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Journal of Virology, September 1998, p. 6997-7004, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Persistent RNA-DNA Hybrid
Structures within the Origin of Replication of Human
Cytomegalovirus
Mark N.
Prichard,1
Sanju
Jairath,2
Mark E. T.
Penfold,3
Stephen St.
Jeor,4
Marlene C.
Bohlman,4 and
Gregory
S.
Pari4,*
Iconix Pharmaceuticals,
Inc.,1 and
Aviron,
Inc.,3 Mountain View, California 94043;
Hybridon, Inc., Cambridge, Massachusetts
021392; and
Department of Microbiology,
School of Medicine, University of Nevada
Reno, Reno, Nevada
895574
Received 11 February 1998/Accepted 22 May 1998
 |
ABSTRACT |
Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates
at the cis-acting origin of replication, oriLyt. oriLyt is
a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA
segment. These alkali-sensitive regions were the result of the
degradation of two RNA species embedded within oriLyt and covalently
linked to viral DNA. The virus-associated RNA, vRNA, was identified by
DNase I treatment of HCMV DNA obtained from sucrose gradient purified
virus. This heterogeneous population of vRNA was end labeled and used
as a hybridization probe to map the exact location of vRNAs within
oriLyt. vRNA-1 is localized between restriction endonuclease sites
XhoI at nucleotide (nt) 93799 and SacI at nt
94631 and is approximately 500 bases long. The second vRNA, vRNA-2,
lies within a region which exhibits a heterogeneous restriction pattern
located between the SphI (nt 92636) and BamHI
(nt 93513) and is approximately 300 bases long. This region was
previously shown to be required for oriLyt replication (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). RNase H analysis determined that vRNA-2 forms a persistent RNA-DNA hybrid structure in the context of
the viral genome and in an oriLyt-containing plasmid used in the
transient-replication assay.
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INTRODUCTION |
Initial reports describing the
identification and characterization of the human cytomegalovirus (HCMV)
origin of replication, oriLyt, have demonstrated that this replication
origin bears little resemblance to other previously identified origins
within the herpesvirus family (1, 2, 7, 8, 10-12, 17, 28, 31,
32). The HCMV core origin spans approximately a 3-kb region from
nucleotide (nt) coordinates 91321 to 93715. Other flanking sequences
which augment origin function have also been defined (1, 12,
18). Within the core origin, multiple consensus transcription
factor binding sites and novel repeat elements have been identified.
However their role in replication has not been determined
(1). Recently, Huang et al. identified an RNA transcript approximately 200 bases long, originating within oriLyt
(14). This observation suggests the possibility that HCMV
has a mode of initiation of DNA replication unlike that of other
herpesviruses. This RNA species, SRT (smallest replicator transcript),
was detected in the presence of phosphonoformic acid and mapped to
regions within the origin shown to be required for oriLyt-dependent DNA replication (14).
In addition to various cis-acting elements within HCMV
oriLyt, another interesting feature is the apparent lack of a
replication initiator protein analogous to the herpes simplex virus
type 1 origin binding protein, UL9 (22, 23). In HSV-1, this
protein binds to the origin of replication and initiates DNA synthesis by an apparent helicase activity (3, 9, 15, 21, 33). Replication at the Epstein-Barr virus oriLyt also appears to be facilitated by the initiator protein, Zta (10). Zta is a
transactivator that binds to sites within the Epstein-Barr virus oriLyt
and is postulated to initiate DNA replication by either the activation of transcription or protein-protein interactions (10).
Origin-dependent replication of HCMV in primary human cells requires
the gene products of the core replication open reading frames in
addition to UL36-38, UL112-113, IE1/IE2 and UL84 (22, 27).
In Vero cells, however, UL84 appears to be the only non-core
replication protein required for oriLyt-dependent replication
(27).
These HCMV characteristics, i.e., (i) the complex structure of oriLyt,
(ii) the presence of an intra-origin RNA transcript, and (iii) the lack
of an apparent trans-acting factor functioning as an
initiator, suggest the possibility of a unique method of DNA
replication for HCMV.
In an effort to define a mechanism of initiation of DNA replication, we
examined DNA structure within oriLyt and identified the presence of
alkali-labile regions. The existence of alkali-sensitive regions
suggests the presence of abasic sites or RNA, which is possibly
embedded within oriLyt. As a consequence of the identification of
NaOH-sensitive regions within oriLyt, we developed a method for
extracting the RNA component associated with packaged virion DNA
(vRNAs). In this report, we show that two vRNAs, approximately 300 and
500 bases long, are present as two stable and persistent RNA-DNA hybrid
regions within oriLyt. Transient-replication experiments show that
vRNAs are incorporated within oriLyt at the time of, or after, DNA
replication and are dependent upon DNA synthesis. One of these RNA-DNA
hybrid regions maps to a previously identified segment of oriLyt that
is indispensable for origin function (1, 18). In addition,
we have identified the presence of variably repeated sequence motifs
within the HCMV origin, corresponding to one of these RNA-DNA hybrid
regions.
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MATERIALS AND METHODS |
Cells and virus.
