Department of Medical Microbiology and Immunology, College of Medicine, University of South Florida, MDC Box 10, 12901 Bruce B. Downs Blvd, Tampa, Florida 33612-4799, USA1
The H. Lee Moffitt Cancer Center, Tampa, Florida 33612-4799, USA2
Author for correspondence: Peter Medveczky (at the College of Medicine). Fax +1 813 974 4151. e-mail pmedvecz{at}com1.med.usf.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HVS can be isolated from healthy squirrel monkeys (Melendez et al., 1968 ). In contrast, HVS induces CD8 lymphomas in several other species of New World monkeys and rabbits (Fleckenstein, 1980
; Medveczky et al., 1989
; Medveczky, 1995
; Melendez et al., 1968
). HVS also efficiently immortalizes New World monkey and human T cells (Biesinger et al., 1992
; Desrosiers et al., 1986
; Medveczky et al., 1993
; Szomolanyi et al., 1987
). The genome of HVS consists of 113 kb of unique sequences designated as L-DNA (light or low G+C), which contains at least 75 open reading frames (Albrecht et al., 1992
; Fleckenstein, 1980
). These L-DNA sequences are flanked by tandem repeats of non-coding H-DNA (heavy or high G+C) (Fleckenstein, 1980
).
Using the lack of sequence homology in the 2 kb at the left end of the genome as a criterion, the various isolates have been classified into three subgroups: A, B and C (Medveczky et al., 1984 ). Group C strains are the most potent in both immortalization and oncogenesis assays, and are the only strains able to immortalize human T cells (Biesinger et al., 1992
; Medveczky et al., 1993
). HVS group C strain 484-77 is also highly oncogenic in New Zealand White rabbits (Medveczky et al., 1989
).
During latent/persistent infection, the viral genome of herpesviruses exists as a circular episome, and HVS is no exception. Tumour tissues and cell lines established from tumours or by in vitro immortalization carry multiple copies of the viral DNA in covalently closed circular form, and circularization occurs by joining the ends of the viral genome (Fleckenstein, 1980 ; Gardella et al., 1984
; Medveczky, 1995
; Schirm et al., 1984
). No evidence has been published indicating that HVS integrates into the host genome.
Most of the viral genes encoded by HVS are inactive in immortalized T cells, and only a limited number of gene products required for oncogenesis, such as the STP and TIP proteins, and small RNAs encoded by the left end of the L-DNA can be detected (for reviews, see Fleckenstein, 1980 ; Medveczky, 1995
). Extensive methylation of the episomal HVS DNA at CG residues in immortalized T cells has been described and is thought to correlate with the lack of gene expression in mammalian cells (Desrosiers, 1982
; Desrosiers et al., 1979
; Youssoufian & Mulder, 1981
). Consistent with this concept, the left end of the L-DNA is hypomethylated. The high G+C H-DNA sequences are also heavily methylated, and all attempts to find transcription in the repeats have failed (Desrosiers, 1982
; Desrosiers et al., 1979
; Youssoufian & Mulder, 1981
). No known functions of H-DNA have so far been described.
Most KSHV genes are also inactive in immortalized cells. One of the proteins that is expressed in cells latently infected with KSHV is the latency-associated nuclear antigen (LANA), which is encoded by orf 73. LANA and episomes containing H-DNA have been shown to co-localize with mitotic chromosomes (Ballestas et al., 1999 ), and persistence of these episomes in cell culture is mediated by LANA binding to a specific sequence within the H-DNA (Ballestas & Kaye, 2001
). LANA has also been shown to bind to histone H1 (Cotter & Robertson, 1999
). These data suggest a mechanism by which LANA tethers viral DNA to mitotic chromosomes through its interaction with H-DNA, allowing efficient segregation of viral episomes to progeny cells.
HVS encodes a homologue of KSHV LANA, and although there is limited similarity between KSHV and HVS LANA, both proteins contain a central glutamate-rich domain as well as several potential phosphorylation sites (Albrecht et al., 1992 ; Russo et al., 1996
). HVS LANA also localizes in the nucleus in a distinctive speckled distribution, similar to that of KSHV LANA (Hall et al., 2000
). These findings suggest that HVS LANA may play a role in episomal maintenance similar to that of KSHV LANA.
