The nucleotide sequence of the Akv murine leukemia virus genome

134,196-20’7 (1934)

VIROLOGY

The Nucleotide Sequence of the Akv Murine Leukemia Virus Genome M. ETZERODT: T. MIKKELSEN, F. S. PEDERSEN, N. 0. KJELDGAARD, AND P. JBRGENSEN Department

of Molecular

Biology,

Received November

of Aarhus, DK-8000 .&has C, Denmark

University

1, 1989; accepted December IS, 1983

The nucleotide sequence of an infectious molecular clone of the Akv murine leukemia virus has been determined by the dideoxy chain termination method after subcloning in bacteriophage Ml3 vectors. The sequence predicts an RNA genome of 3371 nucleotides containing three large open reading frames corresponding to the gag, pal, and env genes. Signal sequences for transcription, splicing, and translation have been identified. The positions of 95 major RNase T1 resistant oligonucleotides of the Akv RNA genome have been located. INTRODUCTION

Expression of the endogenous, ecotropic murine leukemia virus Akv is associated with the high incidence of T-cell leukemia in AKP mice (Rowe, 1973). The Akv provirus is spontaneously activated shortly before birth and the Akv virus is produced throughout the life of the animal (Rowe, 1972). From this strain of mice a polymorphic group of viruses has been isolated from preleukemic tissue and tumors (Kawashima et aL, 1976; Hartley et aL, 1977; Nowinski and Hays, 1978). These viruses are thought to be the outcome of recombinational events between the Akv virus and other endogenous viral sequences. Several investigators have correlated the leukemogenic potential of viruses from AKR mice with differences in the nucleotide sequences at the 3’ part of the RNA genome (Buchhagen et aL, 1980; Cloyd et aL, 1980; Elder et aL, 1977; Hartley et aL, 1977;Lung et aL, 1980; Nowinski and Hays, 1978; Pedersen et al, 1981, 1982; Rommelaere et aL, 1978; Rowe et aL, 1980). In order to obtain a more detailed knowledge of the leukemogenic virus species of the AKR mice, information about the nucleotide sequence of the Akv virus is essential. We here report the complete i Author addressed.

to whom requests for reprints

0042-6822/84 $3.00 Copyright All rights

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

should be

nucleotide sequence of the Akv virus genome in the infectious molecular clone, XAKR623, of an integrated prototype Akv virus (Lowy et aL, 1980). Partial nucleotide sequences have been published, covering the LTR region (van Beveren et aL, 1982) and the env gene (Lenz et aL, 1982) of the h-AKR623. From a noninfectious Akv clone Herr et al. (1982) have obtained the nucleotide sequence of the 3’ half of the Akv genome. MATERIALS

AND

METHODS

Restriction enzymes were purchased either from New England BioLabs or from Boehringer, Mannheim. T4 DNA ligase and the exonuclease Ba131 were from New England BioLabs. Klenow fragment of the Escherichia coli polymerase I was from Boehringer Mannheim. [32P]dATP was purchased either from New England Nuclear (400 Ci/mmol) or from ICN (2000 Ci/ mmol). Plasmid pAKR59 was grown in E. coli HBlOl (Lenz et ah, 1982) and plasmid DNA prepared according to standard procedures (Birnboim and Doly, 1979). The Ml3 bacteriophage clones were grown in E. coli JM103 cells and the replicative form and single-stranded Ml3 DNA was prepared as described by Messing et al. (1981). The nucleotide sequences of the viral inserts were determined by the dideoxynucleotide method as described by Sanger et 196

Akv

MuLV

NUCLEOTIDE

a& (1981). Acrylamide gels for sequencing (0.2 mm thick, 6 and 8%) were prepared as described by Garoff and Ansorge (1981). The nucleotide sequences read from the autoradiograms were handled using computer programs constructed by Staden (1977). RESULTS