Human foreskin fibroblasts were used for
all experiments and propagated in Dulbecco's modified Eagle's medium
supplemented with 10% (vol/vol) fetal calf serum. HCMV AD169 (ATCC
V-538) was propagated as described previously (29).
Isolation of vRNA.
Human foreskin fibroblasts infected with
HCMV (multiplicity of infection, 1 PFU/cell) from 10 roller bottle
cultures (850 cm2) were harvested 15 days postinfection by
using a cell scraper. Infected cells were transferred to 250-ml
centrifuge bottles. Infected cells were sonicated by using 5 to 10 pulses with a microtip probe to release intracellular virus. Cell
debris was removed by low-speed centrifugation, and the supernatant was
layered over a 35% (wt/vol) sucrose cushion in a 38-ml SW28
ultracentrifuge tube. Virus particles were pelleted (110,000 × g) for 1 h at 4°C. The resulting viral pellet was
resuspended in Tris-EDTA (TE) and centrifuged through a 30 to 60%
continuous sucrose gradient by ultracentrifugation (110,000 × g) for 1 h at 4°C. The virus band was removed, and
the sucrose-virus mixture was diluted with TE. Virions were again
pelleted by ultracentrifugation (110,000 × g) for
1 h, and these final virus pellets were resuspended in 300 µl of
TE.
A 100-µl volume of virus was treated with 5 U of RNase A for 30 min
(final volume, 200 µl) at 37°C in RNase A buffer (100 mM Tris-HCl
[pH 7.4], 150 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol). After RNase A incubation, proteinase K, sodium dodecyl sulfate (SDS),
and EDTA (final concentrations, 1 mg/ml, 1% [wt/vol], and 10 mM,
respectively) were added. This mixture was incubated for 3 h at
60°C. After incubation, sodium acetate (pH 5.5) was added to a final
concentration of 0.3 M and the solution was extracted twice with
phenol-chloroform-isoamyl alcohol (25:24:1) and then twice with
chloroform-isoamyl alcohol (24:1). The aqueous-phase solution was
removed, and viral DNA was precipitated with 2 volumes of 100%
ethanol. Viral DNA was resuspended in TE and adjusted to a final
concentration of 1 µg/ml.
A 50-µg sample of HCMV DNA was heated for 5 min at 95°C and
immediately placed on ice for 3 min. Then 50 U of RNase-free DNase I
was added by using DNase I buffer (100 mM Tris-HCl [pH 7.4], 150 mM
NaCl, 10 mM MgCl2, 1 mM dithiothreitol), and the mixture was incubated for 1.5 h at 37°C. vRNA was isolated with Trizol reagent (Bethesda Research Laboratories) as specified by the
manufacturer. The vRNA pellet was resuspended in 50 µl of TE.
DNA constructs.
The oriLyt-containing plasmid, pOri, was
constructed by subcloning the KpnI (nt
89797)-PvuII (nt 94860) fragment directly from the AD169
genome into the KpnI-EcoRV site of pGEM7zf(
)
(Promega, Madison, Wis.). pOri XhoA and pOri XhoB were made by cleaving pOri with XhoI and ligating the resulting 1.1- and 1.7-kb
fragments, respectively, into XhoI-cleaved pBlueScript
KS() (Stratagene, La Jolla, Calif.). pOri BX was constructed by
cleaving pOri XhoB with XhoI and then subjecting it to
partial cleavage with BamHI, excising the 300-bp fragment,
and ligating it into BamHI-XhoI-cleaved pGEM7zf(). Construction of plasmids containing HCMV replication genes
required for oriLyt-dependent DNA replication was described previously
(22).
The oriLyt subclone used to generate riboprobes was constructed by
cleaving pOri with
SphI (nt 93513) and
BamHI (nt
94636)
and ligating the resulting fragment into
SphI-
BamHI-cleaved pGEM7zf(
).
pOri XS was
constructed by cleaving pOri SB-A with
XhoI and
SacI
and ligating the resulting 830-bp fragment into
XhoI-
SacI-cleaved
pBlueScript SK(
).
The subclone pOri-HT, which contains the heterogeneous region within
oriLyt, was made by PCR amplification of AD169 DNA with
the primers
5'-ATGGAAAACCTATATATAAGGAGGGGT-3' and
5'-CTGGGTGGGGGATCCCCGGTCGCCCAC-3'.
The resulting PCR product
was ligated into pCRII (Invitrogen)
and sequenced with T7 and SP6
sequencing primers and internal
primers when necessary.
NaOH treatment of HCMV DNA.
A 5-µg portion of HCMV DNA
AD169 was cleaved with NheI and subsequently treated with 50 mM NaOH or 50 mM NaCl, heated to 65°C for 15 min, and neutralized
with Tris-HCl (pH 7). Single-stranded fragments were separated through
a 5% denaturing polyacrylamide gel (5 M urea) and transferred to a
nylon membrane. The membrane was hybridized with
32P-random-primer-labeled pOri plasmid.
Mapping of vRNA to HCMV oriLyt: Southern blot analysis.
