JOURNAL OF VIROLOGY, May 1979, p. 637-641 0022-538X/79/05/0637-05$02.00/0 Vol. 30, No. 2 NOTE Detection of Virus-Specific RNA in Simian Sarcoma- Leukemia Virus-Infected Cells by In Situ Hybridization to Viral Complementary DNA STEVEN L. KAUFMAN,t ROBERT C. GALLO,`* AND NANCY R. MILLER2 Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20014,1 and Bethesda Research Laboratories, Rockville, Maryland 208502 Received for publication 4 January 1979 An in situ molecular hybridization system which will detect retrovirus RNA in the cytoplasm of individual virus-infected cells has been developed. The technique was applied to cells infected with simian sarcoma-leukemia virus, where the virusspecific RNA was detected by hybridization to simian sarcoma-leukemia virus 3H-labeled complementary DNA. The system is useful for detecting viral RNAcontaining cells in the presence of an excess of virus-negative cells and for determining which type of cell in a heterogenous population is expressing viral RNA. Cytological or in situ hybridization has been used extensively to find the chromosomal location of DNA sequences complementary to a specific purified cellular RNA (for reviews, see Pardue and Gall [11], Hennig [4], Jones [5], and Wimber and Steffensen [15]; for a quantitative analysis of the system, see Szabo et al. [14]). The technique has also been applied to the analysis of cells infected with DNA viruses. It has been used to detect virus-specific DNA in adenovirus-transformed (9) and in polyomatransformed (10) cells by hybridization of the DNA to complementary viral RNA. Complementary DNA (cdna) from simian virus 40 has been used to localize related sequences in host cell chromosomes (13). The use of in situ hybridization in the detection of viral nucleic acids in cells infected by retroviruses has been limited, however. Loni and Green (7) used viral cdna and RNA to detect viral DNA sequences in nuclei of virus-producing cells transformed by Harvey and Moloney strains of murine sarcoma virus. Haase et al. (2) have used the technique to detect proviral DNA in cells infected with visna virus. In many cells infected with RNA type C viruses, however, the proviral sequences can be present in the cellular DNA at one copy per cell or less. This makes detection difficult, requiring very low backgrounds and quantitation of grains. t Present address: Medical College of Virginia, Richmond, VA 23298. In contrast to proviral DNA, viral RNA may be present in multiple copies in infected cells. The purpose of this report is to describe the development of the technique of in situ hybridization to detect viral RNA in type C virusproducing cells. We have used a modification of the technique developed by Harrison et al. (3) who used in situ hybridization to detect globin mrna in fetal mouse liver cells and in Friend virus-transformed cells treated with dimethyl sulfoxide. A similar technique has also been applied in DNA virus systems by Moar and Jones (9) who detected virus-specific RNA in the cytoplasm of adenovirus type 2-transformed rat cells and by McDougall in a recent demonstration of viral RNA in herpes type 2-infected cells (8) and evidence for such sequences in RNA from several human cervical carcinomas (8a). Our results indicate that in situ hybridization can be used to detect RNA of the woolly monkey (simian sarcoma-leukemia) virus (SiSV/SiSAV), an infectious primate type C RNA tumor virus, in the cytoplasm of infected cells which produce the virus. To prepare cytological slides for in situ hybridization, cells grown in tissue culture were trypsinized, the trypsin was neutralized by media containing 10% fetal calf serum, and cells were washed twice with serum-free medium. The cell concentration was adjusted to 106 cells per ml. Portions (200,u) of this suspension were then pipetted into cytocentrifuge buckets and 637