Here we describe the cloning of an infectious L-DNA restriction fragment of the highly oncogenic HVS strain C484-77 in E. coli. Analysis of T cells infected with progeny viruses recovered from cloned DNA showed a complete absence of episomal viral genomes while recombinants with restored terminal repetitive H-DNA were able to replicate as episomes, indicating that intact H-DNA is essential for latent episomal replication. Moreover, LANA expression vectors containing terminal repeats stably replicated in 293 cells as episomes, indicating that LANA is essential and sufficient for episomal replication.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Infection and immortalization of T cells.
Human peripheral blood mononuclear cells were purified by Ficoll gradients and infected as described earlier (Schirm et al., 1984 ). Briefly, OMK cells were infected with approximately 106 p. f. u. of virus. Two to three days after infection, when early signs of cytopathic effects of the virus were observed, the medium was removed and 2x107 mononuclear cells in 10 ml AIM V medium (Gibco BRL) containing 10% fetal calf serum were added to the cultures. Infected lymphocytes were cultured in the same medium. Control uninfected cells were grown in the same medium, or, in some experiments, supplemented with 100 U/ml human recombinant IL-2.
Transfection of DNA and generation of rescued virus.
The main steps for generating virus and recombinants from pBeloBac-HVS are depicted in Fig. 1. Owl monkey kidney cells were transfected with pBeloBac-HVS clones or co-transfected with pBeloBac-HVS and pL-H, a construct containing 5 kb of L-DNA and attached H-DNA units, by the calcium co-precipitation method, as previously described (Medveczky et al., 1989
, 1993
). Individual virus clones and recombinants were isolated by limiting dilution of virus obtained by the transfection step (Medveczky et al., 1989
, 1993
).
Detection of episomal and linear viral genome.
To detect HVS DNA in infected T cells and to determine the physical state of the viral sequences we used the agarose gel method of Gardella et al. (1984) . Briefly, a suspension of live cells was loaded into a well of a vertical agarose gel. DNA from this cell suspension was gently liberated by an overlay lysis solution containing SDS and Pronase. After slow electrophoresis/lysis, the separation of superhelical episomal and linear viral DNA was accomplished by electrophoresis. Under these conditions, most cellular DNA remains in the loading well while some migrates past the viral episomes (Gardella et al., 1984
). Viral DNA was visualized by Southern hybridization with radiolabelled probes, as previously described (Gardella et al., 1984
; Kung & Medveczky, 1996
). Cloned L-DNA pBeloBac-HVS was used as a probe.
DNA sequencing.
DNA sequencing was performed with an ABI Prism 377 Automated DNA Sequencing system (Applied Biosystems). Templates were used in a cycle sequencing reaction using the ABI Prism BigDye Terminators reaction mix (Applied Biosystems) with the appropriate primers.
Construction of LANA expression vectors containing H-DNA.
Because HVS stain A11 has been completely sequenced (Albrecht et al., 1992), the LANA open reading frame from this strain was used to create LANA expression vectors. A restriction fragment spanning nt 107228105708 was isolated by digestion of viral DNA with the restriction enzymes NarI and EcoRV. Because NarI removes the first six nucleotides of the LANA open reading frame, synthetic DNA was designed to replace these nucleotides as well as to contain a Kozak sequence upstream of the start codon. This fragment was cloned into the SalI site of the expression vector pBKCMV (Stratagene). DNA sequencing was performed to confirm the presence of the Kozak sequence and the start codon, as well as to confirm correct orientation. One, two and three repeats of H-DNA were then inserted into the unique MluI site of pLANA, creating pLANAH1, pLANAH2 and pLANAH3, respectively.
Episomal replication assay.
