Eur. J. Biochem. 64. 167-175 (1976)

Simultaneous Translation of Structural and Nonstructural Proteins from Semliki-Forest-Virus RNA in Two Eukaryotic Systems in vitro Niall GLANVILLE, John MORSER, Pertti UOMALA, and Leevi KAARIAINEN

Department of Virology, University of Helsinki (Received August 18, 197SiJanuary 9, 1976)

The Semliki Forest virus genome, 42-S RNA, and the virus-specific intracellular 26-S RNA were translated in two cell-free protein-synthesising systems, the wheat germ extract, and a partially purified system from mammalian tissues. The 26-S RNA directed the synthesis of structural proteins only, as revealed by tryptic peptide mapping. About 75 - 80 of the radioactivity in the products comigrated with capsid and about 4- 8 with envelope protein peptides. All the capsid peptides and the full-sized capsid protein were found in the products in vitro, whereas no complete envelope protein was formed and fewer than half of the envelope peptides were detected. This result is consistent with reports that there is only one initiation site for the translation of virus structural proteins, and that the capsid protein is N-terminal in the polyprotein followed by envelope proteins. The systems programmed with 42-S RNA yielded virtually the same structural peptides. However, the bulk of the radioactivity was in peptides which did not comigrate with the structural ones. These peptides were mostly associated with relatively small-sized products. This shows that Semliki Forest virus 42-S RNA has at least two initiation sites, one for the structural proteins and the other(s) for the nonstructural proteins.

Alphaviruses, which are lipid-containing RNA viruses, provide a model for the study of the expression of identical genes in RNA molecules of different sizes. In cells infected with Semliki Forest virus (SF virus) there are two major messenger RNA species, the viral genome, 42-S RNA (molecular weight 4 x lo6) and 26-S RNA (molecular weight 1.6 x lo6) [I - 31. The latter is a replica of about one third of the 42-S RNA [4] and is found only in the infected cells [ 5 ] . Recent translation of the 26-5 RNA in vitvo has shown that it is a duplicate of nucleotide sequences coding for the virion capsid and envelope, i.e. structural proteins [6,7]. This RNA is, however, unable to start the infection. For this process the additional coding capacity of the 42-S RNA is needed [5]. At present the products of the nonstructural part of the genome are unknown. Genetic evidence ob-~ __Ahhreviations. SF virus, Semliki Forest virus; p.f.u., plaqueforming units; BHK2l cells, cell line from baby hamster kidneys; sulphonate. Hepes, N-2-hydroxyethylpiperazine-N’-2-ethane

tained from complementation of temperature-sensitive RNA- mutants of Sindbis virus (which are unable to synthesise RNA at the restrictive temperature) [8,9] suggest that at least two, perhaps three, nonstructural proteins are needed for the viral replication. These proteins are only needed early in infection [8,10] but it is difficult to identify them by amino acid labelling because host cell proteins are still actively synthesised. Later in infection, when host protein synthesis is effectively inhibited, mostly structural proteins are detected [ l l ,121, and the synthesis of nonstructural proteins is evidently shut off. Thus translation of the 42-S RNA in vitr-o offers one way of identifying the nonstructural proteins. Here we report the translation of the 42-S RNA in two different cell-free protein-synthesising systems. Tryptic peptides of the products are coinpared with those derived from the virion structural proteins : most of these peptides are shown to be nonstructural, but structural proteins are also synthesised.

168

MATERIALS AND METHODS Isolation of S F Virus 4 2 3 R N A

SF virus, grown in BHK21 cells (baby hamster kidney cell line), was concentrated by vacuum dialysis and purified by two successive density bandings in potassium tartrate (5 to 50 w/v) gradients as described previously [13]. The RNA was released with 2 sodium dodecyl sulphate and isolated by centrifugation in a linear 15% to 30% (w/w) sucrose gradient made in 0.1 M NaCl, 0.05 M Tris (pH 7.4), 0.001 M EDTA, containing 0.1 % sodium dodecyl sulphate. Centrifugation was for 10 h at 24000 rev./min in a Spinco SW27 rotor at 23 "C. The fractions containing 42-S RNA were pooled and the RNA precipitated twice with ethanol and dissolved in distilled water at a concentration of 1 mg/ml. The A,,o/A,80 ratio of this preparation was 2: 1 ; it was stored at - 70 "C. Isolation of 26-S R N A

