[18] Procedures for the measurement of interferon mRNA distribution in induced mouse cells

[18]

IF mRNA DISTRIBUTIONIN MOUSECELLS

125

[18] P r o c e d u r e s f o r t h e M e a s u r e m e n t o f I n t e r f e r o n m R N A D i s t r i b u t i o n in I n d u c e d M o u s e C e l l s By JEAN CONTENT, LUK DE WIT, MARGARET I. JOHNSTON, and ERIK DE CLERCQ Mouse interferon mRNA can be translated successfully in several cell-free systems, including the wheat germ system 1 (see this volume [14]) and the micrococcal nuclease-treated rabbit reticulocyte lysate system. 2 We have shown that total cytoplasmic R N A from Newcastle disease virus (NDV)-induced L-929 cells can program the synthesis of interferon in Xenopus laevis oocytes. Moreover, if this RNA is further purified by sucrose-formamide gradient centrifugation, a linear dose-response relationship is obtained between the amount of RNA injected into the oocyte and the amount of interferon translated? A similar quantitative relationship has also been established for the oocyte and wheat germ systems programmed with human interferon m R N A ? -~ These observations have facilitated a study of the mechanism of superinduction of human fibroblast interferon production. *-s When studying the mechanism of interferon priming on interferon production (see Volume 78 [1]) in NDV-induced L-929 cells, we took advantage of this simple quantitative assay to study the kinetics and intracellular distribution of interferon mRNA (IF mRNA). The conclusion of this study was that priming by interferon did not affect IF mRNA transcription or stability, but rather its rate qf translation in NDV-induced mouse L cells. 9,1o M.-N. Thang, D. C. Thang, E. De Maeyer, and L. Montagnier, Proc. Natl. Acad. Sci. U.S,A. 72, 3975 (1975). " B. Lebleu, E. Hubert, J. Content, L. De Wit, I. A, Braude, and E. De Clercq, Biochem. Biophys. Res. Commun. 82, 665 (1978). P. B. Sehgal, B. Dobberstein, and I. Tarnm, Proc. Natl. Acad. Sci. U.S.A., 74, 3409 (1977). 4 R. L. Cavalieri, E. A. Havell, J. Vil~ek, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 74, 3287 (1977). 5 N. B. K. Raj and P. M. Pitha, Proc. Natl. Acad. Sci. U.S.A. 74, 1483 (1977). 6 p. B. Sehgal, D. S. Lyles, and I. Tamm, Virology 89, 186 (1978). r p. B. Sehgal and I. Tamm, Virology 92, 240 (1979). R. L. Cavalieri, E. A. Havell, J. Vil~ek, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 74, 4415 (1977). .9j. Content, M. I. Johnston, L. De Wit, J. De Maeyer Guignard and E. De Clercq, Biochem. Biophys. Res. Commun. 96, 415 (1980). ~o S. L. Abreu, J. C. Bancroft, and W. E. Stewart, II, J. Biol. Chem. 254, 4114(1979).

METHODS IN ENZYMOLOGY, VOL. 79

Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181979-5

126

INTERFERON MESSENGER RNA

[18]

We present here the methodology used to extract IF mRNA, partially purify IF mRNA, and assay IF mRNA by translation in Xenopus laevis oocytes (see also this volume [10]). Materials

