Characterization of the Cell Type-Specific ... - Journal of Virology

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Aug 10, 1987 - Two strains of minute virus of mice (MVM) show different host cell specificities. ... The autonomous parvoviruses, including the minute virus.
Vol. 62, No. 2

JOURNAL OF VIROLOGY, Feb. 1988, p. 552-557

0022-538X/88/020552-06$02.00/0 Copyright © 1988, American Society for Microbiology

Characterization of the Cell Type-Specific Determinant in the Genome of Minute Virus of Mice JEAN-PHILIPPE ANTONIETTI,* ROLAND SAHLI,t PETER BEARD, AND BERNHARD HIRT Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland Received 10 August 1987/Accepted 19 October 1987

Two strains of minute virus of mice (MVM) show different host cell specificities. The prototype strain MVM(p) grows in fibroblasts, whereas the immunosuppressive variant MVM(i) grows in T lymphocytes. In this study, we have mapped on the viral genome a cell type-specific determinant: it is located between 69 and 85 map units in a region coding for the viral capsid proteins. The DNA of MVM(p) does not replicate in lymphocytes. MVM(i) cannot help MVM(p) grow in lymphocytes; thus the determinant acts in a cis fashion. We did not detect viral mRNA during a restrictive infection of lymphocytes with MVM(p). However, when the same cells were transfected with cloned DNA, both MVM(p) and MVM(i) DNAs were transcribed with the same efficiency from both promoters and the RNA was processed normally. Therefore, the specificity determinant is not a cell type-specific enhancer.

whether it has the properties of a transcriptional enhancer (16, 22) or silencer (6, 23).

The autonomous parvoviruses, including the minute virus of mice (MVM) (8), are nonenveloped, icosahedral viruses which infect mammalian cells (4, 30). These viruses are dependent for their own replication on factors transiently expressed during the S phase of growing host cells (4, 12, 27, 29, 30). In addition, host cells must be in a specific differentiated state to support lytic viral infection (30). The genome of the autonomous parvoviruses is a linear single-stranded DNA molecule that is approximately 5,100 bases long. Upon penetration of the virus into the host cell, the virion DNA is uncoated and converted to double-stranded monomeric replicative-form (RF) DNA. The RF DNA is amplified and serves as template for the synthesis of viral mRNAs and progeny viral DNA. There exist two strains of MVM that show different host cell specificities. The prototype strain, MVM(p) (2, 27, 30), grows in fibroblasts, whereas the immunosuppressive variant, MVM(i) (1, 5, 10, 17, 21), grows in T lymphocytes. MVM(p) and MVM(i) bind to the same receptors, and both MVMs are internalized. Their DNAs are converted to double-stranded RF DNAs in both fibroblasts and lymphocytes (20, 26, 28). In the present study we focused our attention on the lymphocyte cell line EL-4. We wanted to locate the region in MVM(i) DNA which determines whether the virus will grow in lymphocytes. We therefore constructed in vitro recombinant viral genomes in which fragments of MVM(i) and MVM(p) DNAs have been exchanged. By isolating the corresponding recombinant viruses and testing their growth on fibroblasts and lymphocytes, we found that a cell type specificity determinant, located between 69 and 85 map units (m.u.), a region coding for viral capsid proteins, is necessary for the growth of MVM(i) on lymphocytes. To find out more about how the cell type specificity determinant functions and at which stage of the viral replication cycle it acts, we compared the synthesis of the viral mRNAs and DNA during permissive and restrictive infections. We tested whether the determinant encodes a diffusible factor, that is, whether it acts in cis or tr-ans, and