Plasmid pOri was cleaved with various restriction enzymes as described
in the figure legends, separated on a 1% agarose gel, and transferred
to ZetaProbe (Bio-Rad) nylon membrane. vRNA 32P-end-labeled
probes were generated by using 10 U of T4 polynucleotide kinase (New
England BioLabs), 20 µCi of [
-32P]ATP, and
approximately 5 µg of vRNA. The membranes were incubated in a Robbins
Scientific hybridization oven for 14 h at 65°C with labeled vRNA
by using 5 ml of hybridization buffer (1.5× SSPE, 7% SDS, 10%
[wt/vol] polyethylene glycol 4000). The blots were washed twice for
15 min each at room temperature with 2× SSC (1× SSC is 0.15 M NaCl
plus 0.015 M sodium citrate)-1% SDS and then twice with 0.1×
SSC-1% SDS for 45 min each at 65°C. The blots were then exposed to
BioMax X-ray film (Kodak) for 15 h at 80°C.
Northern analysis.
vRNA (20 to 50 µg) was denatured for 15 min at 70°C in RNA sample buffer (50% formamide, 5% formaldehyde,
1× morpholinepropanesulfonic acid [MOPS]), separated on a 3%
formaldehyde-agarose gel (3:1 Nusieve [FMC Corp.] agarose), and
transferred to ZetaProbe Nylon membrane. Membranes were hybridized with
random-primer-generated 32P-labeled plasmid pOri in
hybridization buffer for 14 h at 60°C and washed as described
above.
For RNA probes, hybridization was performed with riboprobe buffer
(1.5× SSPE, 1% SDS, 50% formamide, 100 µg of salmon sperm
DNA per
ml, 20 µg of tRNA per ml). Probe concentrations were adjusted
accordingly so that the same specific activity was used for each
riboprobe representing each RNA strand. The membranes were incubated
for 14 h at 65°C.
RNase H treatment of HCMV DNA.
Sucrose gradient-purified
virus DNA obtained as described above was treated with 10 U of
Escherichia coli RNase H (Boehringer Mannheim) for 1 h
at 37°C. The DNA-RNase H mixture was extracted once with
phenol-chloroform-isoamyl alcohol (25:24:1) and once with
chloroform-isoamyl alcohol (24:1), ethanol precipitated, and
resuspended in TE. DNA samples were then cleaved with BglII and PvuII. Single-stranded DNA fragments were separated on a
1% formaldehyde-agarose gel and transferred to a nylon membrane. The
blots were hybridized with 32P-random-primer-labeled pOri
SB-B.
The transient-replication assay was performed as described previously
(
23). Total cellular DNA was extracted with
phenol-chloroform,
cleaved with
HindIII, and treated
with RNase H. DNA was then treated
in the same as the viral DNA
described above. Duplicate samples
were treated with
HindIII and
DpnI to ensure that replicated
plasmid
DNA was present in samples that included all the required genes
in the cotransfection mixture.
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RESULTS |
Identification of site-specific alkali-labile sites within HCMV
oriLyt.
Viral DNA from the HCMV (AD169) genome was subjected to
mild-alkali hydrolysis in an attempt to detect the presence of abasic sites (i.e., nucleotides in which the base component is missing) in
virion DNA. Unmodified DNA is unaffected under these conditions, but
abasic sites or ribonucleotides will be degraded and the cleavage products can be detected on denaturing gels.
Sucrose gradient-purified virus was isolated and HCMV DNA was extracted
as described in Materials and Methods. HCMV DNA was
cleaved with
NheI and then treated with either NaOH or NaCl.
Single-stranded
fragments were separated through a denaturing gel and
probed with
oriLyt sequences from nt 89797 to 94860 (pOri). Figure
1 is an
autoradiogram of a Southern blot
of HCMV DNA treated with either
NaOH or NaCl. The arrow indicates a DNA
band corresponding to
a fragment of approximately 550 bases in the
alkali-treated sample
which was not detected in the NaCl-treated sample
(Fig.
1). This
result indicated that there were alkali-labile sites
within oriLyt
of HCMV.
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FIG. 1.
Identification of site-specific alkali-sensitive regions
within HCMV oriLyt. Viral DNA isolated from sucrose gradient
centrifugation was cleaved with NheI and incubated at 70°C
in 50 mM NaOH or 50 mM NaCl. Samples were neutralized with Tris-HCl (pH
7.0), separated on a 5% denaturing polyacrylamide gel, transferred to
a nylon membrane, and hybridized with pOri. Lanes: 1, AD169 DNA treated
with NaCl; 2, AD169 DNA treated with NaOH. The arrow indicates the
presence of a 550-base band on NaOH-resistant DNA.
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|
HCMV oriLyt contains two species of vRNA.
The presence of
NaOH-sensitive regions within oriLyt prompted us to investigate if RNA,
instead of DNA or regions containing abasic sites, could be integrated
or embedded within the HCMV genome. To this end we developed a method
to isolate possible RNA components associated with packaged viral DNA.
