Characterization of visna virus nucleic acid

BIOCHIMICA ET B I O P H Y S I C A ACTA

435

BBA 96939

CHARACTERIZATION OF VISNA VIRUS NUCLEIC ACID DONALD H. H A R T E R , J E F F R E Y SCHLOM AND H. SPIEGELMAN Departments o[ Neurology, Microbiology and Human Genetics and Development, and the Institute o[ Cancer Research, Columbia University College o[ Physicians and Surgeons, New York, N . Y . ~roo32 (U.S.A.) (Received April I9th, 1971)

SUMMARY I. Nucleic acid was extracted from radioactively labeled visna virus particles and analyzed by equilibrium zonal centrifugation in glycerol density gradients. 2. The major component recovered from virions has a sedimentation coefficient of 60-70 S; it is single-stranded RNA as shown by its complete sensitivity to ribonuclease and density after isopycnic gradient centrifugation in Cs2SO4. The vifion also contains a slowly sedimenting 5-7-S nucleic acid species. 3. Visna virions contain nucleic acids that resemble in size and composition those present in RNA oncogenic viruses.

INTRODUCTION Visna virus is the cause of a fatal neurological affliction of sheep characterized by subacute inflammation and demyelination 1,*. The disease has been transmitted to sheep by the inoculation of brain extracts from naturally infected animals 1,2 or by virus propagated in tissue culture 3. Since illness does not occur until months or years after inoculation 4, the malady is considered to be a model "slow-virus infection ''5. Visna virus, however, resembles the oncogenic RNA viruses in certain physical characteristics e, ultrastructural appearance 7, development in cellss,9 and DNA polymerase activities 10-12. Although incorporation studies using radioactive nucleic acid precursors indicate that virus particles contain RNA ~3,14,viral nucleic acid has not yet been extracted or analyzed. We report here that visna virus, like the RNA tumor viruses, contains a single-stranded 6o-7o-S RNA as its major nucleic acid component. METHODS

Cell cultures. Sheep choroid plexus cells were prepared by trypsin dispersion of choroid plexuses removed from the brains of exsanguinated domestic Hampshire or Suffolk sheep as previously described 15. Sheep testis cells were the gift of Drs. K. Takemoto and L. Sturman of the National Institutes of Health. Sheep choroid plexus cells were used in their Ioth passage; sheep testis cells in their I l t h to I3th passages. Cells were grown in reinforced Eagle's medium le containing IO % fetal bovine serum in 25o-mi plastic flasks or Ioo-mm plastic dishes and incubated at 36 °. Biochim. Biophys. Acta, 240 (1971) 435-441

