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Structure of Moloney Murine Leukemia Viral DNA: Nucleotide Sequence Of Proc. Nati. Acad. Sci. USA Vol. 77, No. 6, pp. 3307-3311, June 1980 Biochemistry Structure of Moloney murine leukemia viral DNA: Nucleotide sequence of the 5' long terminal repeat and adjacent cellular sequences (strong-stop DNA/recombinant DNA/DNA sequence determination/inverted repeat/transposons) CHARLES VAN BEVEREN*, JANICE G. GODDARD*, ANTON BERNSt, AND INDER M. VERMA* *Tumor Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138; and tLaboratory of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands Communicated by E. Peter Geiduschek, March 31, 1980 ABSTRACT Some unintegrated and all integrated forms tRNAPrO viral DNA contain terminal of murine leukemia long repeats 5' gag pol end @ ib3 (LT s). The entire nucleotide sequence of the LTR and adjacent L-,, i Genomic terminal cellular sequences at the 5' end of a cloned integrated proviral ViralRredundancy DNA obtained from BALB/Mo mouse has been determined. Viral RNA (45-66cbp) It was compared to the nucleotide sequence of the LTR at the Reverse transcription 3' end. The results indicate: (i) a direct 517-nucleotide repeat at yT the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end ent 3' 5 {Cellular DNA of the primer tRNA (strong-stop DNA); (ifi) an li-nucleotide ,-Strong-stop DNA , -Strong-stop DNA inverted repeat is present at the ends of the 5'-LTR and a total (145 bp) of 17 out of21 nucleotides at the termini are inverted repeats; 5'-LTR (145 bp) 3'-LTR-LTR (iv) sequences CAATAAAAG (at positions -24 to -31) and Integrated viral DNA CAATAAAC (at positions +46 to +53) resembling the hypo- thetical DNA-dependent RNA polymerase II promoter site can FIG. 1. Diagram of the viral DNA containing two long terminal be identified in the 5'-LTR; (v) the sequence GAAA appears to repeats (LTRs). Empty box, strong-stop DNA (145 bp); hatched box, be repeated on both sides of the junction of viral and cellular sequences at the 3' end ofthe genome (372 nucleotides); 0, terminal sequences; and (vi) in analogy with the bacterial transposons, redundancy in the genomic RNA (45-66 bp); and A, cellular DNA. the presence of an inverted reat seuence at the termini of 5'-LTR suggests that M-MLV also has the integration properties (13, 14,4i) Thus, if the genomic RNA is about 8.2 kb long, some of a transposon. of the unintegrated and all of the integrated viral DNAs have additional nucleotides at 3' and 5' ends, which appear to be Infection by retroviruses requires the conversion of viral direct repeats (10, 11, 15-18, #) genomic RNA into DNA by reverse transcriptase (RNA-de- In this communication, we report the nucleotide sequence pendent DNA polymerase) (1, 2). Viral DNA synthesis initiates of the entire long terminal repeat (LTR) located at the 5' end at the 3'-OH end of the tRNA primer located near the 5' end of the cloned integrated proviral DNA obtained from BALB/ of the genomic RNA (3). In the case of Moloney murine leu- Mo mouse (19). Furthermore, the nucleotide sequence of the kemia virus (M-MLV), 19 nucleotides at the 3' end of the viral-cellular junction at the 5' end has also been determined. primer tRNAPrO are hydrogen bonded to the genomic RNA (4). In ref. 20, the nucleotide sequence of the LTR at the 3' end is The 3'-A-OH of the primer tRNAPrO is not hydrogen bonded reported. The results indicate: (i) The LTR is 517 nucleotides and is located 146 nucleotides from the 5'-CAP nucleotide of long; over 99% of the sequences at the 5'-LTR are identical to the genomic RNA. It forms a phosphodiester bond with the first the sequence in the 3'-LTR. (fi) Of the 517 nucleotides present deoxynucleotide triphosphate, and the synthesis of the com- at the 5'-LTR, 145 nucleotides are present in the strong-stop plementary DNA proceeds until it reaches the 5' nucleotide of DNA. (iii) Eleven nucleotides at each terminus of the 5'-LTR the genomic RNA. The 145-nucleotide-long cDNA (strong-stop are inverted repeats. (iv) The sequence CAATAAAAG, which DNA), covalently linked to the tRNAPrO, dissociates and hy- resembles the hypothetical promoter site, is present between bridizes to terminally redundant sequences at the 3' end of the -24 and -31 nucleotides from the 5'-CAP nucleotide. (v) The genomic RNA (5). The synthesis of DNA then proceeds from sequence CAATAAAC is present at positions +46 to +53 from the 3' end of the RNA toward its 5' end. A 600 base pair (bp)- the 5'-CAP nucleotide. (vi) The 18-nucleotide complement to long DNA fragment of opposite polarity (+) can be observed