Previous Article | Next Article 
Journal of Virology, December 2001, p. 11401-11407, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11401-11407.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of the Rhesus Macaque
Rhadinovirus Lytic Origin of DNA Replication
Gregory S.
Pari,1,*
David
AuCoin,1
Kelly
Colletti,1
Sylvia A.
Cei,1
Veronica
Kirchoff,1 and
Scott
W.
Wong2
Department of Microbiology, University of
Nevada-Reno, Reno, Nevada 89557,1 and
Division of Pathobiology and Immunology, Oregon Health
Sciences University/Oregon Regional Primate Research Center,
Beaverton, Oregon 970062
Received 9 April 2001/Accepted 13 August 2001
 |
ABSTRACT |
We have identified a lytic origin of DNA replication (oriLyt) for
rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human
herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open
reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an
upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to
the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the
level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in
DNA replication. RRV oriLyt shares a high degree of nucleotide sequence
and G+C base distribution with the corresponding loci in HHV-8.
| |
INTRODUCTION |
Recently, rhesus macaque
rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, was isolated from
a simian immunodeficiency virus-infected animal with a
lymphoproliferative disorder (25, 29). The RRV genome was
shown to contain many of the same genes as HHV-8, and these genes have
a high degree of sequence similarity to HHV-8 (1, 25).
Like HHV-8, RRV encodes many cellular homologs, and the two viruses
share a similar genomic arrangement (1, 25). One defining
feature of RRV, in contrast to HHV-8, is that although it has been
shown that RRV can establish a latent infection in vivo, a lytic
infection occurs in primary rhesus macaque fibroblasts (RFs) (1,
4, 25). The ability of RRV to infect cells in culture and
produce infectious virus is an important attribute that makes RRV a
very attractive model for lytic replication studies.
HHV-8 is a gamma-2 herpesvirus that is the probable cause of Kaposi's
sarcoma (5, 7-9). In tissue culture, HHV-8 is present primarily in a latent form in human B cell lines (15, 16, 20). The lytic replication cycle is induced in a small number of
cells by incubation with the phorbol ester tetradecanoyl phorbol acetate or n-butyrate (20). It was also shown
that the viral lytic cycle could be initiated by transfection of the
virus-encoded open reading frame (ORF) product ORF50 into latently
infected B cells (22, 28). ORF50 is the putative homolog
of EBV Rta and appears to have transactivating properties
(13). These data implicated ORF50 as the key protein
involved in the transition from the latent to the lytic state of viral
replication. However, notwithstanding a demonstrated clear activation
of the HHV-8 lytic replication cycle by either drug treatment or
transfection of ORF50, it is estimated that only a small number of
cells in culture undergo lytic-phase replication when induced by either
method (14, 20).
Herpesvirus lytic replication initiates at distinct
cis-acting regions within the genome that vary in size and
complexity and are usually marked by the presence of a number of
transcription factor binding sites and multiple repeat regions
(2, 3, 12, 24, 30). These herpesvirus replication origins
were identified using a transient-transfection replication assay in which permissive cells are transfected with plasmids containing fragments of the viral genome and then incubated with infectious virus.
Viral infection supplies essential transacting factors required for
efficient origin-dependent DNA replication (3, 10, 12, 26,
27). Initiation of DNA synthesis for many herpesvirus origins is
via the interaction of a virus-encoded factor with a defined
cis-acting element present with the lytic origin. For
Epstein-Barr virus, the lytic origin is composed of four Zta binding
sites that appear to act as initiator sites for replication
(23). Substitutions or deletions of this region either
result in a reduction of or completely abolish origin-dependent replication.
The subcloning of the entire RRV genome into cosmid and plasmid
vectors, coupled with the fact that lytic infection occurs upon
infection of cultured cells, allows the development of a transient-transfection replication assay for the identification of the
RRV lytic origin of DNA replication (oriLyt). To this end, we
transfected cosmid clones of RRV into a new cell line of telomerized RFs to identify the location of an oriLyt. The RRV oriLyt was localized
to a region of the RRV genome between ORF69 and ORF71 (vFLIP). This
region amplified only in the presence of infecting virus and contains
an upstream A+T-rich region and a 300-bp downstream G+C repeat element.
In addition, oriLyt amplification occurred in cells cotransfected with
a complete set of RRV cosmids when an ORF50 expression plasmid was
included in the transfection mixture, allowing the elucidation of the
set of transacting factors required for origin-dependent DNA replication.
| |
MATERIALS AND METHODS |
Cells and virus.
Stock RRV (strain 17577) was propagated in
primary RFs isolated as previously described (25).
Telomerized RFs (telo-RFs) were used for all transfection experiments,
and their development is described elsewhere (V. Kirchoff, S. Wong, S. St. Jeor, and G. S. Pari, submitted for publication).
