Retroviruses. containing 10% fetal calf serum and 0.24 U of insulin (Eli Lilly & Co.) per ml. Viruses. All virus preparations were clarified culture

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1 JOURNAL OF VIROLOGY, Sept. 1982, p X/82/ $02.00/0 Copyright 1982, American Society for Microbiology Vol. 43, No. 3 In Situ Hybridization: General Infectivity Assay for Retroviruses ALAN REIN,* NANCY RICE, STEPHANIE-SIMEK, MAURICE COHEN, AND RICHARD J. MURAL National Cancer Institute Frederick Cancer Research Facility, Frederick, Maryland Received 8 April 1982/Accepted 25 May 1982 We have devised a general infectivity assay for retroviruses. A virus-specific [32P]DNA probe is hybridized in situ to a monolayer culture, and foci of infected cells in the monolayer are detected by exposure of the hybridized culture to X-ray films. The method is quantitative, in that it gives the same titer for Moloney murine leukemia virus as does the standard UV-XC test. The specificity of the assay is indicated by the fact that murine leukemia virus and baboon endogenous virus do not cross hybridize under the conditions used. The assay is completed within 1 to 3 weeks and should be broadly applicable for retroviruses which replicate without altering cellular morphology: its use is demonstrated with mouse mammary tumor virus and the helper virus of the reticuloendotheliosis complex. In general, the replication of retroviruses is not accompanied by any morphological effect upon the host cells. In the absence of any visible effect, it is evidently difficult to design a quantal (as opposed to endpoint dilution) infectivity assay. Consequently, these assays have been virtually limited to those exceptional cell-virus combinations in which a morphological alteration can be observed (e.g., references 1, 10, 11, 15, 16, and 23). We have now developed a new procedure by which any replicating retrovirus can be assayed in any monolayer cell culture system. This procedure is based on the technique of in situ hybridization described by Brahic and Haase (3). In this assay, focal areas of infected cells are detected by hybridization of a virus-specific [32P]DNA probe to the cell monolayer, followed by exposure of the monolayer to X-ray film. Thus, the "foci" visualized by the assay are areas of the culture containing high levels of virus-specific RNA and are independent of any biological effect of the virus upon the host cell, or even of the production in the cell of a recognizable virus-specific protein. The utility of this assay is demonstrated with several retroviruses which have previously been extremely difficult to assay. MATERIALS AND METHODS Cell culture. 3T3FL mouse cells (8) were grown in McCoy 5A medium supplemented with 10% fetal calf serum. NIH/3T3 cells (14) were grown in Dulbecco modified Eagle medium containing 10% calf serum (GIBCO Laboratories); D17 dog cells (22) were passed in Dulbecco medium with 10% fetal calf serum; and CrFK feline cells (6) were grown in Dulbecco medium containing 10% fetal calf serum and 0.24 U of insulin (Eli Lilly & Co.) per ml. Viruses. All virus preparations were clarified culture fluids from virus-producing cells; virus dilutions were made in growth medium. Moloney murine leukemia virus (MuLV) and N-tropic WN1802N MuLV were gifts from Janet Hartley (National Institute of Allergy and Infectious Diseases) and were propagated on 3T3FL cells; baboon endogenous virus (BaEV) was obtained by transfection of BEF-3 cellular DNA onto D17 cells (4); M7 was a gift from Charles Benton, Viral Resources Laboratory, National Cancer Institute Frederick Cancer Research Facility; REV-A, the helper virus in the reticuloendotheliosis virus complex, was isolated from S3D6 chicken tumor cells (12, 20) by limiting dilution on quail embryo fibroblasts; and the C3H strain of mouse mammary tumor virus (MMTV) was obtained from Mm5mt/cl cells (18) and grown on CrFK cells in the presence of dexamethasone (10-5 M). Both Mm5mt/cl cells and CrFK cells