Plasmids were transfected into 293 cells using GenePORTER transfection reagent (Gene Therapy Systems) according to the manufacturers protocol. G418 was added 72 h post-transfection to a final concentration of 850 µg/ml. Cells were trypsinized when confluency was reached, and 20% were passed to new plates with fresh media and G418. Low molecular mass DNA was isolated from the remaining cells by the method of Hirt (1967) . To distinguish replicated DNA from DNA used for transfection, isolated DNA was digested with the restriction enzymes DpnI and MboI. Plasmid DNA amplified in dam methylase-positive E. coli is efficiently digested by DpnI while DNA methylated by mammalian cells is resistant to DpnI digestion. Conversely, the DpnI isoschizomer MboI cleaves DNA that is replicated and methylated in mammalian cells but does not efficiently digest DNA produced by dam methylase-positive E. coli. After restriction digestion, DNA was electrophoresed on a 0·8% agarose gel and the DNA was transferred to nitrocellulose. Plasmid DNA was detected by Southern hybridization using random primer-labelled pBKCMV as a probe. To determine the copy number of episomes per cell, the hybridized blots were analysed with a PhosphorImager (Molecular Dynamics) using the ImageQuant program of the manufacturer.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clones were digested with PstI and SacI restriction enzymes, which cut in both H-DNA and L-DNA (Albrecht et al., 1992; Bankier et al., 1985 ), and compared to viral DNA isolated from virions. The results indicated that five clones contained full-length 484-77 HVS L-DNA, since all unique bands co-migrated with virion DNA. However, no supermolar H-DNA bands were observed. Restriction fragment patterns of a representative clone #3 are shown in Fig. 2(A)
. Supermolar H-DNA bands were absent in the clone, and extra 3·1 and 3·9 kb vector bands appeared in the PstI digest of the clone while a single extra 8 kb band appeared in the SacI digest. An identical banding pattern was obtained with clone #17 (not shown). These data show that clones #3 and #17 contain the entire L-DNA region and remnants of H-DNA adjacent to L-DNA that were not cleaved off by NotI. The additional bands present in the digests of cloned viral DNA are contaminating E. coli DNA that resulted from cloning the viral DNA in E. coli. Viral DNA does not contain these bands because this DNA was isolated from purified virus.
|
Fig. 2(B) shows that the progeny viral genome of clone #3 contained an 8 kb supermolar band when cleaved with SacI, suggesting amplification of the vector DNA. Identical banding patterns of virion DNA recovered from clones #17 and #29 were obtained (not shown).
Fig. 2(C) shows viral DNA that consists of the H-DNA at the left end of the genome and the L-DNA up to the first HindIII site. Multiple bands were present due to varying numbers of terminal repeats among the individual viruses. However, when viral DNA cloned in E. coli and DNA from clone-derived progeny was digested with HindIII, which also cleaves once at the right end of the cloning vector, only two bands were present. The smaller band is the fragment between the HindIII restriction site at the left end of the L-DNA and the HindIII site at the right end of the vector. The large band is the vector, which consists of the fragment between the HindIII site in the vector and the HindIII site at the right end of the L-DNA. In the clone-derived progeny, the vector band was present at a much higher level of intensity, indicating that it had been amplified (Fig. 2C
).
The E. coli plasmids of clones #3, #17 and the virion progeny DNA cleaved with NotI showed the expected 7·3 kb vector band (Fig. 3A). No H-DNA repeats were detected in the E. coli clones. Surprisingly, the clone-derived viral genome contained a supermolar band that hybridized with repetitive H-DNA (Fig. 3B
).
|
Sequence analysis the HVS DNA cloned in E. coli revealed that the remnant at the left end consisted of the sequence between the NotI site and the cleavage site, whereas the remnant at the right end consisted of the sequence between the cleavage site and the NotI site (Fig. 4A). In progeny virus produced from this cloned DNA, the sequence flanking the L-DNA consisted of alternating repeats of H-DNA and vector DNA (Fig. 4B
).
|
|
T cells were infected with these rescued viruses and analysed for viral episomes. The amount of episomal DNA that was detected in all four cell cultures was similar to that found in the established HVS-immortalized cell line 484Th (Fig. 5). The second, faster-migrating band in the control cell line 484Th is a result of development of a deletion in a subpopulation of cells often seen in cell lines serially passaged over time. Viral DNA was also detected in the linear range of the gel. This linear species is always present in cultures infected with HVS for up to 3 months after infection.
