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Detection of recombination events in human cells using cloned cytomegalovirus DNA fragments as probes
83 1988, Gene Anal Techn 5:83-86
~f J --~_-
j_--_
Detection of Recombination Events in Human Cells Using Cloned Cytomegalovirus DNA Fragments as Probes CLAUDE HAMELIN, J O C E L Y N Y E L L E , and MICHEL DION
A method is presented for the detection of virus-host interactions at the chromosomal level. This method relies on the analysis of recombinant plasmids carrying specific regions of a viral DNA molecule. Human cytomegalovirus HindlII subgenomic fragments were used
here as a target for recombination events. The human cytomegalovirus (HCMV), a herpesvirus with oncogenic potential, is known to induce chromosomal aberrations in human cells [12, 17, 22]. Virus-host interactions at the chromosomal level are also reflected by the occasional presence of HCMV DNA in the cell genetic bac ground [1, 2, 6]. Conversely, cell-related DNA sequences are found in the HCMV genome [21, 23, 25, 26]. On the basis of these observations, it seemed interesting to use the transforming region of HCMV DNA [2, 15, 18, 19] as a probe for genetic exc h a n g e s b e t w e e n the viral and the cellular genomes.
Materials and Methods Strain AD-169 of H C M V was propagated in human embryo lung cells as previously described [10]. The viral DNA was extracted from infected cells using the technique developed for herpes simplex virus [7]. Random HindlII fragments obtained after complete digestion of the crude DNA preparations were cloned in the plasmid vector pAT153 (3.7 kb) according to standard procedures, as previously described in detail [9]. Colonies ofEscherichia coli HB 101 harboring plasmids with foreign DNA inserts at the unique HindlII
From the Institut Armand-Frappier, Quebec, Canada. Address reprint requests to: Claude Hamelin, Centre de Recherche en Virologie, Institut Armand-Frappier, C. P. 100, Laval-des-Rapides, Qu6bec, Canada H7N 4Z3. Received January 22, 1988.
site were directly selected on nutrient agar plates containing both ampicillin and tetracycline at a final concentration of 50 I~g/ml and 4 txg/ml, respectively [4]. More than 2000 recombinant plasmids were rapidly screened for the presence of a D N A insert about 21 kb in size, using the Chattanooga agarose gel electrophoresis system [9]. Three plasmids carrying such a large piece of f o r e i g n D N A ( p L C A 3 1 8 , p L C C 8 7 1 , and pLCC1534) were cleaved with restriction enzymes E c o R I , HinclI, and PstI in the appropriate buffer and temperature conditions, then run in 1% agarose for 11 hours at 60 V [8]. A limited HindlII digest of each recombinant plasmid was used to transfect subconfluent mouse NIH 3T3 cell monolayers in 60-mm plastic dishes (20 p~g/105 cells) as previously described [28]. Transfected cells maintained for 48 hours at 37°C in 199 H a n k : M E M Earle medium supplemented with 10% fetal calf serum and 50 txg/ml gentamicin were trypsinized, diluted 1:2 in culture medium with 0.3% agarose, then reincubated at the same temperature for 8 weeks.
Results and Discussion Electrophoretic patterns obtained after digestion of plasmids pLCA318, pLCC871, and pLCC1534 with restriction enzymes EcoRI, HinclI, and PstI are shown in Figure 1. The approximate size of each DNA fragment in the gel was determined using 123-bp and 1-kbp ladders from Bethesda Research Laboratories as markers (Table 1). According to the results, all three pAT153 derivatives were carrying 21-kb long DNA inserts, but only p L C C 1 5 3 4 e v e n t u a l l y s h o w e d the e x p e c t e d H C M V H i n d l I I - E subgenomic region [8, 20]. Both pLCA318 and pLCC871 were structurally distinct from the reference plasmid. Differences in the number and size of DNA fragments were observed following digestion of the recombinant plasmids with the selected restriction enzymes but, in general, the pLCA/C restriction profiles looked alike. Several restriction fragments from either pLCA318, pLCC871, or pLCC1534 were comigrating in the gel. This similitude between the DNA patterns prompted us to compare the transforming capacity of the three HindlII fragments cloned in pAT153. Colonies of mouse cells, able to grow in agarose medium, were obtained only after transfection with plasmid pLCC1534 (Figure 2). R e p e a t e d attempts to
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017 0735-0651/88/$03.50
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•
84
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1988,Gene Anal Techn 5:83-86
Figure 1. Restriction profiles of plasmids pLCA318 (O), p L ~ (©), and pLCC1534 (T). Electrophoresis was carried out in 1% agarose for 11 hours at 60 V. Markers: 1. 123-bp ladder; and 2. 1-kbp ladder.