BHK21 cells grown in 1-1 glass bottles were infected with SF virus at 30 p.f.u./cell. After 60-min absorption, the inoculum was removed and the cells were washed ; Eagles minimum essential medium, 0.2 % bovine serum albumin (Armour) and 0.2 yg/ml actinomycin D (Merck, Sharp & Dohme) were added. Some of the cells were labelled with [3H]uridine (500 yCij50 x lo6 cells, spec. act. 30 Ci/mmol, from the Radiochemical Centre, Amersham) between 3 and 6 h post-infection. At 6 h the cells were washed with cold phosphate-buffered saline and thereafter with a buffer consisting of 0.01 M NaCl, 0.01 M Tris (pH 7.4), 1.5 mM MgCI, and 20 mg/ml polyvinylsulphate. After 3 min swelling the cells were scraped into a small volume of the same buffer and broken in a glass Dounce homogeniser. Nuclei were removed by centrifugation at 250 x g for 5 min at 0 "C and the cytoplasm made immediately 2 % with respect to sodium dodecyl sulphate. The cytoplasmic extract was centrifuged in a 15% to 30% (w/w) sucrose gradient for 10 h under the same conditions as the viral 42-S RNA. The 26-S RNA in the gradient was concentrated by ethanol precipitation and resedimented for 20 h in a similar gradient, but without sodium dodecyl sulphate. The 26-S RNA peak was precipitated twice with ethanol and the pellet was dissolved in distilled water at a final concentration of about 1 mg/ml and stored at -70 "C. The second centrifugation resulted in a 6-fold purification of the 26-S RNA as determined from the change in radioactivity/mass ratio. The proportion of viral 2 6 3 RNA is not contaminated to a detectable degree by host cell mRNA [I]. By assuming that the 42-S and 26-S RNAs had the same specific activities, the 26-S RNA : cellular RNA ratio was calculated to be 1:3.5, the contamination being, presumably, largely ribosomal

Translation of Semliki-Forest-Virus mRNAs in vitro

28-S RNA. The preparation stimulated only the synthesis of viral structural proteins, and no further purification was considered necessary. Wheat Germ Cell-Free System

Extracts were prepared and preincubated as described [14]. Fresh commercial wheat germ was the kind gift of Mr Lamminmaki of Raision Vehnamylly. The ability of the extract post-mitochondria1 supernatant) to support protein synthesis was tested with poly(U) (Boehringer). Standard reactions in vitro contained, per 100 pl, 40 yl preincubated extract and 2-4 pg mRNA. In addition the system contained 19 amino acids each 50 pM, 10 pCi ~-[~'S]methionine (spec. act. 170 Ci/ mmol), 0.2 mg creatine phosphate, 0.02 mg creatine phosphate kinase, 1 mM ATP, 0.1 mM GTP, 20 mM Hepes (Sigma, adjusted to pH 7.6 with KOH), 3 mM magnesium acetate (uncorrected for the chelating activities of ATP and GTP) and 6 mM 2-mercaptoethanol. For the 42-S RNA incubations the KCl concentration was 60 mM while for the 26-S RNA it was 110 mM. Incubation was at 30 "C for 30 min. Partially Purified Cell-Free System

The partially purified cell-free protein-synthesising system was prepared according to the methods described previously [15]. All components of the system (ribosomal subunits from mouse liver, pH-5 enzymes from rat liver and initiation factors derived from rabbit reticulocyte ribosomes by washing with 0.5 M KCl) were stored under liquid nitrogen. The ability of the system to support protein synthesis was tested with poly(U). Standard reactions in vitro contained, per 100 pl, 10 units ribosomal subunits, 25 p1 pH-5 enzymes, 8 yl crude initiation factors and 2 pg mRNA. In addition the system contained 19 amino acids each 50 yM, 10 pCi ~ - [ ~ ~ S ] me th io n(spec. in e act. 170 Ci/ mmol) 0.2 mg creatine phosphate, 0.02 mg creatine phosphate kinase, 1 mM ATP, 0.1 mM GTP and 20 mM Tris (pH 7.6), 130 mM KCl and 6 mM 2mercaptoethanol. For the 42-S RNA incubations the magnesium acetate concentration was 3 mM while for the 26-S RNA it was 4 mM. The reaction mixture was incubated at 34 "C for 30 min. The ionic conditions of all the standard reactions and the period of incubation were optimised to give maximum hot-acid-insoluble radioactivity (5 "/, trichloroacetic acid, 20 min at 90 "C) in both systems for the 42-S and 26-S RNA [16]. Isolation of Labelled SF Virus Proteins

