In vitro translation of virion RNA from Moloney murine sarcoma virus

VntOIBoY

101,91-103

(1980)

In Vitro Translation JACKIE

of Virion RNA from Moloney Murine Sarcoma Virus

PAPKOFF,*,’

TONY

HUNTER,?

AND KAREN

BEEMONt

*Department of Biology, University of Californ&, San Diego, La Jolla, Califoonzia 9Z0097,and tTumor Virology LabomEory, The Salk Institute, Post Office Box 85800, San Diego, Cal@rnia 92198 ALGAL

October 30, 1979

Virion RNA of Moloney murine sarcoma virus (MO-MSV) was translated in the mRNAdependent reticulocyte lysate and the translation products were analyzed by gel electrophoresis. The major products had apparent molecular weights of 62,000, 37,000, 33,000, 24,000, 18,000, and 15,000. None of these proteins was derived from the helper virus RNA present in our Mo-MSV virion preparations. Immunoprecipitation studies showed that the 62K protein was related to the gag gene product of Moloney murine leukemia virus (MoMLV). The 37K, 33K, 24K, 18K, and 15K proteins, on the other hand, were found to be unrelated to either the gug gene or ~WVgene products of Mo-MLV. Tryptic peptide mapp~g showed that the 3’7K, 33K, 24K, and 18K proteins had overlapping amino acid sequences. By this criterion the 37K family of proteins was not related to the 62K protein nor to the 15K protein. Likewise, the 15K protein was not related to the 62K protein. Translation of naturally occurring polyadenyiated fragments of virion RNA showed that the 62K protein was derived fkom RNA of genomic length (30 S). The 15K protein was made from RNA of about 25 S, while the 37K, 33K, 24K, and 18K proteins were synthesized from RNAs ranging from 20 to 17 S. On the basis of these results, we have proposed a model for the expression of the genome of MO-MSV in vitro. The 62K protein would arise from the gag gene at the 5’ end of the MO-MSV genome. The 15K protein would originate from one of the two pot gene fragments in the MO-MSV genome. The 3’7K, 33K, 24K, and 18K proteins would be derived from the WC gene of MO-MSV.

(Van Zaane and Bloemers, 1978). MO-MLV serves as the natural helper virus for MO-MSV. A clone of TB cells (Clone 124) infected with the MO-MSV (MO-MLV) complex was isolated by Ball et al. (1973). This clone yields MO-MSV preparations containing very low levels of the helper MO-MLV component (Beemon et al., 1976; Maisel et al., 19’77). The RNA of this virus has been analyzed by several laboratories to determine the structure of the MO-MSV genome (Canaani et al., 1977; Dina et al., 1976; Hu et al., 1977; Donoghue et al., 1979, Verma, 1979). RNA isolated from MO-MSV virions sediments at about 60 S and appears to consist of two identical 30 S subunits (Beemon et al., 1976; Maisel et al., 1977). The 30 S genomic RNA is about 6000 nucleotides long (Dina et al., 1976). This is considerably shorter than the genomic RNA of the parent MO-MLV which is about 8400 nucleotides (Rothenberg et al., 1978;

Several different murine sarcoma viruses have been isolated from mice and rats injected with murine leukemia viruses (Harvey, 1964; Kirsten and Mayer, 1967; Moloney, 1966). It has been postulated that such sarcoma viruses are generated by recombination between the leukemia virus and host genetic elements (Ball et al., 1973a; Maisel et al., 1973; Scolnick et al., 1973). Moloney murine sarcoma virus (MO-MSV) was derived from a Balb/c mouse injected with Moloney murine leukemia virus (MoMLV) (Moloney, 1966). MO-MSV is capable of transforming fibroblasts in vitro and causing tumors in mice (Aaronson and Rowe, 1970; Aaronson et aE., 1970; Moloney, 1966). All murine sarcoma viruses isolated to date are defective viruses and require a helper leukemia virus for replication I To whom reprint requests should be addressed. 91

0042-6822/80/03OOQl-13$02.00/O Copyright All rights

Q 1980 by Academic Press, Inc. of reproductionin any form reserved.