Sequencing Strategy The X-AKR623 clone of Lowy et cd (1980) contains the entire sequence of an integrated Akv virus together with bordering regions of the mouse genome. Utilizing the unique PstI restriction enzyme site in the LTR region, the entire viral genomic information was subcloned in pBR322 yielding the pAKR59 plasmid (Lenz et cd, 1982). Both the X-AKR623 DNA and the pAKR59 DNA express the Akv virus upon transfection into NIH3T3 fibroblasts (Lowy et al, 1980, Lenz et a& 1982; and our own unpublished observations). The published restriction enzyme map of pAKR59 viral DNA (Lenz et d, 1982) was confirmed except that the XhoI site at position 4095 (see Fig. 2) is 5’ to the adjacent BgflI site at position 4099. The pAKR59 plasmid DNA was digested with the appropriate restriction enzymes

197

SEQUENCE

(BumHI, BgZII, SmaI, or PstI) in order to produce large viral DNA fragments. These fragments were either cloned directly into the bacteriophage Ml3 vectors (Messing et al, 1981; Messing and Viera, 1982) or further digested with other restriction enzymes (HueIII, RsaI, or Saz&A) before cloning into the Ml3 vectors. In total 115 such clones were sequenced. Ml3 clones containing the largest BgZII fragment (nucleotides 314-4099) and the clones containing the PvuII fragment (nucleotides 381’7-5329) were isolated. From the replicative forms smaller fragments of the viral DNA were generated by the use of the exonuclease B&31 as described (Poncz et al, 1982) before recloning into the Ml3 vectors. In total 84 B&31 clones were sequenced. Both strands of the viral PstI fragment in pAKR59 have been sequenced except for the 90 nucleotides next to the 5’ PstI site and the 132 nucleotides next to the 3’ PstI site where for technical reasons only one strand has been sequenced. Figure 1 summarizes the sequencing strategy. The sequence analysis shows that the Akv provirus fragment in the plasmid pAKR59 contains 8303 nucleotides. We have chosen to present the sequence data in Fig. 2 as the DNA (+) strand of the part of the provirus which contains the complete viral transcriptional unit of 8371 nucleotides corresponding to the Akv RNA genome.

0

A-NW623 LTR

.“”

LTR

PAW59

FIG. 1. Physical map of the Akv viral RNA and the Akv provirus. Restriction enzyme cleavage sites used for the cloning of DNA fragments into bacteriophage Ml3 vectors are indicated. The broken lines indicate the large DNA fragments isolated from the plasmid pAKR59 which was used to generate subclones by further restriction enzyme digestion. The B&II fragment and the PvuII fragment used to generate subclones by BoZ31 digestion are indicated by the sawteeth lines. Ba: BornHI, Bg: B&I, Ps: P&I, Pv: PvuII, S: SmaI.

FIG. 2. Nucleotide sequence of the Akv viral genome. Nucleotide number 1 corresponds to the 7 methyl-G cap site on the viral RNA and nucleotide 8371 to the polyA addition site. The R regions are at positions 1-68 and 8304-83’71. The Ur, region is at positions 69-144, the primer binding site at positions 145-162. The gag gene is at positions 636-2246, the pd gene at positions 2250-5837, and the env gene at positions 5726-7786. The Us region is at positions 7822-8303. The inverted repeats at the 3’ end of the U, region and the 5’ end of the US region are at positions 132-144 and 7822-7834. A 99-nucleotide-long repeated sequence in the Us region is found at positions 7932-8030 and 8031-8129. 198

Akv

MuLV

NUCLEOTIDE

FIG. 2-Cmztiaued

SEQUENCE

ETZERODT

200

FIG.

ET AL.

