VIROLOGY

86,297-311

(1978)

Avian Retrovirus RNA-Directed DNA Synthesis: Transcription at the 5’ Terminus of the Viral Genome and-the Functional Role for the Viral Terminal Redundancy MARC S. COLLETT’ Department ofMicrobiology,

ANTHONY

AND

J. FARAS”

University ofMinnesota Medical School, Minneapolis, Minnesota 55455 Accepted November 29, 1977

The size and structure of DNA synthesized on 35 S RNA tRNA”” template. primer complexes in reconstructed reactions by the avian retrovirus RNA-directed DNA polymerase in vitro have been elucidated. Under standard enzymatic reaction conditions three major species of DNA are initiated on tRNA@ at the 5’ end of the viral genome. Two of these are single stranded in nature and exhibit lengths of 70 and 100 nucleotides. The third is hairpin in structure and is 185 nucleotides in length. A precursor-product relationship has been demonstrated between DNAro, DNAlm, and the hairpin DNAw species. Furthermore, it appears that hairpin DNA,“5 is formed during DNA synthesis rather than after DNA synthesis as a consequence of the intramolecular folding of complementary sequences present in the DNA,%5 transcript. Finally, we present data indicating that, in addition to conversion of DNAim to hairpin DNA, the DNA E+,species can attach to the 3’ end of the viral genome presumably by base pairing with the terminally redundant sequences at the 3’ terminus to provide a primer for the continuation of DNA transcription at this end of the viral RNA. These data provide a functional role in proviral DNA synthesis for the terminally repeated genomic sequences. INTRODUCTION

The bulk of the DNA synthesized in vitro by either detergent-disrupted virions of Rous sarcoma virus (RSV) or reconstructed reactions containing 70 S RNA or 35 S . tRNA@ template . primer complexes is initiated upon a tRNAt’?’ primer molecule located near the 5’ end of the viral genome (Faras et al., 1973, 1974; Dahlberg et al., 1974; Harada et al., 1975; Folk and Faras, 1976; Waters et al., 1975; Taylor and Illmensee, 1975; Staskus et al., 1976). Under conventional reaction conditions this DNA product exhibits a nucleotide length which reflects the distance between the tRNALrp primer and the 5’ end of the RNA genome and therefore represents a very small portion (ca. 1%) of the nucleotide sequences contained in the RSV genome (Haseltine et ’ Present address: Department of Pathology, University of Colorado Medical School, Denver, Colorado 80262.

‘To whom dressed.

requests

for reprints

should

be ad-

al., 1977; Shine et al., 1977; Collett and Faras, 1977; Friedrich et al., 1977). In an effort to analyze further in vitro transcription of the viral genome under conditions in which RNase degradation of the template is negligible, we have directed our attention to reconstructed reactions containing purified RNA-directed DNA polymerase and genome RNA (Collett and Faras, 1977). As is the case with reactions containing detergent-disrupted virus (Collett and Faras, 1975; Rothenberg and Baltimore 1976, 1977), increasing the concentration of deoxynucleoside triphosphates in reconstructed reactions also facilitates the elongation of DNA transcripts to lengths of 2500 nucleotides or more, indicating that other regions of the RSV genome can be transcribed into DNA by the RNA-directed DNA polymerase in vitro (Collett and Faras, 1977). This synthesis of DNA transcripts longer than the estimated distance between the tRNAtrP primer and the 5’ end of the viral RNA may involve the termi-

297

0042-6822/7a/0s62-0297$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

298

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AND FARAS

nally redundant sequences of the RSV genome which apparently are also ultimately responsible for circularization of proviral DNA (Collett and Faras, 1976; Junghans et al., 1977; Haseltine et al., 1977; Schwarz et al., 1977; Collett et al., 1977). In addition to its proposed involvement in the synthesis of covalently closed circular forms of viral DNA, the 5’ terminus of the viral genome may be involved in integration of proviral DNA into the chromosomal DNA of the host cell (Collett, Coffin, and Faras, ICN-UCLA Symposium on Animal Virology, 1976; Coffin, 1976; Shine et al., 1977). Furthermore, since transcription and translation of the viral RNA begin at the 5’ end of the viral genome these sequences may serve as a promoter site for RNA polymerase and an attachment site for ribosomes, respectively. Because of these possible functions of the 5’-terminal sequences of RSV RNA we are continuing our studies on the transcription of this region of the viral genome. In this communication we present data on the structure of the DNA transcribed at the 5’ terminus of the viral genome, its mechanism of synthesis, and its involvement in the elongation of the viral DNA at the 3’ terminus of the viral DNA.

