RNA metabolism of murine leukemia virus: Detection of virus-specific RNA sequences in infected and uninfected cells and identification of virus-specific messenger RNA

J. Mol. Biol. (1973) 80, 93-117

RNA Metabolism of Murine Leukemia Virus : Detection of Virus-specific RNA Sequences in Infected and Uninfected Cells and Identification of Virus-spectic Messenger RNA HUNGFmt

AND DAVY, BALTIMORE

Department of Biology Maaeachusetts Institute of Technology 77 Massachusetts Avenue Cambridge, Mass. 02139, U.S.A. (Received 22 March 1973, ati in revised form 17 July 1973) Virus-specific RNA sequences were detected in mouse cells infected with murine leukemia virus by hybridization with radioactively labeled DNA complementary to Moloney murk leukemia virus RNA. The DNA was synthesized in vitro using the endogenous virion RNA-dependent DNA polymerase and the DNA product W&Bcharacterized by size &nd its ability to protect radioactive viral RNA. Virusspecific RNA sequences were found in two lines of leukemia virus-infected cells (JLS-VI 1 and SCRF 60A) and also in an uninfected line (JLS-V9). Approximately 0.3% of the cytoplasmic RNA in JLS-VII cells was virus-specifk and 0.9% of SCRF 6OA cell RNA ww virus-specific. JLS-V9 cells contained approximately tenfold less virus-specific RNA than infected JLS-Vll cells. Maloney leukemia virus DNA completely annealed to JLS-Vll or SCRF 60A RNA but only partial annealing was observed with JLS-V9 RNA. This difference is ascribed to nonhomologies between the RNA sequences of Moloney virus and the endogenous virus of JLS-V9 cells. Virus-specific RNA was found to exist in infected cells in three major size classes: 60-70 S RNA, 35 S RNA and 20-30 S RNA. The 60-70 S RNA was apparently primarily at the cell surface, since agents which remove material from the cell surface were effective in removing a majority of the 60-70 S RNA. The 35 S and 20-30 S RNA is relatively unaffected by these procedures. Sub-fractionation of the cytoplasm indicated that approximately 35% of the cytoplasmic virus-specific RNA in infected cells is contained in the membrane-bound material. The membrane-bound virus-specific RNA consists of some residual 60-70 S RNA and 35 S RNA, but very little 20-30 S RNA. Virus-specific messenger RNA was identified in polyribosome gradients of infected cell cytoplasm. Messenger RNA wss differentiated from other virus-specific RNAs by the criterion that virusspecific messenger RNA must change in sedimentation rate following polyribosome d&aggregation. Two procedures for polyribosome disaggregation were used: treatment with EDTA and in vitro incubation of polyribosomes with puromycin in conditions of high ionic strength. As identified by this criterion, the virus-specific messenger RNA appeared to be mostly 35 S RNA. No function for the 20-30 S was determined.

1. Introduction Although the genetic material of RNA tumor viruses is RNA, considerable evidence argues that the virus genetic inform&ion is trctnsferred to DNA sequences shortly t Present addrem: The Salk Institute for Biological Studies, P.O. Box 1809, San Diego, Calif. 92112, U.S.A. 93

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after infection (Temin, 1964; Bader, 1964; Hill & Hillova, 1972; reviewed by Temin, 1970). The discovery that RNA tumor virus particles contain an RNA-dependent DNA polymerase activity in the virions has given much support to this hypothesis (Baltimore, 1970; Temin & Mizutani, 1970; reviewed by Temin & Baltimore, 1972). In addition, the ability of the RNA-dependent DNA polymerase to dire& the synthesis of DNA complementary to tumor virus RNA in vitro has made possible the synthesis of highly labeled DNA that can be used as a probe for virus-specific nucleic acid sequences in infected cells. RNA tumor viruses contain a single 60-70 S RNA complex, with an estimated molecular weight of approximately 1-2~ lo7 (Robinson et al., 1965) and smaller amounts of RNAs with sedimentation values of 4 S and 7 S (Bishop et al., 197Oa,b). The 60-70 S RNA molecule can be denatured to produce three or four 35 S RNA subunits, each with molecular weight of approximately 3x lo6 (Duesberg, 1968; reviewed by Duesberg, 1970). A majority of the 4 S RNA appears to be cellular transfer RNA (Bonar et al., 1967; Erikson & Erikson, 1971) but the nature of the 7 S RNA is as yet unclear. Cells infected with RNA tumor viruses produce several virus-specific proteins, which include virus envelope proteins, internal core proteins, the RNA-dependent DNA polymerase and the protein (or proteins) responsible for cell transformation. Some of these proteins can be detected immunologically with subgroup-specific, group-specific or interspecies-specific antisera (Geering et al., 1970; Ishizaki & Vogt, 1966 ; Huebner et aZ., 1963). It has also been observed that uninfected cells can express some of these virus antigens (Huebner & Todaro, 1969), and it has recently been shown that these antigens are the expression of genetic information for endogenous RNA tumour viruses that can be induced by various methods (Lowy et al., 1971; Weiss et al., 1972; Hanafusa et al., 1972). In view of the apparently varied modes of virus propagation and expression, characterization of the virus-specific messenger RNA is important. In particular, it should be significant to determine whether any of the RNA species of the genome act as messenger RNA and whether messenger RNAs are found in sizes other than those found in the virion. It might also be that during different states of virus growth or expression different messenger RN.& are expressed. Virus-specific RNA has been detected in cells infected with avian (Leong et al., 1972; Coffin $ Temin, 1972; Hanafusa et aI., 1970) and murine (Green et a& 1971; AxeI et al., 1972) RNA tumor viruses. The virus-specific RNA sequences were detected by hybridization of labeled DNA complementary to tumor virus RNA with infected cell RNA. A size analysis of the virus-specific RNA in cells infected with avian sarcoma virus (Leong et al., 1972) and murine leukemia virus (Tsuchida et al., 1972) has been done by others but virus-specific messenger RNA was not identified. In the work reported here virus-specific RNA was examined in mouse cells chronically infected with murine leukemia virus and virus-specific messenger RNA was identified.

2. Materials and Methods (a) Materials and solutions The following solutions were used and their composition or a reference is given here. RS buffer is 0.01 M-NaCl, O-01 M-Tris (pH 7.4), 1.6 mM-MgCl,. SDS buffer is 0.1 M-N&~, 0.01 u-Tris (pH 7.4), 1 mM-EDTA. SDS was added to this buffer in the concentrations mentioned.

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SSC is 0.16 M-N&~, 0*015 M-sodium citrate. Earle’s solution (Earle, 1943). Phosphate buffered saline with 0.02% EDTA (Dulbecco and Vogt, 1954). Actinomycin D was a gift of Merck, Sharpe C Dohme; puromycin was obtained from Calbiochem; cycloheximide from Nutritional Biochemicals; NP40 from Shell Oil Co. ; proteinase K (chromatogmphically pure), from EM Labs, Elmsford, N.J.; and dextran sulfate from Sigma. 3H-labeled deoxynucleotide triphosphates were obtained from Schwarz-Mann and d-[32P]TTP and [3H]adenosine and [3H]uridine from New England Nuclear Corp. (b) Growth of cells and puri$cdion of virus JLS-VI 1 is a line of cells derived from BALB/c mouse bone marrow and subsequently infected with Moloney murine leukemia virus (Wright et al., 1967). They were kindly provided by Electronucleonics, Inc., Bethesda, Md. The cells were grown as monolayers in 32-0~ glass prescription bottles, with Joklik-modified Eagle’s medium (Eagle, 1959) supplemented with non-essential amino acids (Grand Island Biological) and 7% fetal calf serum (Microbiological Associates, Bethesda, Md). JLS-VQ, the uninfected parent line of JLS-Vll, was kindly supplied by Dr K. Manly and grown in the same medium. SCRF 6OA cells are a permanent line of lymphocytes derived from NZB mice that liberate a murine leukemia virus and were a gift of Dr F. Jensen (Lemer et al., 1972). They were grown in roller bottle suspension culture in the same medium as for JLS-VI I cells. The SCRF 60A cells were maintained in exponential growth at cell densities between 4 x 106/ml and 1 x 106/ml. M-MuL virus? w&s prepared from the supernatant fluid of JLS-VII cells grown to confluence. Two procedures for virus concentration were used. In the first method, supernatant medium from JLS-Vll cells was cleared of cell debris by centrifugation et 800 g for 10 min. The virus was then harvested from the supernatant by centrifugation in a Beckman type 19 rotor for 325 h at 19,000 revs/mm. The virus wss resuspended in 1 to 2 ml 0.01 m-Tris, pH 7.5, transferred to a screw-cap test tube and briefly sonicated in an immersion sonic&or (Raytheon) in order to disperse aggregates. One-ml portions of the virus suspensions were then layered onto 4.5-ml 25% to 45% sucrose gradients in 0.01 MTris, pH 7.5, and centrifuged for at least 3 h at 35,000 revs/min in a Beckman SW50.1 rotor, during which time the virus sedimented to its equilibrium density of I*15 to 1.16 g/ cm3 (approx. 35% sucrose). The virus band was visible and iwas withdrawn through the side of the centrifuge tube with a syringe needle. The virus W&B diluted with 0.01 M-Tris and recovered by centrifugation in a Beckman type 65 rotor for 45 min at 49,000 revs/min. The purified virus pellet was resuspended in 0.01 M-Tris at approximately 1 to 2 mg viral protein/ml and stored at - 70°C. In the second method, the cell supernatant wss clarified as before and the virus was precipitated by the addition of ammonium sulfate to 50% saturation. The precipitate was harvested by centrifugation for 15 min at 8000 revs/min in a Sorvall GSA rotor, resuspended in O-01 M-Tris buffer, and harvested by centrifugation at 29,000 revs/min in a Beckman type 30 rotor for 90 min. The pellet was resuspended in Tris buffer and purified as above. Large quantities of cell supernatant could be more rapidly processed by this method than by the first procedure. However, this method resulted in damage to MuL virus virions obtained from certain cell lines other than JLS-Vll, so the first procedure may be of more general use. (c) Preparation of the M-MuL virus DNA probe Radioactively Iabeled DNA complementary to M-MuL virus RNA was prepared using the “endogenous” DNA polymerase reaction of MuL virus virions (Manly et al., 1971). Briefly, purified M-MuL virus was added to an incubation mixture containing 0.02 Mdithiothreitol, 0.01 aa-Tris (pH 8*3), 6 mM-magnesium acetate, 0.06 M-N&I, 1 mM each dATP, dGTP and dCTP, 25 &!i d-[3H]TTP/ml (18 Ci/ mmol), 0.01 o/oNP40, 9 miM-creatine t Abbrevietions used: MuL virus, mu&e leukemia virus; M-MuL virus, Maloney stmin of murine leukemia virus ; SDS, sodium dodeoyl sulfate.