HVS LANA is sufficient for replication and maintenance of plasmids containing H-DNA
Because KSHV LANA is involved in the maintenance of KSHV episomes through its interaction with the H-DNA (Ballestas et al., 1999 ; Ballestas & Kaye, 2001
), the ability of HVS LANA to support maintenance of plasmids containing HVS H-DNA was tested. A LANA expression vector was constructed by inserting the LANA open reading frame into pBKCMV, designated as pLANA. One, two and three repeats of H-DNA were inserted into pLANA, creating pLANAH1, pLANAH2 and pLANAH3, respectively. KSHV LANA and four units of H-DNA, which were previously shown to be sufficient for replication (M. M. Medveczky, G. Fejer, E. Horvath, B. Lane, Y. Chang, P. S. Moore, B. Chandran & P. G. Medveczky, unpublished results), were used as a positive control. These plasmids contain a G418 resistance marker, and will confer long-term G418 resistance to cells harbouring them if they are maintained in transfected cells. 293 cells were transfected with these constructs as well as pLANA, which expresses LANA but does not contain H-DNA, and pBKH3, which contains three repeats of H-DNA but does not express LANA. Transfected cells were passaged under selection of G418. Low molecular mass DNA was isolated from G418-resistant cells by Hirt extraction (Hirt, 1967
), digested with DpnI and MboI, and the DNA separated by gel electrophoresis.
Fig. 6(A) shows two representative ethidium bromide-stained gels of Hirt-extracted DNA after eight (left blot) and nine (right blot) passages from two independent experiments. Each lane represents DNA extracted from 3·75x105 cells, and equal loading of DNA from each culture was determined by comparing the amount of mitochondrial DNA present. Mitochondrial DNA is replicated by the mammalian replication machinery and is therefore resistant to digestion by DpnI but sensitive to digestion by MboI. The gel indicated no significant difference in the amount of loaded DNA quantities among lanes and showed that MboI completely digested the mitochondrial DNA.
|
To determine the copy number of episomes per cell after nine passages, the amount of episomal DNA present was compared to 0·1 ng of vector DNA (equivalent to approximately 53 copies per cell). Constructs that expressed LANA but contained no H-DNA were detected at less than one copy per cell. Constructs that expressed LANA and contained one or two repeats were present at a copy number of at least 1·6 and 1·3 copies per cell, respectively. Constructs that expressed LANA and contained three repeats were present at a copy number of at least 4·9 copies per cell. These calculations are based on the assumption that all episomal DNA was completely extracted by the Hirt method. Extraction of 100% of the episomal DNA is unlikely; therefore, these figures are probably an underestimate of the actual copy number.
Plasmids that expressed LANA and contained three H-DNA units were maintained in cell culture for much longer. pLANA was undetectable after 10 passages. Plasmids containing one or two repeats could still be detected after 16 passages, whereas plasmids containing three repeats could still be detected after 25 passages. It should be noted that, although the HVS LANA expression vectors were different sizes due to varying amounts of H-DNA, all replicated plasmid DNA migrated the same distance during electrophoresis. This would seem to indicate that rearrangement or amplification of episomal DNA had occurred, although we cannot say for certain what happened. Attempts to clone this replicated DNA in E. coli for further analysis were unsuccessful, possibly due to its large size.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is somewhat surprising that the HVS L-DNA fragment was found to be infectious. Purified HVS DNA digested with SmaI (which cleaves three times in H-DNA but not in L-DNA) was found to be non-infectious (G. Keil & B. Fleckenstein, personal communication). It is plausible that SmaI cleavage leads to loss of essential elements required for packaging and cleavage of replicative intermediates. NotI, which cuts only once in each repeat unit, presumably does not eliminate H-DNA sequences essential for lytic replication.
Another interesting question is the mechanism of amplification of the vector and generation of rearranged H-like DNA repeated between vector sequences during transfection of cloned DNA. Since the length of cloned HVS DNA is shorter than wild-type virion DNA by as much as 40 kb it is likely that amplification of the vector occurred to fill' the mature virion capsid with DNA of required length. Another issue is how the vectorH-DNA repeats were generated. One possible mechanism involves recombination between the H-like DNA in the E. coli clone, which would result in formation of a circular vectorH-DNA molecule. Amplification of this sequence could then occur by generation of concatamers by a rolling circle mechanism followed by joining of these concatamers and the termini of L-DNA.
