VIROLOGY
185,883-887
(199 1)
Is There a Unique LORI Department
D.
of Pathology,
KLAMAN, Tufts
Episome
ELIZABETH
A.
in EBV Transformed
HURLEY,
AND
University
School
of Medicine,
Received
March
4, 199 1; accepted
DAVID
136 Harrison August
A.
B Cells? THORLEY-LAWSON’
Avenue,
Boston,
Massachusetts
02 111
2, 199 1
We have examined the frequency of episome formation in resting B cells 36 hr after infection with Epstein-Barr virus. We have detected an average of 0.65 episomal genomes per cell (SD 0.17). We have used this information to predict the distribution of circularization events in B cell clones derived from such infections. We have observed that in 32 out of 33 cases (with one unresolved) the B cell clone has arisen as a consequence of a single circularization event. This result is highly statistically different from the predicted distribution. We suggest that there is an exclusion or selection mechanism operating that favors either the formation or retention of a single circular genome per cell early in infection. 0 1991 Academic
Press,
Inc.
infection when the viral genome copy number begins to amplify such that long-established lines have anywhere from 1 O-500 copies per cell (13). Thus, amplification is not associated with activation or immortalization. The conclusion, that a single genome was involved in immortalization and the establishment of latency, was based initially on quantitative estimates using cell lines of known genome copy number as standards. This technique can distinguish one from two genome copies per cell (see results section below). We also used a different approach to test the single genome hypothesis. Since every CCC is formed by recombination of varying numbers of terminal repeat sequences, each circularization event generates a characteristic size of joint fragment (1 I). Therefore, newly infected cells were cloned and analyzed for the size and number of viral joint fragments. Analysis of nine such clones revealed that, in every case, the B cell clone contained CCC that had arisen as a consequence of a single circularization event, two or more circularization events were not detected. Based on these results we wondered if there was some exclusion mechanism that allowed only a single genome per cell to circularize. In this paper we have analyzed this phenomenon in more detail. One way to ascertain if there were an exclusion mechanism would be to demonstrate a single circularization event occurring even at high MOI. Our best calculations indicate that we are infecting at a MOI of around 10. However, estimates of MOI require certain assumptions that make them only semiquantitative (see discussion below). By comparison we have observed that we can accurately assess the frequency of circularization events in a freshly infected B cell population prior to the onset of cellular proliferation. Given the dependability of this estimate we can use the Pois-
Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that is thought to play a role in a number of neoplastic and benign diseases in humans (reviewed in (7) and (10)). As is typical of herpesviruses, infection with EBV results in life-time persistence with reactivation. The site of latent infection has not been definitively demonstrated but is thought to involve B cells or epithelial cells or a combination of the two. The virus, whose genome is linear double-stranded DNA in the virion, persists in latently infected cells as multiple covalently closed circular episomes (CCC). ln vitro, the virus will latently infect normal resting B lymphocytes, driving them to become activated into immortalized lymphoblasts (3, 14). This provides, by far, the most amenable in vitro system for studying herpesvirus latency. About 16 hr postinfection, transcription and expression of viral latent gene products expressed in the nucleus (EBNA’s) can be detected (2, 14) along with cell surface activation molecules such as Blast-l (CD48) (15) and CD23 (14). Subsequently, 2 to 3 days postinfection, the cells simultaneously express the latent membrane protein (LMP), undergo blastogenesis and enter into the cell cycle (2, 74). We have recently described a detailed study of the behavior of the viral genome during the process of infection, activation, and immortalization of the cell (5). Although, several hundred viral genomes became cell associated our data was most consistent with the conclusion that a single viral genome circularized per cell at about 16 hr postinfection. This process required cellular activation. Once the cells begin to proliferate, the single viral genome replicates one to one with the immortalized B cell. No further circularization events and no amplification were detected until 7 to 10 days post’ To whom