8--&ttintMd

(for review see Coffin, 1982a): The terminally repeated R sequence present both at The Akv Vim1 Genome the 5’ end (nucleotides l-68) and at the 3’ Figure 2 shows the nucleotide sequence end (8304-8371), the U5 unique sequence corresponding to the Akv viral genome as (69-144), the tRNA binding site (145-162), derived from the plasmid pAKR59. As the gag region coding for the structural found for other retroviruses, the viral ge- core proteins (636-2246), the pal region nome can be divided into specific regions coding for the reverse transcriptase (2250DISCUSSION

Akv

MuLV

NUCLEOTIDE

5837), the env region coding for the membrane-associated spike glycoproteins (X26-7786), and the U, unique sequence (7822-8303). The 3% viral genomic RNA has a 7methyl Gppp cap structure at the 5’ end and is polyadenylated at the 3’ end. By nuclease mapping of the 35 S genomic RNA, Ostrowski et al. (1981) have identified the cap site as the nucleotide denoted as number 1 in Fig. 2. The exact nucleotide to which the poly(A) tail is added has not been precisely identified. The sites for transcription termination and polyadenylation in eukaryotic mRNAs are normally preceded by the poly(A) signal AATAAA (Proudfoot and Brownlee, 1976) close to the CA site for poly(A) addition (Benoist et al, 1980) and the transcription termination signals TGCT, TTGC, and a T cluster (Bogenhagen and Brown, 1981). All these nucleotide sequence signals are found among the 22 terminal nucleotides of the R sequence. The LTR Region The LTR region has the sequence structure Us, R, II, and is found in the X-AKR623 phage bordering the extremities of th,e provirus. Our sequence of the LTR region with a length of 539 nucleotides is identical to that previously published by van Beveren et al. (1982). In the integrated provirus two sequences normally associated with eukaryotic transcription initiation are located upstream from the R region in the II, region. The sequence TATAAAAA located around position 8277 (-26 nucleotides from the cap site) is a 7/8 match with the consensus Hogness-Goldberg or TATA box (Efstratiadis et &, 1980). The CCAAT sequence around position 8225 (-78 nucleotides from the cap site) seems to be a conserved sequence among many eukaryotic promoter structures (Benoist et al, 1980). Furthermore the Us region contains a 99-bp repeated sequence at positions 7932-8030 and 8031-8129. Such repeated elements in connection with eukaryotic promoter structures have received much attention due to their ability of enhancing the transcription

SEQUENCE

201

(Levinson et a& 1982; Laimins et cd, 1982). The 99-bp repeated sequences in the Akv genome contains an element TGGAAAG around positions 7945 and 8044, which is a perfect match to the proposed consensus sequences TGG#G (Weiher et aL, 1983). In a genome as large as 8 kb it is possible to find several sequences that resemble the above-mentioned sequence signals for transcription initiation and termination. In most cases these structures appear isolated and in no reasonable order. However, in the Akv sequence we find a CCAAT-like sequence around positions 7137 and 7161, a TATA box around position 7184, a poly(A) signal around position 7788, and a T cluster and a CA sequence localized around 20 nucleotides 3’ of that poly(A) signal. At the moment it is not clear if a biological function is associated with these consensus signals for transcription start and termination. Open-Reading Frames In the viral RNA reading frame 1, starting the codon triplets at the first nucleotide, we find no open-reading frame longer,than 87 triplets. Reading frame 2 startihg with the second nucleotide has one large openreading frame of 766 triplets from nucleotide 5489 to 7786 corresponding to env proteins. No other open-reading frames larger than 81 triplets are found in this frame. The reading frame 3 contains two large open-reading frames: one of 633 triplets located between nucleotide 348 and 2246 and another of 1196 ‘triplets located between nucleotide 2250 and 5837. These open-reading frames correspond to the coding regions for the gag and the pol proteins, respectively. An open-reading frame of 107 triplets is found in the U, region. The identification of the three long openreading frames as corresponding to the viral genes gag, pal, and env is obtained through comparison with the amino acid sequence data from murine leukemia viruses (Henderson et aL, 1982; Linder et cd, 1982; Chen, 1982) and the predicted amino acid sequence of Moloney murine leukemia virus (MoMuLV) (Shinnick et al, 1981). On

