Istituto di Malattie Infettive Veterinarie, Facoltà di Medicina Veterinaria, Università degli Studi di Parma, 43100 Parma, Italy1
Department of Pathobiology, 264 Greene Hall, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA2
Author for correspondence: Vicky van Santen. Fax +1 334 844 2652. e-mail vvsanten{at}mail.auburn.edu
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Abstract |
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Introduction |
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Cell lines persistently infected with the gammaherpesviruses EpsteinBarr virus (EBV), herpesvirus saimiri (HVS), human herpesvirus-8 (HHV-8), and murine gammaherpesvirus-68 (MHV-68) have been established from cells isolated from infected hosts (Cesarman et al., 1995 ; Jung et al., 1999
; Nilsson, 1979
; Usherwood et al., 1996
). This process has been facilitated by the growth-transforming ability of these gammaherpesviruses (Flore et al., 1998
; Jung et al., 1999
; Miller, 1990
; Moses et al., 1999
). In contrast, no evidence for growth-transformation by BHV-4 has been obtained. None of the genes associated with transformation by other gammaherpesviruses, unique to each individual virus, are in the BHV-4 genome (Lomonte et al., 1996
). In cell lines persistently infected with gammaherpesviruses, the infection is predominantly latent. In the vast majority of cells, viral gene expression is restricted to a specific subset of genes and the cells survive and replicate. In a small subset of cells, the virus is reactivated from latency, resulting in production of infectious virus and death of the cell (Kieff, 1996
; Moses et al., 1999
). In cell lines persistently infected with gammaherpesviruses, the viral genome is maintained as a circular episome (Cesarman et al., 1995
; Decker et al., 1996
; Jung et al., 1999
; Kieff, 1996
; Usherwood et al., 1996
). Origins of replication for the circular viral genomes, oriP, distinct from the origins of replication used during lytic virus replication, have been identified (Ballestas et al., 1999
; Kung & Medveczky, 1996
; Yates et al., 1984
). A viral gene product is required for episomal maintenance of DNA containing oriP (Ballestas et al., 1999
; Kung & Medveczky, 1996
; Lupton & Levine, 1985
; Yates et al., 1985
).
BHV-4 causes cytopathic effect (CPE) and replicates in a broad range of cell lines and primary cultures of various non-human animal species in culture (Peterson & Goyal, 1988 ; Truman et al., 1986
). It also causes CPE and replicates in some human cell lines and primary cell cultures, but 14 of 17 human cell lines and primary cell cultures tested exhibited neither CPE nor virus replication (Egyed, 1998
; Truman et al., 1986
). In the work presented here, we examined the interaction of BHV-4 with a human rhabdomyosarcoma cell line, RD-4, and found that although no CPE is noted, some infectious virus is produced. In addition, persistently infected cell lines that are not carrier cultures could be established.
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Methods |
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Probe labelling.
32P-labelled probes were labelled by random primer extension using the RadPrime kit (BRL).
In situ lysis gel electrophoresis.
In situ lysis gel electrophoresis and gel processing were as previously described (Gardella et al., 1984 ), except that gels were blotted by capillary blotting.
Quantification of DNA and RNA.
Amounts of DNA and RNA detected by Southern or Northern blot analysis were compared using ImageQuant software after detection of radioactivity by a Molecular Dynamics PhosphorImager IS.
Construction of recombinant virus.
Recombination plasmid pR14neoI was used to construct recombinant BHV-4 using a protocol provided by Michael Goltz (personal communication). pR14neoI contains the 4·35 kb BHV-4 strain DN-599 EcoRI H fragment derived from pR14 (van Santen & Chang, 1992 ) and the 2 kb BamHI fragment containing a neomycin-resistance gene expression cassette derived from pRc/CMV (Invitrogen), cloned into the EcoRI and BamHI sites, respectively, of pTZ18U (see Fig. 3
). Plasmid DNA (7·5 µg; linearized with SalI) and BHV-4 strain DN-599 DNA (0·3 µg), prepared as previously described (van Santen, 1991
), were electroporated (Bio-Rad Gene Pulser, 270 V, 960 µF) in DMEM without serum into BT cells from a confluent 75 cm2 flask. Cells were returned to the flask, fed the next day, and split 1:2 when they reached confluence 2 days post-electroporation. When extensive CPE appeared, virus stock was prepared by freezing and thawing the cells three times and removing cell debris by low-speed centrifugation, and was screened for recombinant virus.
