Archives of Virology 50, 45--53 (1976) © by Springer-Verlag 1976

Isolation and Characterization ot Double Stranded RNA Containin 9 Infectious Viral C,enome RNA irom Cells Infected with Semliki Forest Virus By G. WE~GLE~, GISEL~ WENGLER., and K. WA~N Institut fiir Virologic, Justus-Liebig-Universitgt, Giessen, Federal t~epublic of Germany With 5 Figures Accepted September 1, 1975 Summary A double stranded virus specific t~NA sedimenting at about 19S on. sucrose density gradients has been isolated from BHK-21 cells infected with Semliki Forest. virus (SFV). The molecule consists of double stranded RNA (ds I~NA) since it is labeled with aH-uridine, is soluble in 2 • LiCt, resistant against treatment, with DNase and RNase at 2 × SSC, hydrolyzed b y alkali treatment, has a sharp thermal melting point at 89 ° in 1/10 SSC, and an extended appearance under non denaturing conditions in the electronmieroseope. The following findings show that it consists of intact, infectious 42S RNA similar or identieM to the genome RNA of SFV eomplexed to a complementary 42 S minus strand I~NA : 1. Denaturation converts the ds R N A into molecules cosedimenting with 42S I~NA isolated from SFV particles, 2. About 50 per cent of the radioactivity of 3H-uridine labeled 42S I~NA molecules generated from 19S ds I~NA b y denaturation hybridizes to 42S viral RNA. 3. The specific infectivity of denatured 19S ds I~NA is about half of t h a t of similarly treated viral 42 S t~NA. Further properties of this molecule are discussed. Introduction Different species of double stranded RNA (ds I~NA) have been isolated from vertebrate cells infected with the togavirus Semliki Forest virus (SFV) or the closely related Sindbis virus (9, t0, 13, 14). Some of these ds R N A species have been detected in significant amounts only after g N a s e digestion of t~NA extracted from infected cells followed by purification of the double stranded ribonuclease resistant I~NA. The most rapidly sedimenting ds RNA which has been isolated from SFV infected cells sediments slightly faster than 18S ribosomal R N A on sucrose density gradients and it has been suggested from indirect evidence that this molecule consists of 42S genome plus strand RNA of SFV eomplexed to a

46

G. WENGLEtt, GISEL2~WENGLEt~, and K. WA]~N:

c o m p l e m e n t a r y 42 S m i n u s s t r a n d R N A (9, 10). T h e p r e s e n t r e p o r t d e s c r i b e s s o m e p r o p e r t i e s of t h i s ds R N A a n d a s i m p l e p r o c e d u r e for its i s o l a t i o n i n w h i c h n o R N a s e is used. D i r e c t e v i d e n c e is p r e s e n t e d t h a t t h e ds I ~ N A m o l e c u l e i s o l a t e d b y t h i s t e c h n i q u e consists of i n t a c t , i n f e c t i o u s 4 2 S p l u s s t r a n d R N A c o m p l e x e d to i n t a c t c o m p l e m e n t a r y 42 S m i n u s s t r a n d R N A .

Materials and Methods Chemicals, Isotopes, Solution Sl-nuclease, p r e p a r e d according to VoG~ (16) was a kind gift of Dr. W. Rhode. Oligo-dT cellulose was from Collaborative Research, Inc., W a l t h a m , Mass. (U.S.A.) (type T~2). P a n c r e a t i c ribonuclease A was b o u g h t f r o m Boehringer, Ma~nheim, G e r m a n y , and D N a s e I was an RNase-free p r e p a r a t i o n from W o r t h i n g t o n (U.S.A.). SSC consists of 150 m~1NaC1, t 5 m E NaB-citrate, pI-I 7.0. All other chemicals and isotopes used h a v e been described earlier (t8).

Cells and Virus The growth of B H K 21 cell m o n o l a y e r cultures, their infection with Semliki F o r e s t virus and the labeling of cultures w i t h 3H-uridine h a v e been described (17).