92

PAPKOFF,

HUNTER,

Verma, 1918). App~oxima~~~~ ‘IO% of the MO-MSV genome is homologous with a clone of Mo-MLV by hybridization, the remaining 30% being unique to Mo-MSV (Dina et al., 1976). The heteroduplex mapping data of Hu et al. (197’7) and Donoghue eCal. (19791, which are depicted diagrammaticahy in Fig. 6, show that MO-MLV and MO-MSV have a region of homology of about 2 kb at the 5’ end of their respective genomes. Since the likely gene order of MO-MLV from the 5’ end is gag, po1T,env, this region of homolo~ would correspond to the gag gene coding for the internal structural proteins. This conclusion is consistent with the observation that Ma-MSV codes both in vivo and in vitro for a 62,000-dalton protein which is closely related to the gag gene product of MO-MLV (Barbacid et al., 1976; Philipson et al., 1978; Robey et al., 1977). Heteroduplex analysis also shows that most of the sequences presumed to encode the viral reverse transcriptase (poll and the envelope glycoproteins fenv), in the central and 3’ portion of the M~MLV genome, are deleted from Mo-MSV (see Fig. 6) (Hu et al., 1977; Donoghue et al., 1979). MO-MSV RNA has a segment between 0.8 and 2.3 kb from the 3’ end of the genome which is not found in MO-MLV (Hu et al., 1977; Donoghue et al., 1979). This region probably corresponds to the unique sequenees of MO-MSV identified by hybridization. The portion of the genome which is unique to MO-MSV has been isolated intact following RNase H digestion of hybrids between M~-~~SV genomic RNA and an excess of MO-MLV cDNA (Dina, 1973). This 15~0-nu~~eotide RNA fragment was mapped between 0.75 and 2.25 kb from the 3’ end of the MO-MSV genome. Recently, it has been shown that restriction endonuclease fragments of Ma-MSV cDNA, synthesized in vitro, containing the MoMSV specific region of the genome can transform fibroblasts in culture upon transfeetion (Andersson at ai., 1979). Given these data, it seems reasonable to propose that this unique region contains the sar~omageni~ isolation of MO-MSV, We will define this region of Mo-MSV as the arc gene. The gene products of Rous sarcoma virus (RSV) have been identified by in vitro

ANT)

BEEMON

translation of virion RNA preparations containing naturally occurring genomic fragments (Beemon and Hunter, 1977, 1978; Kamine et al., 1978; Purchio et al., 1977, 1978). We have used the same approach to attempt to identify the gene products of MO-MSV, In this paper we present a study of the in vitro translation products of virion RNA of MO-MSV, derived from infected TB cells (Clone 124). The major translation products are a 62,~00-dalton protein (62K) arising from the wg gene and a family of proteins, 37~~~~ (37K), 33,006 (33K), 24,060 (24K), and 18,060 (18K) daltons in size, which appear to be derived from the src gene of MO-MSV. MATERIALS

AND

METHODS

Virus and RNA. The TB (Clone 124) cell Iine {Ball eit al., 1973b) producing the MoMSV (MO-MLV) complex was obtained from Dr. J. Ball. The vies-~onta~ing medium from the producer cells grown in Dulbecco’s mod~ed Eagle’s medium with 10% calf serum was harvested every 24 hr. The virus was concentrated by centrifugation and banding as described (Verma, 19’78). Total RNA was extracted from purified virus and 60 S RNA isolated by sucrose gradient centrifugation. The 60 S RNA was precipitated twice with ethanol, lyophilized from water before being taken up at 1 mglml, and stored in liquid nitrogen. Small aliquots were heated at 100” for 1 min and quick-cooled on ice prior to translation. The RNA used in this study was the kind gift of Dr. Inder Verma. The MO-MLV (Clone 1) (Fan and Paskind, 1974) 70 S RNA used in the experiment shown in Fig. 1 was kindly supplied ,by Steve Edwards. In vitro translation. The mRNA-dependent reticulocyte lysate was used for in, vitro translation as previously described (Beemon and Hunter, 1977, 1978). Viral RNA was used at a final concentration of 30 pglml. [35S]Methionine ~Amersh~Searle >600 Ciimmol) was included at 450 $Xml. m7GTP (P. L. Bio~h~m~~als~ was added to the Iysate at 290 fl for inhibition studies, Completed reactions were incubated for a further 10 min at 30” with 50 pg/ml RNase

MO-MSV

TRANSLATION

A in the presence of 10 mM EDTA before analysis by SDS-polyacrylamide gel electrophoresis. Immunoprt&pitution. Immunoprecipitation of in vitro products was performed as described (Sefton et ccl., 1978). In the experiment shown in Fig. 3,5 ~1 of translation reaction was diluted at least lo-fold with 0.15 M NaCI, 0.01 M sodium phosphate pH 7.2, 1% Nonidet-P40, 1% sodium deoxycholate, 0.1% SDS, 1% Trasylol, and immunoprecipitated with 3 ~1 of antiserum. Where indicated, the antiserum was preabsorbed for 30 min at 0” with purified MO-MLV virions disrupted in the buffer described above (20 pg/pl of antiserum). All the antisera used were kindly provided by Steve Edwards. Size fractionation of poly(A)-containing MO-MSV &ion RNA. Purified MO-MSV