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The second round of screening for recombinant virus utilized PCR and Southern blot analysis of virus originating from single plaques derived from pools of virus selected after the first screening. Dilutions of each pool were added to BT cells in 96-well plates to obtain wells with single plaques. After 8 days, wells containing only one plaque were noted. Cells were disrupted by freezing and thawing three times, and one-fifth of the freezethaw lysate from each well containing one plaque was added to BT cells in a 6-well plate to amplify each plaque-purified virus. However, the cells became confluent before CPE appeared. Therefore, cells were transferred to a 25 cm2 flask and allowed to continue to grow until extensive CPE was apparent. Virus stock and DNA from 200 µl of the virus stock were prepared as described above. The presence of the neomycin-resistance gene was determined by PCR using one-fortieth of the DNA solution as template, and confirmed by Southern blot analysis of one-quarter of the DNA with a neomycin-resistance gene probe. Recombinant virus 26A3neo was propagated as described (van Santen, 1991 ).
Establishment of geneticin-resistant cell lines.
Confluent RD-4 cells were infected with 26A3neo (50 TCID50 per cell). One day p.i., cells were split 1:5 and geneticin (4001000 µg/ml) selection was applied. Medium and geneticin were replaced every 3 days. All doses of geneticin resulted in colonies. After colonies were well-established, cells were trypsinized, allowed to reattach to the same flask, grown to confluence, and tested for the presence of circular and linear viral genomes by in situ lysis gel electrophoresis. Pools of cells selected with and maintained with 1000 µg geneticin/ml were used for all experiments shown.
Immunostaining.
Monolayers were washed three times with PBS, fixed with 4% paraformaldehyde for 1015 min at 37 °C, washed twice with PBS, 0·1% BSA, and incubated 5 min at 20 °C with the same solution. Cells were incubated 5 min at 20 °C with PBS, 0·3% Triton X-100, washed two or three times with PBS, and incubated 10 min at 37 °C with 0·15% H2O2 in PBS. Rabbit anti-BHV-4 hyperimmune serum, diluted 1/500 in PBS, or monoclonal antibody 29 (Dubuisson et al., 1989a ), diluted 1/50 in PBS, was incubated with the cells for 2 h at 37 °C. After three washes with PBS, cells were incubated with peroxidase-conjugated secondary antibody, diluted 1/500 in PBS, for 1 h at 37 °C, and washed three times with PBS. Secondary antibody was detected by development in 250 µg/ml DAB (Sigma), 0·015% H2O2, 50 mM Tris pH 7·4 for approximately 10 min at 20 °C.
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Results and Discussion |
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Viral DNA-replication-dependent late RNA synthesis in RD-4 cells suggested that infectious virus might be produced. Therefore, we determined the titre of BHV-4 8, 24 and 48 h p.i. and found that it increased approximately 2-fold between 8 and 24 h p.i., and a further 2-fold between 24 and 48 h p.i., to a titre of approximately 2x104 p.f.u./ml. This modest virus replication is consistent with the small increase of linear viral DNA in cells between 24 and 48 h p.i.
Based on results described thus far, we concluded that BHV-4 infection of RD-4 cells is predominantly non-permissive and non-cytopathic. The virus enters the cell and the genome is released. Extremely low levels of early and late gene expression and viral DNA replication occur. Some infectious virus is produced, but the RD-4 cells continue to grow without CPE. Dependence of BHV-4 DNA replication on the S phase of the cell cycle (Vanderplasschen et al., 1995 ) is unlikely to be responsible for the limited viral DNA replication and lack of CPE observed in RD-4 cells. Because RD-4 cells do not exhibit contact inhibition, cells in S phase are present at all times. Furthermore, even when infected RD-4 cells were passaged 24 h p.i., no CPE was observed. In contrast, when BT cells infected at an unknown very low m.o.i., such that they reached confluence before plaques formed, were passaged, extensive CPE developed, indicating that the arrest of virus replication due to lack of S phase is readily reversible in BT cells. Therefore RD-4 cells had an outcome of infection distinct from BT cells that was not merely a function of the cell cycle phase at which initial infection occurred.
Persistently infected cells can be selected
Because the amount of circular viral DNA present in infected RD-4 cells steadily declined with passage of the cells, but was still detectable 9 days p.i., after three passages, we reasoned that it might be possible to select cells maintaining the viral genome if the viral genome contained a selectable marker. Therefore, we constructed a recombinant BHV-4 containing a neomycin-resistance gene. We chose to insert the neomycin-resistance gene near the junction of unique and terminally repeated DNA [polyrepetitive DNA (prDNA)]. Insertion of foreign DNA into this region in the HVS genome is more efficient than insertion into the central portion of the genome, presumably because only one recombination event rather than two is required (Desrosiers et al., 1985 ; Grassmann & Fleckenstein, 1989
).