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phosphate, 100 pg creatine phosphokinase/ml, and 20 pg actinomycin D/ml. In standard preparations, approximately O-2 to O-4 mg virus protein was added to a 2.0.ml incubation mixture. The reaction mixture was sealed under nitrogen, and incubated at 37°C for 4 h, after which an equal amount of d-[3H]TTP was again added and incubation was continued for another 4 h. The reaction was stopped by the addition of sodium dodecyl saroosinate to 2% final concentration, and NaOH was added to 0.3 M. The mixture was heated to 100°C for 5 min in a boiling water bath and then neutralized with 0.1 vol. of 3 N-HCl. The reaction mixture was then passed over a Sephadex G50 column equilibrated with O-05 M-triethylammonium bicarbonate buffer, pH 7-O. A small amount of Chelex 100 resin (Bio-Rad Laboratory) was placed at the base of the column in order to remove contaminating heavy metal ions from the DNA product. Portions from the column fractions were assayed for acid-precipitable radioactivity and the DNA product was found to elute in the column flowthrough, well ahead of unpolymerized triphosphates. Those fractions containing the labeled DNA were pooled, and lyophilized to dryness. The lyophilized material was resuspended in 1.0 ml water, NaCl was added to 05 br, yeast tRNA was added to 25 pg/ml as carrier and the product was precipitated with 2 vol. of ethanol. The ethanol precipitate was recovered by centrifugation at 17,000 g for 16 mm and was resuspended at approximately 500 cts/min/pl in 2 x SSC plus 0.1% SDS. The DNA product was stored at -20°C. (d) Preparation

of RNAs

M-MuL virus 60-70 S RNA was obtained by lysis of purified M-MuL virus with a tinal concentration of SDS of 1%. The lysed virus was layered onto an 11.5-ml 15% to 30% sucrose gradient containing 0.5% SDS in SDS buffer and centrifuged for 3.25 h at 35,000 revs/mm and 22°C in a Beckman SW40 rotor. The gradient was fractionated, and the optical density at 260 nm was monitored by a Gilford continuous flow cell spectrophotometer, The 60-70 S RNA appeared as an A 280 peak and corresponding fractions were pooled. NaCl was added to 0.5 M final concentration, and 2 vol. of ethanol were added. The precipitate was then harvested by centrifugation at 35,000 revs/min for 2 h in an SW40 rotor and the pellets were resuspended in 2 x SSC plus O-lob SDS. The amount of RNA present was determined by measuring the Azeo of the sample. Radioactively labeled M-MuL virus RNA was obtained by the addition of 1 mCi each of [eH]adenosine and [3H]uridine to a lo-cm tissue culture dish containing growing JLSVll cells. After 8 h, the medium was removed and discarded and fresh medium with an equal amount of label was added. At 20 h after the first addition of label, the supernatant was removed and clarified of cells as described above. The radioactive virus wss harvested by centrifugation at 60,000 revs/min for 30 min in a Beckman type 65 rotor, and the pellet was resuspended in 0.5 ml SDS buffer containing 0.5% SDS. The lysed virus was then layered onto an SDS-sucrose gradient as described above and centrifuged as described. Portions of fractions from the sucrose gradient were assayed for acid-precipitable radioactivity and 60-70 S RNA could be seen as a peak of radioactivity. The fractions corresponding to the peak were pooled, pelleted and resuspended in 2 x SSC plus 0.1% SDS as described above. The specific activity of the 3H-labeled M-MuL virus RNA was determined by a modiflcation of the method of Gillespie et al. (1972). This procedure uses the fact that RNA tumor virus RNA contains poly(A) sequences and so the amount of RNA can be measured by its capacity to hybridize radioactive poly(U). In a similar manner, the ability of a given amount of labeled RNA to hybridize 3aP-labeled poly(dT) as compared to known amounts of M-MuL virus RNA was determined as shown in Fig. 1. 3aP-labeled poly(dT) was prepared using the puritled DNA polymerase of avian myeloblastosis virus, with a poly(A) template and oligo(dT)8 primer (Baltimore & Smoler, 1972). The 3aP-labeled poly(dT) was purified in a manner similar to the 3H-labeled M-MuL virus DNA product. The specific activity of the 3aP-labeled poly(dT), as determined from that of the input dTTP, was 1700 ots/min/pmol nucleotide. The 32P-labeled poly(dT) was then annealed to known amounts of M-MuL virus RNA in 2 x SSC plus O~l”/o SDS at 53’C and the amount of e2P protected from single-strand nuclease digestion (see below) is shown. The 3H-labeled RNA was also mealed to the 3aP-labeled poly(dT) and the amount of protection resulting

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M- MuL virus RNA nucleotides (pmol)

Fro. 1. Measurement of amount of labeled M-MuL virus RNA. 32P-lebeled poly(dT) (1700 cts/min/pmol), sH-labeled M-MuL virus RNA (120 ots/min/pl) and unlabeled M-MuL virus RNA (15 pg/ml) were prepared as described in Materials and Methods. Poly(dT) (2000 cts/min/assay) was hybridized to varying amounts of unlabeled M-MuL virus RNA for 4 h at 63’C, in a total reaction vol. of 7 ~1. The samples were assayed for amount of poly(dT) hybridized. One and 2 ~1 of 3H-labeled M-MuL virus RNA was also hybridized to poly(dT) and