transform the rodent cells with any of the other two recombinant plasmids failed. M o d i f i c a t i o n of the H C M V o n c o g e n e ( s ) through recombination could well have been at the origin of these negative results, but hybridization experiments revealed that neither pLCA318
nor pLCC871 were carrying viral DNA. The inserted HindlII fragment was, in both cases, of cellular origin. Comigration of DNA fragments in agarose gels was thus only fortuitous. Several reasons were evoked initially to justify the use of the present system. I) Chromosomal interactions between bacteriophages and their hosts have been common knowledge for years [13]. 2) The apparent exchange of genetic material between HCMV and the human cell [1, 2, 6, 21, 23, 25, 26] could, in many ways, be related to the phen o m e n o n of transduction. 3) Regions of the HCMV genome involved in cellular transformation have been precisely mapped [3, 15, 18, 19], and growth in agarose appeared as a convenient phenotypic assay for the cells. 4) The HindlII-E fragment carrying HCMV oncogene(s) could be directly inserted into pAT153, a most useful cloning vector as far as the selection of recombinant bacteria is concerned [4]. 5) This relatively large HCMV subgenomic fragment also appeared as an interesting target for recombination. In principle, the above reasons are still valid. One must note that only three plasmids of this size were found in more than 2000 recombinant bacteria and that problems related to the transfection of E. coli with such large plasmids [11] could be primarily responsible for our negative results. The soundness of our general approach is confirmed by the results shown in Figure 3. A smaller HindllI fragment (12 kb) was used this time as a target. Thirteen recombinant plasmids carrying the HCMV HindlII-L fragment, in the two possible orientations, were found in our bank. One of them, slightly larger in size and with
Table 1. Size of the Restriction Fragments Obtained after Complete Digestion of Three Recombinant pAT153 Plasmids with a 21 kb HindlI D N A Insert at the Respective Site a EcoRl
Hincll
Pstl
pLCA318
pLCC871
pLCC1534
pLCA318
pLCC871
pLCC 1534
pLCA318
pLCC871
pLCC 1534
6.20 4.20 4.10 2.65 1.80 1.70 1.45 0.80 0.39
9.60 5.10 1.95 1.65 1.63 1.55 1.20 0.96 0.95 0.77 0.55 0.45
9.80 5.80 3.70 1.85 1.75 1.40
12.00 10.00
11.00 9.60 2.90
17.00 4.00 1.95 0.32
9.00 3.90 3.75 3.20 2.85
7.00 4.60 4.30 3.95 3.75 0.64
7.20 4.95 4.40 2.85 2.15 1.55 0.67
All sizes are given in kilobases.
:(9 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
-j 1988, Gene Anal Techn 5:83-86
A
13
Figure 2. Normal (A) and pLCC1534-transformed (B) mouse NIH 3T3 cells in semisolid medium after 8 weeks of incubation at 37°C. ( × 1).
additional restriction cleavage sites, had apparently been modified by insertion of a 4-kb piece of foreign DNA. N o w HindlII-P (8 kb) fragments with 500 or 5000 additional bases were also found (results not shown). Unfortunately, precise viral functions have not yet been ascribed to these particular regions of the HCMV genome. All HCMV HindlII subgenomic fragments sufficiently large to allow the detection of recombination events ( 8 - 1 2 kb) are carefully watched. Until now, the D N A of a particular strain of HCMV was considered as structurally stable during in vitro passages. Our results indicate the contrary. The very close relationships that exist Figure 3. Restriction profiles of plasmids pLCC528 (11), pLY7 ([]), and pLCCI50 (A). Electrophoresis was carried out in 1% agarose for 16 hours at 60 V. Markers: 1. 123-bp ladder; and 2. 1-kbp ladder.
between HCMV and the human cell can only lead to this type of variation. Differences eventually found should, however, be less pronounced than those considered possible at the beginning of this work with the HindlII-E fragment in mind. Alterations in restriction patterns of the genomes of different adenovirus or herpesvirus isolates has been reported on several occasions [5, 14, 16, 24, 27]. Deletions or substitutions, ranging in size from a small percentage to over 90% of the genome [16], were detected after serial passage of the viruses at high multiplicities, purification of the variants, and analysis of their D N A in agarose gels. Our results indicate that similar genetic exchanges can be detected using cloned regions of the viral genome as a target.