Furthermore, others have demonstrated that insertion of DNA into this region in BHV-4 has no detectable effect on BHV-4 replication in culture (Keil et al., 1990 ). Accordingly, a recombination plasmid was constructed that contains a neomycin-resistance gene following the BHV-4 EcoRI H fragment, the last EcoRI fragment containing unique DNA at the right end of the genome (Fig. 3
). Recombinant virus 26A3neo, generated by homologous recombination following electroporation of BT cells with recombination plasmid and wt DN-599 DNA, was isolated from a well of a 96-well plate containing a single plaque. PCR and Southern blot analysis (not shown) confirmed that the virus contained the neomycin-resistance gene. As expected, recombinant virus grew to the same titre and produced plaques with the same kinetics and morphology as wt (not shown).
To establish cell lines containing the BHV-4 genome, RD-4 cells were infected with 26A3neo, and split and subjected to geneticin selection (4001000 µg/ml) 1 day p.i. Although RD-4 cells are sensitive to 400 µg/ml geneticin, higher doses were used in an attempt to select cells with the highest copy number of recombinant virus. Pools of cells selected with 1000 µg/ml geneticin (RD4BHV4neo1000) were used for all experiments shown. However, pools of cells selected with lower doses behaved similarly whenever tested. Seven days p.i., microscopic colonies began to appear and were macroscopic by 14 days p.i. The efficiency of establishment of geneticin-resistant colonies selected with 1000 µg/ml geneticin was 5x10-5. Colonies were trypsinized, pooled, and allowed to continue to grow in the presence of geneticin.
Selected cells contain predominantly circular viral genomes
Twenty-four days p.i., selected RD4BHV4neo1000 cells were assayed for circular and linear viral DNA by in situ lysis gel electrophoresis (Fig. 4). Selected cells contained both circular and linear viral DNA. Circular DNA was approximately eight times more abundant than linear. This predominance of circular DNA continued in subsequent passages tested. In contrast, the viral genome was rapidly lost in the absence of geneticin selection and was undetectable 7 days p.i., after two passages (Fig. 4
). The selected cells contained approximately 30-fold more viral DNA 24 days p.i. than they did 1 day p.i., before selection was applied. This result suggests that initially not all cells were infected or that the viral genome was amplified during selection. However, RD4BHV4neo1000 cells (passage 5) contained only approximately one viral genome per diploid genome, as estimated by Southern blot analysis of total DNA (not shown). This result suggests that the viral genome was not amplified during selection, and that therefore initially not all cells were infected. The conclusion that initially not all cells were infected conflicts with the observation (Fig. 1
) that RD-4 cells contain only approximately 2-fold less viral DNA 6 h p.i. than BT cells and suggests that perhaps the m.o.i. was not as high as intended. Nevertheless, it is surprising that only one recombinant viral genome per host cell genome is present in cells selected with a high dose of drug. However, maintenance of a higher number of viral genomes might be incompatible with cell survival and/or multiplication.
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Persistently infected cells grow more slowly than uninfected cells
RD-4 cells persistently infected with BHV-4 took longer to reach confluence than uninfected RD-4 cells, even in the absence of geneticin. An MTT proliferation assay (Fig. 6) confirmed that persistently infected RD-4 cells grew more slowly than uninfected RD-4 cells. This observation suggests why only approximately one viral genome per host cell diploid genome is maintained in RD-4 cells even though the cells were selected and maintained in a concentration of geneticin at least 2·5-fold that necessary to kill uninfected RD-4 cells. The presence of the viral genome, and presumably expression of viral proteins, as indicated by immunostaining, is apparently detrimental to cell growth, as indicated by the slower growth of persistently infected cells. Therefore, any growth advantage to cells containing multiple copies of the neomycin-resistance-gene-containing viral genome might be offset by the disadvantage of containing multiple copies of the viral genome. This is in contrast to the situation for primate T cells infected with recombinant HVS carrying a neomycin-resistance gene, in which 100 copies of viral DNA per cell are maintained (Grassmann & Fleckenstein, 1989
). T cells are the cells normally latently infected and transformed by HVS (Jung et al., 1999
). Cell lines persistently infected with gammaherpesviruses (EpsteinBarr virus, HHV-8, MHV-68, HVS), which have been established without the need for recombinant virus and drug selection, because viral genes contribute to growth-transformation (Jung et al., 1999
; Kieff, 1996
; Moore & Chang, 1998
), also contain numbers of viral genomes much greater than the approximately one copy per host cell diploid genome in RD-4 cells selected for maintenance of recombinant BHV-4 (Drexler et al., 1998
; Sugden et al., 1979
; Tsygankov & Romano, 1999
; Usherwood et al., 1996
). For these other gammaherpesviruses, expression of viral genes promoting cell growth presumably leads to selection of cells with higher numbers of viral genomes. BHV-4 is unique among well-characterized gammaherpesviruses in its lack of association with growth-transformation and malignancy.
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Acknowledgments |
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This is Auburn University College of Veterinary Medicine Publication no. 2576.
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References |
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Received 8 November 1999;
accepted 16 March 2000.