the amount of poly(dT) protected is shown by the arrows. from the addition of 1 and 2 ~1 of radioactively labeled RNA is shown by the arrows. As determined by this method, the specific activity of the 3H-labeled M-MuL virus DNA used was 700,000 cts/min/pg. (e) Preparation of cellular RNA and cell fractionation Cytoplasmic RNA was prepared in the following manner. For JLS-VI1 and JLS-V9, the cell monolayers were rinsed with phosphate buffered saline or Earle’s solution and 3.0 ml RS buffer was added to each bottle of cells. NP40 was then added to 1 y. final concentration and the lysate was collected. Nuclei were removed by centrifugation at 1000 g for 2 min and the supernatant was made 0.4 M-NaCl, 0.01 M-EDTA, and 1% SDS. The supernatant was phenol-extracted 3 times according to the method of Penman (1966), in which 1% isoamyl alcohol in chloroform was added to the organic phase to improve phase separation. The protein interphase was retained and re-extracted along with the aqueous phase each time to avoid the loss of poly(A)-containing RNA species. The extracted RNA was precipitated with 2 vol. of ethanol and harvested by centrifugation. The RNA was resuspended in 2 x SSC plus 0.1 o/oSDS, and its concentration was determined by measuring the Asso. For the SCRF 60A cells a similar method was used, except that the cells were first harvested from the suspension medium by centrifugation at 800 g for 3 min. The cells were then resuspended in RS buffer and lysed as before. All further procedures were the same. For analysis of total RNA species, the cells were processed in the following manner. JLS-Vll and JLS-V9 cells were washed with phosphate buffered saline or Earle’s solut’ion and 3 ml of 0.5% SDS in SDS buffer was added to each bottle. Proteinase K was added to a tIna concentration of 0.5 to 1 mg/ml. The proteinase K had been dissolved at 10 mg/ml in water immediately before use. After approximately 5 min the cell lysate was scraped to the bottom of the bottle with a rubber policeman and collected into a centrifuge tube. The lysate was sonicated in a Branson sonifier at a setting of “5” for 30 s or until the viscosity due to DNA disappeared. The lysate was adjusted to 0.5 M-NaCl and 2 vol. of ethanol were added. The precipitate was collected by centrifugation as described above and then resuspended in 0.5 ml SDS buffer containing 0.5% SDS. The samples were then layered onto an 11.5-ml 15% to 30% sucrose gradient containing 0.1 y. SDS in SDS buffer and centrifuged in an SW40 or SW41 rotor for the times indicated in the Figure legends. The SCRF 60A cells were processed in a slightly different manner. The cells were collected

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from the medium by centrifugation at 800 g for 3 min and then resuspended in 3-O ml of SDS buffer without SDS. After the cells were suspended, SDS was added to 1% final concentration and proteinase K was added to 0.5 to 1 mg/ml. The lysate was then treated as for the JLS-VI1 and JLS-V9 cells. Cytoplasmic extracts of SCRF 60A cells were prepared according to the method of Penman (1966) with minor modifications. The cells were collected as described above and washed once with Earle’s solution. The washed cells were resuspended in 1.5 ml 0.1 concentration RS buffer and dextran sulfate was added to 100 pg/ml. After 10 min at 0°C the cells were homogenized with 7 strokes of an all-glass Dounce homogenizer. The lysed cells were transferred to a centrifuge tube and the homogenizer was rinsed with 0.5 ml full strength RS buffer. Nuclei were removed by centrifugation at 1000 g for 2 mm and the supernatant cytoplasm was saved. The nuclei were washed once with 05 ml RS buffer and the wash combined with the first supernatant. Cytoplasm from SCRF 60A cells was separated into free and membrane-bound components according to the method of Rosbash & Penman (1971). In this method, membranebound components are separated from the free components by differential centrifugation through a sucrose gradient. The pellet, which contained the membrane-bound material, was resuspended in SDS buffer containing 0.5% SDS and extracted with phenol as described earlier. The gradient itself, which contained the free cytoplasmic components, was adjusted to 0.4 M-NaCl, 10 m&r-EDTA and 1% SDS, extracted with 10 ml phenol and ethanol-precipitated as described above. (f) Preparation of polyribosomea and their dkzggregatiun with pzcronayccin Polyribosomes were prepared by layering a SCRF 60A cell extract onto a 36ml 15% to 30% sucrose gradient in RS buffer and centrifuging for 90 min in a Beckman SW27 rotor. The gradient was fractionated and those fractions containing the polyribosomes and monoribosomes were pooled. The pooled fractions were transferred to lo-ml screw-cap polycarbonate centrifuge tubes and underlayed with O-2 ml 60% sucrose in RS buffer. The polyribosomes and ribosomes were then sedimented onto the 60% sucrose cushion by centrifugation in a type 65 rotor for 45 min at 49,000 revs/mm. The supernatant was drawn off and the pelleted polyribosomes and monoribosomes were pooled. Ribosomes were released from polyribosomes with puromycin and high ionic strength according to the method of Blobel & Sabatini (1971). To 0.4 ml of pooled polyribosomes an equal vol. of compensating buffer (1.0 M-KCl, 0.09 M-T& (pH 75), 0.009 M-MgCl,) was added, followed by 40 ~1 of a 10 mg puromycin/ml solution. Following incubation at 0°C for 15 min, the mixture was incubated at 37’C for 3 mm. Two volumes of ice cold RS buffer were then added and the samples were layered onto 10-O-ml 15% to 30% sucrose gradients in RS buffer and centrifuged as described in the Figure legends. (g) HybridizaGm techniques Hybridizations for the C$ curve analyses shown in Fig. 4 were done at 66°C in 2 x SSC plus 0.1% SDS. The standard reaction volume was 5 to 7 ~1 and incubations were oarried out in 50-~1 microcapillary tubes (Manly et al., 1971; Verma et al., 1972). One ~1 of 2 x SSC saturated with diethylpyrocarbonate was added to each reaction to prevent RNA degradation. After incubation at the annealing temperature, the capillaries were broken and the contents of the capillaries were expelled into nuclease reagent, as described below. For analysis of sucrose gradient fractions, the following methods were used. For sampling of fractions from sucrose gradients in RS buffer, 50-d samples were taken from the gradient fractions with an Eppendorf micropipet and placed in 0.5ml sample cups (Technicon Instruments). Five d of compensating buffer (3 M-NaCl, 12 ma6-TES (Sigma) (pH 6*7), 6 mm-EDTA) was added, followed by 5 ~1 of 1y. SDS. Five ~1 of M-MuL virus DNA probe resuspended in 2 x SSC plus O*lo/0 SDS at 100 ctsfmin/~l were added and the cups were sealed with electrical tape and immersed in a 66°C water bath. Following annealing, the samples were processed as described below. For fractions from sucrose gradients in SDS buffer containing 0.1% SDS, 60-r/.1 fractions were taken, and B-PI compensating buffer (2.5 m-NaCI, 10 mM-TES (pH 6.7), 5 mu-EDTA) was added. M-MuL virus DNA probe was added and the samples were processed as above.

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Hybridization of M-MuL virus DNA was measured by resistance to S-l single-strand specific nuclease isolated from Aspergdlw, oryzae (Ando, 1966; Sutton, 1971). The original crude preparation of S-l nucleate was a kind gift of Dr V. Vogt. Subsequent preparation of S-l nuclease was according to V. Vogt (unpublished work) but only partial purification was carried out. Briefly, crude a-amylase powder (Sigma) was dissolved and clarified, incubated at 7O”C, fractionated by ammonium sulfate, and passed over a DEAE-cellulose column. Those fractions with single-strand specific deoxyribonuclease activity were concentrated, and designated nuclease stock. For analysis of samples, nuclease stock was diluted into nuclease buffer (0.25 M-KCl, 0.04 M-ZnS04, 20 pg denatured calf thymns DNA/ml) to give nuclease reagent. The dilution of nuclease stock was determined empirically by the amount necessary to degrade 3H-labeled M-MuL virus DNA probe to acidsolubility using the standard assay conditions. Incubation was for 30 mm at 45°C followed by addition of one drop of carrier yeast tRNA (4 mg/ml) and precipitation with 590 trichloroacetic acid. Fifty ~1 of nuclease reagent per sample was used for both the G,t curve and sucrose gradient samples. Labeled M-MuL virus RNA hybridization to M-MuL virus DNA was measured by resistance of the labeled RNA to ribonuclease digestion. The digestion conditions consisted of 7 ~1 of sample in 2 x SSC plus 0.1% SDS added to 50 4 of 5 rg pancreatic RNase A/ml and 10 pg RN&se T,/ml in 2 x SSC. Digestion was for 1 h at 37”C, followed by trichloroacetic acid precipitation, as above. (h) Quantitation

of virus-specific

RNA in eucroae gradient fractions

Virus-specific RNA in sucrose gradients of cellular RNA was detected by annealing 3H-labeled MuL virus DNA to portions of each fraction as described above. The specific activity of the DNA was sufficiently high so that virus-specific RNA was in excess of the DNA in essentially all of the samples, &s will be discussed in the Results. In conditions of RNA excess the rate of DNA annealing can be expressed as - dCn = kC,J&,, dt where k is the rate constant for the reaction, Cc is the nucleotide concentration of unhybridized DNA and C,, is the concentration of virus-specific RNA, 8 quantity which remains constant. Integration and iteration of this equation yields the relation. fraction hybridized

= 1 - Cn/Cn, = 1 - exp( -kC,,t),

where Cn, is the initial concentration of DNA and t is the time length of annealing. Solving for k at 50% hybridization and substituting this value for k in the previous equation vields fractionhybridized=

I-exp[--s],

where (C&t)+ is the product of C,, and t that gives 60% annealing of the DNA. Fraction hybridized is roughly proportional to C,t from 0 to 50% hybridization, but the relation becomes distinctly non-linear for higher values of hybridization. In the sucrose gradient analyses, the gradient fractions were annealed with 3H-labeled MuL virus DNA for a standard length of time, and the relative amounts of virus-specific RNA were determined from the fraction of 3H-labeled MuL virus DNA probe hybridized using the above relationship.