This work was supported by grant No. A3373 from the Natural Sciences and Engineering Council of Canada and by funds from the lnstitut Armand-Frappier. J. Y. acknowledges a postgraduate scholarship from the Cancer Research Society Inc. We thank L. Cousineau for excellent technical assistance.
References 1. Boldogh, I., Brichacek, B., Gonczol, E., Hirsch, 1., and Vaczi, L. (1985)Acta Microbiol. Hung. 32, 147-154. 2. Boldogh, I., Huang, E.-S., Baskar, J. E, and Vaczi, L. (1985) Acta Microbiol. Hung. 32, 167-173. 3. Clanton, D. J., Jariwalla, R. J., Kress, C., and Rosenthal, L. J. (1983) Proc. Natl. Acad. Sci. USA 80, 3826-3830. 4. Dion, M., Yelle, J., and Hamelin, C. (1986) Anal. Biochem. 154,209-212. 5. Ecker, J. R., Kudler, L., and Hyman, R. W. (1984) Intervirology 21, 25-37. 6. Gadler, H., and Wahren, B. (1983) Arch. Virol. 75, 291-298. 7. Gerson, M., Portnoy, J., and Hamelin, C. (1984) Sex. Transm. Dis. 11, 85-90. 8. Greenaway, P. J., Oram, J. D., Downing, R. G., and Patel, K. (1982) Gene 18, 355-360.
H
9. Hamelin, C., Dion, M., and Yelle, J. (1984) Gene Anal. Techn. 1, 79-83. 10. Hamelin, C., and Lussier, G. (1979) J. Gen. Virol. 42, 193-197. 11. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580. 12. Hartmann, M., and Brunneman, H. (1972) Acta Virol. 16, 176. 13. Hayes, W. (1970) The Genetics of Bacteria and Their Viruses, Blackwell Scientific Publications, Oxford. 14. Hirai, K., Honma, H., Ikuta, K., and Kato, S. (1984) Arch. Virol. 79, 293-298. 15. Kouzarides, T., Bankier, A. T., and Barrell, B. G. (1983) Mol. Biol. Med. 1, 47-58. 16. Larsen, S. H. (1982)Virology 116, 573-580. © 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
--___,,a-
..--- ~__
-f ___...---~
....- --
86 1988, Gene Anal Techn 5:83-86
-._~..~ F'--~ r"
17. Luleci, G., Sakizli, M., and Gunlap, A. (1980) Acta Virol. 24, 341-345. 18. Nelson, J. A., Fleckenstein, B., Galloway, D. A., and McDougall, J. K. (1982) J. Virol. 43, 83-91. 19. Nelson, J. A., Fleckenstein, B., Jahn, G., Galloway, D. A., and McDougall, J. K. (1984) J. Virol. 49, 109-115. 20. Oram, J. D., Downing, R. G., Akrigg, A., Dollery, A. A., Duggleby, C. J., Wilkinson, G. W. G., and Greenaway, E J. (1982) J. Gen. Virol. 59, 111-129. 21. Ruger, R., Bornkamm, G. W., and Fleckenstein, B. (1984) J. Gen. Virol. 65, 1351-1364. 22. Sakizli, M., Luleci, G., and Gunalp, A. (1981) Acta Virol. 25,248-250.
23. Shaw, S. B., Rasmussen, R. D., McDonough, S. H., Strapans, S. I., Vacquier, J. P., and Spector, D. H. (1985) J. Virol. 55, 843-848. 24. Silva, R. E, and Witter, R. L., (1985) J. Virol. 54, 690696. 25. Spector, D., and Vacquier, J. P. (1983) Proc. Natl. Acad. Sci. USA 80, 3889-3893. 26. Strapans, S. I., and Spector, D. H. (1986) J. Virol. 57, 591-602. 27. Studdert, M. J., Fitzpatrick, D. R., Browning, G. E, Cullinane, A. A., and Whalley, J. M. (1986) Arch. Virol. 91, 375-381. 28. Yelle, J,, Dion, M., and Hamelin, C. (1983) J. Virol. Meth. 7,321-326.
