A model structure for Rous sarcoma virus genomic RNA and its implications for various functions of the viral RNA

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Reference

A model structure for Rous sarcoma virus genomic RNA and its implications for various functions of the viral RNA

DARLIX, Jean-Luc, BROMLEY, Peter A., SPAHR, Pierre-François

Abstract

The sequence and structure of RSV genomic RNA leader and gene junctions have been studied. The secondary structure of the leader and interactions of the gene junctions with a viral protein may well control the functions of RSV-RNA in reverse transcription, translation and processing.

DARLIX, Jean-Luc, BROMLEY, Peter A., SPAHR, Pierre-François. A model structure for Rous sarcoma virus genomic RNA and its implications for various functions of the viral RNA.

Molecular Biology Reports , 1981, vol. 7, no. 1-3, p. 127-133

DOI : 10.1007/BF00778743

Available at:

http://archive-ouverte.unige.ch/unige:150495

Disclaimer: layout of this document may differ from the published version.

1 / 1

functions of the viral RNA

J.-L. Darlix, P. A. Bromley & P.-F. Spahr

Department of Molecular Biology, University of Geneva, 30, quai Ernest-Ansermet, 1211 Geneva 4, Switzerland

Abstract

The sequence and structure of RSV genomic RNA leader and gene junctions have been studied. The secondary structure of the leader and interactions of the gene junctions with a viral protein may well control the functions of RSV-RNA in reverse transcription, translation and processing.

Introduction

The genomic RNA of Rous sarcoma virus (RSV) is 10000 nucleotides long and codes for 4 known genes which map in the order 5' to 3': the virus group specific antigen (Gag) the viral DNA poly- merase (Pol), the viral envelope glycoprotein (Env) and the transforming gene (Src). RSV-35S-RNA extracted from mature virions possesses features of both a messenger RNA and a template for reverse transcription: on one hand it is polyadenylated, capped and has the same chemical polarity (positive strand) as the polysomal viral RNA found in infected cells and on the other hand it carries a tRNAtryp hybridized to a region one hundred nucleotides away from its 5' end, that serves as a primer for the synthesis of the minus strand of the proviral DNA (for a review, see 1).

When translated in vitro RSV-RNA promotes the synthesis ofa polypeptide of 76 kilodaltons, the Gag product, identical to that found in vivo (17). In addition, a polypeptide of 180 kilodaltons is synthesized that represents the translation of the first two genes Gag and Pol (12). This polypeptide is also found in infected cells (12).

Recently it has been shown that the subgenomic viral 28S and 22S mRNA coding for the glyco- proteins and the src protein, respectively, have at their 5' end a segment of RNA corresponding to

that found at the 5' end of the genomic RNA (20). It seems likely that the full length viral RNA serves as a precursor to the spliced viral mRNAs (16).

RSV-RNA has a tight structure as revealed by optical means, electron microscopy and by nucle- ases specific for double stranded RNA (3, 7). We have recently reported a detailed analysis of the secondary and tertiary structures of RSV-35S-RNA (8), and on the basis of these results we have proposed a cloverleaf model for its structure. In this paper we present new data on the nucleotide sequence of the leader preceding the first cistron and on possible interactions of viral proteins with the genomic RNA and discuss the implications of the model structure for the various functions of RSV-RNA.

Materials and methods

Cells and viruses, isolation of virion nucleic acids, fingerprint analysis of RNA and sequencing of RNA were as described previously (8).

Enzymes. E. coli RNases H and III were purified and assayed as reported (4, 7). T 4 polynucleotide kinase and RNA ligase were obtained from P.L.

Biochemicals. AMV DNA polymerase and Phys- arum I RNase were gifts of Dr. J. Beard and Dr. J.

P. Bargetzi, respectively.

Molec. Biol. Rep. 7, 127 133 (1981). 0301-4851/81/0073 0127 $1.40.

9 Dr W. Junk Publishers, The Hague. Printed in The Netherlands.

128

Synthesis of specific fragments of viral cDNA Viral cDNA synthesis was initiated using a specific DNA primer (TCCAT, collaborative Re- search) and conducted under conditions previously described (6). Specific cDNA fragments were then obtained by digesting the heterogeneous cDNA product with restriction endonucleases and sepa- rating the various fragments. This procedure and the mapping of the various fragments will be described in detail elsewhere.

