Previous Article | Next Article 
Journal of Virology, June 2000, p. 5182-5189, Vol. 74, No. 11
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Differential Activity of Two Non-hr
Origins during Replication of the Baculovirus Autographa
californica Nuclear Polyhedrosis Virus Genome
Saman
Habib1,* and
Seyed E.
Hasnain2,3
Membrane Biology Division, Central Drug
Research Institute, Chattar Manzil,
Lucknow-226001,1 Eukaryotic Gene
Expression Laboratory, National Institute of Immunology, Aruna Asaf Ali
Marg, New Delhi-110067,2 and Centre
for DNA Fingerprinting and Diagnostics, Nacharam,
Hyderabad-500007,3 India
Received 28 September 1999/Accepted 13 March 2000
 |
ABSTRACT |
The identification of potential baculovirus origins of replication
(ori) has involved the generation and characterization of
defective interfering particles that contain major genomic deletions
yet retain their capability to replicate by testing the replication
ability of transiently transfected plasmids carrying viral sequences in
infected cells. So far, there has not been any evidence to demonstrate
the actual utilization of these putative origins in Autographa
californica multinucleocapsid nuclear polyhedrosis virus
(AcMNPV) replication. By using the method of origin mapping by competitive PCR, we have obtained quantitative data for the ori activity of the HindIII-K region and the
ie-1 promoter sequence in AcMNPV. We also
provide evidence for differential activity of the two ori
in the context of the viral genome through the replication phase of
viral infection. Comparison of the number of molecules representing the
HindIII-K and ie-1 origins vis-à-vis the
non-ori polH region in a size-selected nascent DNA
preparation revealed that the HindIII-K ori is
utilized ~14 times more efficiently than the ie-1 region
during the late phase of infection. HindIII-K also remains
the more active ori through the early and middle replication phases. Our results provide in vivo evidence in support of
the view that AcMNPV replication involves multiple
ori that are activated with vastly different efficiencies
during the viral infection cycle.
| |
INTRODUCTION |
The prototype baculovirus,
Autographa californica multinucleocapsid nuclear
polyhedrosis virus (AcMNPV), has a
double-stranded, closed-circular genome of ~134 kb with a
coding capacity of over 150 polypeptides (2).
AcMNPV gene expression is temporally regulated in
an ordered cascade through early, late, and very late phases. Viral DNA
replication precedes the late phase and initiates late/very late gene
expression that ultimately results in the production of progeny virus
(23).
Interspersed in the AcMNPV genome are nine
homologous regions (hr) that are adenine-plus-thymine-rich
sequences containing two to eight 30-bp imperfect palindromes with an
EcoRI site as the palindrome core (except hr4C)
(7, 15). hrs were initially postulated to
function as viral origins of replication (ori) because of
their symmetric location in the genome, palindromic structure, and high
A+T content (4). Subsequent analysis of these sequences by
transient replication assays supported this hypothesis (1), and a single palindrome with an intact core was shown to be sufficient for hr plasmid replication in
AcMNPV-infected cells (9, 20). Non-hr ori have also been reported in
AcMNPV. These include sequences within the
HindIII-K region (84.9 to 87.3 m.u.) that are
tandemly repeated in defective viral genomes (18). Sequences
within the HindIII-K fragment also support plasmid
replication in transient replication assays (13).
Additionally, early promoter regions of the virus, including the
ie-1 gene upstream region and 11 other early promoter
regions, have been demonstrated to function as plasmid
ori in these assays, suggesting that early viral promoter sequences can also function as putative AcMNPV
ori (35). A number of virally encoded genes
involved in DNA replication have also been identified. These include
five essential (p143, ie-1, lef-1, lef-2, and lef-3) and five stimulatory
(dnapol, p35, ie-2, lef-7, and pe-38) genes from AcMNPV (14,
21, 22).
The identification of baculovirus replication ori has
primarily been carried out by using two strategies. Putative
cis-acting elements that may be involved in the initiation
of DNA replication have been identified by the characterization of
defective viral genomes generated by serial passage of the virus in
tissue culture (11, 17) and by the analysis of the
replication status of plasmids carrying these elements in transiently
transfected cells in the presence of viral infection (9, 15, 19,
26, 27). However, it is still not known whether any of the
putative ori thus identified are essential for or actually
function as ori in vivo. Moreover, the individual roles of
these multiple putative ori in DNA replication and whether
they are active simultaneously and the relative efficiencies of
utilization of these ori in a normal infection cycle have
also not been worked out.
By using the method of origin mapping by competitive PCR, used
previously for mapping mammalian DNA ori (6, 16, 28, 32), we have been able to measure the efficiency of utilization of two putative non-hr origins (HindIII-K
region and ie-1 promoter region) vis-à-vis the control
non-ori sequence within the polyhedrin (polH)
gene of AcMNPV. In this report, we provide in vivo
evidence for utilization of multiple ori by the virus. Our
results also support the view that different
AcMNPV ori may be activated with vastly
different efficiencies during the viral infection cycle.
| |
MATERIALS AND METHODS |
Cells and virus.