Plasmid constructs.
Construction of cosmid clones used for
transfection and cotransfection experiments (cosmids 8, 9, 28a, and 44)
was previously described (25). The initial plasmid
subclone encoding oriLyt was made from cosmid clone 8 as follows: the
XbaI 6.4 subclone, pRRVO, was constructed by cleaving cosmid
8 with XbaI and isolating the 6.4-kb fragment from
nucleotide (nt) 112979 to 119470 and ligating it into
XbaI-cleaved pGEM7zf(
). All other subclones were made from
the plasmid pRRVO. pRRVL1 was constructed by cleaving pRRVL with
BamHI, releasing the 3.2-kb fragment (genomic coordinates 112979 to 116210), and ligating it into BamHI-cleaved
pGEM7zf(). pRRVR1 was constructed by cleaving pRRVO with
BamHI, removing the BamHI fragment from nt 112979 to 116472, and religating the linearized plasmid carrying nt 116210 to
119470. pRRVM1 was constructed by cleaving pRRVO with NarI
and ligating the resulting 1.5-kb fragment into ClaI-cleaved
pGEM7zf(). pRRVR2 was constructed by cleaving pRRVR1 with
EcoRI and ligating the resulting 2.1-kb fragment from nt
117360 to 119470 into EcoRI-cleaved pGEM7zf(). Plasmid
pRRVL6 was made by using PCR primers corresponding to the 5' and 3'
ends (nt 112979 to 114067) of the A+T-rich region and ligating the
resulting product into pGEM T-easy.
RRV oriLyt deletion mutants were made using Erase-a-Base (Promega).
Plasmid pRRVL1 was cleaved with XhoI and SacI and
treated with lambda exonuclease as described by the
manufacturer. Several colonies were picked and analyzed by agarose gel
electrophoresis. Clones were sequenced to determine the extent of the
deleted sequence.
A plasmid construct expressing RRV ORF50, pEXP50, was generated by
using PCR primers complementary to the putative ORF50 ORF
identified
from the previously published sequence (
25). The
PCR
product was ligated into pTargeT (Promega). This vector uses
the
cytomegalovirus (CMV) immediate early promoter to drive the
expression
of an inserted
gene.
Transfection-replication assay.
Telo-RFs (5 × 105) were propagated and plated for transfection
using Dulbecco's modification of Eagle's medium supplemented with
10% fetal bovine serum in 6-cm dishes 16 h prior to transfection. Culture medium was removed and replaced with 3 ml of complete medium
4 h before transfection. Cells were transfected using 8 µl of
Transit-LT (Mirus) and 3 µg of plasmid or cosmid DNA. Cells were
incubated with the Transit-DNA mixture for 15 h, medium was removed, and cells were infected with concentrated RRV. Concentrated RRV was obtained by harvesting infected cells from a 10-day-infected roller bottle culture and pelleting virus using an SW28 rotor (25,000 rpm, 1.5 h). The resulting virus pellet was resuspended in
5 ml of complete medium. Each dish of transfected telo-RFs was
incubated with 300 µl of concentrated virus stock. Transfected cells
were incubated with virus for 5 to 6 days, and then total cellular DNA
was extracted using Puregene DNA isolation solutions (Gentra Systems).
Five micrograms of DNA was cleaved with EcoRI (15 U) and
DpnI (5 U) and separated on a 0.8% agarose gel. The gel was
transferred to a nylon membrane and hybridized with
32P-labeled pGEM probe in order to detect
replicated plasmid DNA.
For the cotransfection replication assay, cells were transfected using
the BES
[
N,
N-bis(2-hydroxyethyl)-2-aminoethanesulfonic
acid] calcium phosphate method similar to the one used for human
CMV (HCMV) (
19). Cells were incubated with a mixture of
the
oriLyt-containing plasmid pRRVL1 (10 µg) and cosmids 9, 8, 44,
and 28a (2 µg each). Total cell DNA was harvested 6 days
posttransfection
and treated as for the infection-replication
assay.
| |
RESULTS |
RRV cosmid 8 contains a lytic origin of replication.
Recently,
as part of a sequencing endeavor for RRV, the entire RRV genome was
subcloned into overlapping cosmid vectors (25). It was
also demonstrated that these cosmids were sufficient to recover
infectious virus upon cotransfection into permissive macaque fibroblasts (S. Wong, unpublished data). These experiments indicated that (i) the RRV cloned cosmids contained a competent lytic origin and
(ii) the genes necessary for replication of that origin were present in
the cosmids and functional upon transfection into permissive cells.
These facts allowed us to develop a transient-transfection replication-infection assay by individually transfecting each cosmid
from a set of four RRV overlapping cosmids (Fig.