MATERIALS AND METHODS Cells and virus. MVM(i), MVM(p), and their host cells, the EL-4 lymphoma line and A-9 fibroblasts, were as described by McMaster et al. (17). The cells were grown as monolayers in Dulbecco modified Eagle medium containing 5% fetal calf serum. For infection, the medium was removed from cultures that were between 30 and 50% confluent and replaced by 0.4 ml of virus suspension (generally an infected cell lysate) per 9-cm-diameter plate. After 90 min at 37°C, 10 ml of medium with 10% fetal calf serum was added and the infection was continued. Nomenclature. Since the MVM(p) and MVM(i) strains were used in all of the experiments described here, we followed the nucleotide numbering system of Sahli et al. (21). Because of small differences between the two strains, this numbering is not identical to the MVM(p) numbering used by Astell et al. (2) but it does agree with their MVM(i) numbering system (1). Molecular cloning. All of the recombinant DNA work was done according to standard procedures (15). The plasmids pEMBL9+-pl and pEMBL9+-il used to construct new cloned hybrid genomes of MVM were obtained by insertion of the RFs of MVM(p) and MVM(i), respectively, into the plasmid pEMBL9+ (9) at the Sall site after addition of Sall linkers to each end of the RFs (21). These clones possess the 3' extremity of the viral genome intact and extend to the last HhaI restriction site of the 5' extremity of the flip wild-type RF (21); thus they contain more than half of the 5' palindrome [0 to 5,067 base pairs [bp] of MVM(p) for pl and 0 to 5,006 bp of MVM(i) for il] (Fig. 1). The plasmid pEMBL9+-p2 was constructed by exchanging an XbaI-BamHI fragment [85 to 98 m.u. of the MVM(p) RF] from the plasmid pEMBL9+-pl with the corresponding XbaI-BamHI fragment of pEMBL9+-il (the BamHI site was located in the polylinker region sequence of the vector). The plasmid pEMBL9+-p3 was constructed from pEMBL9+-il by removing the EcoRI-XbaI fragment [69 to 85 m.u. of the MVM(i) RF] and replacing it with the corresponding EcoRIXbaI fragment from pEMBL9+-pl. pEMBL9+-i2 was con-

* Corresponding author. t Present address: Institut de Microbiologie, Universitd de Lausanne, 1011 Lausanne, Switzerland.

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structed by exchanging an XbaI-BamHI fragment [85 to 98 m.u. of the MVM(i) RF] from the plasmid pEMBL9+-il with the corresponding XbaI-BamHI fragment of pEMBL9+-pl (Fig. 1). mWB239-il was constructed by inserting a SalI-Sall fragment (0 to 5006) from pEMBL9+-il into mWB239 at the Sall site. The polarity of the insertion in the single-stranded DNA of this phage was the same as that of the viral mRNAs. The plasmid pSP64-iHX used as a template for the synthesis of the complementary-strand SP6 probes was constructed by inserting an HaeIII-XhoI fragment (1854 to 2071) from the RF of MVM(i) into pSP64 (18) between the SalI and SmaI

sites. DNA transfections. Cells were transfected as previously described (3, 24). The medium was removed, and the cells were rinsed carefully twice with S ml of Tris-buffered saline (TBS; 25 mM Tris hydrochloride [pH 7.4], 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4). A 1-ml DNA sample (0.1 to 10 jig of DNA in TBS with 0.5 mg of DEAE-dextran per ml) was added to the center of the plate. The plate was kept at room temperature for 45 min with occasional tilting. The liquid was then aspirated and carefully replaced with 10 ml of TBS. The TBS was aspirated and replaced with 10 ml of medium containing 10% calf serum, and incubation at 37°C was continued. DNA extraction. The volumes below are expressed per 63.6 cm2 (9-cm-diameter) petri dish. Medium was removed, and 1 ml of 10 mM Tris hydrochloride (pH 7.4)-10 mM EDTA-0.6% sodium dodecyl sulfate was added. The lysed cells were harvested after 20 min. Pronase (20 mg/ml, 0.025 ml) was added and allowed to digest for 2 h at 37°C. The DNA was then sheared through a 0.5-mm-diameter syringe, extracted two times with phenol-chloroform, and precipitated with ethanol. The ethanol precipitate was vacuum dried and dissolved in 0.1 ml of 50 mM Tris hydrochloride (pH 8.5)-S5 mM EDTA. A 0.001-ml portion of RNase A (10 mg/ml) was then added, and the mixture was incubated for 30 min at 37°C. The DNA was extracted twice more with phenol-chloroform, precipitated with ethanol, and dissolved in 0.1 ml of 10 mM Tris hydrochloride (pH 7.4)-0.1 mM EDTA. Analysis of DNA by slot blotting. For DNA analysis, 40 RI of 3 M NaOH was added to DNA samples in 400 RI of 10 mM Tris hydrochloride (pH 7.0-1 mM EDTA, and the samples

were incubated for 30 min at 65°C. They were then cooled to room temperature and neutralized by adding 1 volume of 2 M ammonium acetate (pH 7.0). The samples were applied to the nitrocellulose with a Schleicher & Schuell Slot-Blotter. The filter was placed in a vacuum oven at 80°C for 2 h, and hybridization was performed by the method of Johnson et al.