BHK21 cells grown and infected as above, were labelled with ~-[~'S]methionine (500 pCi/30 x lo6 cells)

N. Glanville, J . Morser, P. Uomala, and L. Kaariainen

169

from 3 to 10 h post-infection. The released virus was purified in a discontinuous velocity-density gradient as described before [17]. The nucleocapsid and envelope fractions were separated by sucrose gradient centrifugation after disruption of the virus with Triton X-100 as described previously [18]. The nucleocapsid and envelope peaks were pooled separately and 3 mg of bovine serum albumin was added to each pool as carrier. The proteins were extracted with phenol [19]. They were lyophilized and prepared for tryptic peptide analysis as described below. Acrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that the nucleocapsid and envelope fractions were not cross-contaminated. The ts-3-induced common structural precursor protein with a molecular weight of 130000 [12] was isolated as described by Lachmi et al. [20]. Polyacrylamide Gel Electrophoresis The reactions in vitro were terminated by addition of pancreatic RNAse to 10 pg/ml (Worthington) and EDTA to 5 0 m M and incubation at 37°C for 10 min. Reaction mixtures were then made 1 with respect to both sodium dodecyl sulphate and 2-mercaptoethanol, heated for 2-3 min at 90 "C and dialysed against 0.01 M sodium phosphate buffer (pH 7.0) containing 0.1 sodium dodecyl sulphate and 0.1 mercaptoethanol. Electrophoresis was in 7.5 % dodecylsulphate-polyacrylamide gels [21]. [3H]Leucine/isoleucine labelled SF virus proteins were included as internal markers in every gel. The gels were cut into 2-mni slices and counted in NCS/ toluene-based scintillator [22]. Trjytic Peptide Analysis

The reactions were terminated with RNAase and EDTA and the samples precipitated with 5 % trichloroacetic acid. Following performic acid oxidation, tryptic digestion was performed as described earlier 1161. High-voltage paper electrophoresis was on Whatman 3MM paper, in the first dimension at pH 6.5 and 40V/cm for 90min or 120min. The bands were located by autoradiography, cut and sewn onto a new, similar paper which was electrophoresed at pH 3.5 and 60 V/cm for 45 min or 90 min in the same dimension. In all runs 14C-labelled amino acids and [3'S]methionine were included as mobility markers. Papers were autoradiographed using Kodirex or R P 14 films (Kodak). To quantitise the peptides the bands in each sample track (one track corresponds to one sample) were cut and counted in toluene/Permabland scintillator in a Packard Tricarb 3003 at an efficiency of 95 '%,.

[Mg'] (mMI [K'] ( mM 1 Fig. 1. Mg2 arid K+ concentration C U ~ I ' E S f o r tlle Jructionutcd system. Incubations were for 30 rnin at 34 "C. 42-S RNA (A, B) or 26-S RNA (C,D) were used in a final concentration of 20 pglml. In (A) and (C) the K + concentration was 130 mM. Mgz+ concentrations were 3 mM in (B) and 4 mM in (D). Exogenous RNA added (0 --a); no added RNA ( O . - . . . . O ) +

RESULTS In an attempt to characterise the nonstructural products translated from 42-S RNA, we selected two cell-free protein-synthesising systems with low levels of endogenous protein synthesis. This should allow unequivocal identification of the nonstructural proteins by tryptic peptide mapping. The systems used were the wheat germ cell-free extract described by Roberts and Paterson [14] and the partially purified (fractionated) system developed by Schreier and Staehelin [15]. Conditions of Cell-Free Protein Synthesis

As a control for the systems we used the virusspecific intracellular 26-S RNA, which has previously been translated into structural proteins [6,7,23,24]. The ionic conditions resulting in maximum stimulation by the added RNAs are shown in Fig. 1 and 2. From these curves we selected the standard ionic conditions given in Materials and Methods. The systems were also optimised with regard to the amount of added RNA, time and temperature of incubation (data not shown). Fig. 1 and 2 also reveal the levels of stimulation above background which were achieved with each RNA. With the 26-S RNA stimulation was

170

Translation ofSemliki-Forest-Virus iiiRNAs in sitro

B

I

Li

10

1000

-c 'E

3C

20 0'.*

.-...

