Biosynthesis of reovirus-specified polypeptides 2Aminopurine increases the efficiency of translation...

VIROLOGY 147, 9-18 (19%)

Biosynthesis of Reovirus-Specified Polypeptides

The sl mRNA Synthesized in Vivo Is Structurally and Functionally Indistinguishable from

in Vitro-Synthesized sl mRNA and Encodes Two Polypeptides, ula and (TlbNs

BERTRAM L. JACOBS, JONATHAN A. ATWATER, SUSAN M. MUNEMITSU, AND CHARLES E. SAMUEL’

Section of Biochemistry and Molecular Biology, Department of Biological Sciences, University of Califmnia, Santa Barbara, Califwnia 93106

Received January 28, 1985; accepted July 1, 1985

The structural and functional properties of the reovirus serotype 1 (Lang strain) sl mRNA were examined. Reovirus s-class mRNAs, synthesized either in vivo within infected mouse L cells or in vitro by chymotrypsin-derived cores of purified virions, were purified by filter-hybridization using cDNA clones of the S-class genome segments. Sl cDNA- selected mRNA encoded the synthesis of the M, - 12,000 nonstructural polypeptide designated ~~~~~ in addition to the well-established structural polypeptide ol, now designated (TV=. The coding properties of in vivo- and in vitro-synthesized sl mRNA were equivalent: both encoded ala and ~lb~s. Primer extension analysis of sl mRNA revealed a single major 5’ terminus for both in vivo- and in vitro-synthesized sl mRNA. These results suggest that there is a single transcript of the reovirus Sl genome segment which is functionally dicistronic, and likely encodes both bla and ~J~~NS. o 1985 Academic press. IN.

INTRODUCTION

The genome of reovirus consists of ten segments of double-stranded (ds)’ RNA, each of which is transcribed into mRNA by a virion-associated dsRNA-dependent RNA polymerase (Joklik, 1984). It had previously been thought that each reovirus genome segment encoded a single primary translation product (Zweerink et al, 1971; McCrae and Joklik, 1978; Mustoe et ak, 1978; Levin and Samuel, 1980). However, in vitro studies of the protection by 80s ribosomes of reovirus mRNA against degradation by RNase (Kozak, 1982) and the formation of methionyl-X initiation dipeptides (Cena- tiempo et al., 1984) have indicated that the sl mRNA may be dicistronic, because ribosomes protect two sites on sl mRNA and because sl mRNA yields two methionyl-X

’ Author to whom requests for reprints should be addressed.

2 Abbreviations used: AMV, avian myeloblastosis virus; dsRNA, double-stranded RNA, NaDodSO,, so- dium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

dipeptides. Furthermore, a low-molecular- weight reovirus-specific primary translation product (M, = 12,000-14,000), synthesized in vivo in reovirus-infected mouse L cells and in vitro in cell-free protein-synthesizing systems programmed with reovirus mRNA has recently been identified (Ernst and Shatkin, 1985; Jacobs and Sam- uel, 1985). The low-molecular-weight polypeptide is not detectable in preparations of purified serotype 1 or 3 reovirions (Ernst and Shatkin, 1985; Jacobs and Samuel, 1985). The sedimentation properties of the Lang strain mRNA encoding the M, - 12,000 polypeptide led us to suggest that it is likely encoded by the reovirus sl mRNA (Jacobs and Samuel, 1985). The sl mRNA (Levin and Samuel, 1980) and the Sl dsRNA segment (McCrae and Joklik, 1978; Mustoe et aZ., 1978) have previously been shown to code for the M, - 49,000 polypeptide designated ul. Complete nucleotide sequence analysis of the Lang strain Sl gene reveals a long open reading frame of sufficient length to encode al; this open reading frame overlaps with a second

9 0042-6822/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

10 JACOBS ET AL

short open reading frame with the poten- tial to code for a 119-amino acid polypeptide” (Cashdollar et ab, 1985). We have proposed that the newly characterized iVlr 12,000-14,000 polypeptide be designated ClbNS, and that the well-established n1 polypeptide be renamed ula (Jacobs and Samuel, 1985).