(13).

The single-stranded DNA probe used to analyze the viral DNA amplification (Fig. 2) was synthezised by using the

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20h 6h 48h FIG. 2. Analysis of the amplification of hybrid viral RF in lymphocytes and fibroblasts. To determine the phenotype of the hybrid viruses, we infected lymphocytes and fibroblasts with each hybrid virus (about 100 PFU per cell) and extracted the total DNA after 6. 20, and 48 h. A portion (1/100) of each crude DNA extract was loaded onto nitrocellulose paper with a slot-blotter, and hybridization was performed with a probe of the same polarity as that of the single-stranded viral DNA.

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,ug of RNase Ti per ml was added, and the reaction mixtures were incubated for 45 min at 16°C. Proteinase K (50 p.g) and 10 p.l of 20% sodium dodecyl sulfate were then added, followed by incubation for 15 min at 37°C, phenol extraction, and ethanol precipitation with carrier tRNA. The RNA samples were dissolved, denatured, and fractionated on 6% polyacrylamide-urea sequencing gels at 30 V/cm.

Klenow fragment of Escherichia coli DNA polymerase (15), the template being the single-stranded DNA mWB239-il with an M13 single-strand primer. Before use, the probe was heated at 100°C for 5 min and chilled in ice. Restriction endonuclease digestion, electrophoresis, and Southern blot hybridization. Restriction endonucleases were used according to the recommendations of the manufacturer (Boehringer Mannheim Biochemicals), and the DNA products were separated by electrophoresis (5 V/cm, 4 h) in Tris-acetate buffer (40 mM Tris hydrochloride [pH 7.9], 5 mM NaOAc, 1 mM EDTA) on 1% agarose slab gels. The DNA was transferred to nitrocellulose sheets by the method of Southern (25), and hybridization was performed by the method of Johnson et al. (13). The probe, the EcoRI-EcoRI fragment [68 to 98 m.u. of MVM(p) RF] from pEMBL9+-pl (the second EcoRI site was located in the polylinker sequence of the vector), was end labeled by using the T4 DNA polymerase (15). Synthesis of complementary-strand SP6 probes. The plasmid pSP64-iHX described above was linearized with EcoRI before transcription and then phenol extracted and ethanol precipitated. Transcription of linearized plasmid DNA with SP6 polymerase was done as described previously (11), except that the transcription buffer contained no unlabeled UTP and only 2 mM spermidine. kox-32P]UTP (50 p.Ci; 400 Ci/mmol) was used in each 10-,lI reaction mixture, with 0.5 p,g of plasmid DNA used per reaction. The transcription mix was treated with RNase-free DNase as described earlier (11) and extracted with phenol-chloroform. The [32P]RNA was purified from unincorporated nucleotides by centrifugation through a small Bio-Gel P-6DG gel filtration column. Extraction of cytoplasmic RNA. EL-4 cells were lysed with Nonidet P-40, the nuclei were removed, and the cytoplasmic RNA was isolated as previously described (15). RNase protection experiment. The RNase protection experiment was done as described previously (18, 31). Dried RNA samples (5 jig) were dissolved in 30 p.1 of hybridization buffer (80% formamide, 40 mM PIPES [pH 6.4; piperazineN,N'-bis(2-ethanesulfonic acid)], 400 mM NaCl, 1 mM EDTA) containing 2 x 105 to 7 x 105 cpm of probe, heated at 85°C for 10 min, and incubated at 480C for -8 h. A 300-,ul portion of 10 mM Tris hydrochloride (pH 7.5-S5 mM EDTA-300 mM NaCl containing 100 p,g of RNase A and 4