638 NOTE centrifuged against microscope slides in a Shandon Cytospin cytocentrifuge at 500 rpm for 5 min. After air drying for a minimum of 15 min, the cells were fixed on the slides in ethanolacetic acid (3:1) at room temperature for 10 min. The slides were air dried and stored at -70 C. Just before hybridization, the fixed slides were treated with 0.2 N HCl for 20 min at room temperature, rinsed briefly in water twice, dehydrated through three subsequent 5-min, room temperature washes of 70, 70, and 100% EtOH, and air dried. For the hybridization, [3H]cDNA with a 103-fold excess of trna was dissolved in 40% formamide-3x SSC (lx SSC = 0.15 M NaCl-0.015 M sodium citrate) and 2,ul (10,000 cpm, approximately 1 ng of [3H]cDNA) was applied to the spot containing cells. The liquid was covered with a 12-mm-diameter glass cover slip, and the slides were placed in a rack in a light mineral oil bath at 44 C. It is not necessary to seal the cover slips in any manner. Hybridization was carried out for the desired length of time (usually 40 to 44 h, which corresponds to a Cot with respect to cdna of approximately 0.2). The slides were removed from the oil bath, rinsed twice (2 min) in chloroform to remove the oil, and then rinsed three times (5 min) in 2x SSC. The cover slips fall off during the first rinse. The hybrids were heated at 550C in 2x SSC for 1 h to dissociate nonspecific complexes, and then washed in 2x SSC at 4 C for 24 h. The slides were dehydrated through three successive 5-min washes in 70% EtOH, 70% EtOH, and 100% EtOH and were air dried. The presence of a hybrid between the [3H]- cdna and cytoplasmic RNA was detected by autoradiographic exposure to a photographic emulsion which was used to coat the slides. Autoradiography was carried out essentially as described by Pardue and Gall (11) with the addition of the high-speed scintillation method of Durie and Salmon (1). Kodak NTB-3 nuclear track emulsion was heated to 420C and then diluted with an equal weight of water, also at 42 C. The solution was allowed to sit without mixing (which creates air bubbles) for 10 min at 420C, then poured slowly into a Coplin jar (to facilitate slide dipping), and held at 420C. The slides with the hybridized [3H]cDNA were dipped slowly three times into the emulsion and air dried in a vertical position for 1 h. They were then dipped (10 s) in scintillator {5 g of PPO (2,5-diphenyloxazole), 100 mg of dimethyl PO- POP [1,4-bis-(5-phenyloxazolyl)-benzene] dissolved in 500 ml of dioxane}, air dried, sealed in light-proof black slide boxes, and exposed at -70 C for 3 weeks. After exposure, the slides were developed in Kodak D-19 developer at 17 C for 3 min, dipped in water for 10 s, dipped in Kodak fixer for 3 min, washed in water at 170C for 30 min, and air dried. The cells were stained with Wright-Giemsa (Harleco) for 8 min and for an additional 20 min after addition of ph 6.4 phosphate buffer, then rinsed with water, and air dried. It is essential to have adequate quantities of well-characterized cdna for this technique to be useful. For the synthesis of SiSV/SiSAV [3H]cDNA, 2 ml of 1,000x concentrated (1013 focus-forming units per ml) SiSV/SiSAV grown in marmoset cells (71AP1) was pelleted through a 30% glycerol (in 0.1 M NaCl-0.01 M Trishydrochloride, ph 7.4) J. VIROL. cushion at 100,000 x g for 1 h in a Beckman SW41 rotor. The virus pellet was resuspended in 500 yl of buffer: 2 mm dctp, 2 mm dttp, 0.6 mm [3H]dGTP, 0.9 mm [3H]dATP, 0.1% Triton X-100,4 mm magnesium acetate, 0.04 M Tris-hydrochloride (ph 7.4), 6 mm dithiothreitol, and 50 jig of actinomycin D per ml. After incubation on ice for 10 min, synthesis of [3H]cDNA was carried out at 370C for 16 h. The reaction was stopped by making the mixture 1% in sodium dodecyl sulfate and freezing at -70 C. After thawing, the mix was adjusted to ph 9, trna was added, and the cdna was extracted with an equal volume of 0.05 M Tris (ph 9)-saturated phenol-chloroform-isoamyl alcohol (1:1:0.01). After removal of the aqueous layer, the organic layer was repeatedly washed with 0.1 M NaCl-0.05% sodium dodecyl sulfate-0.02 M Tris (ph 9) until the interface collapsed. The combined