To evaluate whether H-DNA is required for episomal replication, recombinant viruses with rescued H-DNA were constructed. T cells infected with rescued viruses contained high copy numbers of episomal DNA. This is the first report showing that intact terminal repeats of a herpesvirus are essential for the maintenance of episomes in latently infected cells.
To determine if LANA is involved in replication and maintenance of the HVS genome, LANA expression vectors were constructed. LANA was able to support maintenance of plasmids that contained H-DNA, and constructs that had three repeats were detected at much higher levels and persisted significantly longer than plasmids containing one or two repeats. Maintenance of plasmids that contained H-DNA but did not express LANA could not be detected. Surprisingly, plasmids that expressed LANA but contained no terminal repeats were also maintained, but were quickly lost through passage in culture. One possible explanation for this is that LANA may non-specifically bind the vector and tether it to the host cell chromosome, but much less efficiently than H-DNA. Because of this inefficient tethering, plasmids lacking H-DNA are not maintained in cell culture over a long period of time.
Data from studies on the closely related virus KSHV suggest that the terminal repeats play a role in anchoring the viral genome in the nucleus. Cloned KSHV terminal repeats can stably persist in KSHV-immortalized B cells over a 5 month period (unpublished). KSHV LANA tethers the viral episome to metaphase chromosomes (Ballestas et al., 1999 ; Cotter & Robertson, 1999
) by binding to a cis-element within the H-DNA (Ballestas & Kaye, 2001
). These data, taken together, are consistent with the working hypothesis that terminal repeats of gamma-2 herpesviruses contain cis-acting sites essential for LANA binding and episomal maintenance.
Maintenance and replication of the latent EBV genome have been studied extensively. Two components required for persistence of EBV episomal DNA have been identified. The EBNA-1 protein interacts specifically with DNA and transactivates a cis-acting element termed oriP (origin of plasmid DNA replication) (Chittenden et al., 1989 ; Reisman et al., 1985
; Yates & Guan, 1991
; Yates et al., 1985
). The cis-acting element consists of a dyad symmetry element (DS) and a family of 20 tandem copies of a 30 bp repeat (FR), which are located near the left end of the viral genome. Oligomers of EBNA-1 bind to both elements resulting in DNA bending and initiation of DNA replication in the dyad symmetry component. At least seven copies of the 30 bp repeat of FR are required for efficient maintenance of the EBV genome (Wysokenski & Yates, 1989
). Because the LANA expression vector containing three terminal repeats was maintained much longer and at higher levels than plasmids containing only one or two repeats, it is tempting to speculate that the terminal repeats of HVS and other gamma-2 herpesviruses serve a function analogous to FR of EBV.
Replication origins contain not only a dyad symmetry element but are also rich in A+T residues. Therefore, it is unlikely that the terminal repeats of HVS and related gamma-2 herpesviruses contain the origin of replication because of their unusually high G+C content. A 2 kb fragment of HVS C484-77 encodes a dyad symmetry element near the left end, and E. coli plasmid clones containing this fragment replicated autonomously in C484-77-transformed T cells (Kung & Medveczky, 1996 ). Therefore, it is possible that the dyad symmetry element contains an origin of replication. On the other hand, deletion mutants lacking the dyad symmetry element still formed episomes in T cells, showing that the dyad symmetry element is not essential (Kung & Medveczky, 1996
). HVS encodes other dyad symmetry elements that may serve as alternative origins (Albrecht et al., 1992
). Replication has also been shown to initiate at multiple sites in the EBV genome, primarily in a large region extending leftward from oriP (Little & Schildkraut, 1995
), and EBV mutants lacking the dyad symmetry element are still capable of establishing latent infection (Norio et al., 2000
). Therefore, although dyad symmetry elements serve as recruitment sites for the host DNA replication machinery, they are different from those found in small DNA viruses. Papovavirus genomes contain precise origins of replication whereas herpesviruses appear to replicate like mammalian cellular DNA, where replication initiates at broad initiation zones (Hamlin et al., 1994
).