were kind gifts from Larry Arthur (National Cancer Institute Frederick Cancer Research Facility). Virus assays. All assays were performed in 60-mm Falcon tissue culture dishes. Cells were treated with 20,ug of DEAE-dextran (Pharmacia Fine Chemicals) per ml immediately before infection with 0.5 ml of virus. In addition, the medium supplied to the cells after infection was supplemented with 2 jig of Polybrene (Aldrich Chemical Co.) per ml where indicated. The UV-XC test (23) was performed as described previously (19). [32P]DNAs. 70S viral RNA was prepared by sucrose gradient centrifugation. As previously described (9), this RNA served as the template for [32P]cDNA synthesis, using avian myeloblastosis virus reverse transcriptase primed by sonicated denatured calf thymus DNA. The specific activity of the cdna was about 7 x 108 cpm/lpg. Some 70S RNA preparations contained a significant amount of rrna, and the resulting ribosomal [32P]cDNA produced a high background on the hybridized plates. In such cases the total [32P]cDNA 1055

2 1056 REIN ET AL. J. VIROL FIG. 1. Titration of Moloney MuLV by in situ hybridization. 3T3FL cells were seeded at 8 x 104 per culture dish. They were infected the following day with dilutions of Moloney MuLV as described previously (19). Five days after infection, the plates shown here were fixed and hybridized with 106 cpm of Moloney MuLV [32P]cDNA per dish. was first hybridized with unlabeled rrna to a Crt of -1, and hybrids were removed by hydroxyapatite chromatography. Nick translation of cloned viral DNA was performed as described by Rigby et al. (21). The specific activity of the product was about 108 cpm/pg. Unlike the procedure of Brahic and Haase (3), we did not attempt to standardize the size of the [32P]DNAs to 50 nucleotides. Hybridization reaction. Conditions influencing the in situ hybridization reaction were studied extensively by Brahic and Haase (3). Except where noted, we have used their procedures without modification. Confluent monolayers of infected cells were initially fixed by treatment with a 3:1 mixture of ethanol and acetic acid for at least 20 min at room temperature and were then air dried. (This and all subsequent treatments used approximately 2 ml per plate, unless otherwise specified.) Plates could be stored at 4 C for several weeks or more at this point in the procedure. The fixed plates were prepared for hybridization by treatment for 20 min at room temperature with 0.2 N HCI, rinsing with deionized water, treatment with 2x SSC (1 x SSC is 0.15 M NaCI plus M sodium citrate) for 30 min at 70 C, rinsing with water, treatment with proteinase K (5,ug/ml) for 15 min at 37 C in 0.02 M Tris (ph 7.4)-2 mm CaC12, rinsing twice with water, and dehydration by two rinses with 70% ethanol and one rinse with 95% ethanol. They were then allowed to dry. Plates could also be stored after this step. The hybridization mixture was essentially as described by Brahic and Haase (3). It contained 50% recrystallized deionized formamide, 10 mm Tris (ph 7.2), 600 mm NaCI, 1 mm EDTA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 1 mg of bovine serum albumin (Pentex) per ml, 0.1% polyethylene glycol, 20 p.g of chicken or dog rrna per ml, and boiled [32P]cDNA at 1 x 106 to 4 x 106 cpm/ml. As the cdna concentration was decreased from 106 to 105 cpm/ml, the film intensity of hybridized areas also declined markedly. Since 107 cpm/ml gave about the same intensity as 106 cpm/ml, we chose 1 x 106 to 2 x 106 cpm/ml as an optimal cdna concentration. A higher level might be required to detect cells producing very little of the nucleic acid in question. In assays in which a nicktranslated probe was used, rrna was omitted from the reaction mixture. One milliliter of hybridization mix was added to each plate. Each plate was sealed by stretching a strip of Parafilm around the edge of the lid, and the plates were rocked gently at room temperature for 1 to 4 days. After the hybridization, the plates were washed twice