DNA in reconstructed reactions. The details of our procedure for the enzymatic synthesis of DNA in reconstructed reactions have been described elsewhere (Collett and Faras, 1977). The viral RNA template. primer complexes used in these studies consisted of 35 S RNA to which either purified tRNAtrP or oligo(dThz-1s had been reannealed (Collett and Faras, 1975, 1977; Faras and Dibble, 1975). Analytic reaction mixtures contained deoxynucleoside triphosphate precursors (dNTPs) at “ratelimiting” concentrations (one dNTP, usually the radiolabeled one, at 6 m and the other three at 60 $I!0 (Collett and Faras, 1977). Preparative reactions contained all four dNTPs at 60 fl. Radiolabeled dNTP precursors used were either r3H]TTP, [3H]dGTP, [a-32P]dGTP, [(r-““P]dATP, or 1251-labeleddCTP. 1251-labeleddCTP was prepared by a modification of the Commerford reaction (1971) as previously described (Kamon et al., 1976). No differences were noted in any of the characteristics of “Hlabeled, %P-labeled, or ‘251-labeled DNA transcripts. All reactions were incubated at 37” for 1 hr and the DNA transcripts were purified as previously described (Collett and Faras, 1975, 1977). Nuclease digestions. S1 nuclease was purified from Sanzyme (Calbiochem) by MATERIALS AND METHODS the method of Sutton (1971). The condiReagents and virus. The sources and tions of Sr digestion were essentially as preparation of most of the pertinent mate- previously described (Leong et al., 1972). rials employed in these studies have been Our S1 nuclease preparations routinely hypreviously described (Faras et al., 1972, drolyzed greater than 95% single-stranded 1973; Collett and Faras, 1975,1977) as have DNA and less than 5% double-stranded the preparation and purification of cells and DNA under these reaction conditions. Douvirus (Faras et al., 1974; Faras and Dibble, ble- and single-stranded controls were 1975). Avian myeloblastosis virus (AMV; in tested each and every time a sample was plasma), avian sarcoma virus (Prague C analyzed. Assays involving Neurospora crassa nuclease were performed by J. Leis strain), and purified AMV RNA-directed (Department of Surgery, Duke University, DNA polymerase were obtained through the auspices of the Office of Resources and Durham, N.C.). Samples were digested in 200 n-d4 NaCl, 30 mM Tris . HCI (pH 8.1), Logistics, Viral Cancer Program, National 5 mil4 MgClz at 37” for 30 min. Exonuclease Cancer Institute. I (Exo I) was a gift from B. Duncan and H. Purification of viral RNA. The purification of radiolabeled and unlabeled viral Warner of the Department of BiochemisRNA (Dahlberg et al., 1974; Faras and Dib- try, University of Minnesota, St. Paul, ble, 1975), poly(A)-containing viral RNA Minn. Digestions were carried out in 20 (Staskus et al., 1976), and the AMV primer n-&Z Tris HCl (pH 8.1), 20 mM MgCh for RNA (tRNA@) (Faras and Dibble, 1975) 30 min at 37”. Polyacrylamide-formamide gel electrohas been previously described. phoresis. The procedures for electrophoreEnzymatic synthesis of virus-specific

AVIAN

RETROVIRUS

RNA-DIRECTED

I L N

‘0 20 x .T n‘9 L 2

1000

IO I00

= P !!I

c)

N ‘0 ; :

2

”

I

400

iiF

100

e 2 2

40

20

40 DISTANCE

60 MIGRATED

80

DNA

SYNTHESIS

299

sis in 5 and 10% polyacrylamide gels containing 98% formamide have been described (Maniatis et al., 1975; Collett and Faras, 1977). After electrophoresis, each cylindrical gel was sliced into l-mm segments and the radioactivity in each slice was analyzed by either gel solubilization (Collett and Faras, 1977) followed by scintillation counting (“H-labeled transcripts), Cerenkov counting (‘“P-labeled transcripts), or gamma counting (12”1-labeled transcripts). In experiments requiring recovery of DNA from gels, DNA was eluted and purified as previously described (Collett and Faras, 1977). RESULTS

(mm)

Frr,. 1. Formamide-polyacrylamide gel electrophoresis of DNA transcripts synthesized from 35 S RNA. tRNAtrP template. primer complexes. (A) The reannealing of purified tRNAtv primer to viral 35 S RNA is described under Materials and Methods. This template. primer complex was used for the enzymatic synthesis of ““I-labeled dCMP-labeled DNA by the viral DNA polymerase under rate-limiting concentrations of dNTPs (see Materials and Methods). After 1 hr at 37”, the reaction was terminated and the DNA was purified. The sample was then subjected to electrophoresis in a 98% formamide-5% polyacrylamide gel (solid lines). 35 S RNA. tRNA’lP complexes were also employed to radiolabel specifically the initial heptanucleotide portion of tRNAL’P-primed DNA transcripts as follows. o-[:‘“P]dATP (1.7 /&f) and unlabeled dGTP and TTP (both at 60 m were included in enzymatic reactions with the reconstituted template primer complex and the viral DNA polymerase. After 15 min at 37”, unlabeled dATP was added to 1.4 mM and dCTP to 60 fl, and enzymatic synthesis continued for 45 min at 37”. Subsequent purification and 98% formamide-5% polyacrylamide gel electrophoresis were as described above (broken lines). The nucleotide-length curves were obtained as described previously (Collett and Faras, 1977) employing the Hoe111 restriction enzyme fragments of phage +X174 DNA, simian virus 40 DNA, and polyoma virus DNA. (B) Covalent attachment of iDNA transcripts to the tRNA”s primer molecule was demonstrated as follows. iDNA was synthesized from reannealed 3.5 S RNA tRNA”” template. primer complexes as described under Materials and Methods. After SDS-Pronase-phenol treatment and ethanol precipitation, the nucleic acids were chromatographed on Sephadex G-50 and concentrated by a second ethanol precipitation. The material was centrifuged and dissolved in a small volume of 0.02 M Tris HCl, pH 7.4, 0.01 M EDTA. One aliquot (broken lines) was treated