Spodoptera frugiperda cells (Sf9)
were grown in TNMFH medium (31) containing 10% fetal bovine
serum as described by Summers and Smith (31). The cells were
infected with AcMNPV (strain E-2) at a
multiplicity of infection (MOI) of 50 PFU/cell for different time
periods before isolation of total cell DNA.
Extraction and purification of nascent DNA.
Total cell DNA
was isolated from AcMNPV-infected cells as
described by Leisy and Rohrmann (19). Briefly, cells from an
infected T75 flask were dislodged, centrifuged at 7,000 rpm for 3 min
(SS-34 rotor, Sorval RC5C centrifuge), and washed twice with
phosphate-buffered saline. The cell pellet was resuspended in 3 ml of
DNA extraction buffer (10 mM Tris [pH 7.8], 0.6% sodium dodecyl
sulfate, 10 mM EDTA) and then 15 µl of a solution containing 1 µg
of RNase A/µl was added. After a 1-h incubation at 37°C, 375 µl
of a 20-mg/ml solution of proteinase K was added, and the mix was
further incubated for 12 to 16 h at 37°C. The samples were
extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) and
once with chloroform. DNA was precipitated with ethanol, was rinsed
with 70% ethanol, was dried, and was resuspended in 300 µl of TE
(0.01 M Tris [pH 8.0], 0.001 M EDTA).
Isolation of nascent DNA was carried out by sucrose gradient
fractionation followed by further size selection of the fractionated nascent DNA by agarose gel electrophoresis (16, 32). Sucrose gradient fractionation was carried out according to the method of Kumar
et al. (16). Briefly, total DNA from infected cells was
denatured by a 10-min incubation in boiling water and was size
separated on 17 ml of 5 to 30% continuous neutral sucrose gradient
(150 µg of DNA per gradient) for 18 to 20 h at 26,000 rpm in a
Beckman SW28 rotor at 15°C. Sucrose gradients were prepared in 10 mM
Tris-HCl (pH 8.0), 1 mM EDTA, and 0.3 M NaCl. The bottom of the tube
was punctured, and 500-µl fractions were collected from each tube.
Fractions containing 0.3- to 1.5-kb segments of nascent DNA were
identified by 1% agarose gel electrophoresis by using a 1-kb DNA
ladder marker. These fractions were pooled and dialyzed against
Tris-EDTA (0.5 M Tris [pH 8.0], 0.01 M EDTA) for at least 8 h.
DNA was precipitated with sodium acetate and ethanol and was rinsed
with 70% ethanol, dried, and suspended in TE. Further size selection
of dialyzed nascent DNA was performed by fractionating the nascent DNA
on a 1% preparative agarose gel and eluting 0.3- to 1.5-kb segments of
DNA from the gel. After purification, the concentration of this DNA was
determined and the preparation was used as template in competitive PCRs.
PCR amplification and competitor construction.
Primers used
for competitor construction and competitive PCRs for the
HindIII-K, ie-1, and polH regions
are shown in Table 1. Competitor
construction for each of these regions was carried out as described by
Diviacco et al. (5). Four specific oligonucleotides (two
external primers, P1 and P2, and two internal primers, P3 and P4) were
synthesized for each DNA region to be amplified (Fig. 1). The external primers were designed to
amplify DNA regions in the range of 150 to 300 bp. The sequence of the
upper (P1) and lower (P2) external primers is identical to the genomic
region to be amplified. The upper (P4) and lower (P3) internal primers have 3' ends identical to contiguous sequences on the upper and lower
genomic strands, respectively, and 5' ends that carry a 20-nucleotide
(nt) tag. The 20-nt tags of the internal upper (P4) and lower (P3)
primers are complementary to each other and are unrelated to the target
sequence to be amplified. For each primer set, competitor DNA segments
carrying the corresponding genomic sequence with the addition of 20 extra nts in the middle were constructed. These would allow gel
electrophoretic resolution of the template and competitor amplification
products. For competitor construction, the four primers were used to
carry out two separate PCR amplifications. Amplification products of
the P1-P3 and P2-P4 reactions, which contain a single overlapping
region of 20 bp, were annealed together by first denaturing at 94°C
for 1 min followed by lowering the temperature to 50°C (over a period
of 10 min). After further incubation for 2 min at 50°C, the annealed
products were extended by incubation at 72°C for 5 min and were
amplified by using the following PCR conditions: cycles 1 to 5, 94°C
for 1 min, 50°C for 1 min, and 72°C for 1 min; and cycles 6 to 30, 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. One or more subsequent reamplification steps of the full-length competitor were
needed to enrich for the competitor product and allow its quantification by radioactive labelling. All amplification reactions were carried out in an advanced version of the ThermostarII thermal cycler (34).