3

8

Q..

I

"0

W

.-C C .Q

D

2A 0 I

.... .... 0 I

50 70

1500

5

3 r-l

v,

m Y)

u

1000 x m I

0,

-._c

500

. E

r v1 3

8 I

w

c

s

._

r

-2

ln 9 1000

500

""9.0.

90 110 1E 500

Fig.2. M $ + and I(+ conwnirution curves ,for rhe isheuf germ extruct. Incubations were for 30 min at 30 ' C. 42-S R N A (A, B) or 2 6 3 R N A (C,D) were used in a final concentration of 20 pg/ml. K + concentration was 60 mM in (A) and I10 mM in (C); Mg2+ concentration was 3 mM in both (B) and (D). Exogenous RNA no added RNA ( O . . . . . . O ) added (@-@);

Table 1 . Percentage disirihution of [3iS]meihionine umong s i x clusses qfproteins synthesised in vitro and unulyzed in polyucrylumide gels

Results are expressed as a percentage of total ["S]methionine label recovered froin the gel Incubation in iitro

35Sin M , range of -

-

~

>15000

33000"

2000030 000

20000

0

1, ~

Wheat germ extract + 4 2 4 RNA + 2 6 3 RNA

16 0 12 5

79 28 1

23 4 35 4

52 7 24 0

Fractionated system + 42-S RNA + 2 6 3 RNA

99 94

25 20 7

38 0 38 3

49 6 31 6

10

20

30 LO Fraction number

50

60

Fig. 3. Dodecylsulphute-ucrylumide gel electrophoresis. Reactions mixtures including ["S]methionine were incubated under standard conditions. (A) The wheat germ extract programmed with 42-S RNA (40 pg/ml) was incubated for 30 min at 30 ' C , Mg2+ was 3 mM and K + 60 mM. (B) The fractionated system programmed with 26-S R N A (40 pg/ml) was incubated for 30 min at 34 "C. ME'+ was 3 mM and K f 130 mM. Reactions were terminated by addition of RNAase and EDTA and further incubation for 10 min at 37 ' C . The mixtures were heated in the presence of sodium dodecyl sulphate and 2-mercaptoethanol and dialysed against electrophoresis buKer; electrophoresis was on continuous 7.5 ?; acrylamide gels. [3H]Leucine/isoleucine-labelledvirion proteins were included as internal markers in each gel. The gels were sliced and counted in NCS-based scintillator. Proteins synthesised in vitro (@ ; marker d virion )proteins, i.e. capsid molecular weight 33000(C) andenvelopeproteins(E)about 5 0 0 0 0 ( 0 ~ . . . . ~ 0 ) ; arrows indicate position of bromphenol blue, migrating from left to right

Size Range of Products * Capsid

10-20-fold, while with the 42-5 RNA it was 2-5fold, in both systems. When the contamination of the 26-S RNA preparation with ribosomal RNA (see Materials and Methods) is taken into account, this difference in activity becomes even more pronounced.

Aliquots were taken from protein-synthesising reaction mixtures containing [35S]methionine, which had been incubated for 30 min. The size range of the products synthesised was determined by electrophoresis on dodecylsulphate-polyacrylamide gels. [3H]Leucine/isoleucine-labelled SF virus proteins were included in each gel as internal markers. The 26-S RNA products contained a clear peak comigrating with the

N. Glanville. I . Morser, P. Uomala, and L. Kiiiiriiiinen

REFERENCES

WG

42 S

26s

171

-

E

130

C

FS

-

26s

42s

Fig. 4. T r y p i c peptides e.keciropl7oresedu t p H 6.5. A portion of'the incubations analysed in Fig. 3 and quantitised in Table 1 was precipitated, washed, oxidised and digested with trypsin. Samples of capsid and unfractionated envelope proteins isolated from the virion, and their common precursor protein (from ts-3 mutant infected cells). all labelled with [35S]methionine,were digested under similar conditions and included as references. The digests were applied to Whatman 3MM paper along 2-3-cm lines and electrophoresed at pH 6.5. Wheat germ extract (WG): 42-S RNA added (42s); 26-S RNA added (26s); no added R N A ( - ) . Reference digests (References): envelope (E); precursor protein (130); capsid (C). Fractionated system (FS); no added R N A (-): 26-S RNA added (26s); 42-S RNA added (42s). Origin at 13; cathode at top; electrophoresis at 40 Vlcm for 120 min. Autoradiography for 3 days. Numbered bands were analysed further, at pH 3.5 (see Fig. 5 )