Size of tRNAtrP-Initiuted DNA Present at the 5’ End of the RSV Genome It has been previously demonstrated that the size of the DNA product synthesized by detergent-disrupted preparations of RSV in vitro is dependent upon the concentration of deoxynucleoside triphosphates (dNTPs) present in such reactions (Collett and Faras, 1975). We have recently demonstrated a similar effect on the size of the DNA product synthesized in reconstructed reactions containing the purified RNA-directed DNA polymerase and 35 S . tRNALrP template . primer complexes (Collett and Faras, 1977). In reconstructed reactions containing low concentrations of dNTPs, three discernible size classes of DNA could be observed when analyses were performed under denaturing conditions in 98% formamide-5% polyacrylamide gels (Fig. 1A). None of the DNA synthesized exhibited a nucleotide length greater than 200 nucleotides. The tRNAtrP-initiated DNA synthesized under these reaction conditions exhibited nucleotide lengths of 185, 100, and 70 and will be referred to as initial with pancreatic RNase (40 pg/ml, 37” for 30 min) and a second (solid lines) was left untreated. After drying both samples down and resuspending each of them in 99% deionized formamide, they were subjected to electrophoresis in 98% formamide-10% polyacrylamide gels at 150 V for 3.5 hr (until the xylene cyan01 FF dye migrated 50 mm into the IOO-mm gels). The numbers represent nucleotide lengths estimated from the nucleotide-length calibration curves.

300

COLLETT

DNA:iDNAle5 (peak A), iDNAlm (peak B), and iDNAT (peak C), respectively. That all of these species of iDNA are covalently linked to the tRNAtrP primer molecule and therefore represent nucleotide sequences initiated at the 5’ terminus of the RSV genome was demonstrated by isolating viral RNA . radiolabeled DNA hybrids from reconstructed reactions containing 35 S. tRNA@ template. primer complexes and comparing the electrophoretic mobility of RNase-treated with untreated DNA product in 98% formamide-10% polyacrylamide gels (Fig. 1B). Three radiolabeled species were identified in the untreated sample exhibiting lengths of 255, 175, and 145 nucleotides. Subsequent to RNase hydrolysis all three of these species exhibited a shift in electrophoretic mobility and migrated at the positions of iDNA,%, iDNAloo, and iDNATO,respectively. The differences in electrophoretic mobility between the untreated and RNase-treated samples are consistent with the removal of approximately 75 nucleotides from each of these DNA transcripts. Since tRNA@ was the only 4 S RNA associated with 35 S viral RNA in these experiments and no detectable breakdown of this template. primer complex was observed during the course of enzymatic synthesis (Collett and Faras, 1977), we conclude that all three iDNA species were covalently attached to the tRNAtrP primer molecule. In a second experiment the three tRNAtrP-initiated DNA species were tested for the presence of the heptanucleotide sequence (A-A-T-G-A-A-G)ou which is the initial deoxynucleotide sequence immediately adjacent to the primer molecule (Taylor et al., 1974; Eiden et al., 1975). In these studies enzymatic synthesis of DNA from 35 S . tRNAt”” template. primer complexes was first conducted in the absence of dCTP, but in the presence of the remaining three dNTPs including radiolabeled dATP, in order to label only the heptanucleotide sequence. Under these conditions of DNA synthesis in which tRNAtrP is the only primer, the initial heptanucleotide sequence is the only DNA product synthesized (Taylor et al., 1974; Eiden et al., 1975; Collett and Faras, 1976). Enzymatic synthe-