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
~f J --~_-
j_--_
Detection of Recombination Events in Human Cells Using Cloned Cytomegalovirus DNA Fragments as Probes CLAUDE HAMELIN, J O C E L Y N Y E L L E , and MICHEL DION
A method is presented for the detection of virus-host interactions at the chromosomal level. This method relies on the analysis of recombinant plasmids carrying specific regions of a viral DNA molecule. Human cytomegalovirus HindlII subgenomic fragments were used
here as a target for recombination events. The human cytomegalovirus (HCMV), a herpesvirus with oncogenic potential, is known to induce chromosomal aberrations in human cells [12, 17, 22]. Virus-host interactions at the chromosomal level are also reflected by the occasional presence of HCMV DNA in the cell genetic bac ground [1, 2, 6]. Conversely, cell-related DNA sequences are found in the HCMV genome [21, 23, 25, 26]. On the basis of these observations, it seemed interesting to use the transforming region of HCMV DNA [2, 15, 18, 19] as a probe for genetic exc h a n g e s b e t w e e n the viral and the cellular genomes.
Materials and Methods Strain AD-169 of H C M V was propagated in human embryo lung cells as previously described [10]. The viral DNA was extracted from infected cells using the technique developed for herpes simplex virus [7]. Random HindlII fragments obtained after complete digestion of the crude DNA preparations were cloned in the plasmid vector pAT153 (3.7 kb) according to standard procedures, as previously described in detail [9]. Colonies ofEscherichia coli HB 101 harboring plasmids with foreign DNA inserts at the unique HindlII
From the Institut Armand-Frappier, Quebec, Canada. Address reprint requests to: Claude Hamelin, Centre de Recherche en Virologie, Institut Armand-Frappier, C. P. 100, Laval-des-Rapides, Qu6bec, Canada H7N 4Z3. Received January 22, 1988.
site were directly selected on nutrient agar plates containing both ampicillin and tetracycline at a final concentration of 50 I~g/ml and 4 txg/ml, respectively [4]. More than 2000 recombinant plasmids were rapidly screened for the presence of a D N A insert about 21 kb in size, using the Chattanooga agarose gel electrophoresis system [9]. Three plasmids carrying such a large piece of f o r e i g n D N A ( p L C A 3 1 8 , p L C C 8 7 1 , and pLCC1534) were cleaved with restriction enzymes E c o R I , HinclI, and PstI in the appropriate buffer and temperature conditions, then run in 1% agarose for 11 hours at 60 V [8]. A limited HindlII digest of each recombinant plasmid was used to transfect subconfluent mouse NIH 3T3 cell monolayers in 60-mm plastic dishes (20 p~g/105 cells) as previously described [28]. Transfected cells maintained for 48 hours at 37°C in 199 H a n k : M E M Earle medium supplemented with 10% fetal calf serum and 50 txg/ml gentamicin were trypsinized, diluted 1:2 in culture medium with 0.3% agarose, then reincubated at the same temperature for 8 weeks.
Results and Discussion Electrophoretic patterns obtained after digestion of plasmids pLCA318, pLCC871, and pLCC1534 with restriction enzymes EcoRI, HinclI, and PstI are shown in Figure 1. The approximate size of each DNA fragment in the gel was determined using 123-bp and 1-kbp ladders from Bethesda Research Laboratories as markers (Table 1). According to the results, all three pAT153 derivatives were carrying 21-kb long DNA inserts, but only p L C C 1 5 3 4 e v e n t u a l l y s h o w e d the e x p e c t e d H C M V H i n d l I I - E subgenomic region [8, 20]. Both pLCA318 and pLCC871 were structurally distinct from the reference plasmid. Differences in the number and size of DNA fragments were observed following digestion of the recombinant plasmids with the selected restriction enzymes but, in general, the pLCA/C restriction profiles looked alike. Several restriction fragments from either pLCA318, pLCC871, or pLCC1534 were comigrating in the gel. This similitude between the DNA patterns prompted us to compare the transforming capacity of the three HindlII fragments cloned in pAT153. Colonies of mouse cells, able to grow in agarose medium, were obtained only after transfection with plasmid pLCC1534 (Figure 2). R e p e a t e d attempts to
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017 0735-0651/88/$03.50
-j
•
84
~<
1988,Gene Anal Techn 5:83-86
Figure 1. Restriction profiles of plasmids pLCA318 (O), p L ~ (©), and pLCC1534 (T). Electrophoresis was carried out in 1% agarose for 11 hours at 60 V. Markers: 1. 123-bp ladder; and 2. 1-kbp ladder.
