characterization of transformation- and replication-specific sequences of reticuloendotheliosis...

VIROLOGY 113,672-685 (1981)

Characterization of Transformation- and Replication-Specific Sequences of Reticuloendotheliosis Virus

ROBERT S. COHEN, TIMOTHY C. WONG, AND MICHAEL M. C. LAP

Department of Microbiology, University of Southern California School of Medicine, Los Angeles, California 90053

Received February 19, 1981; accepted May 6, 1981

Using a combination of hybridization and oligonucleotide fingerprinting techniques we have determined the sequence relatedness of oncogenic avian reticuloendotheliosis virus (REV) to its associated helper virus, REV-A, and to the other members of the reticuloendotheliosis (RE) virus group. Our studies have shown that approximately 30% of the genomic sequences of REV are unrelated to the nononcogenic RE viruses. These REV- specific, and presumably oncogenic, sequences are arranged in a contiguous fashion, possibly interrupted by a short stretch of REV-A-related sequences, localized between 1 and 2.7 kilobases (kb) from the 3’-end on the REV genome. We have also identified two regions of REV-A sequences which are deleted in the REV genome. The first region encompasses 3 kb of sequences in the 5’-half of the genome, presumably corresponding to the gag-pol genes. The second region represents 1.5(kb) of the enw sequences. These deletions could account for the genetic defectiveness of REV. By studying the sequence relatedness between REV-A and the other RE viruses, we have shown that REV is the only RE virus which contains these oncogenic sequences. We have also determined the relatedness between these viruses. In addition, we have identified a hypervariable region which maps in or near the env gene. The possible significance of this region in accounting for both the varied pathogenicity of the RE viruses and the origin of oncogenic REV is discussed.

INTRODUCTION

Avian reticuloendotheliosis virus strain T (REV-T) was originally isolated from the spleen of a leukotic turkey (Sevoian et al., 1964; Robinson and Twiehaus, 1974). Although REV-T is a C-type retrovirus with virion morphology and mode of replication similar to those of the avian leukosis-sarcoma virus (AL-SV) complex, (Zeigel et al., 1966; Peterson et al., 1972) it was found to be unrelated either sero- logically or in nucleic acid sequence to the AL-SV complex (Theilen et al., 1966; Hal- pern et al., 1973; Purchase et al., 1973; Maldonado and Bose, 1972; Kang and Temin, 1973). On this distinction REV-T became the prototype of a new group of avian retroviruses, the reticuloendotheliosis (RE) virus group, which also includes

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0042-6822/81/1206’72-14$02.00/O Copyright 0 1981 hy Academic Press. Inc. All rights of reproduction in any form reserved.

672

spleen necrosis virus (SNV), duck infectious anemia virus (DIAV), and chick syncytial virus (CSV) (Purchase et al., 1973).

Both REV-T and the original stock of SNV have been shown to cause reticuloendotheliosis in ducks, chickens, and turkeys and thus can be considered oncogenic (Purchase and Witter, 1975). In contrast, CSV and DIAV have not been conclusively shown to be oncogenic, but both cause peripheral nerve lesions and, in the case of DIAV, anemia in various species of fowls (Purchase and Witter, 1975). All four RE viruses are antigenically related (Pur- chase and Witter, 1975) and cannot be distinguished by the extent of nucleic acid sequence homology (Kang and Temin, 1973).

REV-T is the only member of the RE group for which the ability to transform hematopoietic cells or fibroblasts in vitro has been demonstrated (Franklin et al.,

GENOMIC SEQUENCES OF REV 673

1974; Hoelzer et al., 1979, 1980). The virus released from these REV-T-transformed hematopoietic cells has been shown to consist of two virus components (Breitman et al., 1980a; Hoelzer et al., 1980; Gonda et al., 1980). The first component is a replication- competent virus, termed REV-A, which contains a 35 S RNA, and can not transform cells in vitro but causes nonprolifer- ative nerve lesions in chickens similar to those caused by CSV and DIAV (Purchase and Witter, 1975). The second component is a replication-defective virus, termed REV, which contains a 28 S RNA, and can transform cells in vitro, but requires the presence of helper virus, REV-A, for replication (Hoelzer et al., 1979, 1980; Breit- man et al., 1980a). Hybridization experiments have shown that approximately 70% of the REV genome is related to REV- A (Breitman et al., 1980a). The remaining sequences are unique to REV, and presumably encode the transformation-specific functions of the virus. These transformation-specific sequences (“rel”) have been shown to be distinct from all the other known viral oncogenic sequences (Wong and Lai, 1981). No such defective and transformation-specific viral component has been found for the other members of reticuloendotheliosis viruses.

In the present study we have mapped the genomic location of the REV transformation-specific sequences by oligonucleotide fingerprinting and hybridization techniques. We have shown that these sequences are not present in SNV, DIAV, and CSV. Furthermore, in an attempt to account for the varied pathogenicities of these viruses, we have studied their sequence relatedness by oligonucleotide mapping.