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 1.
Steps involved in competitor construction (Diviacco et
al. [5]). P1-P3 and P2-P4 primer pairs were used to
amplify DNA sequences adjacent to the target locus (a and b). The two
products were then denatured and cooled, resulting in the annealing of
the complementary 20-nt tail (c). The annealed product was subsequently
extended and amplified by PCR by using primers P1 and P2 (d). The
resultant competitor was 20 bp longer than the target locus.
|
|
Quantification of the competitor template for each DNA region was
obtained by measuring the amount of incorporated
[
-
32P]dCTP in a competitor reamplification PCR cycle.
A small amount
of competitor, picked by touching a needle to the band
on a polyacrylamide
gel and immersing the tip in TE, was used as
template. The PCR
amplification mixture (50 µl) contained the
standard amount of
cold dCTP (10 nmol) and 0.2 µl (0.5 pmol) of
[
-
32P]dCTP (Jonaki, Hyderabad, India) (4,000 Ci/mmol
and 10 mCi/ml),
corresponding to 1.94 × 10
6 cpm as
measured by Cerenkov counting in a
-counter. The amplification
products were resolved on a 8% polyacrylamide gel, and the radioactive
competitor band was eluted in 150 µl of water. Five microliters
of
the eluted DNA was counted, and the concentration of the competitor
(number of molecules per microliter) was determined from the final
specific activity of [
-
32P]dCTP and the number of
nucleotides incorporated. Dilutions of
this competitor preparation were
used as template in competitive
PCRs.
Competitive PCR experiments.
Competitive PCR was first
carried out by using 10-fold serial dilutions of competitor with a
fixed amount of nascent DNA template for each region in the presence of
primers P1 and P2. The range within which the point of equivalence
between competitor and template lay was thus determined. Similar
reactions were then conducted, using further dilutions of the
competitor within the range. Competitive PCR for each region was
carried out in 30 cycles with the following conditions: denaturation,
94°C, 1 min; annealing, 55°C, 1 min; and extension, 72°C, 1 min.
| |
RESULTS AND DISCUSSION |
Evaluation of in vivo ori activity of two
non-hr putative AcMNPV ori by
competitive PCR.
Two putative non-hr origins, the
HindIII-K region (ori K) and the promoter
region of the ie-1 gene, were selected for analysis of in
vivo ori activity by competitive PCR. A region of the
polh gene that does not support replication of transiently
transfected plasmids in AcMNPV-infected cells
(data not shown) was used as a non-ori control region for
measurement of background DNA levels. PCR primers selected for the
HindIII-K region (84.9 to 87.3 m.u.) amplified a 225-bp
sequence within region V and a small portion of region IV
(13) of the HindIII-K fragment (Fig.
2). Since the hr5 element
(87.6 to 88 m.u.), which is known to be a putative viral
ori, is located close to the HindIII-K
region, we ensured that the primers for the HindIII-K
region would not amplify hr5 ori-derived DNA in
the nascent DNA preparation. This was done by specifically selecting
nascent DNA in the size range of 0.3 to 1.5 kb from the sucrose
gradient fractions as well as from the second gel-purification step
(Materials and Methods). Since the distance between the 3' end of the
225-bp HindIII-K region being amplified and the 5' end
of the hr5 element is greater than 2.2 kb (Fig. 2), the size
of selected nascent DNA fragments would ensure that nascent DNA derived
from the hr5 ori is not amplified by the primers
specific for the HindIII-K region. Primers for the
ie-1 locus amplified a 220-bp sequence within the
ClaI-HincII region of the ie-1
promoter while a 211-bp fragment of the polH gene was
amplified by the external primers designed for this locus (Fig. 2).
Again, the size of the selected nascent DNA fragments (0.3 to 1.5 kb)
ensured that nascent DNA derived from the hr1a putative
ori sequence is not amplified by primers specific for the
polH region. The distance between the 3' end of the
polH region amplified by the external primers and the
hr1a element is ~2.5 kb (Fig. 2). Our attempts at
amplification of a portion of the putative hr5
ori for competitive PCR analysis of ori activity were rendered unsuccessful by the generation of multiple bands due to
extensive homologies with other hr.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 2.