the 3' end of the tRNAPrO is present. after the synthesis of 0.5-1.0 kilobase (kb) of the cDNA (-) transcript (6-9). The double-stranded DNA synthesized either MATERIALS AND METHODS in vitro or in vivo has two types of genome-length molecules: Synthesis of Strong-Stop DNA. The synthesis of strong-stop (i) those that contain the 5'-end genomic sequences repeated DNA was carried out as described (21, 22). at their 3' end (i.e., 5'-3'5') and (ii) those that, in addition to Molecular Cloning of DNA Fragments. The isolation of the having 5' genomic RNA sequences repeated at their 3' end, also 24-kb EcoRI fragment containing the endogenous Moloney contain 3' genomic RNA sequences repeated at their 5' end genome from BALB/Mo mouse liver DNA has been described (3'5'-3'5') (Fig. 1) (10-12). Analysis of the integrated murine (19). The 24-kb EcoRI fragment was cleaved with HindIII, proviral DNA shows structures containing 3'5'-3'5' sequences which cuts once in the M-MLV genome (23). A 9-kb HindIII The publication costs of this article were defrayed in part by page Abbreviations: LTR, long terminal repeat; M-MLV, Moloney murine charge payment. This article must therefore be hereby marked "ad- leukemia virus; bp, base pair(s); kb, kilobase pair(s). vertisement" in accordance with 18 U. S. C. §1734 solely to indicate t A. Berns, M.-H. T. Lai, R. A. Bosselman, M. A. McKennett, M. Van this fact. der Putten, and I. M. Verma, unpublished. 3307 Downloaded by guest on October 1, 2021 3308 Biochemistry: Van Beveren et al. Proc. Natl. Acad. Sci. USA 77 (1980) fragment, containing 5.3 kb of proviral DNA, corresponding shows that bands 1, 2 + 3, and 4, upon treatment with alkali, to the 5' portion of the genome with the LTR, and 3.7 kb of migrate faster on the gel. The size difference between the un- adjacent cellular sequences was cloned in the HindIII site of treated and alkali-hydrolyzed bands is about 70-80 nucleotides, phage X21a as described.t The 9-kb fragment was then sub- which is the approximate size of the primer tRNAPro (4). In cloned in the HindIII site of plasmid pBR322 (24). In addition, order to establish that the DNAs in the bands (Fig. 2A) are co- the 2.35-kb Kpn I subfragment, containing 0.4 kb of the 5'-LTR valently linked to the primer tRNA, ai-32P atom transfer ex- with 1.95 kb of cellular sequences, was subcloned in the Pst I periments were performed (27). DNA synthesis was carried out site of pBR322 by the dG-dC-tailing method (25). The National under conditions in which the limiting dNTP was [a-32P]dATP. Institutes of Health guidelines for recombinant DNA research If the first dNTP incorporated is dA, then, upon hydrolysis with were followed during the entire cloning procedure. alkali, the ribonucleotide participating in the formation of the Sequence Determination Procedures. Restriction endo- phosphodiester band will have an a-32P atom transferred to the nuclease-cleaved DNA was labeled at the 5' termini with ribose moiety. Because the 3'-OH of tRNAPrO is A-OH, the polynucleotide kinase, or at the 3' termini with terminal transfer should occur from [a-32P]dATP to rA. Fig. 2C shows deoxynucleotidyltransferase, and sequence analysis was per- that in bands 1, 2 + 3, and 4 only 32P-labeled AMP can be de- formed as described (26). Sequence ladders were displayed on tected. Thus, the synthesis of bands 1, 2 + 3, and 4 is initiated 8% polyacrylamide/8 M urea 20 X 40 X 0.04 cm or 30 X 40 X at the 3'-A-OH of the primer tRNA. Fig. 2D shows the nucle- 0.04 cm gels, or 6% polyacrylamide/8 M urea 18 X 78 X 0.04 otide sequence of band 1. It contains 145 nucleotides and cm gels. reaches to the 5'-CAP oligonucleotide GCG reported earlier (28). Bands 2 + 3 and 4 are premature termination products and RESULTS represent a subset of strong-stop DNA (unpublished data). Structure of the Strong-Stop DNA. Strong-stop DNA rep- Characterization of Cloned DNA Fragments. A 9.0-kb resents sequences between the 3'-OH nucleotide of the primer HindIl fragment obtained from BALB/Mo mouse cellular tRNA and the 5'-CAP nucleotide of the genomic RNA. The DNA was inserted into the unique HindIII site of the X Charon primer tRNA is covalently linked to the strong-stop DNA. If 21A molecularly cloned.t It was subsequently inserted into the the viral DNA synthesis is carried out under conditions of HindIII site of the plasmid pBR322 and subcloned. The re- limiting substrate concentration, a large portion of the DNA combinant plasmid pMLV1nt-1 contained a 5.3-kb viral se- synthesized appears to be strong-stop DNA (21).
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