UV irradiation

of[32p]

[32p]-labeled virions (15 • 106 cpm, 0,1 ml) were put in a petri dish, placed at 35 cm from a UV lamp (255 nm), and irradiated for various periods of time. Following the irradiation, the viruses were disrupted with 1% SDS and 1 mM EDTA (10 min, 37 ~ and 70S RNA was purified. The recovered peak of[32P] 70S RNA was heated 5 min at 65 ~ C in 75 mM NaC1, 1 mM EDTA and 50 mM Tris-HC1 pH 7.5 and extensively digested with Tl RNase.

[32P]-labeled protein-RNA complexes were recov- ered by filtration through a column of Sephadex G75 in 75 mM NaCI, 1 mM EDTA and 50 mM Tris-HC1 pH 7.5. The protein-RNA complexes amounted to 2%-5% of[32p]counts in the 70S peak.

[32p] RNA in the complex was further purified by proteinase K digestion in 0.5% SDS (20 min, 37 ~ and after two ethanol precipitations the RNA recovered (10 to 20% of the counts found in the [32p] protein-RNA complexes) was extensively digested with T~ RNase and fingerprinted.

Results

The leader sequence of RSV-RNA

a) Location of the A UG initiator codon of Gag gene The N-terminal amino-acids of the Gag gene product being Met-Glu both for Pr-C (13) and for LA23 (unpublished results), a pentadeoxyribonu- cleotide complementary to the sequence of the first five nucleotides coding for Met-Glu was syn- thesized. This pentadeoxynucleotide was then hybridized to RSV [32p] 35S-RNA and the hybrid digested with E. coli RNase H (4, 10). The digestion

products were analyzed by polyacrylamide gel electrophoresis in 8 M urea and at 50 ~ (Fig. 1).

The distinct RNA band (arrow) generated upon RNase H digestion was recovered, extensively digested with Tj RNase and fingerprinted (Fig. 1).

Unique T I oligonucleotides 6 and 35 characterize the RNA fragment which have previously been mapped at the very 5' end of RSV-RNA genome (5). It should be pointed out here that the T t oligonucleotide order 5' Cap 6-30-35... estab- lished previously (5) contained an uncertainty in the order of 30 and 35, and according to the results presented above it should be written 5' Cap-6-35- 30...

The size of the RNA fragment has been measured relative to the 5S, 5,8S, 7S and 9S RNAs and estimated to be 370 + 5 nucleotides. From these results, we may conclude that the sequence pre- ceding the initiator AUG of the Gag gene is made of 370 • 5 residues.

b) Sequence of the region preceding the AUG initiator codon of Gag gene

Several of the specific cDNA fragments obtained by the procedure outlined in Materials and Methods were sequenced by the method of Maxam and Gilbert (11). Part of the leader sequence of RSV- RNA could thus be deduced and is given in Figure 2. The following comments can be made:

1. Several termination codons follow the tRNAtryp binding site and should stop translation in all three reading frames. Similarly, termination codons precede the initiator AUG and should also stop translation in all three reading frames. This observation is consistent with the fact that no new polypeptide was detected by in vitro translation of the 370 nucleotide RNA fragment (result not shown).

2. From position 340 to 354 there is a 15 nucleotide sequence 11 of which could form base- pairing with the 3' OH of 18S rRNA and such an interaction appears to be of importance for the initiation of translation (9). It may be recalled here that a sequence of 7 bases (positions 4 to 13) can also interact with the 3' OH sequence of 18S rRNA (6).