Schematic representation of the positions of
hr and the HindIII-K, ie-1, and
polH regions in a linear map of the
AcMNPV genome. The EcoRI-I fragment
containing the polH gene is shown together with the flanking
hr1 and hr1a elements. The 211-bp 3' end of the
polH gene amplified by primers PH-P1 and PH-P2 is
represented as a shaded box. Part of the AcMNPV
genome containing the HindIII-K, -Q, -P, and -G
restriction fragments shows the location of the 225-bp
HindIII-K region amplified by primers HK-P1 and HK-P2 as
well as the 220-bp ie-1 promoter region amplified by primers
IE-P1 and IE-P2.
|
|
Competitive PCR is used for the absolute quantification of low amounts
of DNA and has been used for mapping
ori in mammalian
cells
(
6,
16,
28) as well as for determination of the abundance
of
sequences within origin regions in nascent DNA preparations
(
32). A fixed amount of DNA sample enriched in nascent DNA
(i.e.,
low-molecular-weight DNA emanating from
ori) is
coamplified with
increasing amounts of a quantified reference template
(competitor),
so that the two templates compete for the same primer set
and
subsequently amplify at the same rate. The ratio between the final
amplification products of the two species is evaluated for each
point.
This ratio is a precise reflection of the ratio between
the initial
amounts of the two templates and is used to evaluate
the amount of the
unknown nascent DNA template. For sequences
that are believed to be at
or near
ori, this method of quantification
of nascent DNA
templates has shown a high level of sensitivity
and fidelity (
32,
37). The isolation of nascent DNA in the
size range of 0.3 to 1.5 kb ensures maximal elimination of broken
genomic parental DNA and large
nascent DNA fragments, including
sheared DNA (typically ranging from 25 to 50 kb). As a result,
sequences located at a significant distance
from
ori would not
be detected. Since Okazaki fragments at
mammalian replication
forks range in size from 25 to 300 nt
(
3), it is presumed that
Okazaki fragments from viral
replication forks would also be eliminated
by size selection of
segments of nascent DNA greater than 0.3
kb.
Competitor DNA fragments constructed for the
HindIII-K,
ie-1, and
polH regions were used as competing
templates in competitive
PCRs for each locus. A constant amount of
nascent DNA template
was used for all reactions carried out for the
quantification
of the number of template (nascent DNA) molecules
representing
each region at a particular time postinfection (p.i.). A
fixed
amount of nascent DNA prepared from
AcMNPV-infected cells harvested
at 30 h p.i.
was added to the PCR mix together with increasing
amounts of the
corresponding competitor DNA. The ratio of the
competitor and template
reaction products (C/T) was calculated
by densitometric analysis of the
ethidium-stained gels (ImageMaster
1D Elite software; Amersham
Pharmacia Biotech) and was plotted
against the number of competitor
molecules added to each reaction
(Fig.
3). The number of competitor molecules
when C/T = 1 was
calculated from the plot equation. This value
corresponds to the
precise number of molecules of the target template
(nascent DNA)
added to the PCRs. For nascent DNA isolated 30 h
p.i., competitive
PCR analysis carried out for each region revealed
that the
HindIII-K
and
ie-1 regions were
represented by ~170- and ~12-times-higher
number of molecules
compared to the control
polH region, respectively
(3.7 × 10
6 molecules of the
HindIII-K region and
2.56 × 10
5 molecules of the
ie-1 region
compared to 2.15 × 10
4 molecules of the
polH region) (Fig.
3). Repeat experiments using
another 30-h
p.i. DNA sample gave similar relative values for
the three loci
(
HindIII-K-
ie-1-polH ratio of 120:7.3:1)
(data
not shown). Additionally, comparison of the number of template
molecules from the
HindIII-K
ori and control
polH regions in a
30-h p.i. preparation from cells infected
with
AcMNPV at 10 MOI
gave a ratio of 103:1, thus
demonstrating that the
HindIII-K region
exhibited high
ori activity even at a lower MOI (data not shown).
These
results demonstrate that both the
HindIII-K and the
ie-1 regions function as
ori in vivo, although at
30 h p.i. the
HindIII-K
ori region is
utilized much more efficiently than the
ie-1 locus.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 3.
Competitive PCR reveals greater abundance of
HindIII-K and ie-1 ori regions compared to
the non-ori control polH region in a 30-h p.i.
nascent DNA preparation. Determinations of the numbers of molecules
representing the HindIII-K, ie-1, and
polH regions are shown in panels A, B, and C, respectively.
A fixed amount (1 µl) of 30-h p.i. template DNA was added to
competitive PCRs for the three regions. PCR products were resolved on
an 8% polyacrylamide gel and were stained with ethidium bromide. The
intensity of the bands corresponding to the template target (T) and
competitor (C) DNAs was determined by densitometric analysis. The ratio
between the two PCR products for each reaction (C/T) was plotted
against the number of competitor molecules added to the reaction. A
linear correlation between the C/T ratio and the quantity of competitor
added to the reaction was observed. Correlation coefficients
(R2) are reported for each plot. The number of target
template molecules, that equal the number of competitor molecules when
C/T = 1, was calculated from the equation of the line fitting the
experimental points.
|
|
HindIII-K and ie-1 ori exhibit differential
activities during the AcMNPV infection cycle.