After the isolation of sucrose gradient-purified virus, virus particles
were treated with RNase A to degrade exogenous RNA which could have
copurified with the virus. Viral protein components were eliminated by
incubation of virus with proteinase K and SDS followed by
phenol-chloroform extraction. HCMV genomic DNA was isolated by ethanol
precipitation and resuspended in TE. Viral DNA was heated to 95°C to
denature the DNA strands and subjected to treatment with RNase-free
DNase I. This effectively removed all of the DNA components of the
viral genome. Following DNase I treatment, an RNA isolation procedure was performed. This resultant RNA was termed vRNA, defining the species
associated with viral DNA.
Figure
2 is an autoradiogram of a
Northern blot in which total vRNA was hybridized with the plasmid
containing cloned oriLyt,
pOri. The arrows indicate that two RNA
species were detected (Fig.
2, lane 1). As a control, total vRNA was
treated with RNase A
before being loaded onto the gel and probed along
with untreated
samples (lane 2). Two vRNAs with approximate sizes of
300 and
500 bases were detected. This data indicated that RNA was
associated
with the HCMV genome, was complementary to oriLyt sequences
as
demonstrated by Northern blot hybridization, and was RNase A
sensitive.
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FIG. 2.
Two species of vRNAs exist within oriLyt. vRNA was
isolated from sucrose gradient-purified AD169 DNA as described in
Materials and Methods. vRNA was separated on a formaldehyde-agarose
gel, transferred to a nylon membrane, and probed with pOri. Lanes: 1, vRNA; 2, vRNA treated with RNase A prior to gel loading. The arrows
indicate the presence of a RNA band corresponding to a 500 bases and an
RNA species of approximately 300 bases.
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vRNAs are localized within two regions of oriLyt.
After the
identification of vRNAs within oriLyt, we wanted to define their
approximate location within the origin. pOri was cleaved with various
restriction endonucleases and subjected to gel electrophoresis.
Southern blots of the resulting DNA fragments were probed with
32P-end-labeled vRNA. A schematic representation of the
cloned HCMV oriLyt fragment, pOri, which was used in restriction
endonuclease mapping experiments is shown at the top of Fig.
3. Below the pOri linear map are the
coordinates of six pOri subclones also used in mapping studies.
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FIG. 3.
Linear map of HCMV oriLyt. The relative positions and
nucleotide sequence coordinates of certain restriction endonuclease
sites are shown at the top. Six oriLyt subclones used for mapping
experiments are shown below the map.
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Figure
4 is an autoradiogram of a
Southern blot of pOri fragments probed with vRNA. pOri cleaved with
BamHI and
NotI produces
four fragments, two of
which hybridize with vRNA (Fig.
4, lane
1). One is the 4.3-kb fragment
containing oriLyt sequences from
the
NotI (nt 92888) site to
nt 94861 plus 3 kb of vector sequences.
The other fragment is the
NotI (nt 92888)-
BamHI (nt 93513) 1.1-kp
fragment
(Fig.
3 and
4). This hybridization pattern reveals that
vRNA is
complementary to the region of oriLyt corresponding to
nt 92888 to
94861 and that no hybridization signal was detected
from nt 89796 to
92888 (Fig.
3). To confirm these hybridization
results, a series of
additional DNA hybridization experiments
were performed. For example,
the
SphI-
BamHI cleavage of pOri shows
that the
vRNA probe hybridized only to the 1.4-kb and 900-bp fragments
(Fig.
4,
lane 2) corresponding to nt 93361 to 94861 and nt 92866
to 93513, respectively (Fig.
3, linear map). In addition, cleavage
with
NotI and
NsiI shows that no hybridization signal
was detected
between nt 92399 (
NsiI) and 92888 (
NotI). The 4.9-kb band results
from the
NotI (nt
92888)-
NsiI (nt 94861) fragment plus 3 kb of
vector
sequence. No hybridization signal corresponding to vector
sequences was
detected. A faint 3-kb band was detected in Fig.
4, lane 2. Further
experiments were performed to determine if
this band was the result of
a weak nonspecific hybridization signal
or if vRNAs extend into this
region.
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FIG. 4.
Mapping of HCMV oriLyt vRNAs. Shown is an autoradiogram
of a Southern blot of pOri cleaved with various restriction
endonucleases and probed with end-labeled vRNA. Lanes: 1, BamHI-NotI; 2, SphI-BamHI;
3, NotI-NsiI; 4, XhoI-NotI;
5, XhoI-EcoRI. Arrows indicate the hybridization
of vRNA to the 4.3-kb BamHI fragment and the 900-bp
BamHI-NotI fragment.
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Results from hybridization studies performed on
XhoI-
NotI and
XhoI-
EcoRI
cleavages also indicated that the two species of
vRNA are contained
entirely within the 2-kb region (nt 92888 to
94861) of oriLyt. vRNA
hybridization signals resulting from the
probing of Southern blots of
an
XhoI-
NotI cleavage indicated that
two bands
were detected, corresponding to nt 92636 to 93799 and
93799 to 94861 (Fig.
4, lane 4). vRNA also hybridized to two of
the fragments from an
XhoI-
EcoRI cleavage corresponding to nt
92888 to
93799 and 93799 to 94861 (lane 5).
These results were the first indication that additional DNA sequence
was present within the pOri clone. This extra DNA was
not reported in
the previously published HCMV sequence (
5).