virion RNA, 40 pg;, was heated to 100” for 1 min and cooled rapidly on ice. Poly(A)containing RNA was selected by two cycles of binding to oligo(dT)-cellulose in 0.5 M NaCl, 0.01 M Tris-HCl pH 7.3, 0.001 M EDTA, 0.1% SDS, and elution with 0.01 M Tris-HCl pH 7.3, 0.001 M EDTA, 0.1% SDS. The poly(A)-containing RNA was sedimented through a lo-30% neutral sucrose gradient containing 0.01 M sodium acetate pH 5.2, 0.001 M EDTA, and 0.1% SDS for 3 hr at 20” in a Beckman SW 50.1 rotor at 49,000 rpm. Fractions were collected, adjusted to 0.2 M NaCl, and precipitated with ethanol in the presence of 10 pg yeast tRNA carrier (Boehringer-Mannheim). The RNA was reprecipitated, dissolved in 100 ~1 water, lyophilized, and finally dissolved in 10 ,ul water for translation. One microliter of each fraction was translated in a lo-p1 reaction, and 1 ~1 of each translation product was analyzed by SDS-polyacrylamide gel electrophoresis. Gel electrophoresis. SDS-polyacrylamide gels contained 14% acrylamide and 0.1% bis-acrylamide and were run as described (Beemon and Hunter, 1977; 1978). Radioactive proteins were detected by fluorography. Tryptic peptide mapping. Heat-denatured MO-MSV virion RNA, 6 ,ug, was translated in a 200-~1 reaction with [35S]methionine as outlined above. The entire translation reaction was run on a 2-mm-

PRODUCTS

93

thick slab gel. The gel was treated as described for identification, extraction, and digestion of the radioactive proteins (Beemon and Hunter, 1978). The digests were separated in two dimensions on cellulose thin layer plates as described by Gibson (1974). RESULTS

Translation

of MO-MSV

Virion

RNA

Purified 60 S RNA isolated from MoMSV virions was heat denatured and translated in the mRNA-dependent reticulocyte lysate. The [35S]methionine-labeled translation products were analyzed on SDS-polyacrylamide slab gels and visualized by fluorography. The major in vitro products migrated with apparent molecular weights of 62K, 37K 33K, 24K, 18K, and 15K (Fig. 1, track A). Philipson et al. (1978) have observed similar translation products from MO-MSV genomic RNA. Comparison of the translation products of MO-MLV virion RNA (Fig. 1, track B) with those of MO-MSV virion RNA shows that MoMLV-specific products are not detectable in the products of MO-MSV RNA. Further comparison of the in vitro translation products of MO-MSV and MO-MLV virion RNAs by gel analysis under a variety of conditions showed that none of the major MO-MSV products comigrated with products of MO-MLV RNA (data not shown). Longer exposures of the gel shown in Fig. 1 revealed a number of minor products. On the basis of their apparent molecular weights and the large size of the RNA coding for them (data not shown), we believe that some of these minor products are coded for by the helper virus RNA. Our data support the evidence (Beemon et al., 1976; Maisel et al., 1977) that there are very low levels of helper MO-MLV RNA in preparations of MO-MSV virus produced by the TB (Clone 124) cell line. m’GTP, a cap analog, inhibits the translation of most capped RNAs in the mRNAdependent lysate and has been used to determine whether or not an RNA preparation has more than one initiation site for protein synthesis (Beemon and Hunter, 1977). In the case of MO-MSV, m7GTP

94

PAPKOFF,

A

HUNTER,

6

AND BEEMON

2. In other experiments, however, the synthesis of the 38K protein appeared to be unaffected by m’GTP (data not shown). As measured by densitometry, the percentage inhibition by m7GTP of synthesis of the 62K, 37K, and 18K proteins was 57, 17, and 15%, respectively. Accurate densitometric quantification for the 33K, 24K, and 15K proteins was not possible. The observed inhibition of synthesis of the 62K protein by m’GTP indicates that the in vitro initiation site for the 62K protein

Al3

FIG. 1. Translation products of MO-MSV and MoMLV virion RNAs. Purified 60-70 S virion RNAs from MO-MSV and MO-MLV virions were heat denatured and translated in the mRNA-dependent reticulocyte lysate as described under Materials and Methods. Two microliters of each translation reaction was analyzed by SDS-polyacrylamide gel electrophoresis as described under Materials and Methods. The gel was subjected to fluorography and exposed for 24 hr. Analysis of an incubation to which no RNA had been added showed no discernible radioactive bands in this exposure (data not shown). The positions of the 62K, 38K, 37K, 33K, 24K, 18K, and 15K proteins are indicated. The molecular weights of these proteins were determined from a calibration plot of marker proteins run in parallel. (A) Total translation products of MO-MSV RNA. (B) Total translation products of MO-MLV RNA.

partially inhibited the synthesis of the 62K protein as well as that of several minor proteins between 62,000 and 38,000 daltons, but had little effect on the other in vitro products (Fig. 2). The 38K protein, which is a minor band migrating slightly slower than the 37K protein (see Fig. 1, track A), is not well resolved in the gel shown in Fig.

FIG. 2. Effect of m’GTP on translation of Mo-MSV virion RNA. Purified MO-MSV virion RNA was heat denatured and translated in the mRNA-dependent reticulocyte lysate with or without the addition of 200 PM mrGTP. Of each translation reaction 2.5 ~1 was analyzed by SDS-polyacrylamide gel electrophoresis. The gel was subjected to fluorography and exposed for 72 hr. (A) Translation products of MoMSV RNA. (B) Translation products of MO-MSV -__. RNA synthesized in the presence of m’GTP.