MATERIALS AND METHODS

Cells and viruses. The continuous cell line, SOC6, of transformed bone marrow cells derived from chickens infected with REV-T was the generous gift of Drs. H. R. Bose and J. D. Hoelzer (University of Texas at Austin). Cells were maintained as suspension cultures in F-10 medium supplemented with 10% tryptose phos-

phate broth, 10% calf serum, and 2% chicken serum. Virus released from these cells was identical to the virus released from the BMC cell line, also originally derived by Dr. H. R. Bose, as determined by oligonucleotide fingerprinting analysis (Breitman et al., 1980a; and see Results). The REV-associated helper virus, REV-A, isolated free of oncogenic REV and plaque purified by Martin Breitman (Breitman et al., 1980a) was grown on C/E chicken embryo fibroblasts in F-10 medium supplemented with 10% tryptose phosphate broth and 5% calf serum. SNV, DIAV, and CSV were the generous gifts of Dr. C. Y. Kang (University of Texas at Dallas). These viruses were maintained in the same way as that described for REV-A.

Radiolabeling and preparation of viral RNAs. The REV-transformed SOC6 cell line was incubated with [32P]- orthophosphate (1 mCi/ml) in phosphate- free F-10 medium supplemented with 10% dialyzed calf serum (labeling medium). Supernatants were harvested every 12 hr for 2 days. The cells were collected by centrifugation at 2000 g for 5 min at 5” and further incubated in labeling medium following each harvest. Viral supernatants were stored at -20”. The REV-A, CSV, DIAV, and SNV grown in chicken embryo fibroblasts were labeled and virus supernatants harvested as described above with the exception that supernatants were stored at -20” directly.

Virus purification and extraction of viral RNA were as previously described (Lai, 1976; Breitman et al., 1980a).

Oligonucleotide jingerprinting and map- Iring. The 32P-labeled viral RNA was adjusted to a final RNA concentration of 1 mg/ml by adding yeast RNA, and then digested with RNase T1 (0.5 unit/pg RNA) at 37” for 45 min in 25-50 ~1 of low-salt buffer (LSB) containing 10 mM Tris-HCl, pH 7.4 and 1 mM EDTA. Separation of T1- oligonucleotides on two-dimensional polyacrylamide gels (termed “fingerprinting”) was performed according to a modification of published procedures (Lee and Wim- mer, 1976). Briefly, separation in the first dimension was performed on a 10% polyacrylamide-0.125% bisacrylamide gel slab

674 COHEN, WONG, AND LA1

(10 X 30 X 0.15 cm) in citrate buffer containing 6 M urea, pH 3.3 at 700 V for 4 hr or until bromphenol blue dye had migrated 1’7 cm from the origin. Separation in the second dimension was performed on a 22% polyacrylamide-1.4% bisacrylamide gel slab (30 X 40 X 0.07 cm) in 50 mM Tris-borate buffer, pH 8.0 at 600 V for 16 hr or until the bromphenol blue dye had migrated 22 cm.

Preparation of poly(A)-containing RNA fragments was essentially as described (Wang et al., 1975). Briefly, equal amounts of 32P-labeled viral RNA were incubated at 50’ in 0.05 M N&CO3 for 0,2, or 4 min, respectively, and then neutralized with HCI and precipitated with two and one- half volumes of ethanol. Samples were combined and poly(A)-containing RNA fractions were selected by binding to an oligo(dT)-cellulose column (Aviv and Leder, 1972). Following heat denaturation at 100’ for 45 set, poly(A)-containing RNA was loaded onto a lo-25% linear sucrose gradient made in 0.01 M NaCl, 0.061 M EDTA, 0.01 M Tris-HCl, pH 7.4 and 0.2% SDS and centrifuged in an SW4lTi rotor at 27,000 rpm for 16 hr. The gradient was fractionated and pooled into four to six size classes. Individual size classes were precipitated with two and one-half volumes of ethanol and then washed with 75% ethanol before RNase T1 digestion and fingerprinting.