at room temperature with 50% formamide-0.3 M NaCI-5 mm Tris (ph 7.4)-0.5 mm EDTA. They were then rinsed by submersion in 2 liters of 2x SSC for 1 h at 55 C, with stirring. (This modification of the procedure of Brahic and Haase [3] seemed quite helpful in reducing background radioactivity on the plates.) The plates were subsequently rinsed for 1 h at room temperature with 0.6 M NaCI-10 mm Tris (ph 7.4)-i mm EDTA, rinsed briefly with 2x SSC, dehydrated by rinses with 70 and 90% ethanol, and allowed to dry. The edges were then cut from each plate with scissors, and the flat plate bottoms were wrapped in Saran Wrap and exposed to X-ray film. In some experiments an intensifying screen (Du Pont Cronex Lightning Plus) was also placed in the cassette. Films were exposed for 1 to 4 days at -70 C. RESULTS Our initial tests of the assay procedure were performed with Moloney MuLV, since with this virus the results of the new assay could be compared with those of other, well-established techniques, such as the UV-XC test (23). Parallel plates of 3T3FL cells were infected with dilutions of Moloney MuLV or were mock infected with growth medium. Five days after infection, some plates were fixed and hybridized with a [32P]cDNA probe, as described above, and the remaining plates were irradiated with UV light and overlaid with XC cells for the XC test. Figure 1 shows the autoradiogram obtained after in situ hybridization. The infected cultures gave rise to focal areas of quite intense radioactivity; these "hybridization plaques" were not present in the mock-infected control plate, and their number increased with the virus concentration. A quantitative comparison of the hybridiza-

3 VOL. 43, 1982 GENERAL INFECTIVITY ASSAY FOR RETROVIRUSES 1057 TABLE 1. Titrations of Moloney MuLV by in situ hybridization and the UV-XC test' Virus dilution No. of plaques per plate Hybridization assay XC test , , 6 a Replicate plates from the experiment shown in Fig. 1 were irradiated and overlaid with XC cells as described previously (19). tion assay with the standard UV-XC test is shown in Table 1. It can be seen that the plate infected with a 10-3 dilution of MuLV contained roughly 10 times as many hybridization plaques as did the plate infected with the 10-4 dilution. Thus, the number of hybridization plaques was directly proportional to the amount of MuLV in the inoculum. This shows that each hybridization plaque is initiated by a single virus particle in the inoculum and implies that the number of plaques obtained at a given virus dilution can be used to calculate the titer of the initial virus stock. In addition, Table 1 shows that at each dilution, the number of hybridization plaques is approximately the same as the number of XC plaques; thus, the virus titer obtained in the hybridization assay is the same as the PFU per milliliter determined by the XC test. The specificity of the assay was investigated s. by comparing the hybridization of MuLV-infected plates with that of BaEV-infected plates, using either a BaEV- or an MuLV-specific probe. A BaEV-specific [32P]cDNA probe enabled us to detect BaEV plaques, but showed no reaction with mouse cells which had been infected with MuLV (Fig. 2A). Conversely, when replicate plates were tested with an MuLVspecific probe, hybridization plaques were obtained with the MuLV-infected plates, but not with the BaEV-infected plates (Fig. 2B). Thus, the assay is at least "group specific", in the sense that BaEV and MuLV do not register in MuLV and BaEV assays, respectively. This specificity is consistent with the low degree of overall homology between MuLV and BaEV: their nucleic acids do not cross hybridize to a detectable extent in stringent liquid hybridization assays, and we have estimated that MuLV probes hybridize to BaEV DNA only -1/100 as well as BaEV probes in Southern blotting experiments (5). We have also used the in situ hybridization technique to assay other retroviruses. REV-A has previously been assayed by virtue of