aqueous layers were made 0.2 M with NaCl, 2 volumes of EtOH were added, and the cdna was precipitated overnight. The precipitate was collected by centrifugation, redissolved in 0.1 M NaCl-0.01 M Trischloride (ph 7.4)-0.001 M EDTA, and nucleic acids were re-precipitated with cetyltrimethylammonium bromide (12). After a second EtOH precipitation, the cdna was alkali treated (0.3 N NaOH, 37 C overnight) and neutralized. Because of residual resistance (approximately 20%) to single-strand-specific S1 nuclease, the cdna was further purified by centrifugation on a neutral sucrose gradient (15 to 30% in 100 mm LiCl, 10 mm Tris (ph 7.4), 1 mm EDTA, and 0.2% sodium dodecyl sulfate) at 100,000 x g (30,000 rpm) for 16 h in a Beckman SW41 rotor. The main cdna peak (7S) was pooled, and the fractions at the top of the gradient were discarded. Approximately 4 x 107 cpm of cdna with a specific activity of 1.4 x 107 cpm/,ug was obtained from the synthesis. The cdna hybridized 75% to DNA from SiSV-infected cells at an uncorrected Cot of 8,500, with 6% self-annealing at the same Cot. When hybridized to SiSV ['25I]RNA,
VOL. 30, 1979 the cdna protected 100% of the RNA from RNase digestion at a cdna:rna ratio of 2:1, indicating that it was a representative copy of the entire RNA genome. Figure 1A shows the result of the hybridization of SiSV/SiSAV (71AP1) [3H]cDNA to 71AP1 cells producing SiSV/SiSAV. The grains, indicating the presence of hybridized [3H]cDNA, are dense over all the cells and almost negligible in intercellular spaces. The hybridization is specific: the addition of unlabeled competing SiSV/ SiSAV RNA (Fig. 1B), but not RNA from avian myeloblastosis virus, an unrelated virus (Fig. 1C), eliminates the grains. The hybridization is RNA dependent and is abolished when the cells are pretreated with RNase (Fig. 1D). Denaturation of cellular DNA (2) in 95% formamide- 0.1x SSC at 650C for 2 h before addition of [3H]cDNA resulted in no detectable hybridization, confirming that under the conditions used, htybridization was to cellular RNA. The specificity of the hybridization was also NOTE 639 demonstrated by the lack of hybridization of the SiSV/SiSAV (71AP1) [3H]cDNA to RNA from normal marmoset (HF) or normal human (A204) cell lines (Fig. 2A and 2B, respectively). There was also no hybridization seen between the SiSV/SiSAV (71AP1) [3H]cDNA and RNA of cells producing baboon endogenous virus (Fig. 2C) or cells producing feline leukemia virus (Fig. 2D). A bat cell line producing SiSV/SiSAV was as positive (Fig. 2E) as the 71AP1 (SiSV/SiSAV) cells used for production of the virus from which the cdna was synthesized, confirming that the positive hybridization was virus specific and not due to a marmoset cell-specific contaminant. Figure 3 shows the results of the hybridization of SiSV/SiSAV (71AP1) [3H]cDNA to 71AP1 (SiSV/SiSAV) cells diluted with uninfected feline embryo fibroblasts (FEF). The virus-producing cells are clearly detectable in the midst of the negative, nonproducing FEF cells. It is possible to detect one virus-positive cell on the slide, even if there are 103 or more nonproducing I FIG. 1. In situ hybridization of SiSV/SiSAV[3H]cDNA to the RNA of 71AP1 marmoset cells producing SiSV/SiSAV. Cytological preparations, [3H]cDNA synthesis, the hybridization conditions, and autoradiography were carried out as described in the text. Viral 70S RNA from SiSV/SiSA V and avian myeloblastosis virus was prepared as described in (16). (A) SiSV/SiSAV [3H]cDNA hybridized to 71AP1 cells producing SiSV/SiSAV; (B) same as (A) with the addition of competing, homologous SiSVISiSA V viral RNA; (C) same as (A) with the addition of competing, nonhomologous avian myeloblastosis virus RNA; (D) SiSV/SiSAV [3H]cDNA hybridized to 71AP1 (SiSV/SiSAV) cells which had been pretreated with RNase (1 IL of 1 mg of RNase A [Worthington]per ml and 2 [L of3x SSC were applied to the cells, covered, incubated in mineral oil at 37 C for 1 h, removed from oil, and washed as described in the text).
