flanking
regions.
Transient replication assays.
Transient replication assays
were done essentially as described elsewhere (5, 26, 30) by
a modified calcium phosphate method (9). Each dish received
10 µg of plasmid DNA. Dishes were washed twice with calcium- and
magnesium-free phosphate-buffered saline 20 h posttransfection,
and Dulbecco modified Eagle medium containing 10% (vol/vol) fetal calf
serum was added. Transfected cells were infected with HCMV AD169 at
about 50 PFU per cell, and total-cell DNA was harvested 96 h after
infection. The purified DNA preparations were digested with
DpnI and EcoRI unless otherwise indicated and
subjected to electrophoresis through a 0.8% agarose gel in TAE buffer
(40 mM Tris-acetate [pH 8.0], 1 mM EDTA) and then transferred to a
Zeta probe nylon membrane (Bio-Rad, Richmond, Calif.) and probed with
random primer, 32P-labeled pGEM-7Zf(). Each plasmid was
tested at least three times.
To estimate relative replication efficiencies, pSP54 or pSP50 and
pGEM-7Zf() were cotransfected with the test plasmid as internal
wild-type and negative standards, respectively. The total amount of DNA
transfected per dish was 10 µg, and the molar ratio of pSP50 or
pSP54:test plasmid:pGEM-7Zf() was 1:10:10. As in the qualitative
assay, the transfected cells were infected with HCMV, and 96 h
later total DNA was purified, treated with DpnI and
EcoRI, subjected to electrophoresis, transferred to Zeta
probe nylon membrane, and hybridized with 32P-labeled
pGEM-7Zf(). DpnI-resistant bands were quantified with a
PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). The replication
efficiency of each deletion was estimated relative to those of pSP54
and pSP50 and expressed as the ratio of test plasmid to wild type
following normalization against the ratio of pSP50 to pSP54 wild-type
controls. Each plasmid was tested quantitatively at least three times.
| |
RESULTS |
Kanr cassette insertions defining an oriLyt
core region.
Previous studies established minimal
oriLyt boundaries on the basis of nested exterior
deletions (3, 22); no individual elements that are
mechanistically essential can reside outside the minimal region defined
by such experiments. However, the boundaries defined in these earlier
studies are inadequate descriptions of the replicator, in that
progressive exterior deletions into either side of the
oriLyt region extending from roughly nt 90500 to 93930 produced increasingly defective replication. Moreover, constructs combining the minimal boundaries defined by such exterior
deletions failed to replicate (3, 22). These results
suggested that oriLyt consists of a core functional region
flanked on both sides by auxiliary elements. Replicator auxiliary
elements, like transcription enhancers, can sometimes function when
separated from core elements (11). Therefore, to test this
possibility and to better define the oriLyt core region, we
first made a series of 1.15-kbp Kanr cassette insertions
across the previously defined oriLyt region in the context
of pSP50, which contains the full-length replicator (Fig.
1A). These plasmids were tested several
times for their abilities to mediate HCMV-directed DNA synthesis in a
transient assay (5, 26, 30). The relative replication
efficiency of each plasmid in this experiment was estimated by
measuring band intensities with a PhosphorImager.
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FIG. 1.
Kanr cassette insertion mutants. (A) Plasmid
constructs. Positions of the Kanr cassette insertions are
plotted in relation to landmark features of oriLyt. The
name, insertion coordinate, and relative activity for each mutant are
noted at the right. Features located on the oriLyt line
above the mutants include the UL59 open reading frame and transcript
(16), the 29-bp repeats (triangle), the Y block (hollow
box), and the two large, imperfect dyad symmetries A and B
(3). The core region is shaded and bound by dotted lines,
and the flanking regions within which partially inactivated insertions
are represented by a gradient. (B) Transient transfection assay of the
Kanr cassette insertion constructs. The abilities of the
constructs described in panel A to mediate DNA replication were tested
as described in Materials and Methods (lanes 1 to 14).