AND

FARAS

sis was then continued after the addition of dCTP and a large excessof unlabeled dATP (lOOO-fold) so that the radiolabeled heptanucleotide could be elongated and thus “chased” into larger DNA transcripts. The resultant DNA transcripts specifically labeled in the heptanucleotide position were subjected to electrophoresis in 98% formamide-5% polyacrylamide gels. As seen in Fig. lA, all three tRNAt’P-initiated DNA species contain the labeled heptanucleotide. These data are consistent with the initiation and synthesis of the three species of iDNA at the 5’ end of 35 S. tRNAtW complexes. These conclusions are further supported by nucleotide sequence data indicating that iDNA lo0 and iDNAT represent nucleotide sequences at the 5’ end of the viral genome (Shine et al., 1977; Collett and Faras, 1977; Haseltine et al., 1977) and pulse-chase experiments indicating that iDNAloo and iDNAT can be chased into iDNA& (see below). Secondary Structure of iDNA Secondary structural features of the three species of iDNA were determined with several specific nucleases (Table 1). S1 nuclease and Neurospora crassa nuclease are both single strand-specific endonucleases, whereas exonuclease I, also single strand specific, hydrolyzes DNA progressively from the 3’ terminus only (Sutton, 1971; Linn and Lehman, 1965; Lehman, 1971). Each of the three uniformly labeled iDNA species was examined under conditions that would allow the detection of nondenaturable or “hairpin” DNA and duplex DNA. As shown in Table 1, iDNA1& maintained a high degree (ca. 70%) of resistance to hydrolysis with either S1 or N. crassa nucleases after heat denaturation, indicating rapid, unimolecular reassociation of this DNA species. That the 3’ end of this apparent hairpin DNA was present in a double-stranded conformation was demonstrated by the nearly complete resistance to exonuclease I digestion. In addition,. iDNAlas showed no further increase in duplex structure subsequent to self-annealing. Both iDNAlm and iDNAT,, were nearly completely sensitive to hydrolysis with all three nucleases when assayed for hairpin DNA

AVIAN

RETROVIRUS

RNA-DIRECTED

DNA

301

SYNTHESIS

In addition, since after S1 nuclease treatment this portion of the molecule represents ‘complementary single strands of DNA, the length of the S1 nuclease-resistNature of the Hairpin Structure of iDNAIRs ant iDNAIB5 species should be one-half that The iDNAIa5 transcripts were analyzed of the total nuclease-resistant portion, or in further detail to determine more pre- approximately 65 nucleotides. Confirmacisely the structure of the hairpin species. tion of this prediction was obtained by anThe “snap-back” or hairpin nature of alyzing the size of the S1 nuclease-resistant iDNAlss could be removed after an initial portion of iDNA1% on 98% formamide-5% S1 nuclease treatment by subjecting the polyacrylamide gels (Fig. 2). The compleresistant portion (70%) to a second cycle of mentary strands of the S1 nuclease-resistheat denaturation and S1 nuclease hydrol- ant portion of iDNA1% migrated coincident ysis (Table 1). Furthermore, if S1nuclease- with iDNAT in these denaturing gels, a treated iDNAIB5 was allowed to reassociate result consistent with the prediction of its after heat denaturation, it regained a nearly size from the nuclease studies. Although these studies indicate that the complete double-stranded structure demonstrating that complementary strands S-resistant portion of iDNAlss exhibits a were present after S1 nuclease treatment. size similar to that of iDNATO, they do not Since 70% of the heat-denatured iDNAl% is indicate whether these two DNA species resistant to S1 nuclease, we estimate that represent complete complementary nucleoapproximately 130 of 185 nucleotides con- tide sequences. From the data presented tained in this iDNA species are involved in thus far two physical structures can be posthe duplex portion of the hairpin structure. tulated for the hairpin iDNAIsb species (Fig.

after heat denaturation or for duplex DNA after reassociation indicating their singlestranded nature (Table 1).

TABLE SECONDARYSTRUCTUREOF~DNA iDNA

fraction

1

DETERMINEDRYNUCLEASEDIGESTION" Nuclease resistant

SI

N. crassa

Exo I

-- --.~Self-annealed’ .--___. Si

Label

(%)

Hairpin”

iDNAIHS (peak A)

Uniform Hepta

68.1 14.7

72.0 -

83.5 -

70.5 -

iDNAI(*

(peak Bl

Uniform Hepta

5.5 6.2

0.0 -

11.0 -

10.4 -

iDNAT” (peak Cl

Uniform Hepta

4.2 3.1

0.0 -

0.0 -

12.8 -

Peak A. SI”

Uniform

2.6

-

5.0

76.5

” Uniformly “‘I-labeled, or “‘P-labeled heptanucleotide-labeled (hepta) tRNA’lP-initiated DNA transcripts, were synthesized as described under Materials and Methods, SDS-Pronase-phenol extracted, treated with alkali, and then subjected to electrophoresis in 98% formamidepolyacrylamide gels. The three iDNA species were eluted from the gels and purified. Nuclease digestions were performed as described under Materials and Methods. All values represent percentage resistant to nuclease hydrolysis. Single- and double-stranded DNA standards exhibited greater than 95% and less than 5% hydrolysis, respectively, with both St nuclease and exonuclease I. ’ The presence of nondenaturable or “hairpin” DNA was determined by nuclease treatment subsequent to boiling each of the transcripts in 20 m&f Tris HCI, pH 7.4, for 1 min followed by quenching in ice water. ’ Each of the transcripts (2000 cpm) was self-annealed in 5 /.rl of 0.6 M NaCI, 0.02 M Tris HCl, pH 7.4, 0.01 M EDTA for 18 hr at 68” before being treated with Si nuclease. ‘The S1 nuclease-resistant portion of uniformly “‘P-labeled iDNAlns (peak A. SI) was isolated from a formamide-polyacrylamide gel (Fig. 2).