Identification of transformation-related viral genes and cis-acting elements of HVS episomal maintenance is an essential step for the comprehensive understanding of the molecular mechanisms involved in latency and HVS-induced oncogenesis. The Bac clones of HVS described here can now be efficiently and rapidly mutated in E. coli using a variety of extremely efficient methods (Zhang et al., 1998 ). The HVS clones can also provide a new tool to address general questions about the latent replication of gamma-2 herpesviruses. HVS, unlike KSHV, can be readily mutagenized and propagated in tissue culture. One possible advantage of the HVS system is that it provides all the technical tools to determine whether LANA is required for lytic or latent DNA replication of the virus. Another future use of the HVS system could be the generation of KSHVHVS hybrids to understand the role of LANA and the terminal repeats in the biology of gamma-2 herpesviruses.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ballestas, M. E. & Kaye, K. M. (2001). Kaposis sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mediates episome persistence through cis-acting terminal repeat (TR) sequence and specifically binds TR DNA. Journal of Virology 75, 3250-3258.
Ballestas, M. E., Chatis, P. A. & Kaye, K. M. (1999). Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen. Science 284, 641-644.
Bankier, A. T., Dietrich, W., Baer, R., Barrell, B. G., Colbere-Garapin, F., Fleckenstein, B. & Bodemer, W. (1985). Terminal repetitive sequences in herpesvirus saimiri virion DNA. Journal of Virology 55, 133-139.[Medline]
Biesinger, B., Muller-Fleckenstein, I., Simmer, B., Lang, G., Wittmann, S., Platzer, E., Desrosiers, R. C. & Fleckenstein, B. (1992). Stable growth transformation of human T lymphocytes by herpesvirus saimiri. Proceedings of the National Academy of Sciences, USA 89, 3116-3119.[Abstract]
Chittenden, T., Lupton, S. & Levine, A. J. (1989). Functional limits of oriP, the EpsteinBarr virus plasmid origin of replication. Journal of Virology 63, 3016-3025.[Medline]
Cotter, M. A.II & Robertson, E. S. (1999). The latency-associated nuclear antigen tethers the Kaposis sarcoma-associated herpesvirus genome to host chromosomes in body cavity-based lymphoma cells. Virology 264, 254-264.[Medline]
Delecluse, H. J., Hilsendegen, T., Pich, D., Zeidler, R. & Hammerschmidt, W. (1998). Propagation and recovery of intact, infectious EpsteinBarr virus from prokaryotic to human cells. Proceedings of the National Academy of Sciences, USA 95, 8245-8250.
Desrosiers, R. C. (1982). Specifically unmethylated cytidylic-guanylate sites in herpesvirus saimiri DNA in tumor cells. Journal of Virology 43, 427-435.[Medline]
Desrosiers, R. C., Mulder, C. & Fleckenstein, B. (1979). Methylation of herpesvirus saimiri DNA in lymphoid tumor cell lines. Proceedings of the National Academy of Sciences, USA 76, 3839-3843.[Abstract]
Desrosiers, R. C., Silva, D. P., Waldron, L. M. & Letvin, N. L. (1986). Nononcogenic deletion mutants of herpesvirus saimiri are defective for in vitro immortalization. Journal of Virology 57, 701-705.[Medline]
Fleckenstein, B. C. M. (1980). Molecular Aspects of Herpesvirus Saimiri and Herpesvirus Ateles. New York: Raven Press.
Gardella, T., Medveczky, P., Sairenji, T. & Mulder, C. (1984). Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis. Journal of Virology 50, 248-254.[Medline]
Hall, K. T., Giles, M. S., Goodwin, D. J., Calderwood, M. A., Markham, A. F. & Whitehouse, A. (2000). Characterization of the herpesvirus saimiri ORF73 gene product. Journal of General Virology 81, 2653-2658.
Hamlin, J. L., Mosca, P. J. & Levenson, V. V. (1994). Defining origins of replication in mammalian cells. Biochimica et Biophysica Acta 1198, 85-111.[Medline]
Hirt, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. Journal of Molecular Biology 26, 365-369.[Medline]
Kung, S. H. & Medveczky, P. G. (1996). Identification of a herpesvirus saimiri cis-acting DNA fragment that permits stable replication of episomes in transformed T cells. Journal of Virology 70, 1738-1744.[Abstract]
Little, R. D. & Schildkraut, C. L. (1995). Initiation of latent DNA replication in the EpsteinBarr virus genome can occur at sites other than the genetically defined origin. Molecular and Cellular Biology 15, 2893-2903.[Abstract]
Medveczky, P. (1995). Oncogenic transformation of T cells by herpesvirus saimiri. In DNA Tumor Viruses: Oncogenic Mechanisms , pp. 239-249. Edited by G. Barbanti-Brodano, M. Bendinelli & H. Friedman. New York: Plenum.