202

ETZERODT

the (-) strand of the provirus we find no open-reading frame longer than 130 triplets. The gag Region The gag gene of MuLVs encodes a 65,000Da precursor protein (Pr6!jW). The synthesis of Pr6W is suggested to be initiated at the ATG at position 636 which is the first translation initiation codon present in the sequence 3’ of the cap site. The sequence around this ATG is a perfect match with the proposed consensus sequence for translation initiation signals (Kozak, 1981) (G/A)XXATGG. The Pr65g”g protein is myristylated (Henderson et aL, 1983) and proteolytically processed to the four structural viral proteins ~15, ~12, p30, and ~10. The proteolytic cleavage points are the Tyr-Pro at positions 1020-1025 for the p15C-Np12, the Phe-Pro at positions 1275-1280 for the p12C-Np30, and the Leu-Ala at positions 2064-2069 for the p30C-NplO (Oroszlan et al, 1978). A glycosylated form of Pr65gW(gPrW) has been found on the surface of MuLVinfected cells and in the culture medium but not in the mature virus (Edwards and Fan, 1979; Eisenman and Vogt, 1978; Schultz et c& 1979; Edwards et cd, 1982; Fan et a& 1983). The results of Fan et aL (1983) indicate that the gPr8WW is not required for murine leukemia virus replication. The amino acid sequence of gPr8p seems to be’longer than the Pr658”Q.Since the carboxy terminal proteolytic peptides are shared with Pr658”Q,additional amino acids are believed to be located in the amino-terminal part of the glycoprotein (Schultz et d, 1979; Saris et a& 1983). This correlates with the open-reading frame found to precede the translation initiation signal for Pr65g”g both in the nucleotide sequence of MoMuLV (Shinnick et &, 1981) and Akv. This leader sequence contains several uncharged mostly hydrophobic residues and might be the signal sequence required for the cotranslational translocation across the endoplasmatic reticulum (Blobel and Dobberstein, 1975). In the Akv

ET AL.

sequence there is no ATG triplet between the cap site and the ATG initiating the synthesis of Pr658”g.Two GTG triplets are found at positions 408 and 480 in the leader sequence one of which might serve to initiate the synthesis of gPr808”8 and gPr200@‘@&.However, it is not known if cells infected with X-AKR623- or pAKR59derived virus synthesize these glycoproteins. The gag region of the Akv sequence contains two possible N-glycosylation sites around positions 712 and 1810 with the structure -Asn-Xxx-Ser/Thr-Yyywhere X and Y are not Pro (Bause, 1983). The possible glycosylation sites of Akv in the gag region are homologous to the possible glycosylation sites found in the amino acid sequence of Rauscher murine leukemia virus (RaMuLv) and MoMuLV (Henderson et al, 1982). In gPr8W from RaMuLV Schultz et al. (1981) found that the Asn 177 residue in p30 was glycosylated (corresponding to the nucleotide 1810 in the Akv sequence). Furthermore they mapped a second oligosaccharide in the amino-terminal part. In the p30 region of the MoMuLV gPr8WWanother oligosaccharide was mapped by Saris et al (1983), who also found a gag-pal precursor (gPr2008”g-pd) glycosylated at least once in the pal region. In the pal gene there are four possible glycosylation sites around positions 4450,4714, 4927, and 5294. The pal Region The gag open-reading frame is separated from the pal open-reading frame by a single UAG termination triplet at position 2247. The low amounts of the pd gene products are produced as a polyprotein of 180,000 Da also containing the gag proteins (Pr180B”B-d). It is not known if the translation termination codon is overcome by a read-through mechanism or by splicing. The sequence of the 3’ end of a noninfectious Akv cone has previously been published (Herr et oh, 1982). This sequence begins at position 3306 in the pd gene and extends to position 7862 in the UB region. The two sequences exhibit seven differences. At positions 4683, 5237, 5437, 5882,