MO-MSV TRANSLATION

PRODUCTS

95

is different to those used for the 3?K, 33K, 24K, and 18K proteins and is consistent with the initiation site for the 62K protein being near a 5’ terminal cap structure.

To test whether any of the in vitro products were encoded by the gag or env genes, MO-MSV virion RNA products were immunopreeipitated with specific antisera directed against viral structural proteins. An antiserum directed against ~30, the major internal structural protein of MoMLV, which is derived from the polyprotein precursor coded by the gag gene (Eisenman and Vogt, 197’S), precipitated the 62K protein, as well as all of the minor proteins between 62,000 and 38,000 daltons, including the 38K protein (Fig. 3, tracks C and G). This precipitation was prevented if the antiserum was preabsorbed with a large excess of detergent-disrupted MO-MLV virions (Fig. 3, tracks D and H). The same proteins were also recognized by antisera directed against total MO-MLV virions (Fig. 3, track E). An antiserum directed against ~10, an internal structural protein which is coded at the 3’ end of the gag gene of MO-MLV, did not precipitate the 62K protein (Fig. 3, track I) as efficiently as the anti-p30 serum, although the anti-p10 serum precipitated the gag gene product of several other MLVs as efficiently as our anti-p30 serum (data not shown). None of these three sera recognized any of the 37K, 33K, 24K, 18K, or 15K proteins, although small amounts of the 18K protein were brought down nonspecifically. Antiserum directed against the viral glycoprotein, gp70 (a product of the env gene), did not precipitate any of the in vitro products (data not shown). This is corroborated by our failure to precipitate the 37K, 33K, 24K, 18K, and 15K proteins with anti-whole MO-MLV virion serum, which has a major activity against gp’70 (S. Edwards, personal communication). Our immunoprecipitation results strongly support the conclusion that the 62K protein is encoded by the gag gene of MO-MSV. The failure of any of the sera we have tested

FIG. 3. Immunoprecipitation of MO-MSV virion RNA translation products. Aliquots, 5 4, of total tr~slation products of heat-denatured Mo-MSV virion RNA were immunopreeipitated with 3 ,ul of the appropriate antiserum aa described under Materials and Methods. Where indicated, the serum was preabsorbed with disrupted Mo-MLV virions. One-fifth of each immunopreeipitate was analyzed by SDSpolyacrylamide gel electrophoresis. One-half microliter of total translation products was run in track A. Samples A-F were generated in a single experiment and all run on the same gel. Samples G-J were obtained in a second experiment and run on a separate gel. Both gels were subjected to fluorography and exposed for 7 days. (A) Total translation products of MO-MSV RNA. (B) Immunopreeipitate with normal rabbit serum. (C) Immunoprecipitate with anti-p30 serum. (D) Immunoprecipitate with preabsorbed antip30 serum. (E) Immunoprecipitate with anti-whole MO-MLV virion serum. (F) Immunoprecipitate with preabsorbed anti-whole MO-MLV virion serum. (G) Immunopreeipitate with anti-p30 serum. (H) Immunoprecipitate with preabsorbed anti-p30 serum. (I) Immunoprecipitate with anti-p10 serum. (J) Immunoprecipitate with preabsorbed anti-p10 serum.

to recognize the 37K, 33K, 24K, 18K, or 15K proteins suggests that these proteins are not related to any of the major viral structural proteins.

PAPKOFF,

96 M

621c

-

1

2

3

4

5

HUNTER, 6

7

a

AND BEEMON

9 10 il

--

12 13 14 l!i id 17 1819

29 M

L

FIG. 4. Size fractionation and translation of poly(A)-containing MO-MSV virion RNA. Poly(A)containing Mo-MSV virion RNA was isolated and sedimented through a neutral sucrose gradient as described under Materials and methods. One-tenth of the RNA in each fraction was translated in the mRNA~ependent reticulocyte lysate in a final volume of 10 ~1 and 1 ,ul of each reaction was analyzed by SDS-polyacrylamide gel electrophoresis. The gel was subjected to fluorography and exposed for 24 hr. The two outside tracks (M) show total translation products of unfractionated MO-MSV RNA. Tracks l-20 show translation products from the bottom to the top of the gradient. The positions of 28 and 18 S ribosomal RNA run in a parallel gradient are also shown.