We report here that reovirus serotype 1 Lang strain sl mRNA, synthesized either iv uitm bv reovirus cores or in Vito within reovirus-infected cells and purified by hybridization to cDNA clones of the reovirus serotype 1 Sl gene, indeed directs the synthesis of both flla and (TlbNs in a mouse L cell-free protein synthesizing system. sl mRNA purified from reovirus-infected cells and translated is, cifro yielded the same relative amounts of ala and fll),Ns as sl mRNA purified from in wife core-catalyzed transcription reactions. Mapping of the 5’- end of either in viva or in vifro synthesized Sl gene transcripts by primer extension indicated that only a single sl mRNA transcript exists. These results demonstrate that the single transcript of the Sl gene is functionally dicistronic, both irr lli~~~ and in kfro.

MATERIALS AND METHODS

Muterids. [““S]Methionine (translation grade) and en”Hance were from New En- gland Nuclear. Nitrocellulose (HAWP) was from Millipore. Bovine liver tRNA (type XI) was from Sigma. Capped and methylated reovirus mRNA was synthesized irr 17lfro from virion-derived cores as previously described (Skehel and Joklik, 1969; Levin and Samuel, 1977). Total cytoplasmic RNA was isolated from mouse L cells as described by Kingsman and Samuel (1980), except that the RNA was further purified by LiCl precipitation. Restriction endo- nuclease &oRI was from BRL; HindIII, Z’oqI, and HirLfI were from New England Biolabs. AMV reverse transcriptase was from Life Sciences, Inc.; polynucleotide kinase and calf intestine phosphatase were

a J. A. Atwater, S. M. Munemitsu, and C. E. Samuel, unpublished results.

from Boehringer Mannheim Biochemicals. [y-“‘P]ATP (3000 Ci/mmol) was prepared as described by Maxam and Gilbert (1980).

Cells CX?U! virus. Mouse L cells were main- tained as static monolayer cultures in Ea- gle’s minimum essential medium (MEM) supplemented with 5% fetal calf serum as previously described (Munemitsu and Samuel, 1984). Working stocks and purified virion preparations of the Lang strain of reovirus serotype 1 and the Dearing strain of reovirus serotype 3 were prepared as previously described (Smith ef uI., 1969; Munemitsu and Samuel, 1984).

Hybrirl-seb~>~tiolr of’ wo~irws M RNA with cI)NA. The individual s-class reovirus mRNAs were purified by hybrid selection to cDNA clones representing each of the four S-class genome segments of the Lang strain. cDNA synthesis was carried out using gradient-purified s-class mRNA as a template for first-strand cDNA synthesis; cloning was performed with phage M13mp9, and rccombinants derived from colorless plaques were screened by North- ern hybridization analysis with gel-fractionated genomc dsRNA.” Clone 50 was identified as representative of genome segment Sl, clone 4 of genome segment S2, clones 40 and 64 of genome segment $3, and clone 60 of genome segment S4.”

Two to ten micrograms of reovirus cDNA, from the RF forms of recombinant phage M13mp9 clones, were bound to s-mm pieces of nitrocellulose and used for hybrid selection of reovirus s-class mRNA essentiallv as described by Maniatis cut ~1. (1982). Briefly, DNA was digested to com- pletion with I;=coRI and then denatured by heating to 100” for 10 min followed by incubation at room temperature for 10 min in 0.5 M NaOH at a DNA concentration of 250 &ml. Samples were cooled on ice, neutralized as described, and adsorbed onto nitrocellulose squares at 37”. The filters were washed with 6X SSC, baked, and hy- bridized with 100 &ml unfractionated core-derived reovirus mRNA or 2.5 m&ml total cytoplasmic RNA from reovirus-infected cells in buffer containing 65% for- amide at 50” for 3 hr, as described by Man- iatis et al. (1982), except that tRNA was added to 100 pg/ml. Filters were washed