To
determine whether there were any differences in the relative
utilizations of the HindIII-K and ie-1 ori
during the viral infection cycle, we isolated nascent DNA from infected
cells at different times p.i. and used these as templates in
competitive PCR. Nascent DNA isolated from cells 4, 10, and 18 h
p.i. was evaluated for DNA molecules representing the two non-hr
ori and the control polH region. In
AcMNPV-infected Sf9 cells, DNA replication is
detected by about 6 h p.i. and continues until about 18 h
p.i., after which the level of replication declines (23,
33). Thus, nascent DNA isolated at 4 h p.i. would give
background relative values for each region prior to replication
initiation while DNA isolated at 10 and 18 h p.i. would indicate
relative ori activity of the HindIII-K and
ie-1 regions vis à vis the polH control region when viral DNA replication activity is high in infected Sf9 cells.
Competitive PCR experiments for the three regions, carried out by using
nascent DNA isolated 4 h p.i. as template, gave a
HindIII-K-
ie-1-polH template molecule ratio
of 1.01:1.7:1 (i.e.,
2.12 × 10
5:3.62 × 10
5:2.097 × 10
5 molecules) (Fig.
4). Near-equal representation of the two
ori regions (
HindIII-K and
ie-1)
and the non-
ori control region
polH in this
prereplication DNA preparation confirmed that differences
in the number
of template molecules obtained for other time points
are an actual
indication of their relative
ori activities. Infection
of
Sf9 cells with
AcMNPV at an MOI of 50, prior to
the isolation
of nascent DNA at different times p.i., ensured that all
cells
were infected at the same time. Competitive PCR with nascent DNA
isolated 10 h p.i. yielded a
HindIII-K-
ie-1-polH template molecule
ratio
of 37.6:1.5:1 (i.e., 8.46 × 10
5:3.4 × 10
4:2.25 × 10
4 molecules) (Fig.
5), demonstrating that the
HindIII-K
ori is
active at 10 h p.i.
while the
ie-1 region does not show
ori activity
at this time point. Quantification of nascent DNA isolated 18
h
p.i. revealed slightly higher relative
ori activity of the
HindIII-K
region, although the change in relative
ie-1 ori activity was
insignificant compared to the activity
10 h p.i. The
HindIII-K-
ie-1-polH template molecule ratio obtained at 18 h p.i. was 43.4:1.8:1
(i.e.,
8.74 × 10
4:3.742 × 10
3:2.014 × 10
3 molecules) (Fig.
6). These results indicate that
replication
is initiated at the
HindIII-K region
throughout the viral replication
phase with maximal utilization of
the
HindIII-K
ori in the late
replication phase (30 h p.i.). On the other hand, the
ie-1
promoter
region is utilized as an
ori primarily in the late
phase of replication.
HindIII-K, however, remains the
more active
ori even in the late
replication phase (Fig.
7). The lower
ori activity of
the
ie-1 region suggests that although active as an
ori in the late phase,
ie-1 is not a preferred
origin of
AcMNPV replication.
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 4.
AcMNPV ori and control
regions are represented equally in a prereplication nascent DNA
preparation from infected Sf9 cells at 4 h p.i. Panels A, B, and C
depict the determinations of the numbers of molecules representing the
HindIII-K, ie-1, and polH regions
by competitive PCR, respectively. Evaluation of the number of target
template molecules representing each region was carried out as
described in the legend to Fig. 3. Best curve fit for the experimental
points was obtained by fitting the points to a linear equation. The
number of target template molecules calculated for C/T = 1 from
the equation of the line fitting the experimental points is reported
inside the plot frame.
|
|
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 5.
Competitive PCR analysis reveals abundance of nascent
DNA molecules representing the HindIII-K region in DNA
prepared 10 h p.i. Determinations of numbers of molecules
representing the HindIII-K, ie-1, and
polH regions are shown in panels A, B, and C, respectively.
Best curve fit for the experimental points was obtained by fitting the
points to a linear equation, and the number of target template
molecules when C/T = 1 was calculated.
|
|
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 6.
HindIII-K continues as an active
ori 18 h p.i. Competitive PCR analyses for
determination of the number of molecules representing the
HindIII-K, ie-1, and polH regions
in nascent DNA prepared from AcMNPV-infected Sf9
cells 18 h p.i. are shown in panels A, B, and C, respectively. The
experimental points fitted to a linear (panels A and B) or quadratic
(panel C) equation were used to calculate the number of target template
molecules in the nascent DNA preparation.
|
|
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 7.
Comparison of ori activities of the
HindIII-K and ie-1 regions at different times
p.i. The ratio of number of molecules representing the
HindIII-K and ie-1 regions relative to the
polH control region for 4, 10, 18, and 30 h p.i. is
plotted as relative ori activity.
|
|
The mechanism by which baculoviruses generate mature, circular,
unit-length genomes after replication is still not clear.
Kool et al.