Additional DNA
sequence was also demonstrated when a
XhoI-
EcoRI
cleavage pattern of pOri was examined. When probed with vRNA,
the
predicted hybridization bands should yield
XhoI fragments
of
1.2 and 1.05 kb. However, the 1.05-kb fragment was detected
as an
approximately 1.4-kb DNA band (Fig.
4, lane 5). This was
confirmed when
a
XhoI-
NotI cleavage was performed. A 900-bp band
corresponding to the
NotI-
XhoI fragment (nt 92888 to 93799) is
predicted (
5). However, the DNA band detected
was approximately
1.2 kb (lane 4). Based on these observations, we
conclude that
our subclone of oriLyt, pOri, contained an extra 300 bp
of DNA
sequence between the
NotI site at nt 92888 and the
BamHI site
at nt 93513.
One species of vRNA maps to an essential region of oriLyt.
To
further define the location of HCMV vRNAs within oriLyt, we subcloned
four segments of oriLyt corresponding to sequences from the
NotI site at nt 92888 to nt 94861 (Fig. 3, bottom).
Subclone pOri XhoA was cleaved with
MluI and
EcoRI, and Southern blots of the resulting DNA fragments
were probed with vRNA.
vRNA hybridized to two fragments of an
MluI-
EcoRI cleavage, indicating
that this species
of vRNA spans the
MluI site (Fig.
5A, lane 1,
and Fig.
3, bottom). However,
a
HincII cleavage of pOri SB-A resulted
in hybridization to
the two
HincII fragments and no other region,
indicating
that this vRNA species was localized between the flanking
HincII sites (Fig.
5A, lane 4, and Fig.
3). This species of
vRNA
is referred to below as vRNA-1.
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FIG. 5.
vRNA-2 maps within an essential region of oriLyt. Shown
are subclones of pOri cleaved with various restriction endonucleases
and probed with vRNA. (A) Lanes: 1, subclone pOri XhoA cleaved with
MluI-EcoRI; 2, subclone pOri XhoB cleaved with
BamHI; 3, subclone pOri XhoB cleaved with
NotI-BamHI; 4, subclone pOri SB-A cleaved with
HincII. (B) Subclones of pOri indicating the locations of
vRNA-1 and vRNA-2. Lanes: 1, pOri BX cleaved with
XhoI-SacI; 2, pOri SB-B cleaved with
SphI-BamHI; 3, pOri XS cleaved with
XhoI-SacI.
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The localization of the other species of vRNA was determined with the
subclone pOri XhoB (Fig.
3, bottom). A
BamHI cleavage
of
pOri XhoB produces three fragments. Only one of these was detected
by
the vRNA probe (Fig.
5A, lane 2). This 1.3-kb
BamHI fragment
corresponds to the
BamHI fragment at the left end of the
pOri
XhoB clone (Fig.
3, bottom). This result indicated that this vRNA,
subsequently called vRNA-2, was contained within nt 92636 to 93513
(Fig.
3). To further define the location of vRNA-2, pOri XhoB
was
cleaved with
NotI and
BamHI. The resulting
fragments were
probed with end-labeled vRNA. vRNA-2 hybridized to the
NotI-
BamHI
fragment corresponding to nt 92888 to
93513, indicating that vRNA-2
maps completely within this fragment
(Fig.
5A, lane 3, and Fig.
3, bottom).
In conclusion, vRNA-1 is located between the
XhoI (nt
93799)-
SacI (nt 94636) sites and vRNA-2 is contained
entirely within
the restriction fragment
SphI (nt
92866)-
BamHI (nt 93513), (Fig.
5B, lanes 2 and 3, respectively). Based on these results, we conclude
that the
hybridization signal seen in Fig.
4, lane 2, was the
result of a weak
nonspecific binding to the 3-kb fragment. No
hybridization signal was
detected when vRNA was used as a probe
against sequences within the
restriction fragment
BamHI (nt 93513)-
XhoI
(nt
93799) (Fig.
5B, lane 1, and Fig.
3). The region between nt
92866 and
93513 was previously shown to be essential for oriLyt
function
(
1).
The location of vRNAs also explains the results observed in Fig.
1,
where a 550-bp alkali-resistant fragment was detected
in Southern
blots. This fragment was the result of the degradation
of the two vRNA
segments which flanked an NaOH-insensitive 500-bp
DNA region.
vRNA-2 originates from the same DNA strand as SRT.
Recently, a
transcript of approximately 200 bases was identified within oriLyt
(14). This transcript, SRT, is localized between nt 92431 and 92688 (14). The 5' end of SRT is located approximately
180 nt upstream of the 3' end of vRNA-2. To determine if there could be
some relationship between vRNA-2 and SRT, we investigated if vRNA-2
originated from the same strand as SRT. To this end, we performed
Northern blotting with single-stranded riboprobes specific for vRNA-2.
Total vRNA was probed with riboprobes generated from plasmid pOri SB-B
(Fig. 3). Two riboprobes were made, one generated from the bottom (nt
93513 to 92576) strand and the other from the top (nt 92576 to 93513)
strand. When probes generated from the top strand were used, a broad
band was detected in the autoradiogram of a Northern blot of vRNA (Fig.