To determine the sizes of the RNAs coding for the in vitro products, poly(A)containing RNA was selected from heatdenatured MO-MSV v&ion RNA by two cycles of binding to oligo(dT)-cellulose and fractionated by sedimentation through a neutral sucrose gradient. The RNA from each fraction was translated and the products were analyzed on an SDS-polyacrylamide slab gel (Fig. 4). The results show that the 62K protein is synthes~ed from poly~enylated RNA of approximately 30 S (fractions 6-8), the size of the MO-MSV genome. The several minor proteins between 62,000 and 38,000 daltons, whose synthesis was inhibited by m’GTP and which were precipitated by anti-p30 serum, also appear to be derived from RNA of

about 30 S. This suggests that these minor proteins may be premature termination products of the gag gene whose synthesis is initiated at the same site as that of the 62K protein. The 38K protein is also immunoprecipitated with anti-p30 serum and made from RNA of approximately 30 S. Analysis of the formyf [35S]methioninelabeled N-terminal peptides of the 62K and 38K proteins, however, indicates that they do not share a common N-terminal sequence (data not shown). Therefore, it is possible that the 38K protein is initiated within the gag gene from RNA molecules that are missing a short sequence at the 5’ end of the genome. The 37K and 33K proteins are both synthesized from polyadenylated RNAs of about 20 S (fractions ll-13), while the 24K and 18K proteins are derived from

FIG. 5. Tryptic peptide maps of MO-MSV virion RNA translation products. [3SS]Methionine-Iabeled translation products of MO-MSV virion RNA were isolated and tryptic digests prepared as described under Materials and Methods. Electrophoresis at pH 4.7 was from left to right toward the cathode. Ascending c@omatography was from bottom to top. The chromatograms were exposed to Kodak NS5T X-ray film. The amounts of radioactivity used and the exposure times were as follows: 62K protein, 20,000 epm, 8 days; 37K protein, 40,000 cpm, 8 days; 33K protein, 14,000 cpm, 30 days; 24K protein, 6000 cpm 40 days; 18K protein, 30,000 cpm, 8 days; 15K protein, 5000 cpm, 10 days. 97

98

PAPKOFF,

HUNTER,

RNAs of about 18 S (fractions 12-14) and 17 S (fractions 1%15), respectively. This analysis also revealed that the 15K protein is made from a polyadenylated RNA of about 25 S (fractions 8-10).

Products

To characterize the in vitro products further, two-dimensional tryptic peptide mapping was performed on each of the 62K, 37K, 33K, 24K, 18K, and 15K proteins which had been labeled in vitro with [Y!J]methionine. Where necessary, we ran mixtures of the appropriate digests to identify homologous peptides. The results show that the 37K, 33K, 24K, and 18K proteins have many methionine-containing tryptic peptides in common (Fig. 5). Of a total of 18 peptides reproducibly identifiable in the 37K protein, 17 are present in the 33K protein, 15 are present in the 24K protein, and 9 are present in the 18K protein. The 18K protein has three unique peptides (A-C) not present in the other proteins, while the 24K protein has one unique peptide (D). We conclude that the 37K, 33K, 24K, and 18K proteins form an overlapping set. The 15K protein appears to be unrelated to the 62K protein and the 37K series of proteins. It can be clearly seen that the map of the 62K protein is not closely related to those of the 37K series, although we were unable to distinguish peptide “c” of the 62K protein from peptide 8 of the 37K protein. Peptide 1 of the 62K protein appears to be present in maps of all the in vitro products at low levels and probably represents a small amount of contamination of our protein preparations with incomplete products related to the 62K protein. Translation Virion

of PuriId RNA

30

S MO-MSV

To obtain evidence that the 37K family of proteins is encoded by the MO-MSV genome, we exploited the observation that the reticulocyte lysate is capable of generating translatable fragments from polycistronic mRNAs, thereby permitting identi-

AND

BEE~ON

fication of internally coded proteins (Pelham, 1979; C. Lawrence, unpublished results), Polyadenylated 30 S virion RNA from MoMSV was selected from three successive neutral sucrose gradients and translated in the reticuloeyte lysate. The translation products, analyzed on an SDS-polyacrylamide slab gel, are shown in Fig. 6. The results show that translation of highly purified 30 S virion RNA from MO-MSV in the reticulocyte lysate yields, in addition to the 62K protein from the 5’ Mo-MSV-gag gene, the 37K, 33K, 24K, 18K, and 15K proteins which are presumably encoded by genes within the full-length MO-MSV genome. DISCUSSION

The experimental approach we have used to identify the gene products of MO-MSV by in vitro translation is similar to that previously used to identify the gene products of RSV (Beemon and Hunter, 1977; 1978; Kamine et al., 1978; Purchio et al., 1977, 1978). We expected to identify the product of the gene nearest to the 5’ end of the MO-MSV genome, the gag gene, by translation of intact genomic RNA and to detect products of internal genes by translation of virion RNA shorter than the fulllength genome. Translation analysis of a complete spectrum of polyadenylated fragments of a potentially polycistronic viral RNA provides a method of identifying all the gene products of this RNA and also a means of mapping these gene products to particular regions of the RNA. Translation of MO-MSV virion RNA in the mRNA-dependent reticuloc~e lysate yielded major products with apparent molecular weights of 62K, 37K, 33K, 24K, 18K, and 15K. None of the MO-MLV virion RNA translation products were identified in the translation products of MO-MSV RNA. It, therefore, seems unlikely that any of these proteins arise from the genomic RNA of the MO-MLV helper virus which is present at very low levels in our preparations of MO-MSV virions. Tr~slation of 30 S MoMSV virion RNA, purified on three sequential sucrose gradients, yielded all of the same proteins identified in the products