(
12) demonstrated that a circular topology is a
prerequisite
for the replication of
ori-containing plasmids in
AcMNPV-infected cells, thus suggesting that
baculovirus DNA replication
involves a theta or a rolling circle
intermediate. Replicated
ori-containing plasmids organized
into high-molecular-weight concatemers
containing multiple plasmid
copies in virus-infected cells (
19)
and multimers of viral
DNA were detected in infected cells (
25),
indicating that
AcMNPV replication may use a rolling circle
mechanism.
A role for recombination has also recently been suggested
for
baculovirus replication (
35). Irrespective of the
mechanism
of replication, a population of viral genomes may utilize
multiple
ori with differing levels of initiation efficiency.
Rapid initial
amplification of circular templates (by the theta or the
rolling
circle mode) could take place by replication initiation
primarily
at the
hr origins. The non-
hr HindIII-K
ori is utilized both in
the early and late replication
phases. As replication proceeds
and factors required for the specific
initiation of replication
become limiting, additional
ori
such as the
ie-1 region may also
be activated. Differential
activity of the
HindIII-K and
ie-1 ori in the
viral genome context confirms that multiple
ori are
utilized
during
AcMNPV replication in a temporally
regulated manner.
Determination of the activation profile of other
non-
hr putative
ori sequences (
35) by
using the competitive PCR method could
help delineate the order and
efficiency of activation of these
origins. Activation of
ori
could be regulated by the interaction
of a specific
viral-origin-binding protein(s) such as the
ie-1 gene
product that binds to
hr (
8,
24,
29,
30) and host
factors such as the 38-kDa protein that interacts specifically
with
hr1 (
10). The regulated activation of multiple
ori represents
an interesting molecular event in baculovirus
pathogenesis.
| |
ACKNOWLEDGMENTS |
We thank Anshu Chaturvedi, R. K. Srivastava, and Divya Singh
for technical assistance; Pramod Upadhyay for the gift of the thermal-cycler; and Amit Misra for help in manuscript preparation.
This work was supported by a Young Scientist Grant of the Indian
National Science Academy to S.H.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Membrane Biology
Division, Central Drug Research Institute, Chattar Manzil, Post Box 173, Lucknow-226001, India. Phone: 91-522-212-411, ext. 4282. Fax:
91-522-223-405. E-mail: samamit{at}lw1.vsnl.net.in.
This is CDRI communication no. 5989.
| |
REFERENCES |
| 1.
|
Ahrens, C. H.,
D. J. Leisy, and G. F. Rohrmann.
1996.
Baculovirus DNA replication, p. 855-872.
In
M. L. DePamphilis (ed.), DNA replication in eukaryotic cells. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
|
| 2.
|
Ayres, M. D.,
S. C. Howard,
J. Kuzio,
M. Lopez-Ferber, and R. D. Possee.
1994.
The complete DNA sequence of Autographa californica nuclear polyhedrosis virus.
Virology
202:586-605[CrossRef][Medline].
|
| 3.
|
Burhans, W. C.,
L. T. Vassilev,
M. S. Caddle,
N. H. Heintz, and M. L. DePamphilis.
1990.
Identification of an origin of bidirectional DNA replication in mammalian chromosomes.
Cell
62:955-965[CrossRef][Medline].
|
| 4.
|
Cochran, M. A., and P. Faulkner.
1983.
Location of homologous DNA sequences interspersed at five regions in the baculovirus AcMNPV genome.
J. Virol.
45:961-970[Abstract/Free Full Text].
|
| 5.
|
Diviacco, S.,
P. Norio,
L. Zentilin,
S. Menzo,
M. Clementi,
G. Biamonti,
S. Riva,
A. Falaschi, and M. Giacca.
1992.
A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates.
Gene
122:313-320[CrossRef][Medline].
|
| 6.
|
Giacca, M.,
L. Zentilin,
P. Norio,
S. Diviacco,
D. Dimitrova,
G. Contreas,
G. Biamonti,
G. Prini,
F. Weighardt,
S. Riva, and A. Falaschi.
1994.
Fine mapping of a replication origin of human DNA.
Proc. Natl. Acad. Sci. USA
91:7119-7123[Abstract/Free Full Text].
|
| 7.
|
Guarino, L. A.,
M. A. Gonzalez, and M. D. Summers.
1986.
The complete sequence and enhancer function of the homologous DNA regions of the of Autographa californica nuclear polyhedrosis virus.
J. Virol.
60:224-229[Abstract/Free Full Text].
|
| 8.
|
Guarino, L. A., and W. Dong.
1991.
Expression of an enhancer binding protein in insect cells transfected with the Autographa californica nuclear polyhedrosis virus ie-1 gene.
J. Virol.
65:3676-3680[Abstract/Free Full Text].
|
| 9.
|
Habib, S.,
S. Pandey,
U. Chatterji,
S. Burma,
R. Ahmad,
A. Jain, and S. E. Hasnain.
1996.