marker capsid protein (molecular weight 33000) as shown for the fractionated system in Fig. 3 B. The percentage distribution of radioactivity in the different size classes of products directed by the 26-S RNA, in both systems, is given in Table 1. The products of the 42-S RNA were more heterogenous in size (Fig. 3A). No readily identifiable peak at the position of the capsid protein was present and

more small-sized material was synthesised than in the 26-S-RNA-directed products (Table 1). Tryptic Peptide Analysis Tryptic peptides were prepared from the same material which was analysed on gels. The digested samples were analysed using high-voltage paper electro-

172

Translation of Semliki-Forest-Virus mRNAs in vitro

A

B REFERENCES

WG

42s 2 6 s C

-

E 130 C

-

FS

2 6 s L2S

REFERENCES

WG

42s 26s

-

E

130 C

-

FS

26s 42s

D

Fig. 5. Electrophoresis in the second dimension of'somr tryptic pepptides. Peptide hands numbered in Fig. 4 were cut from the paper, sewn onto another similar paper and re-electrophoresed at pH 3.5. Band 3 in Fig.4 is shown in (A), band 4 in (B), band 11 in (C) and band 14 in (D). Sample tracks are labelled as in Fig. 4. Peptides quantitated are indicated with letters. Electrophoresis was for 45 min (A and B), or 90 min (C and D), at 60 V/cm. Arrows indicate origins, cathode at top. Autoradiography Tor 14-21 days

phoresis, first at pH 6.5 and thereafter at pH 3.5. For comparison digests of virion-derived capsid and envelope proteins were included. As a further control, a tryptic digest of the common precursor of capsid and envelope proteins was used. This protein was isolated from our SF virus ts-3 mutant-infected cells, and has a molecular weight of 130000 [12]. Samples applied to Whatman 3MM paper were electrophoresed at pH 6.5. The peptide bands were visualised by autoradiography and cut parallel to the origin line (the bands cut are numbered in Fig.4). They were then sewn onto a second paper and reelectrophoresed at pH 3.5. This procedure simplifies the identification of comigrating peptides, since those to be compared are

run on the same paper, under the same conditions [16]. Comigration in two dimensions was taken as the criterion for identity of the peptides. Examples of peptides comigrating with those from the capsid protein are shown in Fig.5C,D; some peptides comigrating with virion-derived envelope peptides were also detected in the products in vitvo (Fig. 5 D). As can be seen in Fig. 5 B (peptides b and c), not all the peptides from capsid and envelope were separated under the conditions used. To quantitise the radioactivity in each sample tract the peptides migrating as clear, well-defined bands in two dimensions (e.g. Fig.5D peptides a to f, were cut from the paper and their radio-

N. Glanville, J. Morser, P. Uomala, and L. KBBriHinen

173

Table 2. Percentage disiribuiion of [35S]methionine radioactivity arnonR tryptic peptides derived,from products svnthesised in vitro The columns headed ‘peptides’ give the number major peptides detected. The radioactivity is expressed as a percentage of total migrating as bands in each sample track. The maximum number of nonstructural peptides detected was 18. Of the radioactivity applied, the percentage migrating in bands was: (a) 46.6‘j/,, (b) 49.7%, (c) 32.2%, (d) 70.2”/,, (e) 75.1 System

RNA

Peptides comigrating with those derived from virion ~~~

~~

capsid

envelope

not resolved

peptides radio(max. 9) activity

peptides radio(max. 16) activity

peptides

;4

Yo a) b) c) d) e)

Fractionated system Wheat germ extract Fractionated system Wheat germ extract expt 1 Wheat germ extract expt 2

263 26-S 42-S 42-S 42-S

9 9 9 9 9

74.2 79.0 32.9 18.7 21.3

activities determined by liquid scintillation counting. Those bands which were also found in the incubation without added RNA were excluded, as were ‘smeary’ peptides and those remaining at the origins.