Isolation o / 1 9 S ds R~TA /rom In/ected Cells T o t a l R N A was e x t r a c t e d w i t h phenol at 60 ° C in 10 m ~ N a - a e e t a t e , p H 5.2 f r o m infected B H K - 2 1 cells 6 hours p.i. as described b y S e~ER~E~ (12) and p r e c i p i t a t e d from the aqueous phase w i t h ethanol. The precipitate was suspended in buffer consisting of 2 M LiC1, 0.1 per cent SDS, I0 m~a triethanolamine, pI-I 7.4, and transferred into a loosely fitting D o u n c e homogenizer. The R N A soluble in the buffer was e x t r a c t e d b y intermitten~ gentle h o m o g e n i z a t i o n of t.hc suspension for a b o u t 6 hours in ~he cold (4 ° C). The suspension was centrifuged for 30 m i n u t e s at 16,000 r p m in the SorvalI SS34 rotor and the e x t r a c t e d R N A was precipitated from the s u p e r n a t a n t b y a d d i t i o n of three v o l u m e s of ethanol. The 19S ds R N A was isolated from this R N A by sucrose d e n s i t y g r a d i e n t eentrifugation either directly (Fig. 1A) or after treatment" w i t h 10 ~g/ml of D N a s e I in 10 r e ~ N a - a e e t a t e , 2 m E MgC12, p H 5.2, for 10 m i n u t e s at 37 ° C (Fig. 1 B). A b o u t 20 ixg of the ds R.NA can be isolated f r o m 108 infected cells.

Analysis o/ Thermal ~elting by S1-Nuclease Digestion 3H-Uridine labeled ds R N A was dialyzed into 1/10 SSC and 20 ~1 aliquots sealed in capillaries were h e a t e d at different t e m p e r a t u r e s for 5 minutes and rapidly cooled to - - 2 5 ° C. The a m o u n t of I~NA remaining double s t r a n d e d was d e t e r m i n e d b y digestion w i t h Sl-nuclease (2 hours, 50 ° C, in 300 m ~ NaC1, 3 mE ZnSO4, 10 ~g/ml d e n a t u r e d calf t h y m u s D N A , 30 m E N a - a c e t a t e , pJ:t 4.5) followed b y TCA precipitation a n d c o u n t i n g of t h e precipitate.

Electronmicroscopy o] ds R N A A b o u t 0.I y.g of ds R N A in 10 ~xl w a t e r were added to 15 tzl of a solution p r e p a r e d by m i x i n g 100 ~1 c y t o c h r o m e C (1 m g / m l in water), 25 ~1 E D T A (200 mE, pI-I 7.4) and 500 ~1 a m m o n i u m - a c e t a t e (1 ~, pI-I 7.4). A drop consisting of 190 ~1 of 200 m E a m m o n i u m - a c e t a t e containing 0.2 per cent f o r m a l d e h y d e was layed on a teflon dish and 10 al of t h e ds g N A solution were i n j e c t e d into the drop. A f t e r i n c u b a t i o n for 5 minutes a t r o o m t e m p e r a t u r e m a t e r i a l was picked up f r o m t h e surface of t h e drop w i t h a collodion-covered grid and r o t a r y s h a d o w e d w i t h P t - P d at an angle of 20 °. L e n g t h m e a s u r e m e n t s were done b y comparison w i t h a grating replica.

Oligo-dT Cellulose Chromatography This analysis was done as described b y A w v and LEDE~ (1) b u t using NaC1 instead of KCI and in t h e presence of 0.1 per cent SDS in all buffers.

Double Stranded I{NA from Semliki Forest Virus Infected Cetts

47

In/ectivity o~ R N A Infectivity was analyzed on chick embryo fibroblasts 24 hours after seeding either by the hypeI4onie assay system described by t{ICttTER, and W~etCER (11) or by the method using DEAE-Dextran (500 ~g/ml) described by VA~EnI and PAeA~O (15).

Results

B H K - 2 1 cells infected with Semliki Forest virus were labeled with aH-uridine in the presence of a c t i n o m y e i n D, the t o t a l I{NA was isolated a n d salt soluble m a t e r i a l was extracted frost this t~NA with 2 M LiCI, Sucrose d e n s i t y gradient s e d i m e n t a t i o n p a t t e r n s of the LiC1 soluble material are presented in Figure 1.

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Fig. 1. Sucrose density gradient centrifugation of I~NA soluble in 2 5~ LiC1 BHK-21 cells infected with SFV were labeled with SH-uridine (10 FCi/ml) from 0 to 6 hours p.i. in the presence of actinomycin D (1 Bg/ml), the total I%NA was extracted from the culture at 6 hours p.i. and the RNA soluble in 2 M LiC1 isolated as deseribed in the NIaterials and Methods section. This RNA was fraetionated b y sucrose density gradient fractionation (6 hours, 40,000 rpm, 2° C, S W 4 t Spinco rotor, linear gradients from 10 to 30 per cent sucrose in 50 m ~ KC1, 10 mN K-acetate, 0.1 msI EDTA, pH 5.2) either directly (part A) or after treatment, with DNase I (10 Fg/ml in 10 m ~ Naacetate, 2 ms~ MgC12, pH 5.2, 37 ° C, 10 minutes) (paxt B) or after treatment with KOt~ (300 ms~ KOH, 60° C, 60 minutes) (part C). Total radioactivity in the gradient fractions was determined in a Triton X-100--toluene scintillation mixture (17). The optical density distribution was measured in a flow through cuvette o - - -- -- ®aH-uridine radioactivity ; - absorbanee at. 260 n m