Frogs and Oocytes. Xenopus laevis were imported from the South African Snake Farm, P.O. Box 6, Fish Hoek, Cape Province, South Africa. Frogs are anesthesized by immersion for 10 min in MS 222 (Sandoz) 1% (w/v) in water. Pieces of one ovary are removed, teared, and free individual oocytes are collected and kept in Barth solution until injection. Frogs are usually sewn and can be used a second time within 1 or 2 weeks. Micropipettes. Drummond microcaps (2/zl) are stretched to 10-30/zm diameter in a Narishige automatic glass microelectrode puller PN-3 (Narishige Sci. Inst. Lab., Tokyo, Japan). Buffers and Reagents Barth medium (modified by Gurdon11): Tris, HCI, 2.0 mM; NaC1, 88.0 mM; KCI, 1.0 mM; Ca(NO3)2" 4 1-120, 0.33 mM; CaCI2" 2 H20, 0.41 mM; MgSO4" 7 HzO, 0.82 mM; NaHCOa, 2.4 mM; penicillin, 0.1 g/liter; streptomycin, 0.01 g/liter Phosphate-buffered saline (PBS): NaCI, 136 raM; Na2HPO4 • 12 H20, 8 mM; KC1, 2.7 mM; KH2PO4, 1.47 mM, pH 7.2 Triton lysing medium (TLM): "Iris. HC1, I0 mM, pH 7.5; KC1, 10 mM; MgC12, 1.5 mM; 2-mercaptoethanol, 6.5 mM; Triton X-100, 0.13% (w/v); sucrose, 13% (w/v) Sucrose lysing medium (SLM)11: Tris- HCI, 50 mM, pH 7.6; KC1, 100 mM; MgC12, 5 mM; sucrose, 27.4% (w/v). LiC1 stock solution (10 M) is used after filtration on a 0.45/~m Millipore filter. Formamide stock: formamide (J. T. Baker Chemicals, Reagent grade) is deionized by stirring 2 hr with 4% (w/v) Bio-Rad mixed-bed resin and stored frozen. Sucrose formamide gradient solution: Sucrose (ultracentrifugation grade, Merck) is dissolved at 5% or 20% (w/v) in a solution containing 50% deionized formamide, 5 mM EDTA, 0.01M Tris • HCI, pH 7.5, 0. I M LiC1, and 0.2% SDS (sodium dodecyl sulfate "specially pure," BDH). Methods

Cells and Viruses L-929 mouse cells are grown in Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum. The Kumarov strain of 1~ j. B. Gurdon, J. Embrvol. Exp. Morphol. 20, 401 (1968).

[18]

IF mRNA DISTRIBUTIONIN MOUSECELLS

127

NDV is used for interferon induction. Stock virus is propagated in 10day-old chick embryos (see also Volume 78 [42]). Vesicular stomatitis virus (VSV) (Indiana strain) serves as the challenge virus for interferon assays. Stock virus is propagated in BS-C-1 cells. Induction of Interferon L-929 cells are grown in half-gallon roller bottles; 5-12 bottles are used for one typical RNA preparation. When confluent, the cells are inoculated with NDV at approximately 1 P F U per cell. After 1 hr of incubation at 37°, the virus inoculum is removed; the cells are further incubated with MEM for 12 hr, at which time the cells are harvested, fractionated, and extracted. The in vivo interferon production is usually controlled by assaying the cell culture fluid for interferon content at regular times (i.e., 4, 8, 12, 16, 20, and 24 hr) after NDV infection (see "Interferon Assay" below). Cell Harvesting and Fractionation For optimal IF mRNA activity, cells are extracted 12 hr after NDV injection. The cells are first washed with ice-cold PBS and then scraped off with a rubber policeman (rubber bottle scraper Bellco, 7731-22222). All subsequent operations are done at 4° . The harvested cells are centrifuged for 2 min at 3000 rpm (1200 g) in a clinical centrifuge and washed three times with ice-cold PBS. The cell suspension is then divided into two parts (A and B). Part A is disrupted and extracted in the presence of detergent and is used to prepare fractions 1, 2, and 3. Part B is disrupted without detergent and is used to prepare fractions 4 and 5 (the whole procedure is summarized in Fig. 1). Part A. The cell suspension is centrifuged in ice-cold PBS for 5 rain at 3000 rpm in a graduated tube, and the packed cell volume is measured. The cells are suspended at 4° in 2.5 volumes of a buffer containing 0.13% Triton X-100 (TLM). After 10 rain the suspension is disrupted by 5 strokes of a tight-fitting Dounce glass homogenizer ( " B " pestle). Nuclei are removed by centrifugation for 2 rain at 2000 rpm (500g) in an SS34 Sorvall rotor. A portion of the S0.5 supernatant is immediately extracted with phenol and further purified by LiCI precipitation and formamide-sucrose gradient centrifugation (see details below); this constitutes fraction I (total cytoplasmic RNA). Fractions 2 and 3 are obtained by processing the rest of the S0.s supernatant for the precipitation of polysomes. It is first centrifuged for 10 min at 12,000 rpm in a SS34 Sorvall rotor (17,300g). The supernatant ($17) is made up to 30 mM MgCI~ (by addition of 1 M MgCI~), incubated for 2 hr at 4°, and centrifuged for 20 rain at 2500 rpm (755g) in a SS34 SorvaU rotor. The pellet from this centrifugation is washed two times with 30 mM MgC12, redissolved in H~O, and phenol-extracted (see