1). Although RRV is permissive in
primary RFs, it became obvious after initial transfection experiments
that the transfection efficiency in these cells was very low. We had
previously developed a line of telomerized RFs, and these cells were
found to be highly transfectable and maintained permissiveness for RRV.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 1.
Schematic representation of the RRV genome and oriLyt.
(A) Location of oriLyt mapping between ORF69 and ORF70 contained within
cosmid 8. (B) Plasmid construct pRRVO containing the 6.4-kb
XbaI fragment. Relative positions of some restriction
endonuclease sites, A+T- and G+C-rich regions, and the positions of two
putative ORFs are shown. (C) Plasmid constructs used in the
transfection-infection assay. The box in the bottom right hand corner
defines the symbols used for transcription factor binding sites
identified within oriLyt.
|
|
To determine the location of oriLyt within cosmid subclones, we
transfected individual cosmid clones into telo-RFs and subsequently
infected these cells with high-titer concentrated RRV (approximate
titer, 10
7 PFU per ml). Total cellular DNA was
harvested and cleaved with
EcoRI and
DpnI, which
cleaves only input unreplicated cosmid DNA
that has been propagated in
a Dam
+ bacterial host. Cosmids 8, 9, 28a, and 44 were individually transfected
into telo-RFs. Of the four RRV cosmids
transfected, only cosmid
8 amplified in the presence of infecting
virus, as demonstrated
by the presence of a
DpnI-resistant
band (Fig.
2A, lane 1). Cosmid
8 spans nt
107105 to 130000 of the RRV genome. This sequence is
located at the
right end of the genome. Present within this region
are a series of
repeats referred to as rDL-E 1 and 2 (
25). The
rDL-E
region, spanning approximately nt 114000 to 117000, encodes
a highly
repetitive G+C-rich DNA sequence. According to the published
map of RRV
ORFs, the rDL-E loci appeared to be devoid of any recognizable
ORFs
(
25). We chose to focus on this region as the possible
location of an oriLyt contained with cosmid 8.
View larger version (82K):
[in this window]
[in a new window]
|
FIG. 2.
Identification of RRV oriLyt using the
transient-transfection infection-replication assay. Total cellular DNA
from transfected cells was cleaved with EcoRI and
DpnI, separated through a 0.8% agarose gel, transferred
to a nylon membrane, and probed with SuperCos (Stratagene). Arrowheads
indicate replication products. (A) Autoradiogram of a Southern blot of
total telo-RF DNA from cells transfected with RRV cosmids and
subsequently infected with RRV. Lanes: 1, cosmid 8; 2, cosmid 9; 3, cosmid 28a; 4, cosmid 44. (B) RRV oriLyt is present within a 6.4-kb
XbaI I fragment subcloned from cosmid 8. DNA was treated
as for panel A except that blots were probed with pGEM7zf(). Lanes: 1 and 2, pRRVO-transfected infected telo-RF DNA; 3 and 4, as lanes 1 and
2 except that cells were treated with PFA; 5 and 6, pRRVR1-transfected
infected telo-RF DNA.
|
|
Several subclones of cosmid 8 were transfected into telo-RFs and
evaluated for their ability to amplify in the presence of
infecting
virus (data not shown). Of these subclones, a 6.4-kb
XbaI
subclone made from cosmid 8 replicated when transfected into
cells that
were subsequently infected with RRV (Fig.
2B, lanes
1 and 2) but not in
cells treated with phosphonoformic acid (PFA)
at the time of
infection (Fig.
2B, lanes 3 and 4). These data
indicated that the
6.4-kb
XbaI fragment contained a lytic origin
of
replication.
RRV oriLyt is composed of an A+T-rich and a short G+C-rich
sequence.
The XbaI subclone pRRVO was cleaved with
BamHI, which essentially cut the 6.4-kb XbaI
fragment in half (Fig. 1). The resulting two BamHI fragments
were subcloned. A plasmid clone, pRRVR1, corresponding to the right
half of pRRVO from nt 116210 to 119470 failed to replicate in infected
telo-RF cells (Fig. 2B, lanes 5 and 6). The failure of this fragment to
amplify may indicate that RRV oriLyt was localized in the right half of
pRRVO. To test this hypothesis, we transfected the subclone pRRVL1,
which carried the left half of pRRVO. Transfection of pRRVL1 resulted
in the detection of a DpnI-resistant band and indicated that
RRV oriLyt was contained in pRRVL (nt 112979 to 116210).
Now that it had been demonstrated that a replication origin was present
within the plasmid subclone pRRVL1, we wanted to identify
essential DNA
elements contributing to origin function and further
localize the
boundaries of oriLyt. The right half of pRRVO contains
a highly
repetitive G+C-rich region spanning approximately 1.7
kb (Fig.