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Virus. Visna virus K 485, kindly supplied by Drs. H. Thormar and P. A. P~lsson, Institute of Experimental Pathology, University of Iceland, was propagated in sheep choroid plexus cells. Second and third passage virus was stored at - - 7 °0 and used as stock virus. In]ection o/cells. Confluent monolayers were incubated with visna virus at a multiplicity of 0.2--0. 5 TCIDs0 per cell. After an adsorption period of 4 h at 36°, maintenance medium containing 2 % heat-inactivated lamb serum was added. Cultures were further incubated at 36°; plate cultures were kept in a humidified atmosphere of 5 % CQ. Addition o/ radioactive precursors. When cytopathic changes first became prominent (usually 3-4 days after inoculation), medium was removed from cultures and replaced with fresh maintenance medium containing either i2 #C/ml E5-3HJuridine (New England Nuclear Corporation) or 65/~C/ml [~2PJorthophosphate (International Nuclear Corporation). Cultures were re-incubated for approximately 40 h and the medium was harvested. If sufficient cells remained attached, fresh medium containing radioactive plecursors was again added and the cnltures were re-incubated for an additional 30-50 h. Harvested material was stored at 4 ° until used for virus purification. Purification o/ radioactively labeled virus. Medium from visna-infected cultures was clarified by centrifugation at 8 700 × g for IO min at 4 °, and concentrated b y centrifugation at 78 o o o × g for 3 h or b y precipitation with (NH4)2SO 4 (30 g/Ioo ml medium) at 4 ° for I h at p H 7.5-8. Precipitates were collected by centrifugation at 6 4 o o × g for 30 rain, dissolved in o.oi M Tris-o.I M NaCl-o.ooI M E D T A (pH 8.3) and centrifuged at 78 ooo × g for 3 h; pelleted material was suspended in 0. 5 ml of o.oi M Tris-o.I M NaCl-o.ooI M E D T A (pH 8.3) containing 0.5 % bovine plasma albumin (Fraction V, Armour Pharmaceutical Co.). Concentrated virus was layered on a preformed linear gradient of 5 % to 4 ° % (w/w) potassium tartrate in o.I M phosphate buffeI (pH 7.0) containing o.ooi M E D T A and centrifuged in an SW 41 swinging bucket rotor at 2Ol ooo × g for 3 h at 4 °. A visible virus band sedimenting between I. 18 and 1.2o g/ml was collected, diluted in a large volume of o.oi M Tris-o.i M NaCl-o.ooI M E D T A (pH 8.3) containing 0. 5 % bovine plasma albumin, pelleted b y centrifugation (78 ooo ×g, 3 h), resuspended and subjected to a second cycle of potassium tartrate density gradient centrifugation. Fractions from the gradient were collected by puncturing the bottom of the tube and a portion of each fraction was delivered to scintillation vials containing NCS solubilizer; toluene-BBOT scintillation fluid was added and radioactivity determined. Density of selected fractions was monitored using an Abbe refractometer. Fractions in the radioactivity peak corlesponding to the density of visna virions were pooled 15, diluted with o.oi M Tris-o.I M NaCl-o.ooI M E D T A (pH 8.3), pelleted b y centrifugation and resuspended in 2.0 ml of the same buffer. Extraction o/viral nucleic acid. Nucleic acid was extracted from purified radioactive visna virions b y treatment with 1 % sodium dodecyl sulfate in the presence of pronase (0.5 mg/ml), o.I M NaCl, o.oi M Tris (pH 7.4), o.oi M E D T A and 1 % mercaptoethanol at 360 for 30 rain. The reaction mixture was then extracted twice with equal volumes of cresol-phenol (pH 8.4) and chloroform-isoamyl alcohol at 4 ° for IO rain. Nucleic acid was precipitated from the aqueous phase b y the addition of EscheBiochim. Biophys. Acta, 24o (197 I) 435-441

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richia coli carrier RNA (IO/*g/ml), NaC1 (o.4 M final concentration) and 2 vol. of ethanol, and stored at --20 °.

Glycerol gradient sedimentation analysis. Nucleic acid to be examined was pelleted by centrifugation at 23 oooXg for 3 ° min at o °, resuspended in 0. 5 ml o.oi M Tris-o.I M NaCl-o.ooI M EDTA (pH 8.3) and layered onto a preformed density gradient of io % to 30 % (v/v) glycerol in the same buffer. Gradients were centrifuged at 2Ol ooo x g for 2.5 h at 4 ° in a SW 41 swinging bucket rotor. 24-drop fractions were collected from below; 5o-/.1 portions were mixed with 0.5 ml NCS reagent (Amersham/ Searle) and radioactivity was counted after addition of toluene-BBOT. I8-S and 28-S RNA extracted from rat embryo fibroblast cells were used as markers in sedimentation analysis. Cs2SO4 density gradient centri/ugation. 32P-labeled viral nucleic acid, resuspended in 0.002 iV[ EDTA, was mixed with saturated Cs,SO 4 to a density of 1.55 ° g/cm 3 and centrifuged at 31 ooo rev./min at 20 ° for 60 h in a SW 56 rotor. Fractions were collected from the bottom and 3 vol. of trichloroacetic acid mixture (equal volumes of IOO % trichloroacetic acid, saturated sodium orthophosphate, and sodium pyrophosphate) were added in the presence of E. coli RNA as carrier. After IO min, the precipitable material was collected on a nitrocellulose filter and dried, and the radioactivity was determined in a liquid scintillation counter using BBOT scintillation fluid.