transform the rodent cells with any of the other two recombinant plasmids failed. M o d i f i c a t i o n of the H C M V o n c o g e n e ( s ) through recombination could well have been at the origin of these negative results, but hybridization experiments revealed that neither pLCA318
nor pLCC871 were carrying viral DNA. The inserted HindlII fragment was, in both cases, of cellular origin. Comigration of DNA fragments in agarose gels was thus only fortuitous. Several reasons were evoked initially to justify the use of the present system. I) Chromosomal interactions between bacteriophages and their hosts have been common knowledge for years [13]. 2) The apparent exchange of genetic material between HCMV and the human cell [1, 2, 6, 21, 23, 25, 26] could, in many ways, be related to the phen o m e n o n of transduction. 3) Regions of the HCMV genome involved in cellular transformation have been precisely mapped [3, 15, 18, 19], and growth in agarose appeared as a convenient phenotypic assay for the cells. 4) The HindlII-E fragment carrying HCMV oncogene(s) could be directly inserted into pAT153, a most useful cloning vector as far as the selection of recombinant bacteria is concerned [4]. 5) This relatively large HCMV subgenomic fragment also appeared as an interesting target for recombination. In principle, the above reasons are still valid. One must note that only three plasmids of this size were found in more than 2000 recombinant bacteria and that problems related to the transfection of E. coli with such large plasmids [11] could be primarily responsible for our negative results. The soundness of our general approach is confirmed by the results shown in Figure 3. A smaller HindllI fragment (12 kb) was used this time as a target. Thirteen recombinant plasmids carrying the HCMV HindlII-L fragment, in the two possible orientations, were found in our bank. One of them, slightly larger in size and with
Table 1. Size of the Restriction Fragments Obtained after Complete Digestion of Three Recombinant pAT153 Plasmids with a 21 kb HindlI D N A Insert at the Respective Site a EcoRl
Hincll
Pstl
pLCA318
pLCC871
pLCC1534
pLCA318
pLCC871
pLCC 1534
pLCA318
pLCC871
pLCC 1534
6.20 4.20 4.10 2.65 1.80 1.70 1.45 0.80 0.39
9.60 5.10 1.95 1.65 1.63 1.55 1.20 0.96 0.95 0.77 0.55 0.45
9.80 5.80 3.70 1.85 1.75 1.40
12.00 10.00
11.00 9.60 2.90
17.00 4.00 1.95 0.32
9.00 3.90 3.75 3.20 2.85
7.00 4.60 4.30 3.95 3.75 0.64
7.20 4.95 4.40 2.85 2.15 1.55 0.67
All sizes are given in kilobases.
:(9 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
-j 1988, Gene Anal Techn 5:83-86
A
13
Figure 2. Normal (A) and pLCC1534-transformed (B) mouse NIH 3T3 cells in semisolid medium after 8 weeks of incubation at 37°C. ( × 1).
additional restriction cleavage sites, had apparently been modified by insertion of a 4-kb piece of foreign DNA. N o w HindlII-P (8 kb) fragments with 500 or 5000 additional bases were also found (results not shown). Unfortunately, precise viral functions have not yet been ascribed to these particular regions of the HCMV genome. All HCMV HindlII subgenomic fragments sufficiently large to allow the detection of recombination events ( 8 - 1 2 kb) are carefully watched. Until now, the D N A of a particular strain of HCMV was considered as structurally stable during in vitro passages. Our results indicate the contrary. The very close relationships that exist Figure 3. Restriction profiles of plasmids pLCC528 (11), pLY7 ([]), and pLCCI50 (A). Electrophoresis was carried out in 1% agarose for 16 hours at 60 V. Markers: 1. 123-bp ladder; and 2. 1-kbp ladder.
between HCMV and the human cell can only lead to this type of variation. Differences eventually found should, however, be less pronounced than those considered possible at the beginning of this work with the HindlII-E fragment in mind. Alterations in restriction patterns of the genomes of different adenovirus or herpesvirus isolates has been reported on several occasions [5, 14, 16, 24, 27]. Deletions or substitutions, ranging in size from a small percentage to over 90% of the genome [16], were detected after serial passage of the viruses at high multiplicities, purification of the variants, and analysis of their D N A in agarose gels. Our results indicate that similar genetic exchanges can be detected using cloned regions of the viral genome as a target.