MO-MSV TRANSLATION

of denatured 60 S virion RNA, which contained many genomic fra~ents (Fig. 6, also see Fig. 4, track 7). This indicates that the sequences codings for the 62K, 37K, 33K, 24K, 18K, and 15K proteins are most likely contained within the full-length MSV genome and are being expressed by fragmentation of the 30 S RNA either prior to or during its translation. This phenomenon has been observed for other viral RNAs, and it appears that the retieulocyte lysate is capable of generating translatable RNA fra~ents from polycistronic mRNAs (Pelham, 19’79; C. Lawrence, unpublished results). Although all of the in vitro products can be expressed from fulllength MSV virion RNA, most of these proteins appear to be translated from the subgenomic fragments already present in our total virion RNA preparation (Fig. 4). Having identified these proteins as products of the MO-MSV genome, we sought to characterize them and to identify the genes coding for them. Several lines of evidence suggest that the 62K translation product is derived from the gag gene at the 5’ end of the MO-MSV genome. The cap analog, m7GTP, partially inhibited the synthesis of the 62K protein, suggesting that synthesis of the 62K protein is initiated at a site adjacent to the 5’ terminal cap, which is presumably present on the genomic RNA of MO-MSV. A 5’ coding location for the 62K protein is also indicated by the fact that the 62K protein is only made from RNA of genomic length (about 30 S) and not from smaller polyadenylated RNA molecules found in the virion. Antisera directed against total MoMLV virions as well as against ~30, the major MO-MLV gag protein, recognize the 62K protein synthesized in vitro. Similar immunoprecipitation results have been obtained by Philipson et al. (1978) with in vitro products of MO-MSV virion RNA. A 62K protein apparently identicai to that synthesized from MO-MSV virion RNA can be immunoprecipitated both from MO-MSV infected TB cells and from the in vitro translation products of polysomal poly(A)containing RNA of approximately 30 S isolated from such cells (unpublished results). The heteroduplex analysis of Mo-

PRODUCTS

99

FIG. 6. Translation of purified 30 S MO-MSV virion RNA. Poly(A)-cont~~ng MO-WV virion RNA was isolated and sediments through a neutral sucrose gradient as outlined under BIateriaIs and Methods. The 30 S region of the gradient was ethanol precipitated and resedimented twice as described. Onehalf of the 30 S RNA from the final gradient was translated in the mRNA-dependent reticulocyte lysate in a final volume of 10 ~1. Unfractionated, heatdenatured 60 S virion RNA from MO-MSV was translated in parallel as described under Materials and Methods. Two microliters of each translation reaction was analyzed by SDS-poly~rylamide gel electrophoresis. The gel was ~uoro~aphed and exposed for 7 days. (A) Products formed in an incubation to which no RNA was added. (B) Total translation products of unfractionated, denatured 60 S MO-MSV virion RNA. (C) Total translation products of purified 30 S MO-MSV virion RNA.

MSV carried out by Hu et al. (1977) and Donoghue et aE. (1979) indicates that the MO-MSV genome has extensive homology with MO-MLV in the gag gene, but also

100

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HUNTER,

AND BEEMON

FIG. 7. A model for the expression of MO-MSV genomic RNA in z&o. The MO-MLV genome is represented di~mmatical~y to scale at the top of the figure, as it would appear in a hete~uplex with Mo-MSV cDNA (Huet a&, 1977; Donoghue et al., 1979), with the 5’ end on the left and the poly(A) on the right. The approximate locations of the gag, pal, and enu genes are shown as thin lines above the MO-MLV genome, the gene boundaries being marked by bars. These locations were deduced from the known size of the gag gene product (Eisenman and Vogt, 1978) and the size of the body of the env gene mRNA (Rothenberg et al., 1978). The Ma-MSV genome is depicted below the MO-MLV genome. The region unique to MO-MSV determined by heteroduplex analysis is boxed and labeled SRC. Distances from the 3’ end of the genome are marked in kilobases. The in vitro protein products of MO-MSV RNA are shown as thick lines. The nenomic RNA fragments from which these proteins originate are drawn as lines of intermediate thickness.