6, lane 2). However, no distinct band was
detected when riboprobes made from the bottom strand were used to probe
vRNA (Fig. 6, lane 1). This indicated that vRNA-2 originated from the
top strand, which is the same strand as the previously identified SRT
(14).
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FIG. 6.
Detection of vRNA-2 with strand-specific probes. Shown
is an autoradiogram of a Northern blot where vRNA was separated on a
formaldehyde gel and probed with strand-specific riboprobes. Lanes: 1, vRNA hybridized with a riboprobe generated in the nt 92576-to-93513
direction (top strand); 2, vRNA hybridized with a riboprobe generated
in the nt 93513-to-92576 direction (bottom strand). The arrow in lane 2 indicates the broad band detected when probes generated from the top
strand were used.
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vRNA-2 is present as an RNA-DNA hybrid.
vRNA was isolated from
packaged viral DNA and was mapped to regions within oriLyt where
NaOH-sensitive structures were shown to exist. To show that vRNA was
integrated in the HCMV genome and to confirm that the alkali
sensitivity was due to the presence of an RNA-DNA hybrid, we used the
bacterial enzyme RNase H, an endoribonuclease which specifically
degrades the RNA component of an RNA-DNA hybrid structure, to locate
hybrid structures within oriLyt.
Viral DNA was isolated as described in Materials and Methods, cleaved
with
BglII and
PvuII, and treated with 10 U of
RNase
H. DNA fragments were separated as single-stranded DNA on a
denaturing
gel and hybridized with pOri SB-B. Figure
7A is an autoradiogram
of a Southern blot
of RNase H-treated HCMV DNA. When HCMV DNA
was treated with RNase H, a
3.2-kb band was detected in addition
to the expected 5.9-kb band (Fig.
7A, lane 2). In samples that
were not treated with RNase H or in
samples where a cosmid containing
the HCMV oriLyt region(pCM1029) was
treated with RNase H, no additional
band was detected. Detection of the
3.2-kb band is consistent
with the cleavage of oriLyt DNA near the
NotI site at nt 92888
(Fig.
3). However, since RNase H
degrades the entire RNA strand,
it is not possible to determine the
exact cleavage area.
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|
FIG. 7.
vRNA-2 is integrated in the genome as an RNA-DNA hybrid.
(A) Viral DNA was treated with RNase H, cleaved with BglII
and PvuII as described in Materials and Methods, and
separated under denaturing conditions. The samples were hybridized with
the subclone pOri SB-B. Lanes: 1, AD169 DNA cleaved with
BglII and PvuII; 2, AD169 DNA cleaved with
BglII and PvuII and treated with RNase H; 3, cosmid pHC1029 cleaved with BglII and PvuII and
treated with RNase H. The arrows indicate the full-length 5.9-kb
fragment and the 3.2-kb fragment resulting from an RNase H cleavage.
(B) OriLyt plasmid reacted with RNase H after the
cotransfection-replication assay. Total-cell DNA was cleaved with
HindIII, which cuts pOri only once. Lanes: 1, total-cell
DNA from a cotransfection using the required replication genes along
with pOri and probed with pOri SB-B; 2, total-cell DNA from a
cotransfection using the required replication genes along with pOri,
except that the plasmid encoding UL105 was omitted. The arrow indicates
a 3.2-kb cleavage product originating near the NotI (nt
92888) site.
|
|
RNase H sensitivity was also observed in oriLyt when plasmid pOri was
used in the transient-replication assay. pOri was cotransfected
along
with plasmids containing all of the required replication
genes
(
22). Hirt extracts of plasmid DNA were treated with RNase
H. In samples where all of the required genes were present in
the
cotransfection, RNase H cleavage of pOri was detected (Fig.
7B, lane
1). However, when a plasmid encoding UL105, a protein
required for
transient replication, was omitted from the transfection
mixture, no
RNase H-cleaved DNA could be detected (lane 2). Duplicate
samples were
cleaved with
DpnI to ensure that replicated plasmid
was
present (
26). We have performed experiments where we have
omitted each replication gene from the transfection mixture, and
we did
not detect RNase H cleavage in any of these experiments
(
26). These results indicated that the incorporation of
vRNA-2
within oriLyt takes place at the time of or after the
replication
process in the transient-replication assay but does not
demonstrate
an absolute requirement for DNA replication in the
formation of
hybrid structures. Figure
8
is a schematic of HCMV oriLyt indicating
the locations of vRNA-1 and
vRNA-2.
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|
FIG. 8.
HCMV oriLyt region from nt 89796 to 9486. Shown are the
relative positions of vRNA-1 and an expanded view of the region where
vRNA-2 maps. Also shown is the relative position of the SRT. The shaded
areas are regions which were previously shown to be indispensable for
origin function. Also shown is the region where an extra 300 bp of DNA
sequence is located which is composed of reiterated repeated sequence
elements.
|
|
vRNA-2 localizes to a region of oriLyt which exhibits a
heterogeneous restriction pattern.