1).
Immediately upstream of this G+C repeat region is
an A+T-rich region
spanning approximately 900 nt (Fig.
1). We
constructed a series of
subclones with portions of the repeat
regions eliminated. These plasmid
subclones and their location
are illustrated in Fig.
1. The plasmid
subclone, pRRVL1, encoding
the 3-kb region from nt 112979 to 116210 was
fully replication
competent (Fig.
3A,
lane 2). This clone contained both A+T and
G+C repeat regions. When the
DNA sequence in pRRVL1 was subjected
to analysis using software
designed to search for putative ORFs,
two ORFs not previously
identified in the original published sequence
of either RRV strain were
identified (
1,
25); the locations
of these ORFs are shown
in Fig.
1.
View larger version (61K):
[in this window]
[in a new window]
|
FIG. 3.
RRV oriLyt contains two essential repeat elements.
Autoradiograms of Southern blots of DNA from transfected infected cells
treated as described in Materials and Methods are shown. Arrowheads
indicate replication products. (A) DNA from cells transfected with
pRRVL3 (lane 1), pRRVL1 (lane 2), pRRVM1 (lane 3), pRRVL4 (lane 4), or
pRRVL2 (lane 5). (B) Replication of a plasmid construct containing both
intact G+C- and A+T-rich genomic sequences. Lanes 1 and 2, transfection
with plasmid pRRVL5 in mock-infected cells and RRV-infected cells,
respectively. (C) The A+T-rich region plus 200 bp of the G+C-rich
genomic sequence is required for plasmid replication. Cells were
transfected with oriLyt plasmids containing variable amounts of the
G+C-rich region or the A+T sequence alone. Lanes: 1, pRRVL7 (nt 112979 to 115016); 2, pRRVL6 (nt 112979 to 114067); 3, pRRVL8 (nt 112979 to
114371).
|
|
A transfected subclone containing the complete G+C repeat region but
only a portion of the A+T repeat region failed to replicate
(Fig.
3A,
lane 5). This subclone, pRRVL2, contains genomic sequences
from the
KpnI site at nt 113582 to the
NarI site at nt
115688
(Fig.
1). pRRVL2 deletes part of the A+T repeat DNA sequence
region
and truncates the putative 2.0-kb ORF that goes through the
G+C-rich
region (Fig.
1). Similarly, plasmids pRRVL3 and pRRVL4 failed
to replicate (Fig.
3A, lanes 1 and 4, respectively). pRRVL3 contains
only a small portion of the G+C repeat region and truncated portions
of
both potential ORFs. pRRVL4 extends further 3' than pRRVL3
but still
does not include the complete G+C repeats or putative
ORFs. Failure of
these subclones to replicate indicated that the
left boundary of oriLyt
extended to the original
XbaI fragment
at nt 112979 (Fig.
1). Another subclone, pRRVM1, also failed to
replicate, indicating that
an oriLyt does not lie in the middle
sequence of the 6.4-kb
XbaI region (Fig.
3A, lane 3). In order
to test where the
right boundary of oriLyt was, we generated a
subclone that extended to
the
NarI site at nt 115688 and tested
it in the replication
assay. This subclone, pRRVL5, amplified
in the presence of RRV,
indicating that the DNA sequence located
between the
NarI
(115668) and the
BamHI (116210) sites was dispensable
(Fig.
3B, lane
2).
In order to further define the boundaries of RRV oriLyt, we generated
several plasmid constructs that deleted various amounts
of the G+C-rich
genomic sequence (Fig.
1). In addition, we subcloned
the A+T-rich
region alone and tested this plasmid, pRRVL6, along
with the G+C
deletion constructs. The plasmid construct, pRRVL7,
which removes 650 bp from the 3' end of pRRVL5 replicated, which
indicated that the
entire G+C-rich region was not necessary for
efficient replication
(Fig.
3C, lane 1). The A+T region alone,
however, did not replicate,
indicating that additional sequence
was required (Fig.
3C, lane 2).
Several other deletion constructs
were tested (data not shown), and it
was determined that the smallest
clone that was replication competent,
pRRVL8, contained approximately
300 bp of the 3'-flanking G+C sequence
(Fig.
3C, lane 3). Replication
of pRRVL8 was somewhat decreased
compared to that of larger clones;
nevertheless, this construct defines
the minimal boundaries of
RRV
oriLyt.
RRV cosmids contain all the genes required for origin-dependent
lytic DNA replication.