has a deletion which may extend into the 3’ end of the gag gene. Consistent with this study, our results suggest that the 62K MO-MSV gag precursor protein may be missing part of ~10, the C-terminal portion of the MO-MLV gag precursor (Barbacid et al., 1976). Anti-p10 serum fails to precipitate efficiently the 62K protein synthesized in vitro even though the in vitro gag gene products of other MLVs are efficiently recognized by this serum. In this respect, the MO-MSV arising from the TB (Clone 124) cell line is similar to the 60,~0-dalton gag-related protein (~608~~) characterized in cells infected with other clones of MSV which also appear to lack ~10 sequences (Robey et al., 1977; Barbacid et al., 1976; Oskarsson et al., 1978). The 37K, 33K, 24K, and 18K proteins form an overlapping set translated from an overlapping set of 3’ genomic fragments. For several reasons, it seems possible that these proteins are encoded by the SW gene of MO-MSV. First, this set of proteins appears to be unrelated to the 62K protein

both by tryptie peptide mapping and by immunoprecipitation and, therefore, is not encoded by the gag gene. The 37K series is also not obviously related to the MO-MLV env gene product by immunoprecipitation and is, therefore, probably not encoded by the env gene. Second, these proteins are only synthesized from polyadenylated RNAs whose sizes range from 20-17 S (ZSOO1800 nueleotides). Assuming that protein synthesis is initiated near the 5’ end of these subgenomic RNAs, the 37K, 33K, 24K, and 18K proteins must be derived from sequences within the WC gene of MO-MSV. There is good agreement from several lines of physical, biochemical, and biological evidence that the WC gene of MO-MSV is about 1200- 1500 nucleotides in length with its 3’ end located about 700800 nueleotides from the poly(A) (Dina and Beemon, 1977; Hu et al., 1977; Dina, 1978; Andersson et al., 1979; Donoghue et al., 1979; Verma, 1979). Since a region of 12001500 nucleotides could code for a protein of 40K-50K, it is possible that the 37K

MO-MSV TRANSLATION

protein and other members of the family could be coded in their entirety by the MO-MSV arc gene. ~eliminary results of hybrid arrest translation using cloned recombinant DNA containing Mo-MSV-specific sequences support the conclusion that the 37K, 33K, 24K, and 18K proteins are encoded by the MO-MSV STC gene. While the 37K, 33K, 24K, and 18K proteins appear to be unique MO-MSV coded products, their definitive identification as MO-MSV arc proteins awaits an antiserum which recognizes the MO-MSV sarcoma-specific products. If these proteins are encoded by the src gene of MO-MSV, we would expect to find one or more of them in MSV infected cells. A 21s MSV-specific RNA has been isolated from MO-MSV infected cells and translated in vitro to produce a 37K protein as well as small amounts of 33K, 24K, and 18K proteins (M. Jones, H. Fan, and I. Verma, manuscript in preparation). In vitro translation of MO-MSV virion RNA also yielded a 15K protein coded for by a polyadenylated RNA of about 25 S. This protein was not recognized by antisera directed against p30 or whole MO-MLV vu-ions, and its peptide map was unrelated to that of the 62K protein. The 15K protein is, therefore, unlikely to be encoded by the gag or env genes. From the size of the RNA coding for the 15K protein and its unrelatedness to the 3’7K family, it seems unlikely that the 15K protein is encoded by the src gene. By elimination, the 15K protein may originate from either of the pal gene fragments remaining in the MO-MSV genome (see Fig. 7). Either of the two fragments is large enough to code for the 15K protein (Hu et al., 1977; Donoghue et al., 1979). Donoghue et al. (1979) have identified a 22 S subgenomic RNA in cells transformed by MO-MSV (Clone 124), which contains a 5’ derived leader sequence spliced to the 3’ portion of the MSV genome at the beginning of the pal region. This RNA, which is found on polysomes, may code for the 15K protein. From the data presented in this paper, we propose that the genome of MO-MSV is expressed in vitro as depicted by the model in Fig. 7. The 62K protein is expressed

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only from intact genomic RNA containing the 5’ gag gene whereas the 37K, 33K, 24K, 18K, and 15K proteins would be generated from overlapping 3’ fragments of genomic RNA, The almost complete overlap of amino acid sequences of the members of the 37K family deduced from their tryptic peptide maps, coupled with the fact that the sizes of coding RNA decrease with the sizes of the proteins in the family, suggests that these proteins all share a common C-terminal region as indicated in Fig. 6. This organization predicts that the Nterminal sequences of the 37K, 33K, 24K, and 18K proteins should all be deferent from each other in addition to being different from the 62K and 15K proteins. Examination of the tryptic peptides of these proteins labeled specifically at the N terminus with formyl [YS]methionine shows that this is indeed the case (data not shown), The 800-nucleotide region lying to the 3’ side of the src gene is common to MO-MSV and MO-MLV. The function of this region is not known. So far, we have not identified a protein coded by this region. ACKNOWLEDGMENTS This work was supported by Grants CA-14195 and CA-17036 from the National Cancer Institute. K. B. was the recipient of a postdoctoral fellowship from the National Institutes of Health, and J. P. was supported by a National Institutes of Health predoctoral training grant to the University of California, San Diego. We would like to thank the members of the Tumor Virology Laboratory for helpful discussions during the course of this work and constructive criticisms of the manuscript. We are grateful to Chris Roberts for her help in isolating the MO-MSV virion RNA and to Judy ~einkoth for pe~orm~ng the m’GTP ex~~ments. REFERENCES AARONSON, S. A., JAINCHILL, J. L., and TODARO, G. J. (1970). Murine sarcoma virus transformation of Balb/3T3 cells: Lack of dependence on murine leukemia virus. Proc. Nat. Acad. Sci. USA 66, 1236-1243. AARONSON, S. A., and ROWE, W. P. (1970). Nonproducer clones of murine sarcoma virus transformed Balbi3T3 cells. Virology 42, 9-19. ANDEKSSON, P., GOLDFARB, M. P., and WEINBERG, R. A. (1979). Ch~cte~zation of in vitro synthesized Maloney sarcoma virus DNA: Use of trans-