Medveczky, P., Szomolanyi, E., Desrosiers, R. C. & Mulder, C. (1984). Classification of herpesvirus saimiri into three groups based on extreme variation in a DNA region required for oncogenicity. Journal of Virology 52, 938-944.[Medline]
Medveczky, M. M., Szomolanyi, E., Hesselton, R., DeGrand, D., Geck, P. & Medveczky, P. G. (1989). Herpesvirus saimiri strains from three DNA subgroups have different oncogenic potentials in New Zealand white rabbits. Journal of Virology 63, 3601-3611.[Medline]
Medveczky, M. M., Geck, P., Sullivan, J. L., Serbousek, D., Djeu, J. Y. & Medveczky, P. G. (1993). IL-2 independent growth and cytotoxicity of herpesvirus saimiri-infected human CD8 cells and involvement of two open reading frame sequences of the virus. Virology 196, 402-412.[Medline]
Melendez, L. V., Daniel, M. D., Hunt, R. D. & Garcia, F. G. (1968). An apparently new herpesvirus from primary kidney cultures of the squirrel monkey (Saimiri sciureus). Laboratory Animal Care 18, 374-381.[Medline]
Messerle, M., Crnkovic, I., Hammerschmidt, W., Ziegler, H. & Koszinowski, U. H. (1997). Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proceedings of the National Academy of Sciences, USA 94, 14759-14763.
Norio, P., Schildkraut, C. L. & Yates, J. L. (2000). Initiation of DNA replication within oriP is dispensable for stable replication of the latent EpsteinBarr virus chromosome after infection of established cell lines. Journal of Virology 74, 8563-8574.
Reisman, D., Yates, J. & Sugden, B. (1985). A putative origin of replication of plasmids derived from EpsteinBarr virus is composed of two cis-acting components. Molecular and Cellular Biology 5, 1822-1832.[Medline]
Russo, J. J., Bohenzky, R. A., Chien, M. C., Chen, J., Yan, M., Maddalena, D., Parry, J. P., Peruzzi, D., Edelman, I. S., Chang, Y. & Moore, P. S. (1996). Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proceedings of the National Academy of Sciences, USA 93, 14862-14867.
Schirm, S., Muller, I., Desrosiers, R. C. & Fleckenstein, B. (1984). Herpesvirus saimiri DNA in a lymphoid cell line established by in vitro transformation. Journal of Virology 49, 938-946.[Medline]
Shizuya, H., Birren, B., Kim, U. J., Mancino, V., Slepak, T., Tachiiri, Y. & Simon, M. (1992). Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proceedings of the National Academy of Sciences, USA 89, 8794-8797.[Abstract]
Szomolanyi, E., Medveczky, P. & Mulder, C. (1987). In vitro immortalization of marmoset cells with three subgroups of herpesvirus saimiri. Journal of Virology 61, 3485-3490.[Medline]
Wysokenski, D. A. & Yates, J. L. (1989). Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of EpsteinBarr virus. Journal of Virology 63, 2657-2666.[Medline]
Yates, J. L. & Guan, N. (1991). EpsteinBarr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells. Journal of Virology 65, 483-488.[Medline]
Yates, J. L., Warren, N. & Sugden, B. (1985). Stable replication of plasmids derived from EpsteinBarr virus in various mammalian cells. Nature 313, 812-815.[Medline]
Youssoufian, H. & Mulder, C. (1981). Detection of methylated sequences in eukaryotic DNA with the restriction endonucleases Smai and Xmai. Journal of Molecular Biology 150, 133-136.[Medline]
Zhang, Y., Buchholz, F., Muyrers, J. P. & Stewart, A. F. (1998). A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics 20, 123-128.[Medline]
Received 25 January 2002;
accepted 3 May 2002.