Once an oriLyt had been identified for RRV,
we evaluated the ability of our overlapping cosmid library to
complement origin-dependent lytic replication. These cosmids should
encode all the necessary transacting factors for efficient oriLyt
replication. Our initial attempts to get oriLyt-dependent DNA
replication from cotransfection of four overlapping cosmids plus oriLyt
(pRRVO) failed to produce a detectable replicated product (Fig.
4, lane 1). We assumed that this might be
due to a low level of expression of certain viral transactivators,
which then activate genes required for replication. In order to
overcome this, we added increasing amounts of an expression construct
encoding RRV ORF50 to the transfection mixture. For HHV-8, ORF50 was
shown to activate the entire lytic cycle (11, 13).
Introduction of an HHV-8 ORF50-expressing plasmid into latently
infected cells resulted in the initiation of the lytic cycle of the
virus, as evidenced by the appearance of viral lytic markers.
Consequently, we chose to deliver additional RRV ORF50 by adding an
ORF50 expression plasmid to the cotransfection mixture already
containing the four overlapping cosmids and oriLyt. Transfections containing increasing amounts of pEXP50 yielded a detectable
replication product (Fig. 4, lanes 2 through 4). The oriLyt band also
appeared to be more intense as more pEXP50 was added, suggesting a
higher degree of replication correlated to increasing levels of ORF50 protein (Fig. 4, lanes 2 through 4).
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 4.
Addition of an ORF50 expression construct facilitates
origin-dependent DNA replication in the cotransfection replication
assay. A Southern blot of the cotransfection replication assay is
shown. Telo-RFs were transfected with all four overlapping
cosmids plus pRRVO alone (lane 1) or pRRVO and 0.5, 1, or 2 µg of
pEXP50 (lanes 2 through 4, respectively). The arrowhead indicates the
replication product.
|
|
Inspection of the oriLyt DNA sequence.
We compared the
relative positions of RRV oriLyt and the same locus in HHV-8. RRV
oriLyt maps to the region between ORF69 and the vFLIP (ORF71) gene
locus mapping approximately between nt 110000 and 120000. Homologs of
these genes are located in the same relative position in the HHV-8
genome (Fig. 5A). It was suggested that
this region, in HHV-8, might contain a lytic origin (18). When the HHV-8 and RRV genomes within the genomic regions
between ORF 69 and vFLIP are compared, several similarities are
apparent. This region in HHV-8 is a highly repetitive G+C-rich DNA
sequence and contains a number of transcription factor binding sites.
In addition, except for one ORF identified by DNA sequence analysis and
not described in the original report of the HHV-8 genomic sequence,
this region apparently does not contain any identifiable ORFs
(21).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 5.
Comparison of RRV oriLyt and the corresponding region in
HHV-8. (A) Alignment of RRV and HHV-8 genomes, showing the genomic
regions between ORF69 and vFLIP. (B) Comparison of the 2-kb region from
RRV oriLyt and the similar region in HHV-8. The relative positions of
two putative ORFs within each region are shown. (C) Schematic
representation of the oriLyt region of RRV and the positional homolog
of HHV-8. The G+C base pair distribution across each region is shown.
|
|
RRV oriLyt has an overall G+C content of 61%; a region of similar size
and position in the HHV-8 genome was calculated to
have a 59% G+C
content. Between these two regions (HHV-8 putative
oriLyt and RRV
oriLyt) there is 47% nucleotide sequence homology.
Several similar or
identical repeat elements are present within
RRV and HHV-8 regions; one
example is the DNA sequence CCGGGG,
which is repeated 21 times in RRV
oriLyt and 15 times in the putative
HHV-8 oriLyt region (Fig.
5B). As
with RRV, we were also able
to identify a small ORF (800 bp) within the
G+C repeat region
of the putative HHV-8 oriLyt. This putative ORF is
located in
a similar place in the RRV oriLyt, which is within the
G+C-rich
repeat regions (Fig.
5B). Distribution of G+C base pairs also
had similar patterns in HHV-8 and RRV homologous regions. There
appears
to be the same A+T-rich region followed by a downstream
G+C-rich region
(Fig.
5C). Although the G+C-rich region appears
to be longer in RRV,
the pattern is similar in HHV-8 (Fig.
5C).
Most striking is the
similarity of the pattern of base distribution,
where two A+T-rich
regions flank a highly repetitive G+C-rich
domain.
| |
DISCUSSION |
Lytic replication of HHV-8 occurs in a small number of
persistently infected B cells upon treatment with tetradecanoyl phorbol myristate or sodium butyrate or transfection with the viral
transactivator ORF50 (11, 13, 28). This is not the case
for the rhesus macaque homolog, RRV, where a lytic infection occurs
upon incubation with infectious virus in primary RFs. This feature
lends itself to the development of a transient-replication assay
similar to those used to identify lytic origins of other herpesviruses
(3, 12, 26, 27). RRV shares most of the genes with HHV-8,
including the cellular homologs for interleukin 6, cyclin D, and Bcl2.