DICISTRONIC sl mRNA OF REOVIRUS 11

ten times at 60” in isotonic wash buffer containing 0.5% NaDodSOl and three times at 55” for 1 min per wash in 2 mM EDTA, pH 7. When selections were performed at decreased stringency, the 2-mM EDTA wash was at 50”. Specifically bound mRNA was eluted by incubating the filters for 60 set at 100” in 1 mMEDTA, pH 7, containing 100 pg/ml rRNA. Samples were rapidly cooled by immersion into a slurry of dry ice/ethanol, and RNA was collected by ethanol precipitation. The amount of translatable RNA recovered was propor- tional to the number of filters added to the hybridization reaction mixture.

Protein synthesis in vitro. Protein synthesis catalyzed by micrococcal nuclease- treated S-10 extracts prepared from uninfected mouse L cells was performed ex- actly as previously described (Miyamoto and Samuel, 1980; Jacobs and Samuel, 1985).

Gel electrophoresis. Radioactive ?S-labeled polypeptides were analyzed by dis- continuous NaDodSO,-PAGE on 15% polyacrylamide gels and fluorography or autoradiography as previously described (Samuel, 1981; Jacobs and Samuel, 1985). Where indicated, electrophoresis was through a lo-20% polyacrylamide gradient gel. Gradient gels were composed of a 5- ml layer of 10% polyacrylamide over a lo- ml linear lo-20% polyacrylamide gradient.

Primer extension analysis of reovirus sl mRNA. DNA primers corresponding to the TaqI-Hind111 fragment (positions 337-419) and the HindIII-EcoRI fragment (positions 419-503) were isolated from clone 50 DNA, a M13mp9 reovirus clone of Lang strain genome segment Sl. Clone 50, which represents nucleotides 331 to 1458 of genome segment Sl (Cashdollar et ah, 1985), was digested with EcoRI and Hind111 and the digestion products were fractionated on a nondenaturing 12% polyacrylamide gel. The gel was stained with ethidium bromide and fragments of 84 and 95 bp were electroeluted into 3 MNa Acetate, di- luted, and recovered by several precipita- tions from 67% ethanol. The 95-bp fragment was digested with Tag1 to remove vector sequences. The TaqI-digested DNA was purified by extraction with phenol:

chloroform:isoamyl alcohol and recovered by precipitation from 67% ethanol.

The purified TaqI-Hind111 and HindIII- EcoRI reovirus Sl DNA fragments were dephosphorylated by treatment with calf intestine phosphatase according to procedures provided by the supplier. Dephos- phorylated DNA fragments were phos- phorylated using polynucleotide kinase and [T-~P]ATP (3 mCi/ml) according to procedures provided by the supplier. The sp act of the 32P-DNA obtained was approximately lo7 cpm/pg. DNA primer was denatured and annealed to various RNA preparations as described by Berk and Sharp (1978). Annealing reactions contained either 50 ng of primer and 10 pg of reovirus serotype 1 unfractionated mRNA synthesized in vitro by chymotrypsin-derived cores; 10 ng of primer and 25 pg of total cytoplasmic RNA isolated from reovirus-infected L cells; or 10 ng of primer and 1 pg of in vitro-synthesized reovirus serotype 1 mRNA which was recovered from an in vitro translation reaction mixture by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. Nu- cleic acids were isolated from the annealing mixture by ethanol precipitation. Annealed DNA primer was extended with AMY reverse transcriptase essentially as described by Maniatis et al. (1982). Briefly, RNA:DNA hybrids were incubated at 45” for 1 hr with 50 U of reverse transcriptase. The reaction was stopped by addition of NaDodSOl to 0.2% and EDTA to 20 mM. Nucleic acid was purified by extraction with phenol:chloroform:isoamyl alcohol and recovered by precipitation with ethanol. Precipitated material was dissolved in 50 mM NaOH and RNA was hydrolyzed by incubation at 65” for 1 hr. The solution was neutralized and DNA was recovered by precipitation with ethanol. Precipitated material was dissolved in sequencing gel loading buffer (96% formamide, 10 mM NaOH, 10 mM EDTA, 0.1% bromphenol blue, 0.1% xylene cyanol) and fractionated by electrophoresis through a 6% polyacrylamide sequencing gel containing 8 M urea. Radiolabeled products were visualized by autoradiography at -20” with an intensifying screen. Markers were HinfI-digested pBR322

12 JACOBS ; ET AL.

fragments which were radiolabeled using the Klenow fragment of E. coli DNA polymerase I to fill in 5’ overhanging DNA.