DpnI-resistant products of replication were detected by
Southern blotting; a replica of the resulting autoradiogram is shown.
The tested plasmids are indicated at the top of the panel. The marker
(lane 16) contains 0.1 ng each of EcoRI-treated plasmids
SP54, SP50, and pGEM7Zf(). Plasmid SP50 (lane 15), the parent to most
of the insertions, and the vector pGEM7Zf() (lane 17) were
transfected in parallel as wild-type and negative standards,
respectively, for comparison. (C) Transient assay of the
Kanr cassette insertion constructs from which the insertion
was deleted by PstI treatment, leaving a residual
PstI linker insertion. Each plasmid was tested in duplicate.
The autoradiogram is reproduced here. The deletion constructs, which
correspond to the Kanr insertions described in panel A, are
indicated at the top of each lane.
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|
All tested Kanr cassette insertions from nt 91835 to 92890, inclusive, reproducibly reduced replicator activity to levels
indistinguishable from that of the pGEM vector control in our transient
assay and, therefore, were scored replication negative (Fig. 1B, lanes
1, 5, 6, 8, 9, and 14). The two largest gaps between insertionsfrom nt 92246 to 92747 and from nt 92890 to 93299 (Fig. 1A)overlap critical segments defined by deletions described below. We did not
systematically test for differences between oppositely oriented insertions, but in the cases examined, both insertion orientations inactivated. Insertions between 91475 and 91835 on the left and between
92890 and 94098 on the right reproducibly produced increasing defects
as they approached the center of the oriLyt region (Fig. 1B,
lanes 7 and 10 to 13). Insertions around the boundary were less
deleterious than exterior deletion of flanking sequence to corresponding positions. For example, deletions of right flanking sequence extending past the SacI site at nt 93715 fail to
replicate (3, 22), but an insertion at nt 93299 retained
activity (Fig. 1B). Insertions to the left of nt 91561 or to the right
of nt 93561 had no significant effect on replicator activity, in the context of pSP50 (Fig. 1A and B, lanes 2 and 4; data not shown). Moreover, in the few examples that we tested, insertion of other, unrelated sequences within the core also disrupted the replicator (31). Thus, it is unlikely that the Kanr
cassette itself specifically inhibits replicator function.
To test whether small insertions at corresponding positions similarly
disrupted oriLyt replicator function, the Kanr
cassette was excised with PstI, the proximal flanking
restriction sites, leaving linker-size insertions of approximately 12 nt (depending on the restriction sites used for insertion). In all
cases in which insertions eliminated or greatly reduced replicator
activity, excising the Kanr cassette with PstI
restored replicator function (Fig. 1C and data not shown). These
results demonstrate that loss of function was not due to accidental
changes elsewhere in oriLyt and argue that loss of function
subsequent to Kanr cassette insertion is not simply due to
disruption of an essential protein binding site. Thus, we conclude that
these mutants define a core replicator domain between nt 91751 and
93299, which is slightly smaller than the minimal oriLyt
region defined by our previous exterior deletions (3).
Deletions spanning the oriLyt core region.
To
identify essential oriLyt components, we systematically
examined the core domain using a series of overlapping, roughly 200-bp
deletions across the core domain. Again, each of the deletions was made
in the context of the fully active replicator (Fig.
2A). The replication competence of
each deletion mutant was assessed by a quantitative assay. For these
experiments, we cotransfected each deletion plasmid with a wild-type
oriLyt-containing plasmid as the positive internal standard
and with the parent vector pGEM-7Zf() as the negative internal
standard. The intensity of each replicated signal was measured
with a PhosphorImager, and the relative replication efficiency
was calculated as described in Materials and Methods. Plasmid SP50
served as the internal positive control for most test plasmids.
However, pSP54 was used as the internal positive standard for the
pSP50-derived plasmids SP90, SP72-24, LH13, LH34, and LH50;
thus, for these plasmids, the test signals were at the pSP50
position (Fig. 2B, lanes 16, 17, 21, 22, and 23). Also, the internal
standard for pSP68 comigrated with pGEM because that sample was treated
with both EcoRI and HindIII as well as DpnI (Fig. 2B, lane 14). The quantitative assays were each
repeated at least three times, and the relative replication
efficiencies of each plasmid are summarized to the right in Fig. 2A.