There are, however, some notable differences. For example, RRV lacks the genes for kaposin and nut1 and some of the vMIP-encoding loci. Despite these differences, RRV is still a good model for the study of
gammaherpesvirus lytic replication. All of the core replication proteins along with the viral transactivators have a high degree of
nucleotide sequence and positional homology between the two viruses.
This may allow a hybrid replication assay where proteins from one
system will be used to complement origin-dependent replication for the other.
We developed a transient-replication assay using RRV where infecting
virus supplies all of the necessary transacting factors for lytic
replication. For these studies we generated an RF cell line by
introducing the gene expressing the catalytic subunit of telomerase
(6, 17). These cells, telo-RFs, are highly transfectable
and are much more robust than primary RFs. In addition to using these
cells for our assay, we found it necessary to use concentrated virus
for infection. Regular stock virus was not of a high enough titer to
efficiently infect all of the cells in the culture dish. The
replication-infection assay revealed that a lytic origin was present in
RRV cosmid 8, and subsequent subcloning identified a 2.3-kb region
containing an A+T-rich region followed by a downstream 300-bp G+C-rich
region. Transfection data suggest that both the A+T-rich and the short
(300-bp) G+C-rich regions are essential for efficient replication. This
region would include any putative promoter elements required for the
transcription of mRNA containing one or both of the potential ORFs
found in the downstream region. This RRV oriLyt region closely
resembles the corresponding HHV-8 loci. In addition, a recently
identified lytic origin for the bovine rhadinovirus bovine herpesvirus
4 shows positional homology to the genomes of RRV and HHV-8
(30). The bovine herpesvirus 4 oriLyt also has a G+C-rich
region flanked by an A+T-rich region (30). The
conservation of these structures may reflect a distinct mechanism for
the initiation of DNA replication for this subset of herpesviruses.
We also demonstrated that a set of overlapping cosmids for RRV can
complement origin-dependent DNA replication. We found it necessary to
add additional ORF50 protein in the form of a plasmid construct under
the control of the HCMV major immediate early promoter. Additional
ORF50 may be required because expression from the native ORF50 within
cosmid 28a was not sufficient to activate the other replication
factors. A similar scenario was observed for the cosmid cotransfection
assay for HCMV. In the HCMV system, origin-dependent replication was
inefficient unless additional IE2 was added to the transfection mixture
in the form of a plasmid expression vector (19). Another
possibility for both systems is that these transactivators may directly
participate in lytic replication. The cotransfection assay should allow
the elucidation of factors required for oriLyt-dependent DNA replication.
Now that the lytic origin for RRV has been identified and a
cotransfection assay has been established, we can start to compare this
obviously more lytic viral system to HHV-8, a predominately latent system.
| |
ACKNOWLEDGMENT |
This work was supported by a grant from the National Institutes
of Health (R01 CA85164)
| |
FOOTNOTES |
*
Corresponding author. Mailing address: University of
Nevada, Reno, School of Medicine/Dept. of Microbiology, Howard Bldg., Reno, NV 89557. Phone: (775) 784-4824. Fax: (775) 784-1620. E-mail: gpari{at}med.unr.edu.
| |
REFERENCES |
| 1.
|
Alexander, L.,
L. Denekamp,
A. Knapp,
M. R. Auerbach,
B. Damania, and R. C. Desrosiers.
2000.
The primary sequence of rhesus monkey rhadinovirus isolate 26-95: sequence similarities to Kaposi's sarcoma-associated herpesvirus and rhesus monkey rhadinovirus isolate 17577.
J. Virol.
74:3388-3398[Abstract/Free Full Text].
|
| 2.
|
Anders, D. G.,
M. A. Kacica,
G. Pari, and S. M. Punturieri.
1992.
Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication.
J. Virol.
66:3373-3384[Abstract/Free Full Text].
|
| 3.
|
Anders, D. G., and S. M. Punturieri.
1991.
Multicomponent origin of cytomegalovirus lytic-phase DNA replication.
J. Virol.
65:931-937[Abstract/Free Full Text].
|
| 4.
|
Bergquam, E. P.,
N. Avery,
S. M. Shiigi,
M. K. Axthelm, and S. W. Wong.
1999.
Rhesus rhadinovirus establishes a latent infection in B lymphocytes in vivo.
J. Virol.
73:7874-7876[Abstract/Free Full Text].
|
| 5.
|
Birley, H. D., and T. F. Schultz.
1997.
Kaposi's sarcoma and the new herpesvirus.