3. Results (a) Characterization of Mobney

murine leukemia viru-s DNA probe

Radioactively labeled DNA complementary to M-MuL virus RNA was prepared as described in Materials and Methods. The endogenous reaction system (Manly et al., 1971) was used and actinomycin D was added to the reaction mixture in order to inhibit synthesis of double-stranded DNA (McDonnell et al., 1970; Manly et al., 1971).

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TABLE

1

Digestion of DNAs with S-l nucleme Without nuclease 1. 2. 3. 4.

Double-stranded DNA Denatured DNA M-MuL virus DNA product M-MuL virus DNA product to M-MuL virus RNA

following

Radioactivity With llUCl0&S0

425 362 480 480

annealing

395 8 29 469

Percentage resistance 93 2 6 97

Lines 1 and 2 of the Table are taken from data obtained by Dr Volker Vogt, in which trace amounts of ‘*C-labeled native (double-str&nded) and denatured (single-stranded) P22 phage DNAs were incub&ted with 1 ~1 of S-l nuclease stock (approx. 4000 units/ml) for 20 min at 6O’C in (pH 4.5), 0.1 mM-&SO,, 10 pg denatured calf 200 ~1 assay buffer (0.025 M-potassium acetate thymus DNA/ml). Line 3 shows the effect of S-l nuclease reagent (as described in Materials and Methods) on the 3H-l&beled M-MuL virus DNA product. In line 4, the 3H-l&beled M-MuL virus DNA w&s annealed with M-MuL virus RNA at 11 pg/ml before treatment with nuclease reagent as described in Fig. 4.

The purified DNA product was single-stranded, as shown in Table 1, and it pi&s all complementary to virion RNA, as will be shown below. The asymmetry of the DNA product produced in this manner is useful, since MuL virus-specific RNA sequences can be detected by RNA-DNA hybridization in the absence of a competing DNA reannealing reaction.

23s

16S

5-6 S 4s

IO

0 0

IO Fraction

20

c

no.

FIG. 2. Size of the M-MuL virus DNA product. sH-labeled M-MuL virus DNA product (2500 cts/min) was combined with 14C-labeled E. coli RNA in & volume of 0.1 ml and l&yered onto & 4.5ml 6% to 20% sucrose gradient in SDS buffer without SDS. The gredient was oentrifuged for 4.6 h at 46,000 revs/min in an SWKO.l rotor at 4”C, the tube w&s punctured through the bottom and 3.drop fractions were collected. Yeast tRNA w&s added &s carrier, the samples were precipitated with trichloroacetic acid &nd assayed for acid-precipitable radioactivity. -•--~--, sH (cts/min x 10m2); --O--O--, r4C (cts/min x 10e2).

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Sedimentation analysis of the DNA product in a neutral sucrose gradient is shown in Figure 2. In comparison with sedimentation markers of Escherichia wli ribosomal and transfer RNA, the M-MuL virus DNA had a sedimentation value of 4 to 8 S, with the peak at 5 to 6 S. The size of this DNA product is similar to that of the DNA product from an endogenous reaction incubated in the absence of actinomycin D (Manly et al., 1971), although the extent of RNA transcription is quite different for these two DNAs (Duesberg & Canaani, 1970; Gelb et al., 1971; Varmus et al., 1971; see below). The extent of transcription of viral RNA into DNA is critical to interpretation of hybridization data involving the M-MuL virus DNA. It has been shown that DNA products obtained from endogenous reactions of Rauscher MuL virus and SchmidtRuppin Rous sarcoma virus carried out in the absence of actinomycin D are extremely uneven transcripts of the viral RNA, with large excesses of DNA product necessary to completely cover the viral RNA (Duesberg & Canaani, 1970). However, it has also been shown that the presence of actinomycin D in the endogenous reaction of Rous sarcoma virus results in a DNA product that is much more representative of the viral RNA (Garapin et al., 1973). An analysis of the M-MuL virus DNA product similar to the analysis carried out by Garapin et al. (1973) for the avian Rous sarcoma virus DNA product, is shown in Figure 3. 32P-labeled M-MuL virus DNA was annealed

I

0

1

I 0

I IO

1

I 20 DNA/RNA

I

I

30

I

I

40

FIN. 3. Protection of M-MuL virus RNA with M-MuL virus DNA product. ssP.labeled M-MuL virus DNA was prepared in the same manner described for 3H-labeled DNA, but with a specific activity of 80 cts/min/pmol. aH-labeled M-MuL virus RNA, whose specific activity was 220 cts/min/pmol as determined from Fig. 1, was prepared as described. 3Hlabeled M-MuL virus RNA (340 cts/min) was hybridized to 3aP-labeled M-MuL virus DNA at different DNA/RNA ratios for 72 h in 2 x SSC plus O-1y0 SDS at 66°C in a &al reaction volume of 7 ~1. After the incubation the samples were digested with ribonuclease reagent. Percentage ribonuclease resistance of sH radioactivity is shown as a function of the DNA/RNA ratio.

to 3H-labeled M-MuL virus RNA at various DNA/RNA ratios for 72 hours. At least 85% of the 3H-labeled viral RNA was protected from ribonuclease digestion by the DNA product at a DNA/RNA ratio of approximately 8. This result is similar to that obtained by Garapin et al. (1973), although in the avian case the DNA product protected 100% of the viral RNA. The failure of the M-MuL virus DNA product to protect 100% of the viral RNA from ribonuclease digestion could be due to non-viral RNA contaminating the 3H-labeled viral RNA preparation or it might reflect incom-

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plete transcription of the viral RNA. The transcription of the viral RNA into DNA was probably not completely uniform, since an eightfold excess of DNA to RNA was needed to protect maximally the M-MuL virus RNA. Although even longer incubations may have resulted in complete RNA protection at lower DNA/RNA ratios, this transcription is considerably more uniform than that from reactions not containing actinomycin D (Duesberg & Canaani, 1970; Gelb et al., 1971; Varmus et al., 1971; Garapin et al., 1973). (b) Detection of vim-speciJic

sequencesin infected and uninfected cells

Virus-specific sequences in RNA of infected cells or M-MuL virus virions was detected by hybridization of M-MuL virus probe to the extracted RNAs in conditions of RNA excess. Under these conditions, DNA annealing is a first-order reaction with respect to RNA concentration but is independent of the DNA concentration (Birnstiel et al., 1972). A useful parameter for these analyses is C,t, the product of the RNA concentration and the time of hybridization. In a manner similar to sequence cornplexity analysis for double-stranded DNA reannealing (Britten & Kobne, 196g), it can be shown that the C,t value at 50% hybridization ( (CJ)*) is inversely proportional to the concentration of the hybridizable RNA in the RNA preparation (Leong et al., 1972; Hayward & Hanafusa, 1973). Hybrid formation was measured by the protection of labeled M-MuL virus DNA probe from digestion by a nuclease specific for single-stranded nucleic acids. In these experiments the S-1 enzyme obtained from Aspergillus oryzue (Ando, 1966; Sutton, 1971) was used. Its specificity for single-stranded DNA has been shown previously (Leong et al., 1972). As shown in Table 1, our preparation of S-l nuclease does not degrade double-stranded P22 phage DNA but it does render single-stranded P22 DNA better than 95% acid-soluble. As is also shown, the M-MuL virus DNA probe was nuclease sensitive but prior annealing to an excess of M-MuL virus RNA converted it to better than 95% S-l nuclease resistance. Previous experiments from this laboratory (Manly et al., 1971; Verma et al., 1972) used a similar nuclease specific for single-stranded DNA, which was obtained from conidia of Neurospora crmsa (Rabin et al., 1971) and both enzymes detect hybrids equally efficiently (our unpublished results; Leong et al., 1972). S-l enzyme was used in these experiments because of its rapid and simple preparation and because it is much less sensitive than the Neurospora enzyme to the salt concentrations used for RNA-DNA hybridization. Figure 4 shows the results of hybridization of M-MuL virus DNA probe to RNA extracted from M-MuL virus virions and to cytoplasmic RNA from infected cells producing MuL virus and uninfected cells. Hybridization to 70 S RNA obtained from M-MuL virus virions yielded a curve that closely resembles the theoretical curve for RNA excess hybridization of DNA to a unique species of RNA. The (C&+ value for the M-MuL virus RNA curve was ten units (the unit is defined in the legend to Fig. 4). Hybridization of M-MuL virus DNA to the RNA of JLS-Vll cells produced a similarly shaped curve but with a (CA)* value of 3900 units. Since the shape of the hybridization curve for JLS-Vll cells was identical to that for purified M-MuL virus RNA, all of the virus-specific sequences detectable by the M-MuL virus DNA were apparently present in the cell cytoplasm in equal concentrations. By comparison of the (C,t), values of the JLS-Vll cells with that of purified M-MuL virus RNA, approximately O-25o/oof JLS-Vll cell cytoplasmic RNA was virus-specific.