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FIG. 2.
Overlapping deletions across HCMV oriLyt. (A)
A schematic of the PvuII (nt 89796)-to-KpnI (nt
94860) fragment encompassing oriLyt and deletion constructs.
The deleted region of each plasmid is indicated by nucleotide
coordinates and by a gap in the line corresponding to the position in
the oriLyt core. The open box in the line of pYZ20+1r
represents the reiterated dyad sequence (2). Relative
replication efficiencies are given at the right; ND, none detected. (B)
Quantitative replication assay. The indicated test plasmids were
cotransfected with pSP50 or pSP54 and pGEM-7Zf() as described in
Materials and Methods. An autoradiogram of the resulting Southern blot
is reproduced here. Deletion mutants pSP90, pSP72-24, pLH13,
pLH34, and pLH50 were cotransfected with pSP54 as the positive
internal standard. The other deletion mutants were pSP54 derived, and
SP50 was used for the internal wild-type comparison. The pSP68 sample
was treated with DpnI plus EcoRI and
HindIII because pSP68 lacks an EcoRI site,
and thus the pSP50 internal standard migrated to the position of
pGEM-7Zf(). (C) Qualitative assay. The indicated test plasmids were
assayed for replication competence by transient transfection as
described in Materials and Methods. A representative autoradiogram is
reproduced here; only the region of the autoradiogram containing
replicated (rep'd) signals is shown.
|
|
The deletions in plasmids pYZ15, pYZ16, pYZ17, and pYZ18
reduced replication to 1 to 6% of that of the wild type (Fig.
2B, lanes 5 and 9 to 11), whereas deletions further to the left,
outside the core domain defined by the Kanr cassette
insertion, reduced replication to 13 to 47% of the wild-type level as
shown by pYZ13 and pYZ14 (Fig. 2B, lanes 3 and 4). Likewise, the
deletion in pYZ22 reduced oriLyt function to 5% of the
wild-type level (Fig. 2B, lane 20), and deletions further to the right, outside the core domain, had little or no effect on replication (Fig.
2B, lanes 21 to 23). Expansions of the deletions in pYZ15 and pYZ18,
namely, pYZ15L, pYZ15R, pYZ15LR, and pYZ18L and pYZ18LL, each further
reduced replication as compared to their respective progenitors but
nevertheless retained minimal activity (Fig. 2B, lanes 6 to 8, 12, and
13 and data not shown). Therefore, even though these two regions are
important for oriLyt activity, they were not scored as
essential sequences.
In contrast, pSP68, pYZ19, pSP90, pYZ20, and pYZ20+1r reproducibly
replicated at levels less than or equal to those for the internal
vector control (Fig. 2B, lanes 14, 15, 16, 18, and 19). A drawback of
the quantitative assay is that cotransfection with replicating plasmids
enhanced the background DpnI-resistant signal produced by
nonreplicating plasmids, including the vector control (e.g., Fig. 2B,
lane 2), to as much as 0.1% of pSP50. As a result, plasmids
replicating at very low efficiency were sometimes difficult to
distinguish from nonreplicating plasmids. Therefore, the replication competence of each mutant plasmid was also examined by a qualitative replication assay without cotransfected control plasmids.
Deletions pYZ15 and pYZ18, which in quantitative assays were estimated
to retain only about 1% of pSP54 replicator activity (Fig. 2B, lanes 5 and 11), gave readily detectable replicated signals in the qualitative assay (Fig. 2C, lanes 5 and 8). These assays confirmed the subtle differences between weakly replicating constructs (Fig. 2C, lanes 5 to
9). Plasmids SP68, pYZ19, pSP90, and pYZ20 did not replicate detectably
in the qualitative assays (Fig. 2C, lanes 9, 10, 11, and 13). Plasmids
that failed to replicate detectably in both quantitative and
qualitative assays were given scores of ND (none detected). Plasmids
that gave relative replication efficiencies of less than 10% were
rescued by restoring the wild-type sequence to the manipulated regions
to ensure that the observed phenotypes were not due to unanticipated
changes elsewhere in oriLyt (data not shown).