128

INTERFERON MESSENGERRNA NDV Induced L- 929 cell pellet

[18]

I

I

3 W ~ in ice cold PBS

I Dotlnce homogeaization in TLM (0.13% Triton X-100)

Doun¢~ h ~ i z a t i o n in SLM (0.8 M Sucrose)

I

I

centrifug. 500 g

centriftlg, at 1000 9

i

'

~

. . . . ifu9. 17,3Q0 g

Phenol extraction

Phenol extraction

Phenol extraction

I Ethanol precipitation I LiCI 2M precipitation

I Ethanol precipitation I LiCI 2M precipitation

(3 cycles)

(3 cyclesl

I Ethanol precipitation

I

Socrose formamide gradient ultracentrifugation

~ Ehtanol precipitation + 3 washes

Ethanol precipitation

I

Ethanol precipitation

Phenol extraction

Ethanol precipitation

Ethanol precipitation

~ Ehtanol precipitation +3 washes

!

Phenol extraction

I

SuCrose formamide gradient ultracentrifugation

centrlfug, at 17,300 g

~

I

ultracentlrifu~tion ~ Ehtanol Sucrose formamide gradient

precipitation + 3 washes

FIG. 1. Flow diagram for the preparation of five classes of RNA by cell fractionation. NDV, Newcastle disease virus; TLM, Triton lysing medium; SLM, sucrose lysing medium; centrifug., centrifugation. below). This R N A constitutes fraction 2 (total polysomal RNA). The supernatant from the last centrifugation is phenol-extracted and further purified by LiC1 precipitation and formamide-sucrose gradient centrifugation (see below) and yields fraction 3 (free cytoplasmic RNA ). Part B. The washed cells are lysed by 15 strokes of a tight-fitting

[18]

IF mRNA DISTRIBUTIONIN MOUSECELLS

129

Dounce homogenizer in a buffer containing 0.8 M sucrose (SLM). ~2 The lysate is centrifuged for 5 min at 1000 g, and the resulting supernatant is centrifuged for 10 rain at 12,000 rpm (17,300g). The resulting $17 is layered onto 2 ml of SLM and centrifuged at 60,000g for 20 min in the SW41 rotor of a Beckman ultracentrifuge. The pellet is phenol-extracted and constitutes the membrane-bound fraction (fraction 4). The supernatant ($60) is further centrifuged for 2 hr at 160,000 g in a Beckman type 65 or 75 Ti fixed-angle rotor. The resulting pellet is phenol-extracted and further purified by formamide-sucrose gradient centrifugation (see below). This RNA constitutes fraction 5 (free polysomal RNA ).

Interferon mRNA Extraction Fractions 1-5 described above are deproteinized immediately by three extractions with an equal volume of phenol, chloroform, and isoamyl alcohol [25 : 25 : 1 (v/v/v)] in 100 mM Tris HC1, pH 8.9, and 1% SDS. The resulting aqueous phase is further extracted with chloroform, isoamyl alcohol [25:1 (v/v)]; after addition of 0.2 M NaC1, the RNA is precipitated with two volumes of ethanol at - 2 0 °. When required, the RNA is redissolved in water and precipitated in 2 M LiCI at 4° for 6-16 hr. The LiC1 precipitate is redissolved in water, and the precipitation is repeated 2 or 3 times. After the last precipitation the RNA is precipitated with 2 volumes of ethanol, washed twice in 75% ethanol, and dried under vacuum before it is finally dissolved in water.