I t can be seen t h a t besides slowly s e d i m e n t i n g m a t e r i a l molecules s e d i m e n t i n g slightly faster t h a n 18S ribosomal I{NA have been extracted ~dth 2 ~ LiC1 (Fig. 1 A). The labeling of these molecules with s t t - u r i d i n e a n d their solubility i n 2 M LiC1 suggests t h a t t h e y consist of double s t r a n d e d R N A (2). The d a t a presented i n Figure 1 B show t h a t DNase t r e a t m e n t does n o t alter their s e d i m e n t a tion b e h a v i o u r or their specific activity. T r e a t m e n t with alkali hydrolyses the molecules to small molecular weight material (Fig. 1 C). B a n d eentrifugation of the t~NA i n the a n a l y t i c a l ultracentrifuge gives a s e d i m e n t a t i o n coefficent of I 9 S (data n o t shown). The 19S m a t e r i a l is resistant

48

G. WENGLE~, GISELA WENGLEn, and K. WA~N:

a g a i n s t digestion w i t h p a n c r e a t i c g N a s e in 2 x SSC a n d has a s h a r p m e l t i n g profile (Tin =- 89 ° C in 1/10 SSC) as e x p e c t e d for double s t r a n d e d R N A molecules (Fig. 2).

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Fig. 2. Melting curve and g N a s e resistance of the 19S ds R N A 3tt--urid:ine-labeled ds R N A was isolated from a gradient similar to t h a t shown in Figure 1A. P a r t A : The melting curve was determined b y measurement of the acid preeipitable radioactivity remaining after 5 minut.es incubation of the ds R N A at the temperature indicated on the abscissa followed b y digestion of the ss t £ N A with $1 nuclease ( , - - , - - . - - . ) . P a r t B: RNase resistance was determined as follows: 14Curidine labeled 42 S ss R N A from SFV was mixed with the ds R N A and the mixture was incubated in 1 × SSC in the presence of RNase (10 ~g/ml) a¢ 37 ° C. The TCA preeipitable radioactivity was determined in aliquots at the times indicated on the abscissa •- • TCA precipitable 3H-uridine radioactivity; . - - • TCA preeipitable ~4Curidine radioactivity

L i n e a r molecules of r a t h e r homogeneous l e n g t h are seen in the electronm i c r o g r a p h of t h e 19 S ds I~NA shown in F i g u r e 3. The m e a n l e n g t h of these molecules is 3. i ix a n d t h e i r e x t e n d e d a p p e a r a n c e is t y p i c a l for double s t r a n d e d nucleic acids. Single s t r a n d e d nucleic acids w o u l d form collapsed bushes u n d e r t h e conditions used. A s e d i m e n t a t i o n a n a l y s i s of d e n a t u r e d ds R N A is p r e s e n t e d in F i g u r e 4 A : A f t e r d e n a t u r a t i o n t h e m a j o r i t y of the m a t e r i a l eosediments w i t h laC-uridinel a b e l e d a u t h e n t i c 42 S single s t r a n d e d R N A (ss R N A ) i s o l a t e d from S F V particles. This suggests t h a t t h e 19S ds t~NA consists of 42S plus s t r a n d t~NA e o m p l e x e d to c o m p l e m e n t a r y molecules of 4 2 S m i n u s s t r a n d R N A . I n o r d e r to show t h a t t h e 42S R N A g e n e r a t e d b y d e n a t u r a t i o n from the 19S ds I~NA consists of a m i x t u r e of 4 2 S p l u s a n d m i n u s s t r a n d s , this 4 2 S R N A , l a b e l e d w i t h SH-uridine, has been i s o l a t e d from sucrose d e n s i t y g r a d i e n t s a n d h y b r i d i z e d to a n excess of u n l a b e l e d 42S plus s t r a n d s i s o l a t e d from S F V p a r t i c l e s (Fig. 4B). The results show t h a t in a c c o r d a n c e w i t h t h e a b o v e e x p e c t a t i o n a b o u t 50 p e r cent of t h e 3ttu r i d i n e r a d i o a c t i v i t y of this 42S m a t e r i a l is i n c o r p o r a t e d into h y b r i d molecules u n d e r these conditions. Since t h e 42 S plus s t r a n d I~NA contains s o m e w h a t m o r e