Analysis of 26-S R N A Products The complete analysis of the translational products of the 26-S RNA in the two systems in vitro revealed that all [35S]methionine-labelledtryptic peptides found in the capsid protein were also synthesised in vitro (Table 2). This result indicates that translation of the 26-S RNA was in frame in both the systems in vitro. Under the conditions used, the translational capacity of the systems was limited as shown by the incompleteness of translation of the envelope proteins. The quantitative analysis showed that the bulk of the radioacitivy migrating as bands was confined to capsid peptides (Table 2). The polyacrylamide gel electrophoresis of the 26-S RNA products revealed that 20 - 30 of the material comigrated with the virion capsid protein (Table 1).We eluted this material from a slab gel and the [35S]methionine-labelled tryptic peptides from it were compared with those derived from capsid and envelope proteins after electrophoresis at pH 6.5. The peptide bands were cut and quantitised by liquid scintillation counting. From these measurements we estimated that more than 60 of the material consisted of capsid peptides. However, some envelope peptides distinguishable already in the first dimension (Fig.4, peptides 20 and 22) were also detected. Similarly, the peak with a molecular weight of about 28 000 (Fig. 3) was analyzed for its tryptic peptides. Again the bulk of the peptides were of capsid specificity but envelope peptides were also detected (data not shown).

(x

___________

~~~~~

4 7 4 7 10

4.6 7.5 5.7 6.4 9.7

radioactivity

Nonstructural peptides

peptides radio(max. 18) activity I,

/”

:4 2 2 1 1 2

12.0 4.6 3.2 2.7 3.5

1 3 7 18 16

I

9.2 8.9 57.7 72.2 65.5

Analysis of the 42-S R N A Products Virtually the same capsid and envelope protein peptides found in the 2 6 3 RNA products were present also in the 42-S-RNA-directed products. All the capsid peptides were found in both systems but fewer envelope peptides were synthesised in the fractionated system than in the wheat germ extract (Table 2). The capsid-peptide/envelope-peptide radioactivity ratio was, however, smaller in the 42-S RNA products than in the 26-S RNA products. The major difference between the products of the 42-S and 26-S RNAs lies in the number of nonstructural peptides, e . g . peptides a, b and c in Fig.5A. Altogether 18 nonstructural peptides were detected in the wheat germ extract programmed with 42-S RNA (Table 2). Interestingly most of the radioactivity was confined to five peptides (Table 3) which represented close to 50% of the total radioactivity migrating as bands. Another analysis of the products synthesised in the wheat germ extract programmed with a different batch of 4 2 3 RNA confirmed the reproducibility of the nonstructural peptides. The first dimension of the analysis is shown in Fig.6 together with second dimension electrophoresis of the characteristic nonstructural peptides. A summary of the complete quantitisation is presented in Tables 2 and 3. Material migrating with the peak at a molecular weight of about 28000 (see Fig. 3A) in the 4 2 3 RNA product was eluted from a slab gel and the tryptic peptides analysed (data not shown). This material contained capsid peptides together with the nonstructural peptide bands 3 , 8, 18 (cf. Fig.4 and 6A). The result shows that the most intensively synthesised nonstructural peptides are associated with proteins having a molecular weight close to 30000, suggesting that they are not peptides carrying the N-terminal initiator methionine [25].

Translation o f Semliki-Forest-Virus mRNAs in 1itr.o

174

E

C

+

+

-

C

+

E

C E

+

C

E

+

C

E

Fig. 6. Second analysis of tryptic peptides ,fi.om 42-S-RNA-directed product svntkrsised in whaar germ extract. The [35S]methionine-labelled tryptic peptides were separated initially at pH 6.5 (A). The second dimension analysis (at pH 3.5) of peptides 3, 8, 18 and 20 (the major nonstructnral peptides) are shown in (B), (C), (D) and (E), respectively. The same code assigned to the peptides in the previous analysis (see Fig.4 and 5) was used here. Sample tracks: capsid (C), envelope (E), extract programmed with 42-S RNA (+), control incubation (-). Electrophoresis was for 120 min at pH 6.5 and subsequently for 90 min and 60 Vlcm at pH 3.5 (B- E). Arrows indicate origins, cathode at top. Autoradiography was for 4 days (A) and 14-21 days (B-E)

Table 3. Distrihution of' radioactivity mnong muior nonsttuctural tryptic peptides derived,frorn 4 2 3 R N A products Radioactivity is expressed as a percentage of total migrating as bands in each sample track (see text) Peptide code

Radioactivity in .~

~~

~-

wheat germ extract ~-

.~

Expt 1

Expt 2

fractionated system

10 ~.