reprint
requests
should
be addressed. 883
0042.6822/91
$3.00
CopyrIght 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
884
SHORT COMMUNICATIONS Genome Equivalents
A 1
IR
Genome Equivalents
Experiment&
114
l/a
A
B
C
D
r
0.2 I
0.5 I
1.0 I
0-
118
l/4
1R
1
Genome Copy #
Genome Copy # 0.1 I
E
2. I x
20 -
0.1 I
0.2 I
0.5 I
1.0 I
21 I
x
II
2-
I2 -
x 1
0.002
12’
CL16
RAJI
FIG. 1. (A) Gardella gel analysis of viral genomes in newly infected B cells at 36 hr postinfection. Purified populations of resting B cells were obtained (6) infected with the 895-8 strain of EBV, and subjected to Gardella gel analysis at 36 hr postinfection as described previously (4, 5). Experiments A and B represent infections of B cells from two different donors with the same virus preparation and experiments C, D and E represent infection of the same donor’s B cells with three separate virus preparations. These virus preparations were preselected for optimal levels of transforming virus and have been characterized previously (5). All had T.D.,, = 5 X 1 05/ml when tested by assaying serial dilutions of the virus on fixed numbers of target cells (14). For estimates of actual MOI see text. The average number of CCC was estimated by comparison to a standard curve generated by serial dilution of the RAII (50 CCC per cell) (I), or CL1 6 (11 CCC per cell) (7) with the EBV negative lines RAMOS or BL-2 (6). The results for the RAJI cells are shown. (B) Sample calibration curves of serially diluted RAII and CL1 6 cells. Serial dilutions of RAJI or CL1 6 cells were prepared as described above, fractionated on Gardella gels, and the presence of circular genomes was assessed by Southern blotting as described previously (4, 5). The resulting hybridization signals, as exemplified for RAJI In (A), were scanned with a Biorad Videodensitometer Model 620 and a plot of area under the curve versus predicted genome copy number was made. The resulting plot was then used to quantitate the number of CCC in the experiments, examples of which are shown in (A) above. A new calibration curve was generated for each experiment.
son distribution to predict the distribution of circularization events in a subsequently derived collection of B cell clones without the necessity of accurately measuring the MOI. Analysis of a sufficiently large number of such clones should allow the demonstration of either a random distribution or the favoring of a single genome. CCC can be detected in freshly infected B cell populations, prior to the onset of cellular proliferation, by performing Gardella gel analysis (6) which allows the resolution of circular and linear forms of the virus (4). The results obtained for different virus preparations with B cells from several individuals are shown in Fig. 1
and summarized in Table IA. The number of CCC was estimated by comparison to known standards as described previously (5, 6) and demonstrated in Fig. 1. The results show that reproducible values could be derived with a mean of 0.65 CCC per cell. The estimates did not seem to vary strikingly between individuals or virus preparations although the later was expected as virus preparations were preselected for optimal levels of transforming virus (see legend to Table 1A). Cells from infected cultures were subjected to limiting dilution analysis and 20 of the subsequent cultures
SHORT TABLE
COMMUNICATIONS
1A
ESTIMATES OF CIRCULAR VIRAL GENOME COPY NUMBER AT 36 HR POSTINFECTION Dono? 1 1 2 3 3 3 4 5
Virus
preparationb 1 2 3 4 5 6 7 7
Copy
No.
0.50 0.70 0.41 0.49 0.78 0.88 0.78 0.66 x = 0.65 s = 0.17
a CCC copy number was estimated as described in the legend to Fig. 1. Estimates for the same donor of B cells were performed at the same time. Independent calibration curves were established for each donor. b Virus stocks were prepared and characterized previously (5,14). Only preparations with T.D.,, = 5 X 1 05/ml when tested by assaying serial dilutions of the virus on fixed numbers of target cells (14) were used in experiments. For estimates of actual MOI see text.