RESULTS

Translation of Hybrid-Selected Reovirus Serotype 1 s-Class mRNAs

To determine unequivocally which of the four S-class genome segments codes for mlbNS, and to determine whether other s- class mRNAs encode multiple primary translation products, we purified the individual s-class mRNAs by hybrid selection to cDNA clones representing each of the four S-class genome segments of the Lang strain of reovirus serotype 1. Figure 1

ClbNS- am

a bcdefgh ilkI m

FIG. 1. Translation in vitro of hybrid-selected reovirus serotype 1 s-class mRNA. Individual reovirus serotype 1 s-class mRNAs were purified from in vitro core transcription reaction mixtures by hybrid selection to the RF forms of phage Ml3 clones containing cDNA inserts of the reovirus S-class genes. RNA was added to a micrococcal nuclease-treated L cell-free protein-synthesizing system and the products were analyzed by NaDodSO,-PAGE as described under Materials and Methods. Translations were programmed with the following RNAs: lanes a and m, unfractionated reovirus serotype 1 mRNA; lanes band 1, clone 50 (Sl) hybrid-selected RNA; lanes c and k, clone 4 (SZ) hybrid-selected RNA; lane d, clone 40 (S3) hybrid-selected RNA; lanes e and i, clone 60 (54) hybrid-selected RNA; lanes f and h, phage M13mp9 RF DNA (vector control) hybrid-selected RNA; lane g, no added RNA; and lane j, clone 64 (S3) hybrid-selected RNA. The figure is a fluorogram of a 15% gel.

shows the polypeptides synthesized in L cell-free protein synthesizing systems programmed with individual hybrid-selected reovirus serotype 1 s-class mRNAs. Clone 50 DNA, complementary to the Sl segment, selected for an mRNA population which programmed the synthesis of two primary translation products, nla and clbNs. Clone 50 selected mRNA also programmed the synthesis of a small amount of n3, apparently due to a small amount of the highly efficiently translated s4 mRNA (Levin and Samuel, 1980) which was a contaminant in the clone 50 (Sl) selected sl mRNA. mn,Ns could not have been translated from the contaminating s4 mRNA, because s4 mRNA purified by hybrid selection to clone 60 DNA programmed the synthesis of large amounts of cr3 but did not program the synthesis of any detectable mn,Ns. In fact, (TlbNs was not synthesized in response to mRNA selected by clones complementary to the S2 (clone 4), S3 (clones 40 and 64), and S4 (clone 60) segments which programmed the synthesis of (TV, ens and c3, respectively (Fig. 1). These results demonstrate that both nla and UibNs are indeed coded for by the Sl genome segment of reovirus serotype 1 (Lang strain).

Additional minor polypeptides in the mol wt range 20,000-30,000 were also synthesized ix uitro in protein-synthesizing systems programmed with the serotype 1 Lang strain reovirus mRNA (Fig. 1, lanes a and m). Among the most prominent of these minor polypeptides were those coded for by the s4 mRNA; they were detectable with s4 mRNA synthesized either in vitro by chymotrypsin-derived cores of purified virions (Fig. 1, lanes e and i, Fig. 2, lane b) or i71 ,uivo within reovirus-infected mouse L cells (Fig. 2, lane c). Reovirus serotype 3 s4 mRNA, synthesized in vitro by cores of Dearing strain virions and purified by hybrid selection to serotype 1 clone 60 (S4) DNA at reduced stringency, programmed for the synthesis of similar sized minor polypeptides as did clone 60-selected serotype 1 s4 mRNA (Fig. 2, lanes b and d, respectively). The complete cDNA sequence of the S4 genome segment of Dearing strain serotype 3 reveals a single open reading frame extending 1095 nucleotides