As mentioned above, it became
apparent while mapping vRNAs that the origin subclone, pOri, contained
additional sequence which was not represented in the original HCMV
published sequence (5). Mapping experiments had confirmed
that this additional sequence was present between the SphI
(nt 92576) and BamHI (nt 93513) restriction sites. To define
the structure of this region in the context of the viral genome, we
probed an SphI-BamHI restriction digest of AD169
DNA with plasmid pOri SB-B. The predicted size of the
SphI-BamHI fragment is 650 bp. However, when a
SphI-BamHI digest of AD169 DNA was hybridized
with pOri SB-B, a series of bands ranging from 850 to 1,000 bp were
detected (Fig. 9A). This result indicated that this region of oriLyt was heterogenous with respect to the presence of the restriction sites SphI and BamHI.
The heterogeneous section was subcloned from viral DNA by PCR with
primers which flank this area. Several PCR products were sequenced,
including one which included the entire 1,000-bp region. In addition,
fragments cloned directly from viral DNA were sequenced to ensure the
accuracy of the sequence derived from the PCR product. Inspection of
the DNA sequence from this region of oriLyt revealed that three
repeated sequence elements are reiterated a variable number of times
within this region (Fig. 9B).
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|
FIG. 9.
The HCMV oriLyt region contains reiterated repeated
sequence elements. (A) SphI-BamHI heterogeneous
restriction pattern. Shown is an autoradiogram of a Southern blot of
AD169 viral DNA cleaved with SphI-BamHI and
hybridized with pOri SB-B. (B) Three sequence motifs that are variable
reiterated within the SphI and BamHI restriction
sites.
|
|
DNA sequencing of the subclone, pOri also showed that it contained the
same reiterated sequence elements which corresponded
to the extra 300 bp of DNA sequence reported here.
| |
DISCUSSION |
While searching for unusual DNA structures within the HCMV origin
of replication, oriLyt, we observed two alkali-sensitive loci which
flanked an alkali-resistant DNA segment. Initially, we suspected the
presence of NaOH-sensitive abasic regions within oriLyt. However,
through the development of a procedure to isolate RNA associated with
viral DNA, we discovered that alkali-sensitive areas within oriLyt were
due to the presence of RNA-DNA hybrid structures. These vRNAs were
detected by Northern analysis and were degraded by RNase A treatment.
Southern blots of alkali-treated DNA and RNase H experiments revealed
that vRNAs were present as stable and persistent RNA-DNA hybrid
structures flanking a DNA-DNA segment of oriLyt and are covalently
linked to DNA segments.
vRNAs are integrated and packaged into virions, as demonstrated by both
alkali sensitivity and RNase H cleavage of virion DNA. To ensure that
vRNAs were not the result of cellular RNA contamination, purified virus
isolated from a sucrose gradient was treated with RNase A. RNase H
cleavage experiments indicated that not all packaged viral genomes
contained vRNA within oriLyt, in that a percentage of genomic DNA
apparently was not cleaved. It is clear, however, that a large
percentage of packaged viral DNA was cleaved by RNase H. It is
difficult to quantitate the relative amounts of cleavage since
double-stranded probes were used and the uncleaved DNA strand was still
detectable in Southern blots. HCMV genomes may contain variable lengths
and amounts of vRNAs; in addition, some genomes may contain vRNAs that
have secondary structures that make them poor substrates for bacterial
RNase H. There is also the possibility that a percentage of viral
genomes do not have RNA-DNA hybrids. These viral species may be
defective or may not replicate with the same efficiency or by the same
mechanism as hybrid-containing species.
Various attempts to map the exact 3' end of these vRNAs revealed the
absence of a distinct termination site (26). This suggests that vRNAs are integrated with staggered ends during the replication process. Five prime end mapping has been more successful. 5'-RACE (rapid amplification of cDNA ends) data indicates that the 5' end of
vRNA-2 is about 300 bases upstream of the BamHI site at nt
93513 (26). This is consistent with alkali sensitivity data, where a 500-base region separates the two vRNAs. Southern analysis also
shows that vRNA-2 is contained entirely within the
SphI-BamHI fragment, a region required for oriLyt
function (1). Attempts in our laboratory to retain oriLyt
function in constructs where portions of the region between the
NotI (nt 92888) and BamHI (nt 93515) site were
deleted have failed (26). This suggests that the region
where this RNA-DNA hybrid exists is necessary for replication in
the transient-replication assay. In addition, although vRNA-1 maps to a region which is dispensable, oriLyt function is
severely defective when a deletion to the SacI (nt 93715)
site was tested in transient-replication assays (1, 26).
vRNA-2 is within a region of oriLyt containing variably repeated
sequence elements. An SphI-BamHI restriction
digest of AD169 DNA revealed a heterogeneous pattern when hybridized
with plasmid pOri SB-B. When subclones of viral DNA were sequenced, the
numbers of three repeated sequence elements were variable. These
sequence elements accounted for the extra 300 bp observed within the
pOri subclone. These elements were not found anywhere else in the AD169 genome and did not have significant homology to any sequence in the
GenBank database. The role of these reiterated repeat sequence elements
in vRNA formation or in the replication of oriLyt is unknown. In
addition, the presence of these variably repeated regions may not fully
explain the appearance of a DNA ladder, as seen in Fig. 9, when AD169
DNA was cleaved with SphI and BamHI. This
phenomenon could also be due in part to the inefficient cleavage of
this region of HCMV DNA because of the presence of various lengths of
the RNA component of a RNA-DNA hybrid. This structure would be a poor
substrate for restriction endonucleases.