grown up, DNA prepared, and the clonality and number of circularization events was estimated as described in the legend to Fig. 2. Results for six such cultures are shown in Fig. 2. Numbers 1, 4, 5, and 6 have two distinct circularization events whereas numbers 2 and 3 have one. Similarly, cultures 1, 4, and 6 have two cell populations according to the number of rearranged J, chains and cultures 2,3, and 5 have only one. A summary of all the cultures tested is presented in Table 1 B. In most cases the number of circularization events was the same as the number of clones detected in the culture with four exceptions. In the case of cultures 8, 15, and 17 two clones were present but only one size of joint fragment was detected. Subcloning confirmed that they consisted of two independent B cell lines that contained the same size joint fragment (not shown). The only other exception was culture 5 that demonstrated two circularization events but only one detectable clone. It was conceivable that the culture could have contained two clones, one in low abundance with a very high CCC copy number and one in high abundance with a low copy number. Thus, both clones would be detected with the EBV probe but only the later with the J, probe. Attempts to resolve this issue were unsuccessful, as the culture died after several passages and attempts to subclone it were also unsuccessful. Table 1 C summarizes the calculation performed for the expected distribution of circularization events, based on the Poisson distribution and the data in Table 1A and that observed. It is apparent that there is a highly significant bias in our results suggesting that the
885
distribution of genomes is nonrandom (x2 < 0.005). Conversely, with 32 of 33 clones expressing one size of CCC we may use the binomial distribution to calculate the 95% confidence limits on the hypothesis that there is preference for one per cell. This turns out to be 0.91-l .OO, i.e., 95% confidence that >91% of all clones will have only one size of episome. These calculations were performed on the conservative assumption that no. 5 contained two episome populations per cell although this was not definitely resolved. Several explanations can be offered for the observation of a single circularization event in each cell clone. The first is that our predicted distribution is incorrect, the number of circularization events being in reality much lower. In order for this to be true, there would Culture# 123456
FIG. 2. Southern blot analysis of typical lines. Resting B cells were purified, infected, and then subjected to limiting dilutions at 36 hr postinfection as described previously (5). Cultures containing growing cells were picked and grown until 10’ cells were available for DNA extraction. DNA was prepared by standard techniques (8) and subjected to digestion with either BamHI(EBV blot) or Bglll(J, blot). The EcoRl 1 (left hand) probe in the SP65 vector (kind gift from Dr. Elliott Kieff) was used to probe the EBV blot for the number of independent circularization events present. The SaulllA fragment of the 1, region of immunoglobulin genes (kind gift of Dr. Ted Krontiris) was used as a probe to determine the numberof rearranged immunoglobulin heavy chains to estimate the number of cell clones present. The results shown are for the first six lines picked and are representative.
SHORT
886 TABLE
COMMUNICATIONS
1B
SUMMARYOFCELLCULTURESTESTEDAND RESULTS FROMTHESOUTHERN BLOTANALYSIS Culture 1 2 3 4 5’ 6 7 8** 9 10 11 12 13 14 15** 16 17** 18 19 20
No. of Episomes 2 1 1 2 2* 2 2 1** 3 2 1 2 3 1 1** 1 l*' 1 1 1
No. of Clones 2 1 1 2 1* 2 2 2** 3 2 1 2 3 1 2** 1 2” 1 1 1
Note. Cultures derived by limiting dilution of infected B cells were analyzed as to the number of originating episomes, based on the number of CCC joint fragments, and clonality based on the number of rearranged immunoglobulin heavy chains as described in the legend to Frg. 2. * Culture 5 remained unresolved as it died before it could be subcloned. ** Cultures 8. 15, and 17 contain two different clones with identical size joint fragments.