In vivo In vitro

Serotype X’S-

/As-

ulbNS-

abcdef

FIG. 2. Translation in vitro of hybrid-selected reovirus serotype 1 and serotype 3 s4 mRNA. s4 mRNA was purified from in vitro serotype 1 and 3 core-catalyzed transcription reaction mixtures by hybrid selection to clone 60 DNA at reduced stringency as described under Materials and Methods. RNA was translated in a micrococcal nuclease-treated L cell- free protein-synthesizing system and the products were separated by NaDodSO,-PAGE on 15% gels. Translations were programmed with unfraetionated reovirus serotype 1 mRNA, lane a; reovirus serotype 1 s4 mRNA, lanes b and c; reovirus serotype 3 s4 mRNA, lane d; and unfractionated reovirus serotype 3 mRNA, lane e. The translation in lane c was programmed with RNA isolated from reovirus-infected mouse L cells; the translations in lanes a, b, d, and e were programmed with RNA isolated from in vitro core transcription reaction mixtures. No RNA was added to the translation in lane f. The figure is a fluorogram of a 15% gel.

that the M, 20,000-30,000 .s4-encoded polypeptides are not post-translational degradation products of u3. Conceivably these polypeptides are formed because of premature termination in vitro of s4 mRNA translation.

Translation of sl mRNA Puriified from Reovirus-Infected Cells

We and others have previously shown that UlbNS is synthesized in mouse L cells infected with reovirus serotypes 1 and 3 (Jacobs and Samuel, 1985; Ernst and Shat- kin, 1985), as well as in cell-free protein- synthesizing systems programmed with reovirus mRNA prepared in vitro from reovirus-derived core transcription reaction mixtures (Jacobs and Samuel, 1985; Ernst and Shatkin, 1985). However, the amount of BlbNs synthesized in infected

Pulse Imin) 2 5

’ ’ Chase (mini o 3 e 132a5d; 5 to 25 55

from the 5’-proximal ATG (Giantini et aZ., 1984). No additional open reading frames capable of yielding a polypeptide greater than 40 amino acids (Mr < 5000) are revealed upon examination of all other possible reading frames in both the plus and the minus strand. Furthermore, the minor &-encoded polypeptides were synthesized within the first 5 min of translation in vitro (Fig. 3, lanes b and g), and radioactivity was not chased from (r3 into these lower molecular weight polypeptides (Fig. 3, lanes b-f and h-k). These results suggest

abcdefghijk

FIG. 3. Pulse-chase analysis of reovirus serotype 1 protein synthesis in vitro. Protein synthesis in vitro was performed as previously described (Miyamoto and Samuel, 1980; Jacobs and Samuel, 1985), except that the reaction volume was 75 ~1. De nouo incorporation of [%S]methionine was stopped at 2 min (lanes a-f) or 5 min (lanes g-k) by addition of 1 mM nonradioac- tive methionine. Aliquots (10 ~1) were removed after the following chase periods: 0 min, lanes a and g; 3 min, lane b, 8 min, lane c; 13 min, lane d, 28 min, lane e; 58 min, lane f; 5 min, lane h; 10 min, lane i; 25 min, lane j; or 55 min, lane k. Arrowheads mark the low- molecular-weight, s4-encoded polypeptides. The figure is an autoradiogram of a 15% gel.