RESULTS Production of hybrid viruses from MVM(i) and MVM(p). To find out which region on the MVM(i) DNA determines whether the virus grows on lymphocytes, we constructed in vitro recombinant viral genomes in which DNA fragments of MVM(i) and MVM(p) have been exchanged (Fig. 1). The cloned DNAs were linearized with the Sall restriction endonuclease; 2 p.g of each DNA type was transfected into the lymphocyte cell line EL-4 and into the fibroblast cell line A-9, and the cells were continually passaged. From the cultures which lysed, we harvested the supernatants to be used as virus stocks for further infections. We obtained the viruses MVM(pl), MVM(p2), and MVM(p3) from fibroblasts and the viruses MVM(il) and MVM(i2) from lymphocytes after 2 to 4 weeks (about four to eight passages). To determine the phenotypes of the hybrid viruses, we infected lymphocytes and fibroblasts (on 30% confluent cells in 6-cm-diameter plates) with each hybrid virus (ca. 100 PFU per cell) and extracted the total DNA after 6, 20, and 48 h. We blotted part of the DNA in each extract (1/100 of the total) on nitrocellulose paper and performed hybridization with a single-stranded DNA probe of the same polarity as that of the single-stranded viral DNA (Fig. 2). We did not detect the single-stranded DNA of the virions because of the polarity of the probe, so the signals we detected 6 h after infection show that hybrid virus DNA had been converted to a double-stranded DNA in both cell types. The DNAs of the hybrid viruses MVM(pl), MVM(p2), and MVM(p3) were amplified in fibroblasts but not in lymphocytes; thus MVM(pl), MVM(p2), and MVM(p3) have the same phenotype as MVM(p). The DNAs of MVM(il) and MVM(i2) were amplified in T lymphocytes but only slowly amplified in fibroblasts; thus MVM(il) and MVM(i2) have the MVM(i) phenotype. The decrease of the signal for Hindlil

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MVM(il), respectively; we observed the expected bands at 1.1 and 1.3 kilobase pairs (kb) for MVM(p3) and at 2.5 kb for MVM(il) (Fig. 4). When we infected lymphocytes with the same amount of MVM(p3) and MVM(il) (about 100 PFU per cell for each virus), we observed the 2.5-kb band that is specific for the RF MVM(i), but we did not detect any MVM(p3) RF. After decreasing the amount of MVM(il) 10-fold, we still saw only the 2.5-kb band specific to MVM(il). Thus during an infection of lymphocytes with MVM(p3), we were not able to detect the RF of MVM(p3), even if it was coinfected with MVM(il). Likewise, we found that the wild-type MVM(p) was not helped by MVM(i) to replicate in EL-4 cells (data not shown). Comparison of viral transcription in the lymphocyte cell line EL-4 infected with either MVM(il) or MVM(p3). We infected EL-4 cells with MVM(i) and MVM(p3) and extracted the cytoplasmic RNA after 6, 12, and 24 h. We detected the viral mRNAs (7, 14, 19) by hybridizing 5 ,ug of cytoplasmic RNA with the SP6 RNA probe described in Materials and Methods. We digested the RNA-RNA duplexes with RNases A and Ti, denatured them at 100°C for 5 min in formamide, and analyzed the lengths of the protected fragments by electrophoresis on polyacrylamide-urea sequencing gels. The ex-

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MVM(il) and MVM(i2) at 48 h is due to the fact that infected lymphocytes detach from the plate and are lost during the washing step which precedes the DNA extraction. We conclude from this experiment that the cell type-specific determinant is located in the EcoRI-XbaI fragment (from 69 to 85 m.u.), which is in the coding region of the viral capsid proteins. The wild-type MVM(i) does not enhance replication of the hybrid MYM(p3) in the lymphocyte cell line EL-4. We tested whether MVM(il) assists the hybrid MVM(p3), which has the same phenotype as MVM(p) but differs from MVM(il) only in the EcoRI-XbaI fragment, to replicate in the lymphocyte cell line EL-4. We mixed MVM(il) and MVM(p3) in different proportions and then infected lymphocytes with the mixtures. After 24 h we extracted the total DNA, digested 5 ,ug of each DNA with HindIII, separated the fragmgents by electrophoresis, transferred the DNA to nitrocellulose paper, 1and then hybridized the DNA with the EcoRI-EcoRI fragment of pEMBL9+-pl [69 to 98 m.u. of the MVM(p) RF]. There is one additional Hindlll site in the RF of MVM(p3) corppared with MVM(il), so it is possible to differentiate the two REs. By hybridizing with the EcoRlEcoRI fragment of pEMBL9+-pl, we obtained two bands (1,343 and 1,111 bp) that are specific for the RF of MVM(p3) and one band (2,453 bp) that is specific for the RF of MVM(il) (Pig. 3). To show the pattern specific to MVM(p3) and MVM(il), we infected fibroblasts and lymphocytes with MVM(p3) and