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103

1

I

IO

102

IO3

IO4

IO5

IO6

Relative C,l

FIG. 4. Detection of virus-apeciflc RNA sequences in infected and uninfected cells. RNA from the cytoplasm of JLS-Vll, JLS-V9, SCRF BOA cells and from M-MuL virus virions was prepared. Differing dilutions of the RNA samples were hybridized with approximately 600 cts/min sH-labeled M-MuL virus DNA probe in 2 x SSC plus 0.1% SDS at 66°C in a final reaction volume of 7 4. Incubation was for 4 h except for the last 2 points of the JLS V-9 sample. Amount of 3H-labeled M-MuL virus DNA hybridized is shown as a function of the product of the RNA concentration and length of hybridization (Crt). Arbitrary units were used here, since the important results are comparisons between the different curves shown. One unit is defined as hybridization with RNA at a concentmtion of 0.021 pg/ml for 4 h, and would correspond to a standard C$ value of 1.6 x 10v3 mol-s/l aorrected to 0.12 M-N&+ concentration (Britten & Smith, 1970). (a) M-MuL virus RNA; (m) SCRF 60A RNA; (A) JLS-Vll RNA; (x) JLS-V9 RNA; (-----) theoreticel plot for RNA excess hybridization of DNA to RNA with a (C$), value of 10, and reaching 94% hybridization (see Materials and Methods). C,t values at whioh 60% hybridization was reached [(C&l are also shown.

Hybridization to the cytoplasmic RNA of JLS-V9 cells, which is the uninfected parent cell line of the JLS-VI1 cells, also indicated the presence of RNA sequences complementary to the M-MuL virus DNA probe (Fig. 4). However, only 50 to 60% of the M-MuL virus DNA was protected by the JLS-V9 RNA. Although it is not evident in the data, we assume that the 50% to 60% hybridization is a true plateau value because, as mentioned below, in cells induced to produce more RNA a plateau at this level is seen. The (C$) value for the JLS-V9 RNA was therefore approximately 50,000 units. So approximately 0.02% of the RNA in uninfected JLS-V9 cell cytoplasm is RNA tumor virus information, tenfold less than in infected JLS-Vll cells. This virus-specific RNA is presumably an expression of an endogenous RNA tumor virus of the JLS-V9 cells; it has been demonstrated that BALB/c mice contain an endogenous RNA tumor virus that can be activated from tissue culture cells (Lowy et al., 1971; Aaronson et al., 1971). An RNA tumor virus can be readily induced from the JLS-V9 cells by treatment with bromodeoxyuridine (Besmer, McDaniels & Fan, unpublished results), and RNA extracted from the induced cells shows a higher concentration of RNA sequences complementary to the M-MuL virus DNA probe, but the final level of hybridization is still 50 to 60% (our unpublished result). In addition, RNA extracted from the induced virus cannot completely anneal the M-MuL virus DNA probe (Besmer & Haseltine, personal communication). This suggests that the observed level of complementarity between the M-MuL virus DNA probe and JLS-V9 RNA was due to incomplete sequence homology between the endogenous JLS-V9 virus and M-MuL virus rather than incomplete transcription of an endogenous genome that is completely complementary to M-MuL virus.

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In contrast to the JLS-V9 cells, hybridization of M-MuL virus DNA probe to cytoplasmic RNA extracted from SCRF 60A cells showed complete protection of the DNA probe (Fig. 4). SCRF 60A cells are a permanent line of lymphocytes derived from NZB mice; upon establishment of the line, spontaneous production of an RNA tumor virus occurred (Lerner et al., 1972). Comparison of the (C,t), values indicated that approximately 0.9% of SCRF 60A cytoplasmic RNA was virus-specific, approximately three times as much as in JLS-Vll cells. To investigate further the precision of the homology between these two viral RNAs, a melting profile of the hybrid formed between M-MuL virus DNA and SCRF 60A RNA was compared to that of a hybrid between JLS-Vll RNA and M-MuL virus DNA. As seen in Figure 5, the

70

80

90

100

Temperature ( “C)

FIG. 6. Melting curves for hybridization of JLS-Vll and SCRF BOA RNA with M-MuL virus DNA probe. 32P-labeled M-MuL virus DNA (800 cts/min/pmol, 200 cts/min/pl) and 3H-labeled M-MuL virus DNA (600 ots/min/pl) were prepared as described. The [sxP]DNA was annealed with an excess of SCRF 6OA RNA for 4 h at 66°C in 2 x SSC plus O*l% SDS, and the [3H]DNA was annealed to excess JLS-Vll RNA in a similar manner. After annealing, ~-PI portions of each incubation mixture were oombined, sealed in SO-p1 capillary tubes and raised to different temperatures in a water bath. The capillaries were rapidly chilled in ice water and expelled into S-l nuclease reagent. The samples were assayed for amount of hybrid present, and the percentage of hybrid melted is shown as a function of temperature. -a-e-, JLS-VI1 hybrid; --O--O--, SCRF 60A hybrid.

T, values for the two hybrids, as well as the shapes of the melting curves, appeared identical. Therefore, the SCRF 60A virus and the M-MuL virus share extensive sequence homology, and the homology is quite exact. This is of considerable use, since RNA metabolism of the SCRF BOA virus can be studied using the M-MuL virus DNA probe, which has already been characterized. The SCRF BOA cells have two advantages: greater amounts of virus-specific RNA, and the ability to grow in suspension culture. These are of considerable value in more detailed analyses of virus-specific RNAs in infected cells, since the extraction of large numbers of cells is necessary to obtain sufficient virus-specific RNA and rapidity of extraction is essential to avoid RNA degradation.

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(c) Size distribution of virus-specijic RNA from infected cells A rapid and simple method to study the virus-specific RNA from MuL virusinfected cells was developed as described in Materials and Methods. The technique uses SDS and proteinase K, which together are very effective in deproteinizing nucleic acids, inhibiting degradation by nucleases and producing a quantitative recovery of RNA (Firtel & Lodish, 1973). Following centrifugation of the extracted RNA through a sucrose gradient, the fractions were assayed for virus-specific RNA by hybridization with 3H-labeled M-MuL virus DNA probe. The percentage of hybrid formation was related to virus-specific RNA concentration in each sample as described in Materials and Methods. Figure 6 shows the conversion of the percentage of hybrid

IO

20

IO

20

Froctlon no. (0)

(b)

Fm. 6. Correction for relative virus-specific RNA concentration across a sucrose gmdient. RNA (approximately 600 pg) from SCRF 60A cytoplasm was prepared and layered on a 15% to 30% sucrose gradient in SDS buffer containing 0.1% SDS (see Fig. 7). The gradient was fractionated in 0.6-ml fractions and 60-p] portions were prepared for hybridization. Twofold dilutions of the samples were also made into SDS buffer containing O*l”J SDS, and the samples were prepared for hybridization. cH-labeled M-MuL virus DNA was added to the samples, ctnd annealing w&s carried out for 8 h. The amount of hybrid formed was determined in each sample. (a) Percentage M-MuL virus DNA hybridized across the gradient. (b) Values from (a) converted to relative virus-specific RNA concentration (see Materials and Methods). A value of 1 corresponds to & virus-specifle RNA concentration that will anneal 60% of the M-MuL virus DNA probe in the incubation conditions. -m--a-, Values for undiluted fraction; -x-x-, values for 2.fold diluted fractions; --O-O-, values for undiluted fractions multiplied by 0.5.

formation to relative virus-specific RNA concentration for a sucrose gradient of cytoplasmic RNA from SCRF 60A cells. Each gradient fraction was analyzed and a twofold dilution of each fraction was also processed. It can be seen that the concentrations of virus-specific RNA in each fraction as determined from the original or diluted fraction agreed reasonably well. It should be noted that this analysis assumed that each fraction had the capacity to protect 100% of the M-MuL virus DNA probe. In order to achieve 100% hybridization, two criteria must be fulfilled : a.11of the RNA sequences detectable by the MuL virus DNA probe must be present and these sequences must be in excess of the DNA probe. In gradients similar to those shown in Figure 6, annealing of M-MuL virus DNA to more highly concentrated

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fractions resulted in maximal hybridization of the DNA in all fractions (unpublished result), so all detectable virus-specific RNA sequences were indeed present in each gradient fraction. Furthermore, it can be calculated from the results of Figure 4 and the total amount of RNA analyzed in the sucrose gradient that all of the samples analyzed in Figure 6 contained at least a tenfold excess of virus-specific RNA over M-MuL virus DNA. Therefore, the above criteria were met and the conversion of percentage hybridization to relative virus-specific RNA concentration should be valid. The results shown in Figure 6 also support this method of analysis, since otherwise the relative virus-specific RNA concentrations determined from the two dilutions would not have agreed. Since different samples from a sucrose gradient contain different amounts of sucrose, the viscosity of the samples varies. The rate of hybridization is inversely proportional to the viscosity (Wetmur & Davidson, 1968), so that this factor should be taken into account. However, at 66°C the viscosity of 30% sucrose is only approximately one-third greater than for 15% sucrose (Wetmur 6 Davidson, 1968), and these are the two extremes in sucrose concentration. Since the viscosity effect on hybridization across the sucrose gradient is relatively small, no corrections for it were made in these experiments.