14 JACOBS ET AL.

mouse cells, relative to the other reovirus- encoded proteins, is apparently less than the amount of ClbNs synthesized in cell-free protein-synthesizing systems derived either from mouse L cells (Jacobs and Sam- uel, 1985) or wheat germ or rabbit reticu- locytes (Ernst and Shatkin, 1985). To determine if this difference is due to a functional difference between sl mRNA svnthesized in vitr*o and sl mRNA synthesized in infected cells, we have isolated cytoplasmic RNA from reovirus serotype l-infected mouse L cells and analyzed the efficiency with which in V~VO sl mRNA pro- grams a cell-free protein-synthesizing system prepared from uninfected mouse L cells to make ula and UlhNs. The unfractionated mRNA preparations which were added to the cell-free translation reactions contained comparable amounts of Sl-encoded RNA as measured by dot-blot hybridization analysis (data not shown). As shown in Fig. 4, a cell-free protein-synthesizing system prepared from uninfected L cells catalyzed the translation of the unfractionated reovirus RNA isolated from reovirus-infected L cells (Fig. 4, lane e) as efficiently as unfractionated, capped, and methylated reovirus RNA synthesized i?~ citro by cores (Fig. 4, lane d). Furthermore, there was no significant difference between unfractionated in vitro- and ilz Gz?o-synthesized rcovirus mRNAs in the amount of the (TlbNs product synthesized in vitro (Fig. 4). We also purified sl mRNA by hybrid selection from unfractionated RNA isolated either from infected cells or from in dro core transcription reactions, such that equal amounts of RNA were selected (RNA excess). sl mRNA purified from infected cells (Fig. 4, lane a) was translated with approximately the same efficiency as sl mRNA purified from core transcription reactions (Fig. 4, lane b), yielding similar amounts of PlbNs and ula. Furthermore, the addition of 500 FM S-adenosylhomocys- teine to the in aitro protein-synthesizing system did not have a detectable effect on the synthesis of reovirus polypeptides (data not shown). The apparent reduced efficiency of synthesis of (TIbNs in reovirus-infected mouse L cells as compared to in vitro

ala u2 oNS a3

a IbNS

abcde

FIG. 4. Translation in 11ifro of sl mRNA isolated from reovirus-infected cells or from irr. vitro core transcription reaction mixtures. Total cytoplasmic RNA was isolated from reovirus serotype l-infected mouse L cells as previously described (Kingsman and Samuel, 1980) and under Materials and Methods. RNA isolated from infected cells contained approximately 0.4% sl mRNA, as determined by dot-blot hybridization analysis (data not shown). Genome Sl-specific RNA was purified by hybrid-selection to clone 50 DNA, in RNA excess, as described under Materials and Methods. Translations were programmed with the following RNAs: clone 50 hybrid-selected RNA isolated from infected cells, lane a, or from an i?/ vifro core transcription reaction, lane b; 4 &ml of unfractionated i?~ vitro RNA, lane d; 100 &ml of total cytoplasmic RNA isolated from reovirus-infected cells, lane e. No RNA was added to the translations for lane c. Thr figure is a Huorogram of a 10-20’3 polyacrylamide gradient gel. Lanes a-e are from the same gel; however, lanes d and e are a shorter exposure than lanes a-c.

protein-synthesizing systems, therefore is presumably not due to differences in reovirus mRNA made in vitro as opposed to in 7nao.


Primer Extension Mapping of sl mRNA

We have previously shown that the mRNA which codes for (Tn,Ns sediments with the mRNA that codes for cla during sucrose gradient velocity sedimentation, and have suggested that both ula and (TlbNs are translated from the same species of mRNA, which may be identical to the plus strand of the Sl genome segment (Jacobs and Samuel, 1985). In order to more defin- itively establish the nature of the 5’-terminus of reovirus sl mRNA, we have mapped the 5’ end of the reovirus serotype 1 sl mRNA by primer extension using two different restriction fragments prepared from clone 50 (Sl) DNA. A TaqI-Hind111 fragment (T-H), mapping from nucleotide 337 to nucleotide 419 of the Sl dsRNA segment (Cashdollar et aZ., 1985), and a Hind111 to EcoRI fragment (H-R), mapping from nucleotide 419 to nucleotide 503 of the Sl dsRNA segment were isolated, end labeled with 32Pi, annealed to various preparations of RNA, and then used as primers for reverse transcriptase. Figure 5 is an autoradiogram of a 6% gel showing the extension products. Use of cytoplasmic RNA isolated from reovirus serotype l-infected L cells (Fig. 5, lanes d and e) or reovirus mRNA isolated from in vitro core- catalyzed transcription reactions (Fig. 5, lanes f, g, h, and i) as template led to the extension of both primers and the produc- tion of a single predominant extension product in each case. The apparent lengths of the extension products obtained with the TaqI-Hind111 and the HindIII-EcoRI primers were consistent with the predicted lengths for a single 5’ terminus of a full length sl transcript, 419 and 503, respectively (Cashdollar et ah, 1985). As a control, the primers remained unextended when total cytoplasmic RNA isolated from uninfected cells was used as template (Fig. 5, lanes b and c). Furthermore, no additional primer extension products were detected when reovirus sl mRNA which was recovered from an in vitro translation reaction was used as template (Fig. 5, lanes h and i) as compared to when reovirus al mRNA which had not been preincubated under