Sucrose Gradient Purification of IF mRNA IF mRNA samples from fractions 1, 3, and 5, containing 150-800 tzg of RNA, are first dissolved in water, then brought to 50% formamide, incubated for 2 rain at 37°, and layered onto a linear 5 to 20% w/v sucrose gradient in 5 mM EDTA, 0.01M Tris HC1, pH 7.5, 0. I M LiC1, 0.2% SDS, and 50% formamide. The gradients are centrifuged at 20° for 16 hr at 40,000 rpm in a Beckman SW60 Ti rotor. [zH]Uridine-labeled cytoplasmic mouse cell RNA is analyzed in a parallel gradient in order to localize the position of the major (18 S and 4 S) RNA components and to deduce the position of the IF mRNA peak. The latter is reproducibly found at approximately 11 S. 2 Usually, each gradient is divided into 18 fractions. Fractions corresponding to the 9-12 S region, containing most of the IF mRNA activity, are pooled, made up to 0.2 M NaCI, precipitated with two volumes of ethanol, incubated for at least 16 hr at - 2 0 °, centrifuged, washed 3 times with 75% ethanol, dried, and finally redissolved in water at 2 mg of RNA per milliliter. ~ G. E. Sonenshein and G. B r a w e r m a n , Biochemistry 15, 5501 (1976).

130

INTERFERON MESSENGER RNA

[18]

TYPICAL DISTRIBUTION OF RNA AND IF mRNA ACTIVITY IN NEWCASTLE DISEASE VIRUS-INDUCED L-929 CELLS Fraction 1: total cytoplasmic RNA Amount of RNA (/zg) obtained from 1 ml of packed cells Amount of interferon (units/ml) translated in oocytes when injected with 100 ng of RNA per

Fraction 2: Fraction 3: Fraction 4: total free membranepolysomal cytoplasmic bound RNA RNA RNA

Fraction 5: free polysomal RNA

148

354

141

I1

30

1584

500

316

22a

158

oocyte Upon injection of 50 ng of RNA per oocyte.

Translation of Interferon mRNA in Xenopus laevis Oocytes Xenopus laevis oocytes are microinjected with the mRNA preparations as described previously. 13 RNA samples are dissolved in water at 0.5-2 mg/ml. This corresponds to 25-100 ng of RNA per oocyte, for each oocyte is injected with 50 nl. Within this range of RNA concentrations the amount of biologically active interferon translated is directly proportional to the amount of RNA injected per oocyte, thus allowing a direct estimation of IF mRNA content in a given RNA preparation, z This quantitative relationship is also observed for both polysomal and membrane-bound RNA (fractions 2 and 4, respectively). In contrast, fractions 1, 3, and 5 require the sucrose formamide gradient purification step to show the same linear response. 9 Ten oocytes are injected for every determination of IF mRNA activity. After injection they are incubated in 0.2 ml of Barth's medium in 1.5-ml Eppendorf plastic tubes for 16 hr at 19°. After 120/~1 of the incubation medium have been discarded, the oocytes are homogenized in the remaining medium with a small Teflon pestle. The homogenates are centrifuged at 10,000 g for 2 min in an Eppendorf microcentrifuge. The supernatants are carefully removed (to avoid contamination by the fat layer) and assayed for interferon either directly or after storage at - 7 0 °. 13 j. B. Gurdon, C. D. Lane, H. R. Woodland, and G. Marbalx, Nature (London) 233, 177 (1971).

[18]

IF mRNA DISTRIBUTIONIN MOUSECELLS

131

Interferon Assay Mouse interferon is assayed by inhibition of VSV-induced cytopathogenicity in L-929 cells. The assay is done in duplicate in 96-well microtiter plates. A standard calibrated against the National Institutes of Health mouse interferon reference standard G-002-904-511 is included in each assay. Results The results of a representative experiment are presented in the table. They indicate the amounts of RNA obtained for each of the five fractions as well as the amounts of interferon translated in oocytes upon injection of a standardized amount of RNA. Acknowledgments These investigations were supported by grants from the Belgian F R F C (Fonds de la Recherche Fondamentalc Collective), F G W O (Fonds voor Geneeskundig Welenschappelijk Onderzock), and Geconcerteerde Onderzoeksactics, and by a U.S. National Research Service Award (I F32 AI05662 to M. I. J.).