2-

I,

28s

18s

w o-

I 0

I IO Fraction no.

I

I 20

FIQ. 7. Distribution of virus-speci6c RNA in JLS-VI1 cells. The medium from one bottle (approx. 2.6~ lo7 cells) of JLS-Vll was decanted and the cells were weshed with Earle’s s&ne. The cells were then Iysed with SDS and proteinase K as described previously and prepared for analysis on sucrose gradients. The sample (containing approx. 400 pg RNA) w8s layered on 8 16% to 30% suorose gradient in SDS butler containing 0.1% SDS and centrifuged for 4 h at 36,000 revs/min in an SW40 rotor. The gradient w8s fraotionated and the relative amount of virus-specific RNA in each fraction w8s determined, 8s in Fig. 6. Location of 28 S 8nd 18 S ribosomal RNA optical density peaks is also shown. Sedimentetion values were extrapolated from the 28 S and 18 S RNA positions and the designation of the 70 S sedimentation value w8s confirmed by centrifugation of 8H-18beled M-MuL virus RNA in a parallel gradient (not shown).

Sucrose gradient analysis of an SDS-proteinase K lysate of JLS-Vll is shown in Figure 7. Three major classes of virus-specific RNA were evident: 60-70 S RNA, approximately 36 S RNA, and a broad distribution of 20-30 S RNA (not clearly present in this gradient, but more evident in Figs 8 and 9). The 60-70 S and 35 S

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RNA species correspond in size to the virion RNA and virion RNA subunits and may at least in part represent virion precursors or mature virions. Since the 60-70 S RNA in infected cells was the same size as the RNA in the virion, the possibility that this RNA might represent mature virus particles attached to the cells was investigated. MuL virus particles mature by a process of budding at the cell membrane, so cells were treated with agents that remove material from the cell surface (Fig. 8). Comparing Figures 7 and 8, it can be seen that washing _

EDTA + Trypsin

EDTA Wash

28s I88 k.4’ -

C*-%.,/’

i I IO

28s 18s I

I 20

4

Fraction no.

(a)

(b)

Fm. 8. Effect of EDTA and trypsin on virus-specific RNA distributions. (a) The medium from 1 bottle of JLS-Vll aells was decanted, and the cells were washed with phosphate buffered saline containing 0.02% EDTA. The cells were then lysed with SDS and proteinese K and prepared for analysis on sucrose gradient. The sample was layered onto a sucrose gradient and analyzed as described in the legend to Fig. 7. Annealing w&s for 16 h. (b) The modium from 1 bottle of JLS-Vll cells was decanted, the cells were washed with phosphate buffered saline containing 0.02% EDTA and treated with 6 ml 0.25% trypsin-EDTA solution (Grand Island Biological Co.) for 10 min at 0°C. The oells were lysed with SDS and proteinase K in a mamer similar to that desoribed for SCRF 60A and analyzed as above.

0

IO Fructm

20 no

Fm. 9. Virus-specific RNA in SCRF 60A cells. 4 x lo7 SCRF 60A cells were lysed with SDS and proteinase K. The lysates were prooessed for analysis on SDS-sucrose gradients and analyzed as in Fig. 8. Approximately 250 pg RNA was applied to the gradient. Annealing was for 18 h.

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JLS-Vll cells with EDTA resulted in the removal of some of the 60-70 S RNA from the virus-specific RNA pattern and treatment with trypsin resulted in further removal of this species. The amount of 35 S and 20-30 S RNA remained relatively unchanged by these treatments. It thus appears that EDTA and trypsin liberated mature or nearly mature virus particles from the cell surface into the supernatant medium. The resistance of the 35 S and 20-30 S RNA.s to these treatments would suggest that these species were inside the cell and not at the external cell surface. The fact that the 35 S and 20-30 S RNA did not increase upon EDTA and trypsin treatment further supports the hypothesis that the loss of 60-70 S RNA did not result from its degradation to smaller molecules. Analysis of the virus-specific RNA in SCRF BOA cells is shown in Figure 9. The same type of pattern as observed for JLS-Vll cells was observed. The 35 S RNA was a small but reproducible shoulder on a relatively large peak of 20-30 S RNA. (d) Virus-spe,eciJicRNA in fractionated cytoplasm In order to study the virus-specific messenger RNA in infected cells, the cytoplasmic fraction was further analyzed-since active messenger RNA is located in the cytoplasm of eukaryotic cells. SCRF 60A cells were used in these experiments due to the ease of manipulation of suspension culture cells and also because of the larger amounts of virus-specific RNA in these cells. The first approach to further analysis of the cytoplasm was to examine the virusspecific RNA in subfractions of the cytoplasm. SCRF 60A cytoplasm was divided into membrane-bound and non-membrane-bound (free) components by a brief centrifugation through a sucrose gradient (Rosbash t Penman, 1971). Quantitation of the amount of virus-specific RNA in free and membrane-bound components of SCRF 60A cells is shown in Figure 10. The extracted RNAs were resuspended in

0 ---T-L . .x l ‘x’x\ $ .N 0b2 \\ I 1 I

I

a3 50 F g d

X

x

.

\

100

I 1000

Relative

dhtlon

.

\-

I 100

x

.

I IO

:

. x

I I

I

factor

FIG. 10. Virus-specific RNA in fractionated SCRF 60A cytoplasm. A cytoplasmic extract from 4 x lo* SCRF BOA cells was prepared and separated into membranebound and free components. Approximately 20% of the ribosomea were found associated with the membrane fraction. RNA was extracted from both fractions and each was resuspended in 200 ~1 2 x SSC plus 0.1% SDS. Varying dilutions of each fraction were hybridized to 600 cts/min of 3H-labeled M-MuL virus DNA in a total vol. of 7 ~1. The percentage hybridized as a function of dilution factor is shown. A relative dilution factor of 2 corresponds to 5 ~1 of the undiluted RNA sample hybridized in a total volume of 7 ~1 for 4 h. --@--a--, RNA from the free cytoplasmic fraction; - x-x -, RNA from the membranebound fraction.

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so that an equal volume of each extract would contain RNA from amount of cytoplasm. Increasing dilutions of the RNA preparations were hybridized to M-MuL virus DNA probe and, in analogy to the C,t curve analysis. the dilution factor at which 50% of the M-MuL virus DNA is hybridized is proportional to the concentration of virus-specific RNA in the given fraction of cytoplasm. The membrane-bound and free RNA preparations both completely protected the M-MuL virus DXA, and the dilution factor at which free RNA protected 50% of the M-MuL virus DNA was approximately twice the value for membrane-bound RNA. Therefore, it can be concluded that twice as much virus-specific RNA was present in the free cytoplasm as in the membrane-bound material, or that approximately 35% of virus-specific cytoplasmic RNA is membrane-bound and 65% is free. The fact that the shapes of the two curves are identical and closely approximate the theoretical curve for a single unique RNA species indicates that both the membranebound and free fractions of the cytoplasm contained all species of virus-specific RNA sequences detectable by the M-MuL virus DNA probe, and that they were in approximately equal concentrations. the same volume

an equivalent

(b

70s

355

+

1

) ,

Cd)

28s 18s -

1 ?O

Froctlon no.

FIG. 11. Distributions of virus-specific RNA from SCRF 60A cytoplasm fractions. A cytoplasmic extract from 6 x 10s SCRF 60A cells was prepared. One-third of the oytoplasm was directly extracted with phenol to give RNA from the total cytoplasm. The remaining twothirds was separated into membrane-bound and free cytoplasm. The RNA was extraoted from these fractions and all samples were analyzed on sucrose gradients. Centrifugation was for 3.6 h at 36,000 revs/min in an SW41 rotor and annealing was for 8 h. (a) Total cytoplasm; (b) membrane-bound cytoplasm; (c) free cytoplasm; (d) addition of the values from (b) and (c).