Akv

MuLV

NUCLEOTIDE

SEQUENCE

203

7166, and 7553 our sequence has a G nu- amino terminus. The p15(E) amino tercleotide and at positions 7816-7821 we find minus has been determined as the Glu found at position 7190 (Shinnick et aL, a stretch of six G nucleotides where Herr et al. (1982) report seven G nucleotides. At 1981). positions 5235-5237 the noninfectious clone has a TGA stop codon in the pal reading Akv Transcripticmal Products frame where our sequence exhibits the Trp The gag gene product is translated from codon (TGG). a 35s mRNA. This 35s mRNA might be The reverse transcriptase enzyme con- distinct from the 35s genomic RNA packed sists of a single polypeptide chain of ap- into virions (Levin and Rosenak, 1976). proximately 80,000 Da (Ross et aL, 1971; The possibility exists that the 35s gag-p01 Moelling et al, 1975; Naso et aL, 1975; TronmRNA is a spliced molecule, since candiick et ah, 1972). As in the MoMuLV date splice donor and acceptor sites are sequence (Shinnick et aL, 1981) the pal found in the Akv sequence preceding the reading frame has an additional polypep- pr65uagtranslation initiation site. The setide coding capacity of approximately quence (GAGGTAAGC) around position 50,000 Da. 202 is a 7/9 match with the consensus sequence for donor splice sites (Mount, 1892) and the sequence (GTCTTTGTGTCTThe env Region CAGT) around position 579 is a 12/16 The nucleotide sequence of the Akv onv match with the consensus sequence for gene, obtained from pAKR59, has previ- splice acceptor sites (Mount, 1982). The reously been published (Lenz et aL, 1982). The gion between the candidate splice donor only difference between the sequence pre- and acceptor sites (position 203-583) is part sented here and the one previously pub- of the region that has been implicated in lished is the addition of the T at position packing and dimer formation of the ge5673. nomic RNA (Watanabe and Temin, 1982; The nucleotide sequence of the env gene Mann et aL, 1983). This region contains a and the features of the encoded polypep- 16-bp palindrome (TAG’JTAGCTAACTA) tides have been thoroughly investigated at position 300-315, similar to the palinand compared to other env products from dromes found at analogous positions in the murine leukemia viruses by several au- nucleotide sequencesof MoMuLV (Shinnick thors (Shinnick et al, 1981;Herr et al, 1982; et aL, 1981), spleen focus-forming virus Lenz et aL, 1982; Koch et aL, 1983). The env (Clark and Mak, 1983), and in several of gene products are synthesized as a pre- the mammalian sarcoma- viruses (Reddy cursor glycoprotein (gPr80”“). Translation et aL, 1981; Hampe et aL, 1983; Devare et is believed to initiate at the second ATG aL, 1983; van Beveren et aL, 1983). in the env open-reading frame at position The env gene products are translated 5780 and to terminate at the TAA triplet from a 21s spliced mRNA. Our preliminary at position 7787. There are six possible Northern blot analysis indicates that the N-glycosylation sites in the sequence previous mentioned donor splice site around positions 5910, 6378, 6762, 6858, around position 202 in the RNA sequence 6879, and 6978. Post translational proteo- is used in the generation of the 21s env lytic processing removes the signal peptide mRNA together with an acceptor splice site and cleaves at gPr80en” to give gp70 and located in the region 5493-5831. Two sep15(E) which become covalently linked quences around positions 5513 and 5709 are through a disulfide bond (Pinter et d, candidate acceptor splice sites. 1978). The amino terminus of gp70 from the Akv virus is unknown, but homology to the amino acid sequences of the gp70 Establishment of a Precise Oligmucleotide Map from RaMuLv (Linder et aL, 1982) and Friend murine leukemia virus (Chen, 1982) RNase T1 fingerprint analysis has been suggest the Val at position 5873 as the a useful technique in the comparative