302

COLLETT

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FARAS

tanucleotide label should be present in an S-resistant form. Analysis of heptanucleotide-labeled iDNAlas with S1 nuclease indicated that this specific nucleotide sequence was nearly completely sensitive to hydrolysis suggesting that Model I more closely represents the true structure of the hairpin iDNAlB5 species (Table 1).

Mechanism of Synthesis of the Hairpin iDNAIa Species It has previously been observed that a significant portion of the double-stranded DNA made by in vitro retrovirus reverse transcription exists as intramolecular basepaired or hairpin sequences (Taylor et al., 1972; Green and Gerard, 1974). The exisDISTANCE MIGRATEDtmm) tence of a hairpin-structured DNA species FIG. 2. Formamide-polyacrylamide gel electroamong the products of tRNA@-initiated phoresis of iDNA,s after treatment with S1 nuclease. DNA synthesis at the 5’ end of the RNA (A) ““P-Labeled iDNAIHh (185 nucleotides in length) genome suggests that, subsequent to reachwas isolated from 98% formamide-5% polyacrylamide ing the 5’ terminus of the viral genome, the gels (see Fig. 1). A portion was hydrolyzed with S viral DNA polymerase loops back and trannuclease as described under Materials and Methods, scribes the newly synthesized DNA product followed by treatment with SDS-Pronase, phenol extraction, and ethanol precipitation. The precipitated into a complementary sequence. This is S, nuclease-resistant material was then subjected to indeed a valid consideration as it is known electrophoresis in a 98% formamide-5% polyacrylthat other DNA polymerases are involved amide gel (broken lines) in parallel with a portion of in template switching and fold-back DNA the undigested iDNA]a:, (solid lines). The two electrosynthesis (Kornberg, 1974). Another possipherograms are superimposed for comparative purbility is that some of the tRNAtV-initiated poses. (B) The peak of “‘P-labeled DNA observed in DNA is able to fold back on itself due to the gel described in A of the S, nuclease-treated reversed complementarity within the moliDNAi% (broken lines) was eluted and then subjected ecule itself. However if this were the case to 98% formamide-10% polyacrylamide gel electrophothen the distance between the tRNAtrP resis (broken lines). ‘““I-Labeled iDNA species were included as internal markers (solid lines). primer molecule and the 5’ end of the viral genome would have to be the size of the 3). Structure I suggests that the two 70- hairpin DNA species which is 185 nucleonucleotide DNAs are not completely com- tides in length. We consider this possibility highly unlikely because it is inconsistent plementary in sequence and that their idenwith the known distance between the tity in nucleotide length is only coincidence. Structure II predicts that iDNAT repretRNAtrP primer molecule and the 5’ end of sents a single-stranded component of the S1 the genome which is 101 nucleotides (Shine nuclease-resistant portion of iDNA185. In an et al., 1977; Haseltine et al., 1977). Furthereffort to distinguish between these two more, iDNA synthesized in the presence of models, we again exploited the ability to actinomycin D, which inhibits DNA-dilabel specifically the first seven nucleotides rected DNA synthesis, lacks the iDNA1e6 species (Fig. 4A). Since actinomycin D (heptanucleotide) of the iDNA sequences as described above. If Model I is correct, would be expected to prevent the production of hairpin iDNA if it were formed iDNAla5 labeled in the initial heptanucleotide sequence should not contain radiolabel during transcription but not by folding due complementarity of the in the hairpin portion of the molecule and to intramolecular should therefore be sensitive to S1 nuclease iDNA species, these results support the hydrolysis. Model II predicts that the hep- notion that hairpin iDNAla5 is formed dur-

AVIAN

RETROVIRUS

+70NT-(XINT

RNA-DIRECTED

+I

I

+

IDNA,~

-I

f 45NT

1

-7ONT-

n

FIG. 3. Schematic representation of two possible molecular structures for hairpin iDNA,,. From the data described in Fig. 2 and Table 1, it appears that iDNAl% contains an S1 nuclease-resistant portion that consists of complementary DNA strands of approximately 70 nucleotides in length (70 NT), or the same size as iDNATo. In this figure, the short lines represent nucleotide bases. Base-paired sequences (resistant to S, nuclease) are indicated by pairs of short lines directly opposite one another, while unpaired bases (S, nuclease sensitive) are presented by single short lines. Model I suggests that iDNA-io and the SI nucleaseresistant portion of iDNAIH6 (70 NT) are not completely complementary. This structure is characterized by two regions of SI nuclease sensitivity; one at a small hairpin loop and the other at the region adjacent to the primer molecule (tRNA”P). Together both make up the SI nuclease sensitivity observed in iDNA,ss (45 NT). Model II predicts that iDNAT,, represents a single-strand component of the St nuclease-resistant portion of iDNAIHTr. Here the total SI nuclease sensitivity of the iDNAlxr, molecule is located in a large (45 NT) loop of the hairpin structure. These models may be distinguished by determining if the nucleotide sequences immediately adjacent to the tRNA”r primer molecule (hepta + heptanucleotide or the first seven nucleotides of DNA adjacent to tRNA”“) are sensitive to S, nuclease digestion. OH represents the 3’-hydroxyl group of the iDNAIHs transcript.