Bifunctionality of the AcMNPV homologous region sequence (hr1): enhancer and ori functions have different sequence requirements.
DNA Cell Biol.
15:737-747[Medline].
|
| 10.
|
Habib, S., and S. E. Hasnain.
1996.
A 38 kDa host factor interacts with functionally important motifs within the Autographa californica multinucleocapsid nuclear polyhedrosis virus homologous region (hr1) DNA sequence.
J. Biol. Chem.
271:28250-28258[Abstract/Free Full Text].
|
| 11.
|
Kool, M.,
J. W. Voncken,
F. L. J. van Lier,
J. Tramper, and J. M. Vlak.
1991.
Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties.
Virology
183:739-746[CrossRef][Medline].
|
| 12.
|
Kool, M.,
J. T. M. Voeten,
R. W. Goldbach,
J. Tramper, and J. M. Vlak.
1993.
Identification of seven putative origins of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus DNA replication.
J. Gen. Virol.
74:2661-2668[Abstract/Free Full Text].
|
| 13.
|
Kool, M.,
R. W. Goldbach, and J. M. Vlak.
1994.
A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus.
J. Gen. Virol.
75:3345-3352[Abstract/Free Full Text].
|
| 14.
|
Kool, M.,
C. Ahrens,
R. W. Goldbach,
G. F. Rohrmann, and J. M. Vlak.
1994.
Identification of genes involved in DNA replication of the Autographa californica baculovirus.
Proc. Natl. Acad. Sci. USA
91:11212-11216[Abstract/Free Full Text].
|
| 15.
|
Kool, M.,
C. H. Ahrens,
J. M. Vlak, and G. F. Rohrmann.
1995.
Replication of baculovirus DNA.
J. Gen. Virol.
76:2103-2118[Abstract/Free Full Text].
|
| 16.
|
Kumar, S.,
M. Giacca,
P. Norio,
G. Biamonti,
S. Riva, and A. Falaschi.
1996.
Utilization of the same DNA replication origin by human cells of different derivation.
Nucleic Acids Res.
24:3289-3294[Abstract/Free Full Text].
|
| 17.
|
Lee, H. Y., and P. J. Krell.
1992.
Generation and analysis of defective genomes of Autographa californica nuclear polyhedrosis virus.
J. Virol.
66:4339-4347[Abstract/Free Full Text].
|
| 18.
|
Lee, H. Y., and P. J. Krell.
1994.
Reiterated DNA fragments in defective genomes of Autographa californica nuclear polyhedrosis virus are competent for AcMNPV-dependent DNA replication.
Virology
202:418-429[CrossRef][Medline].
|
| 19.
|
Leisy, D. J., and G. F. Rohrmann.
1993.
Characterization of the replication of plasmids containing hr sequences in baculovirus-infected Spodoptera frugiperda cells.
Virology
196:722-730[CrossRef][Medline].
|
| 20.
|
Leisy, D. J.,
C. Rasmussen,
H. T. Kim, and G. F. Rohrmann.
1995.
The Autographa californica nuclear polyhedrosis virus homologous region 1a: identical sequences are essential for DNA replication and transcriptional enhancer function.
Virology
208:742-752[CrossRef][Medline].
|
| 21.
|
Liu, G., and E. B. Carstens.
1999.
Site directed mutagenesis of the AcMNPV p143 gene: effects on baculovirus DNA replication.
Virology
253:125-136[CrossRef][Medline].
|
| 22.
|
Lu, A., and L. K. Miller.
1995.
The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication.
J. Virol.
69:975-982[Abstract].
|
| 23.
|
Lu, A.,
P. J. Krell,
J. M. Vlak, and G. F. Rohrmann.
1997.
Baculovirus DNA replication, p. 171-186.
In
L. K. Miller (ed.), The baculoviruses. Plenum Press, New York, N.Y.
|
| 24.
|
Okano, K.,
V. S. Mikhailov, and S. Maeda.
1999.
Colocalization of baculovirus ie-1 and two DNA-binding proteins, DBP and LEF-3, to viral replication factories.
J. Virol.
73:110-119[Abstract/Free Full Text].
|
| 25.
|
Oppenheimer, D. I., and L. E. Volkman.
1997.
Evidence for rolling circle replication of Autographa californica M nuclear polyhedrovirus genomic DNA.
Arch. Virol.
142:2107-2113[CrossRef][Medline].
|
| 26.
|
Pearson, M.,
R. Bjornson,
G. Pearson, and G. F. Rohrmann.
1992.
The Autographa californica baculovirus genome: evidence for multiple replication origins.
Science
257:1382-1384[Abstract/Free Full Text].
|
| 27.
|
Pearson, M. N., and G. F. Rohrmann.
1995.
Lymantria dispar nuclear polyhedrosis virus homologous regions: characterization of their ability to function as replication origins.