ala UlbNS ATG ATG Taql

1 /.’ : Hind III ECORI

I

1 14 75 337 419 503 1458

29s- -298

obcdefghij

FIG. 5. Primer extension analysis of genome segment Sl-encoded mRNA. A TagI-Hind111 fragment (T-H), representing sequences from nucleotide 337 to nucleotide 419 of genome segment Sl and a HirLdIII- EcoRI fragment (H-R), representing sequences from nucleotide 419 to nucleotide 503 of genome segment Sl, were isolated from a restriction digest of clone 50 (Sl) DNA as described under Materials and Methods. These fragments were end labeled, denatured, hy- bridized to various RNAs, and used as primers for AMV reverse transcriptase. The extended products were fractionated on a 6% sequencing gel and visualized by autoradiography. The primer-extension reactions in lanes b, d, f, and h were performed with the TaqI-Hind111 fragment; the primer-extension reactions in lanes c, e, g, and i were performed with the HindIII-EcoRI fragment. The following RNAs were used as the template for primer extension: lanes b and c, total cytoplasmic RNA from uninfected cells; lanes d and e, total cytoplasmic RNA from reovirus serotype l-infected cells; lanes f and g, in vitro reovirus mRNA; lanes h and i, in vitro RNA which was preincubated in a cell-free protein-synthesizing system. Lanes a and j contain HinfI-digested pBR322 as markers.

cell-free protein synthesis conditions was used as template (Fig. 5, lanes f and g).

DISCUSSION

Complete nucleotide sequence analysis of the Sl genome segment of reovirus serotype 1 (Lang strain) reveals two open read-

16 JACOBS ET AL.

ing frames (Cashdollar et (xl., 1985). A long open reading frame of sufficient size to code for mla begins at nucleotide 14. A shorter open reading frame, which begins at nucleotide 75 and is sufficient to code for a 119-amino acid polypeptide, overlaps with the longer open reading frame. We and others (Ernst and Shatkin, 1985; Jacobs and Samuel, 1985) have recently identified aMr- 12,000 reovirus-encoded polypeptide which may be the product of the shorter open reading frame. In this manuscript we report that purified sl mRNA, isolated either from reovirus serotype l-infected mouse L cells or from in, l~&o core transcription reaction mixtures, codes for both flla and (JlhNS. We could detect by primer extension only a single 5’ end for sl mRNA svnthesized either iw vitro or iz vi/w. The size of the extended product was consistent with that predicted for a plus-strand tcm- plate sl mRNA equivalent in size to the plus strand of segment Sl dsRNA. These results indicate that only a single reovirus sl mRNA exists in the region extending from the 5’ end of the mRNA through the area of overlap between the two open reading frames, and together with previous results (Jacobs and Samuel, 1985; Ernst and Shatkin, 1985), strongly suggest that both gla and CibNs are translated from the same functionally dicistronic mRNA.