ing DNA synthesis rather than after DNA synthesis. To demonstrate directly that iDNAls5 results from the transcription of the smaller iDNA species and to confirm the presumed precursor-product relationship among the iDNA transcripts, the following pulsechase experiment was performed. Radiolabeled DNA was synthesized from 35 S

DNA

SYNTHESIS

303

RNA. tRNAtrP complexes in the presence of actinomycin D to prohibit the synthesis of iDNAIa5 and allow iDNAloo and iDNA-/o to accumulate (the pulse reaction). Actinomycin D and the labeled precursors were then removed from the reaction mixture by gel filtration and enzymatic synthesis was continued after the addition of unlabeled dNTPs (the chase reaction). Analysis of the products of the pulse reaction and of the pulse-chase reaction after removal of RNA by treatment with pancreatic RNase was performed on 98% formamide-%% polyacrylamide gels. As expected, in the presence of actinomycin, only radiolabeled iDNAloo and iDNAT were present (Fig. 4B). However, when actinomycin D was removed and DNA synthesis was resumed in the presence of unlabeled dNTPs, the bulk of the radiolabel present in iDNAT and iDNA,m appeared as iDNAIX5. Confirmation of the hairpin nature of iDNAjM formed during the chase was obtained by demonstrating that this material exhibited S1 nuclease resistance (ca. 70%) after heat denaturation. An additional, somewhat more direct experiment to establish a precursor-product relationship between iDNAloo and iDNA185 was performed by incubating purified iDNAlcm with the reverse transcriptase under optimum reaction conditions for the synthesis of DNA. As depicted in Fig. 4C, the bulk of the iDNAloo transcripts was converted to iDNA,#, during enzymatic synthesis. The relative ratio of iDNA,, to iDNAT in the pulse reaction product as compared to the chase reaction product in Fig. 4B suggests the conversion of iDNAT,) to iDNAlo. These observations are consistent with hybridization/protection experiments whereby viral genomic sequences complementary to these two iDNA species were analyzed (Collett et al., 1977). Both iDNAIoo and iDNAT species represented identical 5’-terminal nucleotide sequences with the exception of iDNAloo which contained sequences also complementary to the capped, 5’ terminally located Tl RNase oligonucleotide (Collett et al., 1977; Cashion et al., 1976). Collectively these data indicate that iDNAT is a precursor to iDNA,,x, and that both of these iDNA spe-

304

COLLETT

AND

A. 1 I

n ‘0 ; zu

c.l *0 ; z 5

N IO x % ”

DISTANCE

MIGRATED lmml

FIG. 4. Formamide-polyacrylamide gel electrophoresis of tRNAtv-initiated DNA transcripts synthesized in the presence of actinomycin D and precursor-product relationship among the iDNA species. (A) Viral 35 S RNA. tRNA”” complexes were used for the enzymatic synthesis of DNA by the purified viral DNA polymerase as described under Materials and Methods, either in the absence or presence (100 pg/ml) of actinomycin D. After extraction with SDSPronase-phenol and ethanol precipitation, the DNA transcripts were rendered free of RNA by alkali treatment and further purified by Sephadex G-50 chromatography and ethanol precipitated. DNA products were resuspended in 99% deionized formamide, heated to 60’ for 10 min, and then subjected to electrophoresis in 98% formamide-5% polyacrylamide cylindrical gels (0.6 x 10 cm). Subsequent to electrophoresis, gels were sliced into l-mm segments and the ‘“‘I radioactivity was determined in each slice in a Beckman Biogamma II gamma counter. Solid line: tRNAtW-initiated DNA transcripts synthesized in the absence of actinomycin D; broken line: tRNA”P-initiated DNA transcripts synthesized in the presence of actinomycin D. (B) Viral 35 S RNA. tRNALW complexes were used for the synthesis of ““I-labeled DNA transcripts in reconstructed reactions in the presence of actinomycin D and ratelimiting concentrations of dNTPs. After 1 hr at 37”,

FARAS

ties represent nucleotide sequences at the 5’ terminus of the viral genome. The 5’terminal location of iDNAloo has also been recently established by DNA sequence analysis (Haseltine et al., 1977; Shine et al., 1977). Similar hybridization/protection experiments would not be logistically feasible with iDNAIB5 because of its hairpin structure which would exhibit unimolecular reassociation during hybridization.