need to be some fundamental technical artifact such as CCC in newly infected cells entering the gel more efficiently than those from cell lines. This can be tested by analyzing the hybridization signal of the material that fails to enter the gel. Such an analysis does not reveal a selective retention of EBV DNA at the top of the gel for the established cell lines. Another possibility is that only a small fraction of the circularized genomes are transforming. However, we have demonstrated previously that at 36 hr postinfection CD23 expression correlates with the presence of circular genomes (5) and that such cells clone with the same efficiency as a newly established cell line. This indicates that the majority of episomes are indeed transforming. Yet another explanation is that there are multiple circularization events in each infected cell and that one comes to dominate over time. This could only apply if one population of CCC replicated as fast or faster than the cell. All others would have to replicate slower than the cell in order to be lost over time. The explanation that we favor is that there is an exclusion mechanism at work that strongly favors or, perhaps, completely excludes
all but one genome from circularizing during the first 24 hr. Further support for this idea comes from considerations of the MOI. The virus preparations used all had T.D.,, = 5 X 105/ml when tested by assaying serial dilutions of the virus on fixed numbers of target cells (14). Our previous studies indicate that EBV-infected target cells only clone with an efficiency of l-2% (14) thus the actual titer of the virus is probably around l-5 X 1 O’/ml. Since, in the experiments reported here l-2 X lo6 cells/ml were infected, the MOI was about 10. Estimates of total virions in the supernatant by quantitative dot blot (5) indicated 0.5-l X 10’ per ml, suggesting that only 2-3% of virions are transforming. Since we are infecting with about 10 transforming competent virions per cell but only detect 0.65 circularization events it is apparent that only a small fraction of transformation competent virions are circularizing tt%ir genomes under these experimental conditions consistent with the idea that there is some physical constraint on the number of genomes that can form CCC. This argument is further reinforced by the observation that we have consistently seen higher levels of CCC formation when we enrich for the target cell by using purified resting B cells as targets (this study) compared to whole unfractionated B cells (5). This implies that the MOI is indeed greater than 1 and the limitation on CCC formation is the number of available target cells. The exclusive nature of this circularization event may also explain why the CCC replicates exactly one for one with the cellular genome for more than 1 week before amplification begins (5). It is as though the viral DNA during this time period is under the same restraints as the cellular DNA, being able to replicate
TABLE
1C
PREDICTEDANDACTUALOUTCOMEOFTHECLONINGEXPERIMENT For 33 clones derived from cultures with a mean of 0.65 episomes per cell at 36 hr postinfection, and Poisson distribution predicts: No. of episomes 0 Fraction Fraction Expected Observed
of clones of surviving clones for 32 clones
0.522 None*
1 0.339 0.709 23 32
2
83
0.110 0.230 8 (l)**
0.029 0.061 2 0
Note. Difference from random: Comparison of observed versus expected yields a x2 = 91% of all clones will have only one size of episome.
SHORT COMMUNICATIONS
only once per cell division. An investigation of the mechanism that restrains circularization and early replication of EBV DNA may, therefore, have broader significance for understanding the mechanisms that regulate cellular DNA synthesis to occur only once per cell division. ACKNOWLEDGMENTS This work was supported by Public Health Service Grants Al15310 and CA-28737.
REFERENCES A. The state of the virus genome in transformed cells and its relationship to host cell DNA. In “The Epstein-Barr Virus” (M. A. Epstein and B. G. Achong, Eds.), pp. 156-l 78. Springer Verlag, New York/Berlin, 1979. ALLDAY, M. J., CRAWFORD,D. H., and GRIFFIN,B. E. Epstein-Barr virus latent gene expression during the initiation of B cell immortalization. f. Gen. Viral. 70, 1755-l 764 (1989). AMAN, P., EHLIN-HENRIKSSON,B., and KLEIN, G. Epstein-Barr virus susceptibility of normal human B lymphocyte populations. 1. Exp. Med. 159, 208-220 (1984). GARDELLA,T., MEDVECZKY,P., SAIRENJI,T., and MULDER, C. Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis. 1. Viral. 50, 248-254 (1984). HURLEYE. A., and THORLEY-LAWSON,D. A. B cell activation and the establishment of Epstein-Barr virus latency. 1. Exp. Med. 168,2059-2075 (1988).
1. ADAMS,
2. 3. 4.
5.