A plot comparing the relative replicator activities of insertion and
deletion constructs reveals how closely the results of these two
experimental approaches parallel (Fig.
3). Considered together, these results
unambiguously demonstrate that the core region between nt 91751 and
93299 is critical to replicator activity. Moreover, the deletions
identified at least two essential regions in oriLyt:
essential region I situated between nt 92209 and 92573, defined by
pSP68, pYZ19, and pSP90; and essential region II between nt 92979 and
93145, defined by pYZ20 and pYZ20+1r. These two essential regions were
separated by a deletable segment extending from nt 92574 to 92979, which was defined by pSP72-24 (Fig. 2B, lane 17).
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FIG. 3.
Replicator activities of insertion and deletion mutants
relative to those of the wild type. Exterior deletions that impinge
upon the oriLyt region (thick grey bar) progressively reduce
activity (3, 22). The positions and relative replicator
activities of insertions (closed triangles) and deletions (thin grey
bars) are plotted. The trough in the activity plot defines the core
region (cross-hatched rectangle). Essential regions I and II, defined
by deletions that completely abrogated replicator activity (black
rectangles I and II), and the intervening deletable segment (open
rectangle D) are indicated.
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Small deletions within essential region I.
Results from
reconstitution experiments (31), together with the
overlapping adjacent deletion (pYZ18), indicated that no single element
in the pSP68-deleted segment is essential. This region, as well as the
rest of essential region I, contains various previously noted sequence
elements, including two copies of a 29-bp sequence, and we were
interested in their potential roles in oriLyt activity.
Thus, we made several deletions in the context of pSP54, including
either or both of the two 29-bp repeats, a dyad symmetry, G-C- and
A-T-rich stretches, and conjoining sequences (Fig.
4A). The replication efficiencies of
constructs containing these deletions were then assessed both
qualitatively and quantitatively. Plasmids YZ1, YZ3, YZ4, YZ5, YZ6, and
YZ3' all replicated, with efficiencies ranging from 2 to 100% (Fig.
4B, lanes 3 to 7 and 14). Deleting copy B of the 29-bp repeat reduced
replication to about 4% of wild-type activity (pYZ3'; Fig. 4B, lane
14), whereas deleting copy A of the 29-bp repeat did not measurably
affect replication in our assays (pYZ1; Fig. 4B, lane 3). Plasmid YZ3, a version of pYZ3' with a point mutation in the remaining copy of the
29-bp repeat (Fig. 4A, bottom), reduced replication to about the same
level as that for pYZ6, in which both 29-bp repeats were deleted (Fig.
4B, lanes 4 and 7). This result suggests the importance of the presence
of at least one copy of the 29-bp element for oriLyt
activity.
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FIG. 4.
Mutations in essential region I. (A) A schematic of the
mutations in essential region I. Essential region I is enlarged
directly below a schematic of the oriLyt region highlighting
landmark features. For each plasmid, the deleted sequence is indicated
by nucleotide coordinates and by a gap in the line. Relative
replication efficiencies estimated in the quantitative replication
assay are noted at the right; ND, none detected. (B) Quantitative
replication assay of the small deletions. For each test plasmid, pSP50
and pGEM-7Zf() were used as wild-type and negative internal
standards, respectively. The relative replication efficiencies of each
plasmid were measured as described in Materials and Methods and are
indicated at the right of in panel A. Samples for lanes 14 to 17 were
from a transfection experiment and blot separate from those for lanes 1 to 13.