its cytopathic effect on chicken embryo fibroblasts (10, 16). However, it has not been possible to quantitate its infectivity on other cells until now. Figure 3 shows a titration of REV-A on D17 dog cells by means of in situ hybridization. The number of hybridization plaques was directly pro- il A s-. f A... BaEV MuLV A BaEV probe B MuLV probe FIG. 2. In situ hybridization of BaEV and MuLV [32P]cDNAs to BaEV- and MuLV-infected cells. Plates containing 2 x 105 D17 cells were infected with a dilution of BaEV or were mock infected. The cells were fed with medium containing Polybrene and were fixed for hybridization 12 days after infection. Other plates, containing 105 NIH/3T3 cells, were infected with a dilution of N-tropic MuLV or were mock infected; these plates were fixed 5 days after infection. (A) One plate of each type was hybridized with [32P]cDNA made from the M7 isolate of BaEV, using 3 x 106 cpm per dish; (B) a duplicate plate of each type was hybridized with AKR MuLV [32P]cDNA, using 1.5 x 106 cpm per plate. BaEV MuLV

4 1058 REIN ET AL. J. VIROL. * e ' is p '0 * op"p S *% 0 a, _>~~ * yie 0 w FIG. 3. Titration of REV-A by in situ hybridization. Plates containing 2 x 105 D17 cells were infected with serial dilutions of REV-A or were mock infected. The cells were fed with medium containing Polybrene and were fixed for hybridization 13 days after infection. They were then hybridized with REV-A [32P]cDNA, using 106 cpm per dish. portional to the virus concentration; using the number of plaques obtained with the 10-4 dilution of the inoculum, we conclude that this virus stock contained -2.8 x 105 infectious units per ml. Although in this assay a cdna probe was used, essentially similar results were also obtained using a nick-translated REV-A probe (20) (data not shown). Finally, we tested the ability of the hybridization assay to detect MMTV. The lack of an infectivity assay has previously made this virus almost totally unsuitable for many types of biological and genetic experiments. We infected CrFK cat cells with serial dilutions of MMTV; clear hybridization plaques were obtained (Fig. 4). Since 0.5 ml of the 10-3 dilution gave 50 hybridization plaques, the initial virus stock contained 105 infectious units of MMTV per ml. DISCUSSION The results presented in this report show that in situ hybridization can be used as an infectivity assay for retroviruses. The virus titer determined by scoring foci of hybridization was shown (Table 1) to be equal to the true infectivity of an MuLV stock, as determined by the UV- XC test (in turn, the UV-XC test gives the same titer as an endpoint dilution assay for nondefective MuLVs [19]). The fact that the hybridization assay can be used for MuLV titrations on mouse cells, which contain endogenous MuLV-related sequences in their DNA, indicates that the probe is reacting primarily, if not exclusively, with RNA rather than with DNA. This distinguishes this technique from the in situ hybridization assay described by Villareal and Berg, which detects DNA species which replicate to a high copy number in infected cells (28). One important virtue of the hybridization technique is the fact that a culture is unambiguously positive in this assay once a small fraction of the cells is infected. Thus, we have found that it is extremely useful as a rapid, qualitative

5 VOL. 43, 1982 GENERAL INFECTIVITY ASSAY FOR RETROVIRUSES 1059 S i S * * 0 *..4 I 0-3 * 6 % FIG. 4. Titration of MMTV by in situ hybridization. Plates containing 105 CrFK cells were infected with dilutions of MMTV and were overlaid with medium containing Polybrene. The plates were fixed for hybridization 14 days after infection and were hybridized with MMTV [32P]cDNA, using 2 x 106 cpm per dish. The MMTV RNA used for cdna synthesis was a kind gift from Larry Arthur. test for replication of REV-A and BaEV after transfection with either cloned or high-molecular-weight cellular DNA (4). The unique advantage of the present technique is its apparently