The vast majority of eukaryotic mRNAs are monocistronic: ribosomes initiate translation at only a single AUG triplet on most eukaryotic mRNAs (Kozak, 1984). The only well-characterized exceptions to this are the adenovirus Elb mRNA (Bos et ah, 1981), the Sendai virus P/C mRNA (Giorgi et al., 1983), the influenza B virus NA/NB mRNA (Shaw and Choppin, 1984) and the reovirus sl mRNA (Ernst and Shatkin, 1985; Jacobs and Samuel, 1985; Cashdollar et al., 1985; Nagata et al., 1984; this manuscript). In the case of the reovirus sl mRNA, functional dicistrony may be a consequence of the “context” of the sequences surrounding the putative initiation codons for the two primary translation products of this mRNA. Kozak (1981) has proposed that 40s ribosome subunits bind to the Y-terminus of eukaryotic mRNAs

and then “scan” the mRNA for an AUG triplet in the sequence context -“Pu- NNAUGPut4, which represents the site of functional initiation of translation. Non- initiating AUG triplets upstream from initiating AUG triplets usually deviate significantly from the consensus sequence. The 5’-proximal AUG of the sl mRNA of all three reovirus serotypes, which presumably is used for the synthesis of ula, deviates from this consensus sequence in that a pyrimidine (C) is present in the -3 position. The third AUG triplet from the 5’-terminus of the Lang strain sl mRNA, which presumably is used for synthesis of ffli,Ns, conforms to the consensus sequence for initiating codons (Kozak, 1982; Cash- dollar et al., 1985). It is conceivable that, because the 5’-proximal AUG triplet is in less than an optimal context for initiation, only a small percentage of the 40s ribosomes scanning the 5’ end of the sl mRNA pause at this proximal AUG triplet and initiate translation. Most of the 40s ribosomes must presumably pass by the first AUG triplet and initiate at the internal AUG triplet which is in an optimal context for initiation.

Additional minor polypeptides of M,. 20,000-30,000 were synthesized in L cell- free protein-synthesizing systems programmed with reovirus serotype 1 mRNA, and to a lesser extent in systems programmed with reovirus serotype 3 mRNA. Some of these minor polypeptides are clearly coded for by s4 mRNA. The complete sequence of the serotype 3 S4 genome segment is known (Giantmi et al., 1984). Only a single open reading frame longer than 40 codons exists in the reovirus serotype 3 S4 gene. Since the long open reading frame presumably codes for na, the well-established product of the S4 gene, the minor s4-encoded polypeptides are proba- bly related to c3 in amino acid sequence. Our pulse-chase experiments irk vitro do not demonstrate a precursor-product re- lationship between c3 and the minor s4-encoded polypeptides, which suggests that these polypeptides are likely products of premature termination of translation.

Large amounts of flibNs are made in cell-


free protein-synthesizing systems programmed with reovirus mRNA isolated from in vitro core transcription reaction mixtures (Ernst and Shatkin, 1985; Jacobs and Samuel, 1985). However, synthesis of plbNs in vivo, in mouse L cells infected with either serotype 1 or 3, is much less efficient than the synthesis in vitro in cell-free protein-synthesizing systems prepared from uninfected L cells (Jacobs and Samuel, 1985; Ernst and Shatkin, 1985). This apparent difference in translational efficiency does not seem to be due to a gross structural or functional difference between reovirus mRNA made in vitro as opposed to in vivo. Equivalent amounts of in vivo- and in vitro-synthesized, unfructionated reovirus mRNA, as determined by dot-blot hybridization analysis, programmed a cell- free protein-synthesizing system prepared from uninfected mouse L cells to make equivalent amounts of ClbNs. In addition, clone 50 (Sl) DNA hybrid-selected, puriJied reovirus sl-specific mRNA, yielded similar amounts of (TlbNS when translated in vitro, irrespective of the site of synthesis of the sl mRNA, in vivo or in vitro. It is likely, therefore, that the difference between the amount of gn,Ns synthesized in vitro in cell- free systems prepared from uninfected mouse L Ceh, and the amOUnt of fllbNS synthesized in vivo in reovirus-infected mouse L cells, is due to a difference in the translational machinery in the two systems.

ACKNOWLEDGMENT

This work was supported in part by Research Grants AI-12520 and AI-20611 from the National In- stitutes of Health.

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