J. Virol.
69:213-221[Abstract].
|
| 28.
|
Pelizon, C.,
S. Diviacco,
A. Falschi, and M. Giacca.
1996.
High resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.
Mol. Cell. Biol.
16:5358-5364[Abstract].
|
| 29.
|
Rasmussen, C.,
D. J. Leisy,
P. S. Ho, and G. F. Rohrmann.
1996.
Structure-function analysis of the Autographa californica multinucleocapsid nuclear polyhedrosis virus homologous region palindromes.
Virology
224:235-245[CrossRef][Medline].
|
| 30.
|
Rodems, S. M., and P. D. Friesen.
1995.
Transcriptional enhancer activity of hr5 requires dual-palindrome half sites that mediate binding of a dimeric form of the baculovirus transregulator ie-1.
J. Virol.
69:5368-5375[Abstract].
|
| 31.
|
Summers, M. D., and G. E. Smith.
1987.
A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experimental Station bulletin no. 1555.
Texas A & M University, College Station, Tex.
|
| 32.
|
Tao, L.,
T. Nielsen,
P. Friedlander,
M. Zanis-Hadjopoulos, and G. Price.
1997.
Differential DNA replication origin activities in human normal skin fibroblast and HeLa cell lines.
J. Mol. Biol.
273:509-518[CrossRef][Medline].
|
| 33.
|
Tjia, S. T.,
E. B. Carstens, and W. Doerfler.
1979.
Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus II. The viral DNA and the kinetics of its replication.
Virology
99:399-409.
|
| 34.
|
Upadhyay, P.
1999.
Design of a thermocycler based on light and air having optimal heat capacity.
Curr. Sci.
77:515-519.
|
| 35.
|
Wu, Y., and E. B. Carstens.
1996.
Initiation of baculovirus DNA replication: early promoter regions can function as infection-dependent replicating sequences in a plasmid-based replication assay.
J. Virol.
70:6967-6972[Abstract/Free Full Text].
|
| 36.
|
Wu, Y.,
G. Liu, and E. B. Carstens.
1999.
Replication, integration, and packaging of plasmid DNA following cotransfection with baculovirus viral DNA.
J. Virol.
73:5473-5480[Abstract/Free Full Text].
|
| 37.
|
Zimmermann, K., and J. W. Mannhalter.
1996.
Technical aspects of quantitative competitive PCR.
BioTechniques
21:268-279[Medline].
|
Journal of Virology, June 2000, p. 5182-5189, Vol. 74, No. 11
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Hilton, S., Winstanley, D.
(2008). Genomic sequence and biological characterization of a nucleopolyhedrovirus isolated from the summer fruit tortrix, Adoxophyes orana. J. Gen. Virol.
89: 2898-2908
[Abstract]
[Full Text]
-
Hilton, S., Winstanley, D.
(2007). Identification and functional analysis of the origins of DNA replication in the Cydia pomonella granulovirus genome. J. Gen. Virol.
88: 1496-1504
[Abstract]
[Full Text]
-
Carstens, E. B., Wu, Y.
(2007). No single homologous repeat region is essential for DNA replication of the baculovirus Autographa californica multiple nucleopolyhedrovirus. J. Gen. Virol.
88: 114-122
[Abstract]
[Full Text]
-
Viswanathan, P., Venkaiah, B., Kumar, M. S., Rasheedi, S., Vrati, S., Bashyam, M. D., Hasnain, S. E.
(2003). The Homologous Region Sequence (hr1) of Autographa californica Multinucleocapsid Polyhedrosis Virus Can Enhance Transcription from Non-baculoviral Promoters in Mammalian Cells. J. Biol. Chem.
278: 52564-52571
[Abstract]
[Full Text]
-
Jehle, J. A.
(2002). The expansion of a hypervariable, non-hr ori-like region in the genome of Cryptophlebia leucotreta granulovirus provides in vivo evidence for the utilization of baculovirus non-hr oris during replication. J. Gen. Virol.
83: 2025-2034
[Abstract]
[Full Text]
-
Pijlman, G. P., Dortmans, J. C. F. M., Vermeesch, A. M. G., Yang, K., Martens, D. E., Goldbach, R. W., Vlak, J. M.
(2002). Pivotal Role of the Non-hr Origin of DNA Replication in the Genesis of Defective Interfering Baculoviruses. J. Virol.
76: 5605-5611
[Abstract]
[Full Text]
-
Luque, T., Finch, R., Crook, N., O'Reilly, D. R., Winstanley, D.
(2001). The complete sequence of the Cydia pomonella granulovirus genome. J. Gen. Virol.
82: 2531-2547
[Abstract]
[Full Text]
-
Huang, J., Levin, D. B.
(2001). Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication. J. Gen. Virol.
82: 1767-1776
[Abstract]
[Full Text]
Top
Abstract
Introduction