887
6. HURLEY,E. A., AGGER, S., MCNEIL, J. A., LAWRENCE,J. B., CALENDAR, A., LENOIR, G. M., and THORLEY-LAWSON,D. A. When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates. J. Virol. 65, 1245-l 254. 7. KIEFF,E. and LIEBOWIIZ, D. The Epstein-Barr Virus ln B. Fields “Virology” (B. Fields and D. Knipe, Eds.), pp. 1889-1920. Raven Press, New York, 1989. 8. MANIATIS, T., FRITSCH,E. F., and SAMBROOK,J. “Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, NY, 1982. 9. MANN, K. P., STAUNTON, D., and THORLEY-LAWSON,D. A. An Epstein-Barr virus encoded protein found in the plasma membrane of transformed cells. J. Viral. 55, 71 O-720 (1985). 70. MILLER, G. Epstein-Barr virus: Biology, pathogenesis and medical aspects. In “Virology,” 2nd ed. (B. N. Fields et al., Eds.), pp. 1921-1958. Raven Press, New York, 1990. 11. RAAB-TRAUB,N., and FLYNN,K. The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferations. Cell 47, 883-889 (1986). 12. ROONEY,C. M., HOWE, J. G., SPECK,S. H., and MILLER, G. Influences of Burkitt’s lymphoma and primary B cells on latent gene expression by the non-immortalizing PBJHR-1 strain of Epstein-Barr virus. J. Vifol. 83, 1531-l 539 (1989). 73. SUGDEN,B., PHELPS,M., and DOMORADZKI,J. Epstein-Barr virus DNA is amplified in transformed lymphocytes. J. Virol. 31, 590-595 (1979). 14. THORLEY-LAWSON,D. A., and MANN, K. P. Early events in Epstein-Barr virus infection provide a model for B cell activation. 1. Exp. Med. 162, 45-59 (1985). 15. YOKOYAMA,S., STAUNTON, D. E., FISHER,R. C., AMIOT, M., FORTIN, J. J., and THORLEY-LAWSON,D. A. Expression of the Blast-l activation/adhesion molecule and its identification as CD48. J. lmmunol. 146, 2192-2200.
185,883-887
(199 1)
Is There a Unique LORI Department
D.
of Pathology,
KLAMAN, Tufts
Episome
ELIZABETH
A.
in EBV Transformed
HURLEY,
AND
University
School
of Medicine,
Received
March
4, 199 1; accepted
DAVID
136 Harrison August
A.
B Cells? THORLEY-LAWSON’
Avenue,
Boston,
Massachusetts
02 111
2, 199 1
We have examined the frequency of episome formation in resting B cells 36 hr after infection with Epstein-Barr virus. We have detected an average of 0.65 episomal genomes per cell (SD 0.17). We have used this information to predict the distribution of circularization events in B cell clones derived from such infections. We have observed that in 32 out of 33 cases (with one unresolved) the B cell clone has arisen as a consequence of a single circularization event. This result is highly statistically different from the predicted distribution. We suggest that there is an exclusion or selection mechanism operating that favors either the formation or retention of a single circular genome per cell early in infection. 0 1991 Academic
Press,
Inc.
infection when the viral genome copy number begins to amplify such that long-established lines have anywhere from 1 O-500 copies per cell (13). Thus, amplification is not associated with activation or immortalization. The conclusion, that a single genome was involved in immortalization and the establishment of latency, was based initially on quantitative estimates using cell lines of known genome copy number as standards. This technique can distinguish one from two genome copies per cell (see results section below). We also used a different approach to test the single genome hypothesis. Since every CCC is formed by recombination of varying numbers of terminal repeat sequences, each circularization event generates a characteristic size of joint fragment (1 I). Therefore, newly infected cells were cloned and analyzed for the size and number of viral joint fragments. Analysis of nine such clones revealed that, in every case, the B cell clone contained CCC that had arisen as a consequence of a single circularization event, two or more circularization events were not detected. Based on these results we wondered if there was some exclusion mechanism that allowed only a single genome per cell to circularize. In this paper we have analyzed this phenomenon in more detail. One way to ascertain if there were an exclusion mechanism would be to demonstrate a single circularization event occurring even at high MOI. Our best calculations indicate that we are infecting at a MOI of around 10. However, estimates of MOI require certain assumptions that make them only semiquantitative (see discussion below). By comparison we have observed that we can accurately assess the frequency of circularization events in a freshly infected B cell population prior to the onset of cellular proliferation. Given the dependability of this estimate we can use the Pois-
Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that is thought to play a role in a number of neoplastic and benign diseases in humans (reviewed in (7) and (10)). As is typical of herpesviruses, infection with EBV results in life-time persistence with reactivation. The site of latent infection has not been definitively demonstrated but is thought to involve B cells or epithelial cells or a combination of the two. The virus, whose genome is linear double-stranded DNA in the virion, persists in latently infected cells as multiple covalently closed circular episomes (CCC). ln vitro, the virus will latently infect normal resting B lymphocytes, driving them to become activated into immortalized lymphoblasts (3, 14). This provides, by far, the most amenable in vitro system for studying herpesvirus latency. About 16 hr postinfection, transcription and expression of viral latent gene products expressed in the nucleus (EBNA’s) can be detected (2, 14) along with cell surface activation molecules such as Blast-l (CD48) (15) and CD23 (14). Subsequently, 2 to 3 days postinfection, the cells simultaneously express the latent membrane protein (LMP), undergo blastogenesis and enter into the cell cycle (2, 74). We have recently described a detailed study of the behavior of the viral genome during the process of infection, activation, and immortalization of the cell (5). Although, several hundred viral genomes became cell associated our data was most consistent with the conclusion that a single viral genome circularized per cell at about 16 hr postinfection. This process required cellular activation. Once the cells begin to proliferate, the single viral genome replicates one to one with the immortalized B cell. No further circularization events and no amplification were detected until 7 to 10 days post’ To whom
reprint
requests
should
be addressed. 883
0042.6822/91
$3.00
CopyrIght 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
884
SHORT COMMUNICATIONS Genome Equivalents
A 1
IR
Genome Equivalents
Experiment&
114
l/a
A
B
C
D
r
0.2 I
0.5 I
1.0 I
0-
118
l/4
1R
1
Genome Copy #
Genome Copy # 0.1 I
E
2. I x
20 -
0.1 I
0.2 I
0.5 I
1.0 I
21 I
x
II
2-
I2 -
x 1
0.002
12’
CL16
RAJI
FIG. 1. (A) Gardella gel analysis of viral genomes in newly infected B cells at 36 hr postinfection. Purified populations of resting B cells were obtained (6) infected with the 895-8 strain of EBV, and subjected to Gardella gel analysis at 36 hr postinfection as described previously (4, 5). Experiments A and B represent infections of B cells from two different donors with the same virus preparation and experiments C, D and E represent infection of the same donor’s B cells with three separate virus preparations. These virus preparations were preselected for optimal levels of transforming virus and have been characterized previously (5). All had T.D.,, = 5 X 1 05/ml when tested by assaying serial dilutions of the virus on fixed numbers of target cells (14). For estimates of actual MOI see text. The average number of CCC was estimated by comparison to a standard curve generated by serial dilution of the RAII (50 CCC per cell) (I), or CL1 6 (11 CCC per cell) (7) with the EBV negative lines RAMOS or BL-2 (6). The results for the RAJI cells are shown. (B) Sample calibration curves of serially diluted RAII and CL1 6 cells. Serial dilutions of RAJI or CL1 6 cells were prepared as described above, fractionated on Gardella gels, and the presence of circular genomes was assessed by Southern blotting as described previously (4, 5). The resulting hybridization signals, as exemplified for RAJI In (A), were scanned with a Biorad Videodensitometer Model 620 and a plot of area under the curve versus predicted genome copy number was made. The resulting plot was then used to quantitate the number of CCC in the experiments, examples of which are shown in (A) above. A new calibration curve was generated for each experiment.