RESULTS

Analysis o/nucleic acid extracted/rom visna virions. Sedimentation analysis of nucleic acid extracted from visna virions radioactively labeled with E5-3Hluridine is shown in Fig. i. The major component sedimented rapidly with a sedimentation coefficient of approximately 67 S. In addition, a slower sedimenting moiety of approximately 5 S was regularly detected. Analysis of the extract from 32P-labeled virions (Fig. 2) resembled that obtained from 3H-labeled particles with the exception that two small intermediate peaks sedimenting between 3 ° S and 37 S were also present. Ribonuclease treatment o/ nucleic acid extracted ]rom visna virions. Glycerol gradient fractions containing high molecular weight (6o-70 S) and low molecular weight (5-7 S) peaks extracted from 3H-labeled virions were treated with pancreatic A and T 1 ribonuclease (IOO/*g/ml of each) to determine their sensitivity to the enzyme (Table I). The results indicate that the acid-precipitable radioactivity of the 60-7o S material was essentially destroyed by ribonuclease. However, a higher proportion of the radioactivity in the 5-7-S peak remained unaffected by ribonuclease digestion. Sedimentation o] visna virus nucleic acid in Cs2SO~ equilibrium density gradients. The nucleic acid in visna virions was further characterized by Cs,SO 4 equilibrium density centrifugation. This method separates RNA (density, approx. 1.65o) from DNA (density, approx. 1.45o ). DNA-RNA hybrid complexes will band at intermediate densities. The 32P-labeled nucleic acid from visna virions was separated into three regions on the basis of velocitygradient centrifugation in glycerol (Fig. 2), i.e. a high molecular weight (60-70 S), intermediate molecular weight (30-40 S) and low moleculai Biochim. Biophys. Acta, 240 (1971) 435-441

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Fig. I. E q u i l i b r i u m d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of nucleic acid e x t r a c t e d f r o m purified v i s n a virions labeled w i t h [5-SH]uridine. 0. 5 ml e x t r a c t e d R N A w a s l a y e r e d on a i 1.4-ml linear lO--3o % glycerol gradient. T h e g r a d i e n t w a s c e n t r i f u g e d a t 2Ol ooo x g in a Spinco S W 41 r o t o r for 2. 5 h a t 4 °. o.3-ml fractions were collected f r o m below. 5o/,1 f r o m e a c h fraction was a s s a y e d for radioa c t i v i t y in NCS a n d t o l u e n e - B B O T scintillation fluid. I8-S a n d 28-S R N A f r o m r a t e m b r y o f i b r o b l a s t s ( R E F ) were u s e d as e x t e r n a l m a r k e r s . Fig. 2. E q u i l i b r i u m d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of nucleic acid e x t r a c t e d f r o m purified v i s n a virions labeled w i t h [82P]orthophosphate. 0. 5 ml e x t r a c t e d R N A was layered on a I I . 4 - m l linear lO-3O % glycerol gradient. T h e g r a d i e n t w a s c e n t r i f u g e d a t 2Ol ooo × g in a Spinco S W 41 r o t o r for 2. 5 h a t 4 °. o.3-ml fractions were collected f r o m below. 5 ° #1 f r o m each fraction was a s s a y e d for r a d i o a c t i v i t y in NCS a n d t o l u e n e - B B O T scintillation fluid. I8-S a n d 28-S R N A f r o m r a t e m b r y o fibroblasts ( R E F ) were u s e d as e x t e r n a l m a r k e r s . H i g h m o l e c u l a r weight, i n t e r m e d i a t e m o l e c u l a r w e i g h t a n d low m o l e c u l a r w e i g h t regions are d e s i g n a t e d b y H M W , I M W a n d L M W , respectively. TABLE I EFFECT

OF RIBONUCLEASE

ON NUCLEIC

ACID

EXTRACTED

FROM

VISNA

VlRIONS

S a m p l e s were t r e a t e d w i t h p a n c r e a t i c ribonuclease A a n d T t (IOO/~g/ml) in o.i M NaCI, a n d m a i n t a i n e d a t 37 ° for 90 min. R a d i o a c t i v i t y precipitable w i t h 5 °6 trichloroacetic acid w a s determ i n e d a n d c o m p a r e d w i t h a replicate s a m p l e w i t h o u t ribonuclease.

Sample

A cid-precipitable radioactivity (3H counts/rain) Ribonuclease-treated Control % o/control

6o-7o-S v i s n a 5-7-S v i s n a i8-S a n d 28-S r a t e m b r y o fibroblast

14 8o 28

1235 788 51o6

I.I lO.2 o.5

weight (5-7 S). As seen in Figs. 3 and 4, both the high molecular weight and intermediate molecular weight nucleic acid species have densities characteristic of RNA. The low molecular weight (5-7 S) species of the virion, however, contained components that band at the density of RNA as well as components band at densities characteristic of RNA-DNA hybrid complexes (Fig. 5)- These complexes have also been observed in the low molecular weight nucleic acid species of other mammalian RNA tumor viruses (J. SCHLOMand S. SPIEGELMAN, unpublished results). DISCUSSION