J. Med. Microbiol.
46:433-435[Free Full Text].
|
| 6.
|
Bodnar, A. G.,
M. Ouellette,
M. Frolkis,
S. E. Holt,
C. P. Chiu,
G. B. Morin,
C. B. Harley,
J. W. Shay,
S. Lichtsteiner, and W. E. Wright.
1998.
Extension of life-span by introduction of telomerase into normal human cells.
Science
279:349-352[Abstract/Free Full Text].
|
| 7.
|
Cesarman, E.,
Y. Chang,
P. S. Moore,
J. W. Said, and D. M. Knowles.
1995.
Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.
N. Engl. J. Med.
332:1186-1191[Abstract/Free Full Text].
|
| 8.
|
Cesarman, E.,
P. S. Moore,
P. H. Rao,
G. Inghirami,
D. M. Knowles, and Y. Chang.
1995.
In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi's sarcoma-associated herpesvirus-like (KSHV) DNA sequences.
Blood
86:2708-2714[Abstract/Free Full Text].
|
| 9.
|
Chang, Y.,
E. Cesarman,
M. S. Pessin,
F. Lee,
J. Culpepper,
D. M. Knowles, and P. S. Moore.
1994.
Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.
Science
266:1865-1869[Abstract/Free Full Text].
|
| 10.
|
Dewhurst, S.,
S. C. Dollard,
P. E. Pellett, and T. R. Dambaugh.
1993.
Identification of a lytic-phase origin of DNA replication in human herpesvirus 6B strain Z29.
J. Virol.
67:7680-7683[Abstract/Free Full Text].
|
| 11.
|
Gradoville, L.,
J. Gerlach,
E. Grogan,
D. Shedd,
S. Nikiforow,
C. Metroka, and G. Miller.
2000.
Kaposi's sarcoma-associated herpesvirus open reading frame 50/Rta protein activates the entire viral lytic cycle in the HH-B2 primary effusion lymphoma cell line.
J. Virol.
74:6207-6212[Abstract/Free Full Text].
|
| 12.
|
Hammerschmidt, W., and B. Sugden.
1988.
Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus.
Cell
55:427-433[CrossRef][Medline].
|
| 13.
|
Lukac, D. M.,
J. R. Kirshner, and D. Ganem.
1999.
Transcriptional activation by the product of open reading frame 50 of Kaposi's sarcoma-associated herpesvirus is required for lytic viral reactivation in B cells.
J. Virol.
73:9348-9361[Abstract/Free Full Text].
|
| 14.
|
Lukac, D. M.,
R. Renne,
J. R. Kirshner, and D. Ganem.
1998.
Reactivation of Kaposi's sarcoma-associated herpesvirus infection from latency by expression of the ORF50 transactivator, a homolog of the EBV R protein.
Virology
252:304-312[CrossRef][Medline].
|
| 15.
|
Miller, G.,
L. Heston,
E. Grogan,
L. Gradoville,
M. Rigsby,
R. Sun,
D. Shedd,
V. M. Kushnaryov,
S. Grossberg, and Y. Chang.
1997.
Selective switch between latency and lytic replication of Kaposi's sarcoma herpesvirus and Epstein-Barr virus in dually infected body cavity lymphoma cells.
J. Virol.
71:314-324[Abstract].
|
| 16.
|
Miller, G.,
M. O. Rigsby,
L. Heston,
E. Grogan,
R. Sun,
C. Metroka,
J. A. Levy,
S. J. Gao,
Y. Chang, and P. Moore.
1996.
Antibodies to butyrate-inducible antigens of Kaposi's sarcoma-associated herpesvirus in patients with HIV-1 infection.
N. Engl. J. Med.
334:1292-1297[Abstract/Free Full Text].
|
| 17.
|
Nakamura, T. M.,
G. B. Morin,
K. B. Chapman,
S. L. Weinrich,
W. H. Andrews,
J. Lingner,
C. B. Harley, and T. R. Cech.
1997.
Telomerase catalytic subunit homologs from fission yeast and human.
Science
277:955-959[Abstract/Free Full Text].
|
| 18.
|
Nicholas, J.,
J. C. Zong,
D. J. Alcendor,
D. M. Ciufo,
L. J. Poole,
R. T. Sarisky,
C. J. Chiou,
X. Zhang,
X. Wan,
H. G. Guo,
M. S. Reitz, and G. S. Hayward.
1998.
Novel organizational features, captured cellular genes, and strain variability within the genome of KSHV/HHV8.
J. Natl. Cancer Inst. Monogr.
23:79-88.
|
| 19.
|
Pari, G. S.,
M. A. Kacica, and D. G. Anders.
1993.
Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis.
J. Virol.
67:2575-2582[Abstract/Free Full Text].
|
| 20.
|
Renne, R.,
W. Zhong,
B. Herndier,
M. McGrath,
N. Abbey,
D. Kedes, and D. Ganem.
1996.