3. A sequence of 7 nucleotides, CCCUGAC from positions 59 to 65, is repeated just before the initiator AUG of the Gag gene at positions 337 to 343 + 5 from the 5' Cap. c)

1 2 3

Fig. 1. Location of the AUG initiator codon of Gag gene. [rip] LA23-B 35S RNA ( 106 cpm, 1 ~g) was mixed with 0.5 #g TCCAT in 5 #1, heat-denatured, incubated 10 min at 50 ~ in 50 mM Tris-HCl pH 7.9, 10 mM MgCI 2, I mM DTT, then 30 min at 32 ~ and finally 30 rain at 32 ~ in the presence of RNAase H. Electrophoresis in 6% polyacrylamide gels containing 8.3 M urea: without TCCAT and with 1 U RNase H [ 1],, with TCCAT and 1 U R Nase H [2] or 0.5 U RNase H [3]. The [nP]RNA band (arrows) was recovered, extensively digested with T~ RNase and fingerprinted [4]. The size of the RNA was measured relative to 5S, 5.8S, 7S and 9S RNA of Chlarnydomonas reinhardiL

Secondary structure of the leader sequence of RS V-RNA

The leader sequence of RSV-RNA must have a strong secondary structure: For instance the Tj oligonucleotides 6 and 35 markers for that sequence are very susceptible to degradation with RNases III and IV, which nucleases are specific for structured RNA (cf. 7, 8). On the basis of such data we have proposed a secondary structure for the 5' last 100 nucleotides of RSV-RNA (7). In addition we have been able to purify structured RNA fragments 40-50 nucleotides which contain T, oligonucleotide marker 35 (see Fig. 3 and ref. 8). Finally there is a 40

nucleotide sequence (positions 32 to 62) within the 5 last 100 nucleotides and which involves the strong ribosome binding site (6), 30 of which could form base pairs with the 3' end of the leader sequence (positions 308 to 346 + 5) (see Fig. 4).

Study on the structure of the viral genejunctions by U V irradiation of the virus

We have shown previously that sequences in the vicinity of the Gag-Pol, Pol-Env and Env-Sre gene junctions exhibit strong secondary structures that interact with each other (8). Possible interactions of the gene junctions with a viral protein were in-

130

THE LEADER SEQUENCE OF ROUS SARCOMA VIRUS RNA

Portiat nucieotide sequence from the 5' Co__.pp of the RNA to the initiation site for Go__.gg synthesis

I0 3,0 50,

7rnOpppGmCC AUU UGA CCA UUC ACC ACA UUG GUG UGC GCC UGG GUU GAU GGC CGG ACE GAU GAU UCC CUG ACG

A A A

7,0 g0 _i It0 _t

ACU ACG AGC ACA UGC AUG AAG CAG AAG GCU UCA UUU GGU GAC CCC CGA CGU GAU CGU UAG GGA

AUA GUG GGG CC ...

270 290

(T1 oligonucleotide 35 ) ,

... GCUAACAUACCCUACCG ... UUA CAG UGU AGG AAC CAG GAU GGC AAC AAG GCC UAA CGC CCA GGA

310 330 _i 350 _i

AGA GCU CUC CCU UCG GGA CCG UGG CCC GGC CCU GAC CAG CCU GCC GUC UCG UUA GCG AUG ACA 370 AUG GA ...

Fig. 2. Partial nucleotide sequence of RSV-RNA leader sequence. The strategy to sequence the leader of RSV-RNA will be described in detail elsewhere. Sequences of T I oligonucleotide 6 (positions 9 to 24) and of tRNA tryp primer binding site (position 102 to 118) are underlined. The regions between the arrows represent the two fragments protected by 80S ribosomes from T~ RNase digestion (6). The RNA sequence with the dotted line is complementary to the 3' end of the 18S rRNA (9).

ca b

35 Q

30-

Fig. 3. Structured RNA fragments of the leader sequence. Partial nuclease digestion of[3:P] 35S-RNA with Tt RNase and purification of the fragments were carried out as described (8). [32p] RNA fragment No 4 was analyzed by electrophoresis in 8% acrylamide gels containing 7 M urea, 75 mM Trisborate-EDTA pH 8,3 [a] and extensively digested with Tj RNase and fingerprinted [b].

3O 5O

... , . . . . I I .U.

---UUGGuGuGcGCCUGGGuuGAuGG CCGGAC CGA GAUUCCCU---

---GACCA GUCCCGGCCCGG UGCC.A.GGGCUU.c.CCU_

CUCGAGAA---

i

330 310

Fig. 4. Proposed interaction between the 5' and 3' ends of RSV-RNA leader sequence.

vestigated by UV irradiation of the virus. Upon treatment the virus was disrupted and complexes sedimenting at 70S were recovered and subsequent- ly digested with T, RNase (see Fig. 5). Material with an S value greater than 4S was recovered .by gel filtration and further analyzed for its RNA and protein contents (Fig. 5).