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FIG. 5. Coding sequence organization (top) and hybridizing region (bottom) of the four mRNAs. The top part of the figure shows how blocks of coding sequence are assembled by splicing into the four viral mRNAs which code for the four viral polypeptides (14). Vertical markers indicate the positions of the initiation codons used for each protein, and triangles mark the cap sites of the mRNAs. The end of the first intron of the mRNA NS2 (arrow 1) and the cap sites of the mRNAs coding for VP (arrow 2) are indicated. The bottom part of the figure shows an enlargement of the region in which the Haelll(1854)-Xhol(2071) RNA probe hybridizes with the mRNAs. A fragment of 217 nucleotides is protected by the NS1 mRNA, a fragment of 80 nucleotides is protected by the NS2 mRNA (from the end of the first intron [arrow 1] to the end of the probe), and a fragment of about 60 nucleotides is protected by the VP1 and VP2 mRNAs (from their cap site [arrow 2] to the end of the probe). The region containing the cell type-specific determinant is also indicated (region D).

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pected protected fragments are shown in Fig. 5. A fragment of 217 nucleotides should be protected by the NS1 mRNA, a fragment of 80 nucleotides should be protected by the NS2 mRNA, and a fragment of about 60 nucleotides should be protected by the VP1 and VP2 mRNAs. The results are shown in Fig. 6. During the infection of EL-4 with MVM(il), we did not detect any mRNA at 6 h, but at 12 h the viral mRNAs were visible and increased amounts were seen at 24 h. After the infection of lymphocytes with MVM(p3), we did not detect any viral mRNA. This could be due to a block either in transcription or in replication. In the latter case, not enough template DNA would be present to produce detectable amounts of RNA. Transcription of transfected cloned DNA of MVM(il) and MVM(p3) in EL-4 cells. In addition to the DNA clones of p3 and il, a clone of MVM(i) DNA, iA, was constructed by deletion of the determinant fragment (69 to 85 m.u.). We transfected EL-4 cells with 2 ig of linearized DNA of il, p3, and iA by the DEAE-dextran method. After 24 h we extracted the cytoplasmic RNA and analyzed for the presence of viral mRNAs by the RNase protection method described above using 5 pLg of each sample (the probe was the same as that described above). The autoradiogram (Fig. 7) shows MVM(ii)

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accompanied by one or two additional bands one to five nucleotides shorter. These could be due to inhomogeneous RNase digestion of the RNA-RNA duplexes at their extremities due to partial denaturation. The uppermost band present at 243 nucleotides is an artifact, since it is observable even in an RNase protection experiment performed on noninfected lymphocyte total cytoplasmic RNA (Fig. 7, lane -). It is probably due to the presence of undigested probe (the whole-length RNA probe contains, in addition to the MVM sequences, about 25 nucleotides of pSP6 vector). The molecular weight markers (M) were fragments of simian virus 40 DNA digested with Hinfl. Their lengths (in nucleotides) are indicated to the right of the gel.

FIG. 7. Transcription of different transfected cloned DNAs in lymphocyte cell line EL-4. We transfected EL-4 with 2 ,ug of linearized DNA of il, p3, and i/v by the DEAE-dextran method. After 24 h we extracted the cytoplasmic RNA and analyzed 5 ,ug of each RNA using the RNase protection assay as described in the legend to Fig. 6. Lanes: 1, lymphocytes transfected with the il cloned DNA; 2, lymphocytes transfected with the p3 cloned DNA; 3, lymphocytes transfected with the iA cloned DNA that does not possess the cell-type determinant; -, nontransfected lymphocytes; +, lymphocytes infected with MVM(il) (see Fig. 6). The molecular weight markers (M) were as described in the legend to Fig. 6.

that all three DNAs were transcribed with the same efficiency. The mRNAs of NS1, NS2, VP1, and VP2 were synthesized in the same proportions.