640 NOTE J. VIROL. ^S. A B V-.- :.. j....f. :. it v. 1.1... - w C 1O.O.Aw. D Downloaded from http://jvi.asm.org/ FIG. 2. In situ hybridization of SiSV/SiSAV [3H]cDNA prepared from virus produced by marmoset cells to the RNA of cells not producing SiSVISiSA V or to nonmarmoset cells producing SiSV/SiSA V. (A) Normal marmoset cells (HF); (B) human rhabdomyosarcoma cells (A204); (C) A204 cellsproducing baboon endogenous virus (M); (D) FEF cells producing feline leukemia virus; (E) bat cells (B88) producing SiSV/SiSA V. cells also present. In situ hybridization of viral [3H]cDNA to cellular RNA should be useful for detecting cells replicating type C viruses in a mixed population or cells synthesizing viral RNA in a nonproducing system. The method is specific for viral RNA, has a low background that does not interfere with the detection of low levels of positive hybridization, and is independent of the number of virus-negative cells in the population. This makes it a very sensitive method to use in cell culture systems (viral induction, for example) where there is a small number of cells or where only a few cells might be expected to produce virus. Standard liquid hybridization systems might not be sensitive enough to detect such small amounts of viral RNA. We have also successfully hybridized SiSV/SiSAV [3H]cDNA to RNA from SiSV/SiSAV-infected cells which were grown directly on microscope slides (unpublished data). This technique has the advantage of making it possible to examine all the cells in a given population without the possibility of damage or loss of cells during cell washing and cytospin procedures. Perhaps the most interesting application of in on July 20, 2018 by guest
VOL. 30, 1979 -..'. *~~~~~~~~~~~. *-w.*. : %.,i.\..,%6 FIG. 3. In situ hybridization of SiSV/SiSAV [3H]cDNA to a mixture of virus-producing 71AP1 (SiSV/SiSAV9 cells and uninfected, nonvirus-producing FEF cells. Positive hybridization to the viruspositive cell is evident. situ hybridization in the RNA tumor virus system, however, is the examination of fresh cells from animals with virus-induced leukemia or lymphoma. It should be possible, by hybridizing viral cdna to tissue slices as well as to cytospins of peripheral blood and bone marrow samples, to identify tissues or cell populations which are sites of viral replication. The course of primary virus replication and subsequent spread could be followed during the course of such diseases as AKR leukemia and Friend erythro-leukemia in mice, feline leukemia virus-induced leukemia in cats, or natural and gibbon ape leukemia virus-induced leukemia in gibbon apes. It will hopefully be a useful tool in detecting cells which are expressing viral RNA in leukemia animals, or possibly in humans, when no replicating virus can be detected. Several SiSV-transformed nonproducer cell lines (obtained from C. Bergholz and S. Aaronson) are currently under examination to explore this possibility. The method also lends itself to the use of subfractionated cdna, enriched for specific regions of the viral genome, to investigate viral RNA expression in infected cells not producing virus. ACKNOWLEDGMENTS We thank Wolf Prensky and Paul Szabo of Sloan Kettering Institute and Linda Vaught and Brian Durie of the University of Arizona for information on the technical aspects of in situ hybridization and high-speed scintillation autoradiography, respectively; F. Ruscetti and M. Reitz for helpful discussion; R. Williams for excellent technical asistance; and A. 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