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Sucrose gradient analysis of the free and membrane-bound fractions of the cytoplasm as compared to that of the total cytoplasm is shown in Figure 11. A portion of SCRF 60A cytoplasm was separated into membrane-bound and free fractions and the fractions were then phenol-extracted. The remainder of the cytoplasm was also phenol-extracted, and all of the samples were layered onto SDS-sucrose gradients and analyzed as above. Most of the 60-70 S RNA in the cytoplasm was located in the membrane fraction and some 35 S RNA was also present there (Fig. 11(b)). The free cytoplasm contained little 60-70 S RNA and a majority of the 35 S RNA (Fig. 11(c)) . In this preparation relatively little 20-30 S RNA was observed but that which was present was localized in the free cytoplasm. Addition of the values in Figure 11(b) and (c) yielded a profile similar to that obtained from unfractionated cytoplasm, as seen in Figure 11(d). This indicates that the fractionation procedure did not result in the degradation of virus-specific RNA and that the absence of 60-70 S RNA from the free cytoplasm was probably not artifactual. (e) IdentiJication of virus-speci;fic messenger RNA in virus infected cells Experiments by other workers have indicated that only RNA containing identical sequences to virion RNA is detectable in RNA tumor virus infected cells (Cofhn & Temin, 1972; Leong et al., 1972). Therefore, virus-specific messenger RNA must share nucleotide sequences with virion RNA and virion RNA precursors. In order to identify virus-specific messenger RNA it is therefore important to differentiate actually functioning messenger RNA from virion precursor and virion RNA. In particular, demonstration that virus-specific RNA co-sediments with polyribosomes is not suflicient to identify it as messenger RNA, since non-polyribosomal viral RNA complexed with protein or other materials might have the same sedimentation value. Therefore, additional criteria are necessary and in the experiments described in this section the criterion used was that virus-specific messenger RNA must change in sedimentation value if polyribosomes are disrupted. Polyribosomes can be disrupted in a number of ways and the two methods used here were treatment with EDTA, and combined puromycin and high salt treatment. In Figure 12(a), cytoplasm from SCRF 60A cells was analyzed on a sucrose gradient. Before lysing, the cells were treated with 1 tug cycloheximide/ml for 30 minutes at 37°C. Cycloheximide is a protein synthesis inhibitor that works at the level of ribosome translocation (Wettstein et al., 1964; Colombo et al., 1965; Willems & Penman, 1966) and at low levels has been shown to increase the number of ribosomes in polyribosomes and also the size of the polyribosomes (Stanners, 1966; McCormick & Penman, 1969 ; Hogan, 1969 ; Fan & Penman, 1970). The treatment was used here in order to increase the size of the polyribosomes as much as possible; the lack of small polyribosomes would then suggest that ribonuclease degradation of polyribosomes is not a serious problem, since even relatively low levels of ribonuolease would result in the presence of many small polyribosomes as well as monoribosomes. As can be seen from the optical density profile, few monoribosomes and relatively few polyribosomes containing two to four ribosomes were present, so relatively little degradation of the polyribosomes took place during extraction. Portions from each of the gradient fractions were assayed for the amount of virus-specific RNA present by the addition of M-MuL virus DNA and annealing. Virus-specific RNA was found in a broad distribution over the polyribosome region peaking in the area of light

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polyribosomes, 150 to 200 S. Analysis of an equal amount of cytoplasm in which the polyribosomes were disaggregated with EDTA before sedimentation is also shown. The polyribosomes were completely converted to 50 S and 30 S ribosomal subunits (not shown) and increased virus-specific RNA was found in the 50-200 S region. The difference between the two curves in the polyribosome region represents

(al

?EDTA tEDTA

BOS

i0

'0

0

I

4

t

) ;’ “ a : : I :

2 Puromycin

(b)

D P

5

0

)5

I

5

IO Fraction no.

15

)

FIG. 12. Virus-specific RNA in polyribosome gradients of SCRF 60A cells. (a) lo@ SCRF 60A cells were treated with 1 pg cycloheximidejml for 30 min at 37°C and then 8 cytoplasmic extra& wsa prepared. The extraot was divided into 2 equal portions end EDTA at 10 rnM was added to 1 portion. The samples were layered on 16% to 30% sucrose gradients in RS buffer and aentrifuged for 106 min at 27,000 revs/min in en SW27 rotor with large buckets. The gradients were fractionated and portions were processed for hybridization. Annesling was for 16 h. -e-O--, Virus-specifio RNA in oontrol sample; --O--O--, virus-specifIa RNA in EDTAtreated sample; (-----) Asa0 of control sample (A,,, of EDTA-treated sample not shown). (b) Polyribosomes were prepared from 6 x 10s SCRF 608 cells. The polyribosomes were divided into 2 equal portions and 1 portion was diseggregated by puromycin treatment. The remainder was meintained at 0°C in compensating buffer. The samples were then prepared for centrifugation as desoribed and centrifuged for 46 min at 39,000 revs/mm in an SW41 rotor. The gradients were fractionated and the fractions were assayed for virus-specifio RNA. -m-O--, Untreated polyribosomes; --O--O--, puromycin-released polyribosomes; (-----) A,,, of the untreated polyribosomes. (The Aaao of the puromyoin-disaggregated polyribosomes showed optical density only in the monoribosomes and ribosomal subunits and is not shown.)

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possible messenger RNA, since this RNA was moved out of the polyribosome region during polyribosome disaggregation. The “ polyribosome ” region of the EDTA gradient still contained considerable virus-specific RNA, and this RNA was therefore in ribonucleoprotein particles that were not polyribosomes. It should be noted that

Region I

35s

(a)

275 1 .

Region I (contrail-

Cc) (EDTAI

Region II (EDTA)-(Control)

(dl

Fraction no

FIQ. 13. Sedimentation analysis of RNA from SCRF 60A polyribosome gradients. Fractions corresponding to regions I and II for the control and EDTA gradients of Fig. 12(a) were pooled and extracted with phenol. Portions containing equal amounts of the samples were layered onto sucr0e.e gradients and centrifuged for 10 h at 28,000 revs/min in an SW40 rotor. The gradient fractions were assayed for virus-specific RNA as before. Annealing was for 24 h and the values shown are the averages of 2 separate assays. (a) Virus-specific RNA distributions in sucrose gradients of RNA extracted from region I of the control (-@-a-) and EDTA (--O--O--) gradients. (b) Similar analysis to (a) for region II of the same gradient. (c) Subtraction of the EDTA values from the control values for the gradients shown in (a). (d) Subtraction of the oontrol values from the EDTA values for (b).

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by this criterion no more than approximately 20% of the virus-specific RNA in the sucrose gradient was active mRNA. It is possible that virus-specific RNA-containing structures other than polyribosomes are sensitive to EDTA, so another method of polyribosome d&aggregation was also used, in which puromycin, an inhibitor of protein synthesis, was used. It, has been shown that polyribosomes can be d&aggregated in vitro by treatment with puromycin in conditions of high ionic strength (Blobel & Sabatini, 1971). Figure 12(b) shows an experiment similar to that of Figure 12(a) but in which the polyribosomes were disaggregated by an in vitro puromycin treatment. Puromycin treatment altered the sedimentation of a similar proportion of the virus-specific RNA in the polyribosome region, as did EDTA treatment. The agreement of Figure 12(a) and (b) indicates that the majority of the virus-specific RNA whose sedimentation rate changes upon polyribosome disaggregation by either of these methods is indeed messenger RNA. The changes in sedimentation of virus-specific RNA upon polyribosomes disaggregation shown in Figure 12(a) and (1,) were consistently observed in such experiments. In order to analyze the size of viral messenger RNA, the remainders of the fractions of the sucrose gradients shown in Figure 12(a) were pooled as indicated. The RNA was phenol-extracted from these pooled samples and analyzed on SDS-sucrose gradients. The results are shown in Figure 13(a) and (b). In region I of the sucrose gradient of the untreated extract, 35 S RNA was the predominant RNA species, although some 20-30 S RNA was also evident. In contrast, region I of the SUGMSC gradient containing EDTA-treated cytoplasm showed a selective removal of the 35 S RNA, with relatively little effect on the 20-30 S material. Similarly for region 11, the control sucrose gradient appeared to contain mostly 20-30 S RNA, while pretreatment of the cytoplasm with EDTA resulted in a large increase of 35 S RNA in this region. Subtractions of these curves are shown in Figure 13(c) and (d), where it can be seen that the large majority of viral messenger RNA has a sedimentation value of 35 S. The SDS-sucrose gradients in these analyses were run so that 60-70 S RNA, the other major virus-specific RNA species, would have appeared in the pellet. However, since the 60-70 S RNA appears to be mostly at the cell surface, this should not be an important consideration. Similar results were obtained with JLS-Vll cells (not shown), although the levels of both messenger and non-messenger virus-specific RNA were lower.