204

ETZERODT

analysis of murine leukemia virus genomes (Buchhagen et aL, 1980; Lung et &, 1980; Pedersen et aL, 1981,1982; Rommelaere et d, 1978). The assignment of the critical oligonucleotide markers to specific regions of the genome is essential for such studies. The availability of the complete nucleotide sequence of the Akv virus genome and the previous detailed analysis of the RNase T1 fingerprint of this virus (Pedersen and Haseltine, 1980) make it possible to conTABLE

ET AL.

struct a precise oligonucleotide map of the viral genome as shown in Table 1. This table presents the location of 95 major RNase T1 resistant oligonucleotides on the RNA genome of Akv virus. The number of markers imply that on the average one unique oligonucleotide can be found per approximately 90 nucleotides. The close relationship between Akv virus and endogenous viruses of other inbred strains of mice (Coffin, 1982b) makes this oligo1

SEQUENCE MAP LOCATION OF MAJOR RNase T, OLIGONUCLEOTIDE OF THE Akv GENOME Oligonucleotide No. 64A 59 67 68 70A 51A 58 66A 1 12 2 40A 41 32 81 40B 54 60 55 15 3 35 61A. 23B 76 62 75 49 ‘79 16 43 83

Position 136-146 267-2’79 484-492 529-536 544-550 625-638 797-809 840-849 937-965 966-983 1027-1051 1052-1067 1177-1191 1201-1215 1240-1248 1255-1270 1273-1285 1320-1332 1333-1345 1347-1371 1408-1424, 1532-1544 1564-1575 1591-1666 1652-1662 1818-1828 1873-1882 1920-1933 2268-2277 2467-2487 25262541 2582-2589

Oligonucleotide No. 19A 29 5 26A 99A 65A 26B 73 13B 36 7 70B 28 64B 6 45A 50 33A 37B 37A 4B 13c 19B 52 22 27 30 98A 88 46 17 20

Position 2748-2765 2770-2784 2787-2807 298042994 2997-3019 3070-3079 3086-3100 3182-3193 3582-3599 3646-3656 4051-4070 4190-4196 4438-4451 4477-4487 4606-4626 4630-4646 4651-4668 4702-4715 4782-4795 4797-4809 4873-4890 5307-5324 5359-5367 5476-5488 5589-5605 5688-5700 5769-5782 5795-5818 5890-5899 5909-5916 6057-6076 6146-6162

Oligonucleotide No. 56 21 13A 51c 89 11 10 24 33B 9 45B 4A 23A 61B 8 45c 47 31 44 18 69 74 42 57 98B 78 63 80 25 99B 34

Position 6282-6294 63706386 64246441 65476560 6587-6596 6650-6671 6674-6692 6753-6768 68116824 6844-6866 6888-6904 6948-6965 6972-6988 7001-7012 7063-7082 7130-7146 7152-7166 7295-7309 7346-7361 7416-7433 7471-7478 7601-7612 7613-7627 7649-7664 7736-7759 7785-7792 7793-7803 7912-7920 8134-8148 8208-8230 82898301

Note. The position of previously identified RNase T1 resistant oligonucleotides of the Akv genome was determined. The assignment was based on complete oligoribonucleotide sequences (Pedersen and Haseltine, 1980), on partial oligoribonucleotide sequences (unpublished), and on the base composition of the oligonucleotides as predicted from their two-dimensional electrophoretic mobilities. For the oligonucleotides 33A, 35, 40A, 62, 66A, and 75 the RNA sequences deviate from the presented DNA nucleotide sequences at one or two positions.

Akv MuLV NUCLEOTIDE

nucleotide map immediately useful for rapid analysis of expression and genomic variation of this virus family. ACKNOWLEDGMENTS We thank Dr. J. Lenz for the plasmid pAKR59, Dr. D. Lowy for the X-AKR623, Dr. J. Messing for the bacteriophage M13, mp’7, mp8, and mpg, and Dr R. Staden for the computer programs. This work was supported by grants from the Danish Cancer Society, the Danish Natural Science Research Council, the Danish Medical Research Council, and from the Novo Foundation. REFERENCES

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