Lytic growth of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in culture.
Nat. Med.
2:342-346[CrossRef][Medline].
|
| 21.
|
Russo, J. J.,
R. A. Bohenzky,
M. C. Chien,
J. Chen,
M. Yan,
D. Maddalena,
J. P. Parry,
D. Peruzzi,
I. S. Edelman,
Y. Chang, and P. S. Moore.
1996.
Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).
Proc. Natl. Acad. Sci. USA
93:14862-14867[Abstract/Free Full Text].
|
| 22.
|
Sarid, R.,
O. Flore,
R. A. Bohenzky,
Y. Chang, and P. S. Moore.
1998.
Transcription mapping of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) genome in a body cavity-based lymphoma cell line (BC-1).
J. Virol.
72:1005-1012[Abstract/Free Full Text].
|
| 23.
|
Sarisky, R. T.,
Z. Gao,
P. M. Lieberman,
E. D. Fixman,
G. S. Hayward, and S. D. Hayward.
1996.
A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator.
J. Virol.
70:8340-8347[Abstract].
|
| 24.
|
Schepers, A. D.,
D. Pich,
J. Mankertz, and W. Hammerschmidt.
1993.
Cis-acting elements in the lytic origin of DNA replication of Epstein-Barr virus.
J. Virol.
67:4237-4245[Abstract/Free Full Text].
|
| 25.
|
Searles, R. P.,
E. P. Bergquam,
M. K. Axthelm, and S. W. Wong.
1999.
Sequence and genomic analysis of a rhesus macaque rhadinovirus with similarity to Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8.
J. Virol.
73:3040-3053[Abstract/Free Full Text].
|
| 26.
|
Stow, N. D.
1982.
Localization of an origin of DNA replication within the TRs/IRs repeated region of the herpes simplex virus type 1 genome.
EMBO J.
1:863-867[Medline].
|
| 27.
|
Stow, N. D., and A. J. Davidson.
1986.
Identification of varicella-zoster virus origin of DNA replication and its activation by herpes simplex virus type 1 gene products.
J. Gen. Virol.
67:1613-1623[Abstract/Free Full Text].
|
| 28.
|
Sun, R.,
S. F. Lin,
K. Staskus,
L. Gradoville,
E. Grogan,
A. Haase, and G. Miller.
1999.
Kinetics of Kaposi's sarcoma-associated herpesvirus gene expression.
J. Virol.
73:2232-2242[Abstract/Free Full Text].
|
| 29.
|
Wong, S. W.,
E. P. Bergquam,
R. M. Swanson,
F. W. Lee,
S. M. Shiigi,
N. A. Avery,
J. W. Fanton, and M. K. Axthelm.
1999.
Induction of B cell hyperplasia in simian immunodeficiency virus-infected rhesus macaques with the simian homologue of Kaposi's sarcoma-associated herpesvirus.
J. Exp. Med.
190:827-840[Abstract/Free Full Text].
|
| 30.
|
Zimmermann, W.,
H. Broll,
B. Ehlers,
H. J. Buhk,
A. Rosenthal, and M. Goltz.
2001.
Genome sequence of bovine herpesvirus 4, a bovine rhadinovirus, and identification of an origin of DNA replication.
J. Virol.
75:1186-1194[Abstract/Free Full Text].
|
Journal of Virology, December 2001, p. 11401-11407, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11401-11407.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Xu, Y., Rodriguez-Huete, A., Pari, G. S.
(2006). Evaluation of the Lytic Origins of Replication of Kaposi's Sarcoma-Associated Virus/Human Herpesvirus 8 in the Context of the Viral Genome. J. Virol.
80: 9905-9909
[Abstract]
[Full Text]
-
Deng, H., Chu, J. T., Park, N.-H., Sun, R.
(2004). Identification of cis Sequences Required for Lytic DNA Replication and Packaging of Murine Gammaherpesvirus 68. J. Virol.
78: 9123-9131
[Abstract]
[Full Text]
-
Lin, C. L., Li, H., Wang, Y., Zhu, F. X., Kudchodkar, S., Yuan, Y.
(2003). Kaposi's Sarcoma-Associated Herpesvirus Lytic Origin (ori-Lyt)-Dependent DNA Replication: Identification of the ori-Lyt and Association of K8 bZip Protein with the Origin. J. Virol.
77: 5578-5588
[Abstract]
[Full Text]
-
AuCoin, D. P., Colletti, K. S., Xu, Y., Cei, S. A., Pari, G. S.
(2002). Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Two Functional Lytic Origins of DNA Replication. J. Virol.
76: 7890-7896
[Abstract]
[Full Text]
Top
Abstract
Introduction