Similar types of repeat structures have also been found within the
origin of replication of human herpesvirus 6 (HHV-6) (30). In HHV-6, intragenomically amplified sequences within the origin may be
the result of the acquisition of oriLyt by transposition (30). The role of these amplified sequences in HHV-6 is not known. Also unknown is if the oriLyt region of HHV-6 contains RNA-DNA
hybrid structures. In HCMV, regions containing repeated sequences are
necessary for oriLyt function in the transient-replication assay.
The vRNA-2 hybrid structure is in close proximity to a recently
identified transcript within oriLyt, SRT. In addition, although strand-specific probes were not used to define vRNA-1, this vRNA originates from the same strand as vRNA-2, since alkali treatment was
capable of releasing the internal DNA segment. This could occur only if
the vRNAs were on the same strand. vRNA-2 is just upstream of SRT and
originates from the same DNA strand as SRT. SRT itself was not isolated
during the vRNA extraction procedure, indicating that SRT is not
embedded within the viral genome. However, vRNA-2 and SRT may be
related, in that SRT may be a portion of vRNA-2 not embedded within the
HCMV genome. SRT and vRNA-2 could be the result of transcription from
an upstream promoter. Multiple transcription factor binding sites exist
within oriLyt, suggesting the presence of putative promoter regions.
Analysis of regions upstream of vRNA-2 indicate that these regions can
act as promoters in transient-replication assays (26). This
observation is consistent with models where transcription and/or
transcriptional elements play a pivotal role in initiation of DNA
synthesis (6, 16, 20). The formation of persistent RNA-DNA
hybrid structures as a result of transcription has been described
previously (19, 34). In addition, the RNA-DNA hybrid
formation at a bacteriophage T4 replication origin has been described
(4). In this case, the formation of a persistent RNA-DNA
hybrid may be the result of a combination of DNA unwinding and
transcription controlled from a middle-mode promoter during replication
(4). In T4, the unwinding potential of the downstream
promoter region may facilitate RNA-DNA hybrid structure formation by
increasing the opportunity for the 5'-end of the origin transcript to
reassociate with its template strand during transcription
(4).
The formation of an RNA-DNA hybrid structure was detected in the pOri
construct replicated in the transient-replication assay. RNase H
cleavage data from this assay suggests that insertion of vRNA-2 is
dependent on the inclusion of the DNA replication proteins in the
transfection mixture. We cannot determine if RNA-DNA hybrid formation
is related to the activity of UL105 per se or to the transient
replication of the plasmid itself. Nevertheless, the presence of a
RNA-DNA hybrid structure suggests that HCMV DNA replication may involve
an RNase H-like mechanism. If this is the case, an enzyme with this
function must be a component of one of the required replication genes.
One such virus-encoded candidate is the UL84 gene product
(13). Initially, UL84 was shown to be 1 of 11 loci required
for oriLyt-dependent DNA replication (22). More recently,
this observation was underscored by Sarisky and Hayward, who
demonstrated that UL84 plays a critical role in transient replication
(27). The exact function of the UL84 protein in DNA
replication remains unknown. However, in transient-replication assays,
we have observed that the UL84 gene product has an effect on RNA
stability (26).
How might vRNAs play a role in HCMV DNA replication? vRNAs may function
as distinct areas where initiation of DNA replication occurs. The first
step might be RNA-DNA hybrid formation resulting from transcription
originating from an upstream promoter. RNA-DNA hybrid structures could
act as substrates for a specific virus-encoded RNase H-type enzyme.
This virus-encoded enzyme would cause a nick or partial degradation of
the RNA strand of the RNA-DNA hybrid. The remaining RNA portion, or the
free DNA end resulting from the removal of integrated RNA, would
function as a primer from which DNA replication would initiate.
Consistent with this mode of replication, the RNA strand would be
incorporated into a percentage of viral genomes as a consequence of the
normal replication process. This in turn would ensure the initial round
of replication upon new infection independent of a new transcription
event. This type of replication scheme suggests that HCMV DNA
replication takes place on two levels. The first would occur in viral
templates where RNA-DNA hybrids already exist, and the second would be
dependent on a transcriptional event that would cause the formation of
the hybrid structure. The presence of an RNA-DNA hybrid in oriLyt may
not be an absolute requirement for DNA replication; however, they may
facilitate an early or first phase of DNA synthesis. An HCMV
delayed-DNA synthesis phenotype has been described, suggesting that
HCMV DNA replication may occur in two stages (24, 25).
We are currently investigating the function and mechanism of insertion
of vRNAs together with exploring the role of UL84 and its association,
if any, with vRNAs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Microbiology, School of Medicine, University of NevadaReno, Reno, NV
89557. Phone: (702) 784-1383.
| |
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0022-538X/98/$04.00+0
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Top
Abstract
Introduction