The major nucleic acid component recovered from visna virions is a singlestranded RNA with a sedimentation coefficient of 60-70 S. Similar RNA species have Bioch{m. Biophys. Acta, 240 (1971) 435-441

439

VISNA VIRUS NUCLEIC ACID

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Fig. 3. C%SO4 density gradient centrifugation of visna virus high molecular weight nucleic acid. Nucleic acid was extracted from purified a*P-labeled visna virions and sedimented in a lO-3O % {v/v) glycerol gradient as described in MATERIALS AND METHODS, The material sedimenting bet w e e n 6o and 7 ° S was isolated, centrifuged at 23 ooo × g for 3° min, and resuspended in o.oo2 M E D T A . This was mixed w i t h s a t u r a t e d Cs~SO, to a density of 1.55o g/cm 3, and centrifuged at 31 ooo r e v . / m i n at 2o ° for 60 h in a Spinco S W 56 rotor. Fractions were collected from the b o t t o m and processed as described in MATERIALS AND METHODS. Fig. 4" C%SO4 density gradient centrifugation of visna virus intermediate molecular weight n u cleic acid. Nucleic acid w a s extracted from purified, 8*P-labeled visna virions and sedimented in a lO-3O % (V/V) glycerol gradient as described in MATERIALS AND METHODS. The material sedimenting b e t w e e n 30-4 ° S was processed as described in Fig. 3.

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Fig. 5. CseSO4 density gradient centrifugation of visna virus low molecular weight nucleic acid. Nucleic acid was extracted from purified, a2P-labeled visna virions and sedimented in a i o - 3 o % (v/v) glycerol gradient as described in MATERIALS AND METHODS. The material sedimenting between 4-12 S was processed as described in Fig. 3.

been extracted from members of each of the major groups of the oncogenic RNA viruses 17. The 6o-7o-S RNA derived from these RNA viruses can be converted to a 36-S unit by heat or b y treatment with dimethylsulfoxidezs-zl. The minor 37-S RNA peak obtained from zzP-labeled visna particles may represent a disaggregated portion Biochim. Biophys. Acta, 240 (1971) 435-441

440

D.H. HARTERet al.

of the 6o-7o-S RNA. Studies are in progress to determine whether the high molecular weight visna RNA will yield similar 36-S subunits. Like the RNA tumor viruses, visna virus contains a slowly sedimenting 5-7-S RNA. By analogy to the slowly sedimenting tumor virus RNA, 5-7-S visna RNA may be of cellular origin 22-24. This would be in keeping with electron microscopic observations of visna virus development, which appear to show the incorporation of cellular ribosomal material into virus particles °. Although there are many other similarities between visna virus and the RNA tumor viruses, they differ in their antigenic composition*. Antisera to several RNA tumor viruses (Gross, Moloney and Friend murine leukemia viruses and the Bryan strain of Rous sarcoma virus) failed to neutralize visna virus ~5. This distinction is borne out by the recently established absence of antigenic cross-reactivity between visna virus and known oncogenic RNA viruses. Ether-treated purified visna virions did not give precipitates in gel diffusion tests with group-specific (gs) antisera prepared against avian leukosis-sarcoma, murine leukemia-sarcoma, hamster leukemiasarcoma, feline leukemia, mouse m a m m a r y tumor, or simian m a m m a r y tumor (Mason/Pfizer) viruses**. Fulthermore, visna virions did not react with antisera that detects MuLV-gs3 antigen, an antigen that is common to mammalian leukemiasarcoma viruses 26. A more revealing exploration of specific relationships between visna virus and RNA tumor viruses will require studies of hybridization between the RNA of the latter and the DNA product synthesized by the visna RNA-dependent DNA polymerase.

ACKNOWLEDGMENTS

The authors thank Mrs. Viola Mahoney and Miss Sandral Hullett for their excellent technical assistance. This work was supported by grants from the National Institute of Neurological Diseases and Stroke (NS-o6989) and the National Cancer Institute (CA-o2332), a contract from the National Cancer Institute (with the SVCP # 7o-2o49), and a gift from the Miles Hodson Vernon Foundation, Inc. D.H.H. is recipient of Cancer Research Development Award I K3 NS-34,99 o from the National Institute of Neurological Diseases and Stroke.

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VISNA VIRUS NUCLEIC ACID 7 8 9 IO Ii I2 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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Biochim. Biophys. Acta, 24o (1971) 435-441