Double S t r a n d e d R N A f r o m Semliki F o r e s t Virus I n f e c t e d Cells

49

Fig. 3. E l e c t r o m i c r o g r a p h of 19S ds R N A The ds R N A was i s o l a t e d f r o m a g r a d i e n t similar to t h a t s h o w n in F i g u r e 1 B. The p r o c e d u r e u s e d for e l e e t r o m i e r o s e o p y is d e s c r i b e d in t h e Materials a n d M e t h o d s s e c t i o n

Arch. Virol. 50/1--2

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Fig. 4. Analysis of denatured 19 S ds RNA Part A: stt-uridine-labeled 19 S ds t~NA isolated from a gradient similar to that shown in Figure I A was denatured by 3.5 minutes incubation at 80 ° C in 90 per cent formamide containing 1/10 SSC and 0.5 mM EDTA, p H 7.4, After denaturation an aliquot of similarly denatured 14C-uridine-labeled 42 S viral RNA was added and the mixture was fraetionated b y sucrose density gradient eentrifugation (6 hours, 40,000 rpm, 2° C, SW41 Spineo rotor, linear l0 to 30 per cent sucrose gradient in 50 m ~ KC1, 10 m~r t~2-acetate, 0.5 mM EDTA, pI-I 5.2). Acid precipitable radioactivity was determined in each fraction . - - . - - . aI-I-uridine radioactivity; . - - . - - . 14C-uridine radioactivity Part B: Axmther aliquot of the denatured 3t{-uridine-Iabeled 19S ds I~NA, which was fractionated on the gradient presented in part A of the Figure, was fractionated on art identical gradient without addition of 14C-labeled marker RNA. The labeled 42 S t~NA was isolated from this gradient and analyzed for the presence of 42 S minus strand RNA as follows: 20 B1 aliquots of the RNA were mixed with an at least twenty fold excess of unlabeled plus strand I~NA isolated from SFV particles and hybridized for different lengths of time at 80 ° C in 1 × SSC. Acid precipitable radioactivity was then determined without ( . - - . - - . ) or after (A---~--A) l~Nase digestion (10 ~g/ml l~Nase A in 2 × SSC, 30 minutes, 37 ° C)

adenosine t h a n uridine (7) the c o m p l e m e n t a r y R N A contains more uridine t h a n the plus strand. This possibly explains w h y i n this e x p e r i m e n t slightly more t h a n 50 per cent of the 8H-uridine is i n c o r p o r a t e d into hybrids. The b e h a v i o u r of the 19S ds R N A d u r i n g c h r o m a t o g r a p h y on oligo-dT cellulose is shown i n Figure 5. Only a b o u t 50 per cent of the m a t e r i a l b i n d s to oligo-dT cellulose at 500 mM NaC1. Since the i n t a c t plus s t r a n d R N A f o u n d in infected cells a n d in S F V particles is infectious (5), it is possible to d e t e r m i n e the presence of this R N A b y i n f e c t i v i t y assay. Therefore the i n f e c t i v i t y of the 19S ds R N A has been m e a s u r e d before a n d after d e n a t u r a t i o n a n d compared to the i n f e c t i v i t y of 42S plus s t r a n d isolated from S F V particles. The results are presented i n Table 1. D e n a t u r e d double s t r a n d e d 19 S R N A has somewhat less t h a n half the specific i n f e c t i v i t y of d e n a t u r e d single s t r a n d e d plus s t r a n d R N A . U n d e n a t u r e d 19 S ds R N A was n o t infectious. These results are discussed i n detail i n the n e x t section.

Double Stranded t%NA from Semliki Forest Virus Infected Cells

51

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Fig. 5. Oligo-dT cellulose chromatography of 19 S ds tgNA 3tt-uridine-labeled 19 S ds R N A was isolated as shown in Figure 1 A. Oligo-dT cellulose chromatography was performed as described in the MateriMs and Methods section.. Part A: Chromatography of 19S ds t%NA. Parts B and C: l%echromatography of the material not binding at 500 msz NaC1 (B) and of the material Muted at 100 msi NaC1 during the chromatography shown in part A. TotM 3tt-uridine radioactivity was determined in an Miquot of each fraction as described in the legend to Figure I Table 1. Speci]ic in]ectivity o/viral R2%4 and 19S ds R N A I%NA tested (specific infectivities are presented as number of plaques produced by 10.D.260 unit of the I%NA indicated) Assay system

denatured b denatured b 42S viral I%NA 42S viral I%NA 19S dst%NA0, a 19S ds B N A

D E A E - d e x t r a n system~ I{ypertonic system~

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