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Except for pYZ7, which retained about 4% of wild-type activity
(Fig. 4B, lane 8), the tested small deletions in the
pYZ19-deleted portion of essential region I drastically reduced
(pYZ8, pYZ10, pYZ11, and pYZ12) or abolished (pYZ9)
oriLyt activity (Fig. 4B, lanes 9 to 13, and data not
shown). These deletions all fall into the 3' half of the srt
gene. Plasmids YZ9, YZ9R, and YZ9R2 contain a deletion, an insertion,
and a random sequence substitution of the Y block, respectively
(17). The Y block is an oligopyrimidine tract shown to be
essential for oriLyt activity (17). These Y-block
mutants reproducibly gave replicated signals equivalent to the internal
pGEM-negative standard in the quantitative assay (Fig. 4B, lanes 15 to
17) and failed to replicate in the qualitative assay. Rescuing each
mutant by replacing the deleted sequence with wild-type sequence
restored oriLyt activity (data not shown), demonstrating
that any unintended mutations could not be responsible for the observed
phenotypes. Thus, the quantitative results confirmed that the Y block
is essential for oriLyt activity.
| |
DISCUSSION |
The oriLyt core.
We identify a core domain between
nt 91751 and 93299 on the basis of insertions in the context of the
complete replicator. Several lines of evidence support the significance
of these results. First, all 200-bp deletion constructs within the core
region, except pSP72-24, likewise impaired or abrogated replication
(Fig. 3). Second, the core region defined by the work reported here is
similar to but slightly smaller than the minimal replicator defined by
our previous studies (3). The minimal left boundary defined
by insertions is coincident with our previous exterior deletion studies
(see reference 3 and unpublished results). The right
boundary was placed by deletion studies at approximately nt 93715, whereas in the present study a plasmid with an insertion at nt 93299 retained significant activity. Masse et al. (22) reported
that a construct deleted to the left of nt 92210 retained limited
activity, which we have not seen, and defined the minimal replicator as
extending from nt 92210 to 93715. This discrepancy in previous studies
may be due to strain variations in the reiterated sequences overlapping
the large dyad symmetry elements (10, 18) or to
methodological considerations. Regardless of the explanation, the
difference is a minor one, because both prior studies agree that
deletion on the left as far as nt 91750 greatly reduces activity (3, 22), and no essential components were located between nt
91750 and 92210. Our quantitative results demonstrate that the segment
between nt 91751 and 92210 is important to replicator activity.
Finally, alignment of HCMV AD169 and Towne sequences shows that,
excluding the dispensable segment between essential regions I and II,
the core domain is 99.4% identical, which is a level of conservation
much higher than that in the genome as a whole (10, 24) and
even higher than the level of 97.8 to 98.4% observed among AD169,
Towne, and other strains for the major immediate-early
enhancer-promoter region (20). In contrast, the dispensable
segment retains only 91.7% identity.
The finding that insertions anywhere in the oriLyt core
region, including the deletable segment, inactivated the replicator is
of interest because it suggests that essential elements within the core
region act collectively and have strict requirements for functional
interactions. In this regard, we note that even precise substitution of
the deletable segment with a heterologous sequence inactivated the
replicator (31). However, the mechanism(s) by which these
insertions inactivate the replicator is not known and may vary.
Insertions may interrupt clusters of protein binding sites with
cooperative binding interactions or spatially critical protein-protein
contacts. Alternatively, insertion might prevent formation of unusual
nucleic acid structures that could be required for replicator activity
(28) or interrupt essential transcripts or RNA-DNA
interactions that have been suggested to play a role in replicator
activation (17, 27a).
Finally, it is important to note that the oriLyt core region
defined by deletion and insertion studies is not sufficient for replication and does not constitute the minimal replicator because, at
least in transient assays, plasmid constructions containing only the
core do not replicate (3, 22). Replicator activity requires
the core plus flanking auxiliary sequence, even though no essential
elements have been located in the flanking regions. The auxiliary
segments, which presumably act to enhance and regulate the core
replicator mechanism, are functionally distinct from the core. A
limitation of both insertion and deletion approaches is that they
cannot detect widely spaced redundant essential elements. The auxiliary
regions likely contain functional redundancy, because either the
left or the right auxiliary domain sufficed to activate the core in
transient assays and because small deletions adjacent to the core were
less defective than the corresponding insertions (Fig. 3). Such
redundancy may also explain why some of the core failed to score as
essential by deletion criteria.