the reaction was terminated by adjustment of the reaction mixture to 20 mAf EDTA. The entire reaction mixture was then subjected to gel filtration in 20 mM Tris HCl (pH 7.4) on a column of Sephadex G-100 (0.6 x 7 cm) to remove the actinomycin and dNTP precursors. The excluded material was pooled and an aliquot (5500 cpm of ““I-labeled DNA) was used in a second enzymatic reaction for the continued synthesis of (unlabeled) DNA in the absence of actinomycin D. Fresh viral DNA polymerase was added to the reaction and the concentration of the four dNTPs was adjusted to 500 @. Under these conditions there was no detectable increase in radioactivity incorporated into the chase product indicating the complete arrest of incorporation during the chase reaction. After 1 hr at 37”, this chase reaction was terminated by adding EDTA to 20 mM. This mixture (pulse-chase reaction) and an aliquot of the column-excluded material (pulse reaction) were each boiled for 1 min, treated with 40 gg/ml of pancreatic RNase for 30 min at 37”, dried, and subjected to electrophoresis in 98% formamided% polyacrylamide gels. Solid line: ‘““I-labeled DNA from the pulse reaction; broken line: ““I-labeled DNA from the pulse-chase reaction. (C) Viral 35 S RNA tRNAtq complexes were used for the synthesis of ‘H-labeled iDNA transcripts in reconstructed reactions in the presence of rate-limiting concentrations of dNTPs. The DNA product was purified and subjected to polyacrylamide-formamide gel electrophoresis, and iDNAloo was eluted from the gel and purified as described (Collett and Faras, 1977). A portion of the iDNAloo transcripts was then incubated with the viral DNA polymerase in a reaction mixture (10 ~1) containing 500 fl each of unlabeled dATP, dGTP, and dTTP, and ““I-labeled dCTP (3 c1M). After 1 hr at 37’, the reaction was terminated by adjustment of the mixture to 20 nu+f EDTA, 0.5% SDS and heated to 100’ for 60 sec. The mixture was then subjected to gel filtration in water on a column of Sephadex G-100 (0.6 x 7 cm), the excluded material was pooled, and a portion was used for electrophoretic analysis on a 5% polyacrylamide-98% formamide gel (solid line). Another portion of the iDNA,m was incubated in a similar reaction mixture without deoxynucleotide triphosphates and served as a control (broken line).

AVIAN

RETROVIRUS

RNA-DIRECTED

Elongation of iDNA at the 3’ Terminus of the RSV Genome In addition to the iDNAI% hairpin species, the iDNAlm transcript is also a precursor of long (ca. 200-2500 nucleotides) DNA transcripts which have been postulated to represent nucleotide sequences at the 3’ end of the viral genome (Collett and Faras, 1977; Junghans et al., 1977; Leis et al., 1978). This elongation reaction may occur by means of the terminally redundant nucleotide sequences in the viral RNA and the iDNAlm transcripts at the 5’ end of the viral genome. In this proposed model the iDNAloo nucleotide sequences complementary to the terminal sequences at the 5’ end of the viral genome base pair with identical sequences at the 3’ end of the viral genome (Fig. 5A). We have performed an experiment to demonstrate directly that the iDNAloo species can be elongated at the 3’ end of the viral genome. In this study radiolabeled iDNAloo was hybridized to small poly(A)-containing fragments of the viral RNA genome representing approximately 300-500 nucleotides at the 3’ end. The resultant complexes were then employed in an enzymatic reaction to determine if the iDNAlm transcripts originating from the 5’ end could be elongated on the 3’-end RNA. From an analysis of the resultant DNA product on 98% formamide-5% polyacrylamide gels it is apparent that a significant portion of the iDNAloo transcripts was elongated into DNA chains greater than 150 nucleotides in length (Table 2). That the elongation reaction was occurring on 3’poly(A)-containing fragments of the viral genome and not on a low level ofcontaminating 5’ sequences was established by demonstrating (1) that iDNAT,,, which does not contain the terminally redundant nucleotide sequences of the viral genome (ca. 20 nucleotides) (Haseltine et al., 1977; Schwarz et al., 1977; Collett et al., 1977) cannot be elongated subsequent to annealing with these poly(A)-containing 3’-terminal RNA fragments (Table 2); (2) that the tRNAtrP primer RNA cannot bind to these poly(A)-containing 3’-terminal RNA fragments (data not shown); and (3) that cDNA probes representing nucleotide sequences contained elsewhere on the viral genome

DNA

SYNTHESIS

305

(cDNA,,,, cDNA,,,) cannot hybridize to these small poly(A)-containing RNA fragments (Leis et al., 1978). The reason that only a portion of the iDNAloo was elongated in these studies is not clear but may reflect the fact that only 20% of the iDNAloo transcript contains nucleotide sequences complementary to the terminal redundancy (i.e., 20 nucleotides). Since this sequence is approximately 50% AMP/TMP, it may not form sufficiently stable complexes with the small poly(A)-containing RNA fragments of the viral genome to serve as a primer for reverse transcription of the 3’ end under our in vitro reaction conditions. DISCUSSION