~~~

18 b 3a 20 g 8a 3b Others

18.9 11.0 7.1 6.0 5.3 23.9"

15.7 17.4 9.4 7.2 4.7 11.1

~~

15.8 15.4 9.1 3.6 5.7 8.1

Maximally 18 nonstructural peptides were detected (see Table 2).

DISCUSSION Here we report translation of the Semliki Forest virus genome, 42-S RNA, and the 26-S RNA isolated

from infected cells in a wheat germ cell-free extract and in a partially purified system from mammalian tissues. Analysis of [35S]methionine-labelled tryptic peptides from the translational product of 26-S RNA revealed that in both systems in vitvo mostly capsid protein was synthesised. About a third of the capsid protein peptides were in the full-sized protein while the rest were evidently derived from smaller incomplete capsid polypeptides. The number of envelope peptides in the products varied between four and seven out of 16 [30,31] showing that the envelope proteins were not completely translated. The capsid-peptide/envelope-peptide radioactivity ratio of 10-20: 1 (Table 2) is in good agreement with recent reports that all the structural proteins are translated as a polyprotein [6,12,20, 23,261 and that capsid protein is N-terminal in this polyprotein [32]. Since complete translation of capsid protein is necessary before the translation of the envelope proteins can take place, one would expect envelope peptides to be found only in material larger than the capsid protein. However, we detected envelope peptides in proteins with molecular weights from 25 000

N. Glanville, J. Morser, P. Uomala, and L. KBiiriiinen

to 35000. A plausible explanation for this finding would be the cleavage of completed capsid protein from the growing polypeptide chain [32]. This same cleavage evidently takes place in other systems as well [6,7,24,33]. If the cleavage of the capsid protein in vitro is taken into account, the presence of envelope peptides in the eluted material with molecular weights of 25 000 to 35 000 indicates that polypeptides with molecular weights up to 60000 to 70000 were synthesised. This means an elongation rate of 20-23 amino acids per min, which is comparable to that reported for the Krebs ascites cell-free system [34]. The limited translational capacity of our systems has to be remembered when the results of the translation of the 42-S RNA is considered. The high proportion of capsid peptides compared to the envelope peptides resembles the result obtained with the 26-S RNA, i.e. translation of N-terminal capsid protein prior to envelope proteins. Because about half of the envelope peptides were not detectable, only small amounts, if any, of the full-sized polyprotein is synthesised. Thus ribosomes starting from the initiation site of the structural polyprotein cannot possibly continue to translate the nonstructural sequences of the 42-S RNA. The same applies if the order of the sequences for the structural and nonstructural proteins in the 42-S RNA is reversed as indicated by the recent work of S. I. T. Kennedy (personal communication). The nonstructural peptides in the 42-SRNA-directed product must therefore arise from a different initiation site or sites from that for the translation of the structural proteins. In this respect the amplification of the structural genes in SF-virus-infected cells is analogous to the situation in brome mosaic virus infection [35]. One of the brome mosaic virus RNAs (RNA 3) has two cistrons : one codes for a nonstructural protein, the other, which is equivalent to RNA 4, codes for the virus coat protein. Both cistrons of RNA 3 are translated in the wheat germ cell-free system but the nonstructural protein is the major product. The RNA 4 yields only coat protein and is an extremely efficient messenger, as is the SF virus 26-S RNA [7,241. We have recently found two nonstructural proteins with molecular weights of 78000 and 86000 in cells infected with a temperature-sensitive mutant of SF virus [20]. Comparison of the tryptic peptides from the 42-S RNA product made in vitro with those from the nonstructural protein is now in progress. We thank Mrs Mirja Langer and Miss Ritva Saarlemo for excellent technical assistance. This work was supported by grants

175 from the Sigrid Juselius Foundation and the Finnish Academy; one of us (J. M.) was the recipient of an EMBO fellowship.

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N. Glanville, P. Uomala, and L. KPLriPinen, lnstitutum Virologicum Universitatis, Haartmaninkatu 3, SF-00290 Helsinki 29, Finland J. Morser, Department of Biological Sciences, University of Warwick, Coventry, Warwickshire, Great Britain, CV4 7AL