Essential regions I and II.
Deletions that abrogated
replicator activity identified segments that likely include
mechanistically essential components. By this definition, we found two
essential segments in the replicator core. Essential region I, which is
situated between nt 92209 and 92573, contains the previously identified
Y-block element and overlaps the srt gene (17),
as well as several noted reiterated sequences (3, 13, 22).
Our results suggest that the 29-bp repeated sequence is the most
important contributor to replicator activity in the left half of
essential region I. This element consists of an inverted pair of
ATF-CREB consensus sequences, which are separated by an overlapping
directly repeated sequence (3, 13, 22). Oligonucleotides
containing this element are specifically bound in vitro by the ATF-CREB
present in cellular extracts (31), but whether those
interactions are relevant to replicator function as well as whether
other proteins also bind is not known. The finding that several smaller
deletions in the right half of essential region I that overlap the 3'
half of the srt gene either abolished or greatly reduced
oriLyt activity is consistent with our previous hypothesis
that the essential Y block and SRT may cooperate to promote
oriLyt initiation (17) and argues that the entire
segment that overlaps the 3' half of SRT is crucial to replicator
function. Further mutation analysis suggests that formation of a
specific DNA-RNA structure may be essential to activity
(31).
Essential region II, extending from nt 92979 to 93145, was not studied
further, but several features are noteworthy. First, there are five
consensus SP1 binding sites. SP1 binds to three sites in the
Epstein-Barr virus oriLyt downstream component
(12), although whether these SP1 binding sites contribute to
Epstein-Barr virus oriLyt activation is not known. Second,
essential region II overlaps a portion of the highly conserved large
dyad A sequence that is variably reiterated in our laboratory strain
AD169 line (2). The failure of a construct that retained a
complete copy of this dyad (pYZ20+1r) to replicate shows that this dyad
sequence itself is not sufficient to perform the essential region II
function. However, the function of essential region II may require
cooperation of the dyad element with adjacent sequences that are
deleted in pYZ20. Sequences to the right of essential region II contain
another large, conserved dyad sequence that is reiterated in other HCMV laboratory strains (10, 18). Neither of these two reiterated dyad sequences was found to be individually essential, but deleting the
segment spanning both dyads inactivated the replicator. Third, essential region II is upstream of the srt gene, and
sequences including essential region II have been shown to have
promoter activity in transient assays (17). It is possible
that essential region II controls or contributes to expression of a
transcript that participates in oriLyt function. Finally,
essential region II overlaps a region recently found to contain an RNA,
termed vRNA, covalently incorporated into packaged HCMV genomes; this vRNA has the same sense as SRT (27a). The features of this
vRNA suggest that it is the remnant of an initiating RNA. SRT and the vRNAs may functionally link essential regions I and II, but it remains
to be established whether SRT and the newly observed vRNAs are
independently transcribed or are derived by processing of a common
precursor.
| |
ACKNOWLEDGMENTS |
We thank Suzanne M. Punturieri, Mary Beth Kinoshita, and David
Franchi for excellent technical assistance; Tim Moran and Matt Shudt of
the Wadsworth Center Molecular Genetics Core Facility for preparing
oligonucleotides and DNA sequencing; and members of the lab for
valuable discussions. Marilyn A. Kacica made pSP90 and pSP72-24.
This work was supported by NIH grant AI 31249.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The David
Axelrod Institute, Wadsworth Center for Laboratories and Research, and
Department of Biomedical Sciences, P.O. Box 22002, State University of
New York at Albany, Albany, NY 12201-2002. Phone: (518) 474-8969. Fax:
(518) 474-3181. E-mail: anders{at}wadsworth.org.
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J Virol, June 1998, p. 4989-4996, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