The data presented in this communication indicate that the DNA product initiated upon the tRNAL” primer molecule in reconstructed reactions and representing sequences at the 5’ end of the RSV genome contains three structurally distinct initial transcripts. iDNAT is a single-stranded transcript lacking the nucleotide sequences complementary to the terminal 30 ribonucleotides of the viral genome, whereas iDNAlm, which is also single stranded in nature, represents a transcript complementary to the entire distance between the tRNAtrP primer and the 5’ terminus (Haseltine et al., 1977; Shine et al., 1977; Collett et al., 1977). Although iDNAloo accumulates as a consequence of the reverse transcriptase reaching the 5’ terminus of the viral genome, the reasons for the accumulation of iDNAT are unclear; however, it is conceivable that secondary structure at the 5’ terminus of the viral RNA may be involved. The largest DNA transcript synthesized in reconstructed reactions under standard conditions is 185 nucleotides in length and assumes a hairpin structure. A precursor-product relationship exists between iDNAT and iDNAlm, and between iDNA,, and iDNAISS. Furthermore, we have presented data indicating conclusively that iDNAI, can be elongated at the 3’ end of the viral genome presumably by involving the terminal redundant nucleotide sequences. All of the iDNA species have also been detected in reactions containing detergent-disrupted virus, however the pro-

Provirol

t DNA

FIG. 5A. Models of the initial steps in oncornavirus proviral DNA synthesis. Several models of DNA synthesis from 35 S RNA tRNA”1’ complexes in reconstructed reactions are presented. One of the models illustrates a possible mechanism of hairpin DNA synthesis. The other two possible ways in which DNA transcripts longer than the apparent distance between the tRNA”I’ primer molecule and the 5’ end of the viral synthesized on the terminally redundant RNA genome. One model proposes that such synthesis takes place at the 5’ and 3’ ends of the same molecule (circularization) while the other employs the 5’ and 3’ ends of different RNA subunits.

A

template. primer models illustrate genome may be genomic subunit

AVIAN

3’ 5’

RETROVIRUS

RNA-DIRECTED

obcdefgh

DNA

307

wxyzabcdefgh

l

FGH

ABCDEFGH

3’ gbcdefgh

SYNTHESIS

wxyzmc’++% wxyrobcdef

.

5’ ED=-

-

4

3’ obc defph 5’ ---ABCDEFGH

3’ gh (-)

Ti!EEEG(+) wxyzabcdefgh

I-)

WXYZABCDEFGH( +) 4

abcdefgh --

1

wxyzabcdefgh

(-)

WXYZAECDEFGH(t)

abcsefgh

wxyzabc%efph(-)

TCDEFGH

WXYZABCDEFGH(+) 4

c

(Recombination Sticky Ends)

Or

FIG. 5B. Proviral DNA synthesis involving end-to-end transcription. This model of proviral DNA synthesis involves the initial transcription of DNA on the 5’ end of one viral RNA subunit followed by continuation on a second RNA subunit. Elongation of this transcript results in the synthesis of a unit-length minus-strand DNA. The possible involvement of hairpin DNA in the replication of the end of the linear DNA molecule is shown. Additional aspects of the various steps depicted in this model are discussed in the text.

portion of the total DNA product represented by these species is less presumably because of the preponderance of DNA species in addition to iDNA synthesized by detergent-disrupted virus (Taylor et al., 1972; Haseltine et al., 1976; Smith, and Faras, unpublished observations). Since iDNAloo appears to be a precursor to both hairpin iDNA1s5 and longer DNA transcripts representing the 3’ end of the viral genome, once the DNA polymerase reaches the 5’ terminus of the viral genome one of two reactions can occur (Fig. 5A). Either the DNA polymerase employs iDNAloo transcripts as template primer for complementary strand transcription result-

ing in the production of hairpin DNA, or the iDNA]M) transcripts juxtapose with the terminal redundant sequences at the 3’ end of the same or of a second viral RNA subunit to continue transcription and elongate the 5’ end-initiated DNA. Both the appearance of hairpin DNA and the elongation of iDNAIw transcripts at the 3’ end of the viral genome could be the result of viral RNase H hydrolysis of the 5’-terminal ribonucleotides (Collett et al., 1978). Removal of RNA genomic sequences from the hybrid structures by RNase H would result in a single-stranded iDNAI, transcript which either could be converted into hairpin DNA during continued synthesis or

308

COLLETT

AND

FARAS

TABLE 2 could base pair with the terminal redunELONGATIONOF~'-TERMINAL DNA TRANSCRIPTS dant sequences at the 3’ end of the genome. ONE'-ENDVIRALRNA FRAGMENTS" However, it is also conceivable that both events could reflect RNA strand displaceDNA transcripts* Percentage of product > 150 nucleotides in length’ ment during enzymatic synthesis which would still allow the iDNAloo transcripts to Alone Reannealed form one or both of the aforementioned with 3’-end RNA structures. Studies are currently in progress to distinguish between these two possibiliiDNAT 1.0 2.5 3.6 25.1 iDNAlw ties and to elaborate further on the synthedTDNA