son distribution to predict the distribution of circularization events in a subsequently derived collection of B cell clones without the necessity of accurately measuring the MOI. Analysis of a sufficiently large number of such clones should allow the demonstration of either a random distribution or the favoring of a single genome. CCC can be detected in freshly infected B cell populations, prior to the onset of cellular proliferation, by performing Gardella gel analysis (6) which allows the resolution of circular and linear forms of the virus (4). The results obtained for different virus preparations with B cells from several individuals are shown in Fig. 1
and summarized in Table IA. The number of CCC was estimated by comparison to known standards as described previously (5, 6) and demonstrated in Fig. 1. The results show that reproducible values could be derived with a mean of 0.65 CCC per cell. The estimates did not seem to vary strikingly between individuals or virus preparations although the later was expected as virus preparations were preselected for optimal levels of transforming virus (see legend to Table 1A). Cells from infected cultures were subjected to limiting dilution analysis and 20 of the subsequent cultures
SHORT TABLE
COMMUNICATIONS
1A
ESTIMATES OF CIRCULAR VIRAL GENOME COPY NUMBER AT 36 HR POSTINFECTION Dono? 1 1 2 3 3 3 4 5
Virus
preparationb 1 2 3 4 5 6 7 7
Copy
No.
0.50 0.70 0.41 0.49 0.78 0.88 0.78 0.66 x = 0.65 s = 0.17
a CCC copy number was estimated as described in the legend to Fig. 1. Estimates for the same donor of B cells were performed at the same time. Independent calibration curves were established for each donor. b Virus stocks were prepared and characterized previously (5,14). Only preparations with T.D.,, = 5 X 1 05/ml when tested by assaying serial dilutions of the virus on fixed numbers of target cells (14) were used in experiments. For estimates of actual MOI see text.
grown up, DNA prepared, and the clonality and number of circularization events was estimated as described in the legend to Fig. 2. Results for six such cultures are shown in Fig. 2. Numbers 1, 4, 5, and 6 have two distinct circularization events whereas numbers 2 and 3 have one. Similarly, cultures 1, 4, and 6 have two cell populations according to the number of rearranged J, chains and cultures 2,3, and 5 have only one. A summary of all the cultures tested is presented in Table 1 B. In most cases the number of circularization events was the same as the number of clones detected in the culture with four exceptions. In the case of cultures 8, 15, and 17 two clones were present but only one size of joint fragment was detected. Subcloning confirmed that they consisted of two independent B cell lines that contained the same size joint fragment (not shown). The only other exception was culture 5 that demonstrated two circularization events but only one detectable clone. It was conceivable that the culture could have contained two clones, one in low abundance with a very high CCC copy number and one in high abundance with a low copy number. Thus, both clones would be detected with the EBV probe but only the later with the J, probe. Attempts to resolve this issue were unsuccessful, as the culture died after several passages and attempts to subclone it were also unsuccessful. Table 1 C summarizes the calculation performed for the expected distribution of circularization events, based on the Poisson distribution and the data in Table 1A and that observed. It is apparent that there is a highly significant bias in our results suggesting that the
885
distribution of genomes is nonrandom (x2 < 0.005). Conversely, with 32 of 33 clones expressing one size of CCC we may use the binomial distribution to calculate the 95% confidence limits on the hypothesis that there is preference for one per cell. This turns out to be 0.91-l .OO, i.e., 95% confidence that >91% of all clones will have only one size of episome. These calculations were performed on the conservative assumption that no. 5 contained two episome populations per cell although this was not definitely resolved. Several explanations can be offered for the observation of a single circularization event in each cell clone. The first is that our predicted distribution is incorrect, the number of circularization events being in reality much lower. In order for this to be true, there would Culture# 123456
FIG. 2. Southern blot analysis of typical lines. Resting B cells were purified, infected, and then subjected to limiting dilutions at 36 hr postinfection as described previously (5). Cultures containing growing cells were picked and grown until 10’ cells were available for DNA extraction. DNA was prepared by standard techniques (8) and subjected to digestion with either BamHI(EBV blot) or Bglll(J, blot). The EcoRl 1 (left hand) probe in the SP65 vector (kind gift from Dr. Elliott Kieff) was used to probe the EBV blot for the number of independent circularization events present. The SaulllA fragment of the 1, region of immunoglobulin genes (kind gift of Dr. Ted Krontiris) was used as a probe to determine the numberof rearranged immunoglobulin heavy chains to estimate the number of cell clones present. The results shown are for the first six lines picked and are representative.