universal applicability. We have demonstrated its use here with two retroviruses which have, in general, been extremely difficult to assay: REV-A (Fig. 3) and MMTV (Fig. 4). Hopefully, the availability of an infectivity assay will permit genetic approaches to the study of these viruses. We would emphasize that the assay can in principle be used to assay the spread in virus particles of any sequence for which one has a radioactive probe, on any host cell. Since the specificity of the technique (Fig. 2) enables the detection of one virus in the presence of another, it should be readily applicable to the study of replicationdefective viruses, including those which do not code for any known products (such as the mouse and rat 30S sequences [2, 13, 24, 26]), and in tests for the incorporation of nonviral sequences into packageable retrovirus genomes (29). This property distinguishes it from other assays which detect focal areas containing virus-specific antigen (7, 17, 27). It may also be of use in analyzing the genetic requirements for packaging (25). ACKNOWLEDGMENTS We thank Melody McClure, Anne Soria, and Marilyn Powers for excellent technical assistance, Larry Arthur, Robert Bassin, and Raymond Gilden for suggestions on the manuscript, and Jeannie Clarke for preparation of the manuscript. LITERATURE CITED 1. Bassin, R. H., N. Tuttle, and P. J. Fischinger Rapid cell culture assay for murine leukaemia virus. Nature (London) 229: Besmer, P., U. Olshevsky, D. Baltimore, D. Dolberg, and H. Fan Virus-like 30S RNA in mouse cells. J. Virol. 29: Brahic, M., and A. T. Haase Detection of viral sequences of low reiteration frequency by in siti hybridization. Proc. Natl. Acad. Sci. U.S.A. 75: Cohen, M., A. Rein, R. M. Stephens, C. O'Connell, R. V. Gilden, M. Shure, M. 0. Nicolson, R. M. McAllister, and N. Davidson Baboon endogenous virus genome. III. Molecular cloning and structural characterization of nondefective viral genomes from the DNA of a baboon cell strain. Proc. Nati. Acad. Sci. U.S.A. 78: Cohen, M., N. Rice, R. Stephens, and C. O'Connell DNA sequence relationship of the baboon endogenous virus genome to the genomes of other type C and type D retroviruses. J. Virol. 41: Crandell, R. A., C. G. Fabricant, and W. A. Nelson-Rees Development, characterization, and viral susceptibility of a feline (Felis catims) renal cell line (CRFK). In Vitro 9: Fitting, T., and D. Kabat Protein A-coated erythrocyte binding to cell surface antigens: application to quantitate retrovirus infectivity in vitro. Virology 111: Gisselbrecht, S., R. H. Bassin, B. I. Gerwin, and A. Rein Dual susceptibility of a 3T3 mouse cell line to infection by N- and B-tropic murine leukemia virus: apparent lack of expression of the Fv-1 gene. Int. J. Cancer 14: Gonda, M. A., N. R. Rice, and R. V. Gilden Avian reticuloendotheliosis virus: characterization of the highmolecular-weight viral RNA in transforming and helper virus populations. J. Virol. 34: Graf, T A plaque assay for avian RNA tumor viruses. Virology 50: Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. Rowe A new class of murine leukemia virus associated with the development of spontaneous lymphomas. Proc. Natl. Acad. Sci. U.S.A. 74: Hoelzer, J. D., R. B. Lewis, C. R. Wasmuth, and H. R. Bose, Jr Hematopoietic cell transformation by reticuloendotheliosis virus: characterization of the genetic defect. Virology 100: Howk, R. S., D. H. Troxler, D. Lowy, P. H. Duesberg, and E. M. Scolnick Identification of a 30S RNA with properties of a defective type C virus in murine cells. J. Virol. 25: Jainchill, J. L., S. A. Aaronson, and G. J. Todaro Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J. Virol. 4: McCarter, J. A Genetic studies of the ploidy of Moloney murine leukemia virus. J. Virol. 22: Moscovici, C., D. Chi, L. Gazzolo, and M. G. Moscovici A study of plaque formation with avian RNA tumor viruses. Virology 73: Nexo, B. A A plaque assay for murine leukemia

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