T~ oligonucleotide markers for the Gag-Pol (No 3), Pol-Env (No 2 and 4) and Env-Src (No 13b) are present in good yield in the fingerprint analyses. T~

oligonucleotide 14a which maps in the Gag gene is also recovered. The protein content of the material was analyzed by polyacrylamide gel electrophoresis and found to contain multiple bands migrating similarly to viral p19, which do not appear to be phosphorylated (results not shown) (15).

Discussion

The data on the leader sequence and on the gene junctions of RSV-RNA may shed some light on the function of the viral RNA in translation, reverse transcription and RNA processing.

The leader sequence does not seem to participate in tertiary structures although it has a strong sec- ondary structure characterized by T~ oligonucleo- tides 6 and 35 (7, 8). This finding is expected since the 5' end of the leader sequence should be accessible for the initiation of cDNA synthesis, and for the in vitro binding of the ribosomes (6). Initiation of Gag gene translation takes place at the AUG at position 370 - 5, thus at more than 300 nucleotides downstream from the ribosome binding site (6).

The potential interaction between the 3' end of the ribosome binding site and a sequence at the 3' end of the leader may explain how the ribosome bound around GUG at position 26 starts translation of the Gag gene at position 370 + 5. This potential

interaction could also play a role in the discrimina- tion between translation and transcription.

The region of the Gag-Pol gene junction was thought to play an important role in the expression of the Pol gene which appears as a fused Gag-Pol product of 180 K (12, 17). We have been able to synthesize cDNA fragments mapping at the 3' end of the Gag gene and in the vicinity of the Gag-Pol gene junction. The complete nucleotide sequence of this region will be reported elsewhere, but it shows that at the 3' end of the Gag gene only one reading frame is open, and around the Gag-Pol gene junction the 3 reading frames are blocked by UAA, UAG and UGA termination codons. Consequently translation of the Pol gene should be conducted by a specific mRNA which has been spliced in that region, since suppressor tRNAs do not enhance the in vitro synthesis of the 180 K protein in the RSV system (19) as they do in the MuLV system (14).

Interaction of the viral gene junctions with each other might be important in the splicing process that gives rise to subgenomic 28S and 22S RNA, the messenger RNAs for the Env and Src proteins (16).

In this respect the binding of viral core protein p19 (possibly involved in viral maturation (2)) could well impair the splicing process as well as trans- lation of RSV-RNA (18). This possible negative control of p19 on two functions of RSV-RNA is now under investigation.

Acknowledgements

We thank Dr. J. Beard for a generous gift of AMV-DNA polymerase. We thank O. Jenni for the drawings. The excellent technical assistances of M.

Schwager and F. Steimer are gratefully acknow- ledged. This work was supported by grant No 3.310.78 from the Swiss National Science Foun- dation.

132

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0 ~' "J 0~'"~':~"-' 0~" ',-~ 0~'~ "+;

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e ORDER OFT10LIGONUCLEOTIDES OF LA23 RNA

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5' end =. 6 ii ~i ~. 8 ~ 40b+ 14a. 33.14b. 34.29.5+ 26b. 18. 26.3136+ 23.24.22,43.41~ 37. t7.31

Gag gene t Pot gene 1 42

3' end, poty(A) + 27.2 I+ 260. |4r IOa .td)a.32,~4.13b. lob. 20.28. I 1.19.2.15+ 9.4.13o +38

C t Src gene t Env gene

Fig. 5. Interaction of the gene junctions within the virus. UV irradiation of [32P]-labeled virus and isolation of the protein-RNA complex are described under methods. Fingerprint of LA23-B 35S RNA (b) and numbering of the large'r] oligonucleotides (a). No UV irradiation (c) and upon 2 min UV irradiation (d). No increase in the recovery of the T l oligonucleotides was noted after 2 min UV irradiation. 5'to 3" ordering of the Tt oligonucleotides of LA23-B genomic RNA has been published (5). Arrows point to regions believed to be gene junctions (1, 3, 5) (e).

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