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AND BEE~ON Hu, S., DAVIDSON, N., and VERIA, I. M. (1977). A heteroduplex study of the sequence relationships between the RNAs of M-MSV and M-MLV. Cell 10, 469-477. KAMINE, J., BURR, J. G., and BUCHANAN, J. M. (1978). Multiple forms of sarc gene proteins from Rous sarcoma virus RNA. Proc. Nat. Aead. Sci. USA 75, 366-370. KIRSTEN, W. H., and MAYER, L. A. (1967). Morphological responses to a murine erythroblastosis virus. J. Nat. Cancer Inst. 39, 311-319. MAISEL, J., DINA, D., and DUESBERG, P. (1977). Murine sarcoma viruses: The helper-independence reported for a Moloney variant is uncon~~ed; distinct strains differ in the size of their RNAs. Virology 76,295-312. MAISEL, J., KLEMENT, V., LAI, M. M. -C., OSTERTAG, W., and DUESBERG, P. (1973). Ribonucleic acid components of murine sarcoma and leukemia viruses. Proc. Nat. Acad. Sci. USA 70, 3536-3540. MOLONEY, J. B. (1966). A virus-induced rhabdomyosarcoma of mice. Nat. Cancer Inst. Monogr. 22, 139-142. OSKARSSON, M. K., ELDER, J. H., GAUTSCH, J. W., LERNER, R. A., and VANDE WOUDE, G. F. (1978). Chemical determination of the ml Moloney sarcoma virus pP608@J gene order: Evidence for unique peptides in the carboxy terminus of the polyprotein. Proe. Nat. Acad. Sci. USA 75, 4694-4698. PELHAM, H. R. B. (1979). Translation of fragmented viral RNA in vitro. Initiation at multiple sites. FEBS Lett. 100, 195-199. PHILIPSON, L., ANDERSSON, P. OLSHEVSKY, U., WEINBERG, R., BALTIMORE, D., and GESTELAND, R. (1978). Translation of MuLV and MSV RNAs in nuclease-treated retieulocyte extracts: Enhancement of the gag-@ polypeptide with yeast suppressor tRNA. Cell 13, 189-199. PURCHIO, A. F., ERIKSON, E., BRUGGE, J. S., and ERIKSON, R. L. (1978). Identification of a polypeptide encoded by the avian sarcoma virus src gene. Proc. Nat. Acad. Sci USA 75, 1567-1571. PURCKIO, A. F., ERIKSON, E., and ERIKSON, R. L. (1977). Translation of 35s and of subgenomic regions of avian sarcoma virus RNA. Proc. Nat. Acad. Sci. USA 74, 4661-4665. ROBEY, W. G., OSKARSSON, M. K., VANDE WOUDE, G. F., NASO, R. B., ARLINGHAUS, R. B., HAAPALA, D. K., and FISCHINGER, P. J. (1977). Cells transformed by certain strains of Moloney sarcoma virus contain murine p69. Cell 10, 79-89. ROTHENBERG, E., DONOGHUE, D. J., and BALTIMORE, D. (19’78). Analysis of a 5’ leader sequence on murine leukemia virus 21s RNA: Heteroduplex mapping with long reverse transcriptase products. Cell 13, 435-451.

MO-MSV TRANSLATION SCOLNICK, E. M., RANDS, E., WILLIAIS, D., and PARKS, W. P. (1973). Studies on the nucleic acid sequences of Kirsten sarcoma virus: A model for formation of mammalian RNA-containing sarcoma virus. J. Viral. 12, 458-463. SEF~ON, B., BEEMON, K., and HUNTER, T. (1978). A eomparison of the expression of the src gene of Rous sarcoma virus in vitro and in wivo. J. Viral. 28, 957-971. VAN ZAANE, D., and BLOEMERS, H. P. J. (1978). The

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genome of mammalian sarcoma viruses. Biochim. Biqhys. Acta 516, 249-268. VERMA, I. M. (1978). Genome organization of RNA tumor viruses. I. In vitro synthesis of full-genomelength single-stranded and double-stranded viral DNA transcripts. J. Virot. 26,615-629. VERMA, I. M. (1979). Genome organization of retroviruses. III. Restriction endonuelease cleavage maps of mouse sarcoma virus double-stranded DNA synthesized in vitro. Nucleic Acids Res. 6, 18631868.