4. Discussion Virus-specific RNA sequences in cells producing murine leukemia virus were studied in these experiments. Characterization of these RNAs is limited by the nature of the M-MuL virus DNA probe used. As shown in Figures 2 and 3, the DNA product synthesized in the presence of actinomycin D is of the size to which most workers shear DNA for hybridization studies and it is able to protect a large fraction, but perhaps not all, of the M-MuL virus RNA genome. The uniformity of transcription is considerably better than for the DNA product synthesized in the absence of actinomycin D but some areas of the genome may still be copied more frequently than others. Therefore, regions of the MuL virus genome that are relatively infrequently transcribed into DNA would not have been detected in these experiments. All conclusions dram from hybridization data involving the M-MuL virus DNA probe must therefore be qualified accordingly. 8

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The C$ curve analysis of Figure 4 reveals several points about the general virusspecific RNA metabolism in infected cells. First, the fact that the curves for JLS-Vll and SCRF 60A cells have the same shape as the curves for purified M-MuL virus RNA suggests that there be may little preferential expression of virus-specific RNA sequences in cells producing virus. Although this might seem surprising, two factors must be considered. First, the cells are producing virus particles, so a considerable, and perhaps the predominant, portion of the virus-specific RNA may be virion precursor or mature virion RNA. It would be expected that these virion precursor RNAs would contain all viral RNA sequences in equal amounts. Second, it is possible that viral RNA sequences infrequently transcribed during preparation of the DNA probe do occur in frequencies different from virion RNA. Another point that can be drawn from Figure 4 is that at least certain lines of uninfected cells, such as JLS-V9 cells, contain significant amounts of virus-specific RNA. A similar situation has been observed for uninfected chicken cells, in which some cells have been shown to have RNA sequences complementary to avian tumor viruses (Bishop et al., 1973 ; Hayward & Hanafusa, 1973). A third conclusion that results from Figure 4 is that the M-MuL virus DNA product is completely protected by SCRF 60A cell RNA. This indicates that considerable, if not complete sequence homology is shared by these two viruses. This is somewhat unexpected since the two viruses have quite different origins. It has been shown with picornaviruses that closely related strains rapidly diverge in RNA sequences (Young et al., 1968) and it might be expected that a similar situation would occur for RNA tumor viruses. In fact, work with MuL virus has indicated partial non-homology between the RNAs of the Friend-Moloney-Rauscher group of MuL virus and Grosstype MuL virus (Gallo et al., 1973). The difference between the results of Gallo et al. (1973) and of Figure 4 for SCRF 60A and M-MuL virus may reside in the virus strains used. On the other hand, Figure 4 also suggests that non-homology does exist between the RNA of M-MuL virus and the endogenous JLS-V9 virus. This is supported by data obtained from induction experiments in which the JLS-V9 cells were induced to produce endogenous virus by treatment with bromodeoxyuridine. No increase was found in the fraction of M-MuL virus DNA product that could be protected, even when virus was being produced. Virus-specific RNA is found in infected cells in three size classes: 60-70 S, 35 S and 20-30 S RNAs. Similar patterns for Rous sarcoma virus-infected chick cells have been obtained by Leong et al. (1972), although virus-specific RNA with a sedimentation value greater than 70 S was also observed in many chick RNA preparations. In the MuL virus infected cells studied here, such RNA was not observed, but a minor class of RNA with this size would not have been detected. Tsuchida et al. (1972) found 35 S and 20 S virus-specific species in RNA extracted from MuL virus infected mouse cells if the RNA was denatured in dimethyl sulfoxide before analysis. However, mouse cells infected with murine sarcoma virus were found to lack a 20 S component (Tsuchida et al., 1972). The 60-70 S RNA appears to be in particles at or very near to the cell surface, as seen in Figure 8, and may represent mature or nearly mature virions. This is an interesting finding in light of the fact that in chick cells infected with avian sarcoma viruses, virions obtained by washing cells at extremely short intervals do not contain 60-70 S RNA but rather RNA with a sedimentation value of 36 S (Canaani et al., 1973) or 55 S (Cheung et al., 1972) which can mature into 60-70 S RNA. The

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finding of 60-70 S RNA on the surface of MuL virus infected cells would then seem surprising, but a likely explanation of these two findings is that virus particles normally may stick to the cell surface after budding and maturation and the frequent medium changes involved in harvesting virus at very short intervals remove such virus particles (and perhaps also some partially immature particles). However, these two lines of evidence both support the view that formation of 60-70 S RNA is a very late step in the virion assembly. No physiological function has been assigned to the 20-30 S RNA. This broad distribution does not appear to be active as messenger RNA, although the data of Figure 13 do not rule out messenger activity of a small component of 20-30 S RNA. One possibility is that the 20-30 S RNA is an in vivo degradation product of 35 S RNA, and has no part in the life cycle of the virus. Another possibility is that the 20-30 S RNA is a defective or deleted 35 S RNA that can neither function as messenger RNA or be packaged into virions. Many stocks of other RNA viruses have been shown to contain such defective RNAs wrapped in virus coats, called defective interfering particles (Huang & Baltimore, 1970). These defective interfering particles generally occur following multiple serial passage of the virus stocks, and it should be noted that both JLS-Vll and SCRF 60A are permanent cell lines that have been passaged for many generations. A rather unlikely possibility is that 20-30 S RNA is a precursor to 35 S RNA. The virus-specific 35 S RNA in infected cells appears to have two functions: precursor to virion 60-70 S RNA and messenger RNA. The role of 35 S RNA as precursor to virion 60-70 S RNA is concluded by analogy to the data of Canaani et al. (1973), in which Rous sarcoma virus harvested from infected chick cells at very short intervals contains 35 S RNA that can be converted to 60-70 S RNA by incubation in vitro. The role of 35 S RNA as messenger RNA can be concluded from the present work. The finding of 35 S RNA as the virus-specific messenger RNA is particularly interesting since 35 S is the size of the virion 60-70 S RNA subunits. As yet it is unclear if the multiple 35 S subunits of the 60-70 S RNA are identical to each other or are unique and there is indirect evidence supporting either view (Vogt, 1973). If the 35 S subunits are not identical, then the intracellular 35 S RNA could represent as many as four messenger RNAs of similar size. However, if the 35 S subunits are all identical then the intracellular 35 S messenger RNA might represent a single unique virus-specific messenger RNA. A situation similar to the latter possibility is found in the case of poliovirus. In poliovirus-infected cells, the entire viral genome (also a 35 S RNA) is translated into a single protein, followed by cleavage into the various virus-specific proteins (Jacobson $ Baltimore, 1968). However, if this situation is true for the RNA tumor viruses, differential expression of virus genes would be difficult to explain. In the experiments shown in Figures 12 and 13, cell extracts were prepared in the absence of detergents in order to minimize RNA degradation from lysozomal nucleases. The membrane structures were therefore not disaggregated. As seen in Figure 10, the membrane-bound material contains significant amounts of virusspecific RNA and, in particular, it is possible that membrane-bound ribosomes also contain virus-specific messenger RNA. Furthermore, this messenger RNA might be different from the messenger RNA associated with free polyribosomes. It should be noted that EDTA treatment releases a fraction of the membrane-associated messenger

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RNA into the free cytoplasm (Attardi et al., 1969; Rosbash & Penman, 1971) so this portion was included in Figures 12 and 13. Also, as seen in Figure 11, only 35 S and 60-70 S RNA are found in the membrane-bound fraction of the cell. As discussed earlier, the 60-70 S RNA probably represents mature virus particles on or in vesicles of plasma membrane produced upon cell lysis. Therefore, the only detectable virus-specific messenger RNA in themembrane fraction would again be 35 S RNA. However, these experiments do not determine if the 35 S messenger RNA of the membrane and free cytoplasm are different or identical.

This work was supported by grants no. AI-08388 and no. CA-14051 from the National Institutes of Health, no. VC-4D from the American Cancer Society and a contract from the Special Virus Cancer Program of the National Cancer Institute (no. 71-2149). One of us (H. F.) was supported by a fellowship from the Helen Hay Whitney Foundation; the other author (D. B.) was supported by a Research Professorship from the American Cancer Society. The expert technical sssistance of Patricia McDaniels and Michael Paskind is gratefully rtcknowledged. We thank Dr Volker Vogt for originally providing us with S-l nuclease and Dr Richard Firtel for assistance in preparation of s, later stock. We thank Drs Robert Weinberg, Inder Verme and Richard Firtel for useful discussions.

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