DISCUSSION The hybrid virus MVM(p3) that we constructed contains a genome identical to that of MVM(il) except for the EcoRIXbaI fragment (69 to 85 m.u.), which is derived from MVM(p). This is the region which contains the highest degree of divergence between MVM(i) and MVM(p) (a 34-nucleotide and 8-amino-acid difference). Thus the short hairpin structures at each end of the single-stranded DNA of the virion, the two promoters and their upstream regulatory sequences, the splice sites, and the nonstructural proteins NS1 and NS2 are identical for both MVM(il) and MVM(p3) (Fig. 5). Nevertheless, the striking result is that, whereas MVM(il) grows lytically in T lymphocytes, MVM(p3) cannot grow in lymphocytes and grows only in fibroblasts (Fig. 2). We therefore conclude that the region between 69 and 85 m.u., which codes for capsid proteins, contains a determinant which is necessary for the growth of MVM(i) in lymphocytes. It cannot yet be concluded that this region is sufficient to specify viral growth in lymphocytes for the following reason. Despite several attempts we did not succeed in growing a virus with a genome complementary to that of MVM(p3), i.e., the region from 69 to 85 m.u. from MVM(i) and the rest of MVM(p), which would grow on

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lymphocytes. Thus it remains possible that other regions of MVM(i) contribute to the specificity for lymphocytes. On the other hand, we can conclude that the virus MVM(p3) has all the information necessary for efficient growth on fibroblasts. When lymphocytes are coinfected with MVM(il) and MVM(p3), MVM(il) is unable to help MVM(p3) to replicate. This means that there is no diffusible factor produced by MVM(il) which could replace a nonfunctional or absent factor necessary for the replication of MVM(p3). We conclude that the determinant is acting in cis. A possible cis-acting element would be a transcriptional enhancer. To test this possibility, cloned DNA from MVM(il) and MVM(p3) was transfected into EL-4 lymphocytes and the production of viral mRNAs was monitored. mRNA production was comparable in both cases: both promoters were active, and the RNA was efficiently spliced. Even the deletion of the determinant DNA fragment (69 to 85 m.u.) had no influence on transcription. Therefore, the determinant of cell type specificity cannot be an enhancer. The determinant, since it is part of the coding region of the genome, could act through the coat proteins. For instance, the coat proteins of MVM(p3) could stay associated with the infecting DNA in lymphocytes and exert a cis-acting inhibition on transcription or replication. This would explain why we could not detect viral RNA after the lymphocytes were infected with MVM(p3), but found high amounts of transcripts after transfection of the same type of cells. An alternative possibility is that replication of the viral DNA in lymphocytes requires the binding of a cell-specific factor to the determinant DNA. ACKNOWLEDGMENTS We thank B. Bentele for careful technical help and V. Jongeneel for useful discussions. This work was supported by the Fonds National Suisse de la Recherche Scientifique and the University of Lausanne. LITERATURE CITED 1. Astell, C. R., E. M. Gardiner, and P. Tattersall. 1986. DNA sequence of the lymphotropic variant of minute virus of mice, MVM(i), and comparison with the DNA sequence of the fibrotropic strain. J. Virol. 57:656-669. 2. Astell, C. R., M. Thomson, M. Merchlinsky, and D. C. Ward. 1983. The complete DNA sequence of minute virus of mice, an autonomous parvovirus. Nucl. Acids Res. 11:999-1018. 3. Banerji, J., L. Olson, and W. Schaffner. 1983. A lymphocytespecific cellular enhancer is located downstream of the joining region in immunoglobulin heavy-chain genes: Cell 33:729-740. 4. Berns, K. 1984. The parvoviruses. Plenum Publishing Corp., New York. 5. Bonnard, G. D., E. K. Manders, D. A. Campbell, R. B. Herberminsn, and M. J. Collins. 1976. Immunosuppressive activity of a subline of the mouse EL-4 lymphoma: evidence for minute virus of mice cau§ing the inhibition. J. Exp. Med. 143:187-205. 6. Brand, A. H., L. Breedeh, J. Abraham, R. Sternglanz, and K. Nasmyth. 1985. Characterization of a "silencer" in yeast: a sequence with properties opposite to those of a transcripDr4A tional enhancer. Cell 41:41-48. 7. Cotmore, S. F., and P. Tattersall. 1986. Organization of nonstructural genes of the autonomous parvovirus minute virus of mice. J. Virol. 58:724-732. 8. Crawford, L. V. 1966. A minute virus of mice. Virology 29:605612. 9. Dente, L., G. Cesareni, and R. Cortese. 1983. pEMBL: a new family of single-stranaed plasmids. Nucleic Acids Res. 11:16451655.

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