This work was supported by grant No. A3373 from the Natural Sciences and Engineering Council of Canada and by funds from the lnstitut Armand-Frappier. J. Y. acknowledges a postgraduate scholarship from the Cancer Research Society Inc. We thank L. Cousineau for excellent technical assistance.
References 1. Boldogh, I., Brichacek, B., Gonczol, E., Hirsch, 1., and Vaczi, L. (1985)Acta Microbiol. Hung. 32, 147-154. 2. Boldogh, I., Huang, E.-S., Baskar, J. E, and Vaczi, L. (1985) Acta Microbiol. Hung. 32, 167-173. 3. Clanton, D. J., Jariwalla, R. J., Kress, C., and Rosenthal, L. J. (1983) Proc. Natl. Acad. Sci. USA 80, 3826-3830. 4. Dion, M., Yelle, J., and Hamelin, C. (1986) Anal. Biochem. 154,209-212. 5. Ecker, J. R., Kudler, L., and Hyman, R. W. (1984) Intervirology 21, 25-37. 6. Gadler, H., and Wahren, B. (1983) Arch. Virol. 75, 291-298. 7. Gerson, M., Portnoy, J., and Hamelin, C. (1984) Sex. Transm. Dis. 11, 85-90. 8. Greenaway, P. J., Oram, J. D., Downing, R. G., and Patel, K. (1982) Gene 18, 355-360.
H
9. Hamelin, C., Dion, M., and Yelle, J. (1984) Gene Anal. Techn. 1, 79-83. 10. Hamelin, C., and Lussier, G. (1979) J. Gen. Virol. 42, 193-197. 11. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580. 12. Hartmann, M., and Brunneman, H. (1972) Acta Virol. 16, 176. 13. Hayes, W. (1970) The Genetics of Bacteria and Their Viruses, Blackwell Scientific Publications, Oxford. 14. Hirai, K., Honma, H., Ikuta, K., and Kato, S. (1984) Arch. Virol. 79, 293-298. 15. Kouzarides, T., Bankier, A. T., and Barrell, B. G. (1983) Mol. Biol. Med. 1, 47-58. 16. Larsen, S. H. (1982)Virology 116, 573-580. © 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
--___,,a-
..--- ~__
-f ___...---~
....- --
86 1988, Gene Anal Techn 5:83-86
-._~..~ F'--~ r"
17. Luleci, G., Sakizli, M., and Gunlap, A. (1980) Acta Virol. 24, 341-345. 18. Nelson, J. A., Fleckenstein, B., Galloway, D. A., and McDougall, J. K. (1982) J. Virol. 43, 83-91. 19. Nelson, J. A., Fleckenstein, B., Jahn, G., Galloway, D. A., and McDougall, J. K. (1984) J. Virol. 49, 109-115. 20. Oram, J. D., Downing, R. G., Akrigg, A., Dollery, A. A., Duggleby, C. J., Wilkinson, G. W. G., and Greenaway, E J. (1982) J. Gen. Virol. 59, 111-129. 21. Ruger, R., Bornkamm, G. W., and Fleckenstein, B. (1984) J. Gen. Virol. 65, 1351-1364. 22. Sakizli, M., Luleci, G., and Gunalp, A. (1981) Acta Virol. 25,248-250.
23. Shaw, S. B., Rasmussen, R. D., McDonough, S. H., Strapans, S. I., Vacquier, J. P., and Spector, D. H. (1985) J. Virol. 55, 843-848. 24. Silva, R. E, and Witter, R. L., (1985) J. Virol. 54, 690696. 25. Spector, D., and Vacquier, J. P. (1983) Proc. Natl. Acad. Sci. USA 80, 3889-3893. 26. Strapans, S. I., and Spector, D. H. (1986) J. Virol. 57, 591-602. 27. Studdert, M. J., Fitzpatrick, D. R., Browning, G. E, Cullinane, A. A., and Whalley, J. M. (1986) Arch. Virol. 91, 375-381. 28. Yelle, J,, Dion, M., and Hamelin, C. (1983) J. Virol. Meth. 7,321-326.
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017