SHORT
886 TABLE
COMMUNICATIONS
1B
SUMMARYOFCELLCULTURESTESTEDAND RESULTS FROMTHESOUTHERN BLOTANALYSIS Culture 1 2 3 4 5’ 6 7 8** 9 10 11 12 13 14 15** 16 17** 18 19 20
No. of Episomes 2 1 1 2 2* 2 2 1** 3 2 1 2 3 1 1** 1 l*' 1 1 1
No. of Clones 2 1 1 2 1* 2 2 2** 3 2 1 2 3 1 2** 1 2” 1 1 1
Note. Cultures derived by limiting dilution of infected B cells were analyzed as to the number of originating episomes, based on the number of CCC joint fragments, and clonality based on the number of rearranged immunoglobulin heavy chains as described in the legend to Frg. 2. * Culture 5 remained unresolved as it died before it could be subcloned. ** Cultures 8. 15, and 17 contain two different clones with identical size joint fragments.
need to be some fundamental technical artifact such as CCC in newly infected cells entering the gel more efficiently than those from cell lines. This can be tested by analyzing the hybridization signal of the material that fails to enter the gel. Such an analysis does not reveal a selective retention of EBV DNA at the top of the gel for the established cell lines. Another possibility is that only a small fraction of the circularized genomes are transforming. However, we have demonstrated previously that at 36 hr postinfection CD23 expression correlates with the presence of circular genomes (5) and that such cells clone with the same efficiency as a newly established cell line. This indicates that the majority of episomes are indeed transforming. Yet another explanation is that there are multiple circularization events in each infected cell and that one comes to dominate over time. This could only apply if one population of CCC replicated as fast or faster than the cell. All others would have to replicate slower than the cell in order to be lost over time. The explanation that we favor is that there is an exclusion mechanism at work that strongly favors or, perhaps, completely excludes
all but one genome from circularizing during the first 24 hr. Further support for this idea comes from considerations of the MOI. The virus preparations used all had T.D.,, = 5 X 105/ml when tested by assaying serial dilutions of the virus on fixed numbers of target cells (14). Our previous studies indicate that EBV-infected target cells only clone with an efficiency of l-2% (14) thus the actual titer of the virus is probably around l-5 X 1 O’/ml. Since, in the experiments reported here l-2 X lo6 cells/ml were infected, the MOI was about 10. Estimates of total virions in the supernatant by quantitative dot blot (5) indicated 0.5-l X 10’ per ml, suggesting that only 2-3% of virions are transforming. Since we are infecting with about 10 transforming competent virions per cell but only detect 0.65 circularization events it is apparent that only a small fraction of transformation competent virions are circularizing tt%ir genomes under these experimental conditions consistent with the idea that there is some physical constraint on the number of genomes that can form CCC. This argument is further reinforced by the observation that we have consistently seen higher levels of CCC formation when we enrich for the target cell by using purified resting B cells as targets (this study) compared to whole unfractionated B cells (5). This implies that the MOI is indeed greater than 1 and the limitation on CCC formation is the number of available target cells. The exclusive nature of this circularization event may also explain why the CCC replicates exactly one for one with the cellular genome for more than 1 week before amplification begins (5). It is as though the viral DNA during this time period is under the same restraints as the cellular DNA, being able to replicate
TABLE
1C
PREDICTEDANDACTUALOUTCOMEOFTHECLONINGEXPERIMENT For 33 clones derived from cultures with a mean of 0.65 episomes per cell at 36 hr postinfection, and Poisson distribution predicts: No. of episomes 0 Fraction Fraction Expected Observed
of clones of surviving clones for 32 clones
0.522 None*
1 0.339 0.709 23 32
2
83
0.110 0.230 8 (l)**
0.029 0.061 2 0
Note. Difference from random: Comparison of observed versus expected yields a x2 = 91% of all clones will have only one size of episome.
SHORT COMMUNICATIONS
only once per cell division. An investigation of the mechanism that restrains circularization and early replication of EBV DNA may, therefore, have broader significance for understanding the mechanisms that regulate cellular DNA synthesis to occur only once per cell division. ACKNOWLEDGMENTS This work was supported by Public Health Service Grants Al15310 and CA-28737.
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