Synthesis of cDNA. Synthesis of DNA complementary (cDNA) to REV-A and REV-T viral RNAs was performed using an exogenous reverse transcriptase reaction. The reaction was performed in a 30 ~1 reaction mixture containing 3-5 pg of viral RNA, 50 mM Tris-HCl, pH 8.0,8 mM MgC12, 40 mM KCl, 2 mM dithiothreitol, 0.1% NP-40, 1 mM each of dATP, dCTP, dGTP, and TTP, 0.01 mM r3H]TTP (80 Ci/ mmol, New England Nuclear), 50 units of AMV reverse Transcriptase (obtained from the Division of Cancer Cause and Preven- tion, National Cancer Institute), and 60 rg of calf thymus primer DNA which had been prepared by digesting calf thymus DNA (5 mg/ml) with 70 pg/ml DNase I in 10 mM Tris-HCl, pH 7.4,lO mM MgC12 for 2 hr at 37“ (Taylor et al., 1976). The re-

action mixture was incubated at 37” for 2 hr and the nucleic acids were phenol extracted twice and precipitated with two volumes of ethanol. Following ethanol precipitation, the nucleic acids were redissolved in 0.3 M NaOH and incubated at 37” for 2 hr to degrade the template RNA. The DNA was neutralized with HCl and then adjusted to 0.1 M NaCI, 10 mM EDTA, and 10 mM Tris-HCI, pH 7.4 and precipitated with two volumes of ethanol. The cDNA was selected by passing it through a Seph- adex G-100 column. Only the DNA excluded from the column was collected. These cDNAs are at least 300 nucleotides long. The representativeness of the cDNA prepared by this procedure was examined by hybridization to 32P-labeled viral RNA of the homologous virus strain and then “fingerprinting” the hybrid RNA (see below). All of the T1-oligonucieotide spots of the 32P-labeled RNA were detected at roughly equimolar ratios, suggesting that the cDNA was representative of the entire genome.

Nucleic acid hybridization. Two methods for RNA. DNA hybridization modified from published procedures (Coffin et al., 1978) were employed in this study. The first method was a one-step hybridization which was performed in a 50 ~1 reaction mixture containing 1 X lo6 cpm of 32P-labeled REV (REV-A) RNA(specific activity 5 X lo6 cpm/pg), 2 fig REV-A cDNA, and 20 pg yeast RNA in NTE buffer (0.5 M NaCl, 0.01 M Tris-HCl, pH 7.4, 0.01 M EDTA), in sealed polyethylene Eppendorf tubes submerged in a 66” water bath for 24 hr.

The second method involved two steps of hybridization reactions. The first step was performed in 25 ~1 of a reaction mixture containing 2 bg of REV (REV-A) cDNA and either 15 pg of REV-A RNA plus 45 Nug of yeast RNA or 60 pg of RE virus RNA in NTE buffer. Hybridization was performed at 66” for 24 hr as described above. At the end of the first step of hybridization, 6 X 10’ cpm of 32P-labeled REV (REV-A) RNA (specific activity 5 X lo6 cpm/pg) in 25 ~1 of NTE buffer was added to the hybridization mixture and incubation was continued for an addi-

GENOMICSEQUENCESOFREV 675

tional 4 hr. Following incubation in both the first and second methods, the hybrids were split into two equal aliquots and digested with either 25 units of RNase T1 or 15 units RNase T1 plus 5 pg RNase A for 45 min at 3’7”. Digestions were stopped by adjusting reaction mixtures to 0.2% SDS. The hybridization mixtures were then passed through a Sephadex G-100 column and the hybrids were recovered in the void volume. Following two phenol extractions, the hybrids were precipitated with three volumes of ethanol. After precipitation, the hybrids were redissolved in LSB, heat- denatured at 100” for 45 sets, and digested with RNase T1, and then “fingerprinted” on two-dimensional polyacrylamicle gels.

Two control experiments were performed to test the fidelity of this system: (1) When no cDNA was added to the hybridization reactions, the fingerprints of the “hybrids” resolved only a poly(A) spot which was recovered in the void volume of the Sephaclex G-100 column because of its size. (2) When 32P-labeled ribosomal RNA or 32P-labelecl 70 S RNA of Prague strain of Rous sarcoma virus was included in the hybridization reactions, no T1-oligonucleoticle spots derived from these RNAs were detected. This is consistent with the finding that REV and REV-A are not related to avian sarcoma viruses (Kang and Temin, 1973; Wong and Lai, 1981).

RESULTS

Relatedness of REV and REV-A

RNA extracted from virions released from either the BMC (Franklin et al., 1974) or SOC6 (Hoelzer et al., 1980) cell lines contains the RNA of the transforming virus, REV, in a 5- to lo-fold excess over the RNA of the helper virus, REV-A (Breit- man et al., 1980a; Hoelzer et al., 1980). Accordingly, autoradiograms of 32P-labelecl RNA from these virions digested with RNase T, and separated on two-dimensional polyacrylamide gels (“fingerprints”) show the oligonucleotides of REV as dark spots and those of REV-A as either faint spots or not detectable at all (Fig. 1A). This fingerprint is identical to the published fingerprint which was obtained

from the preparations containing mainly 30 S REV RNA (Breitman et al., 1980a). The fingerprints of this RNA preparation will henceforth be referred to as REV fingerprints. Some oligonucleotides, e.g., 2 and 6, were present in variable amounts in different RNA preparations. The reason for the variability was unclear. In order to determine the sequence relatedness of REV to REV-A, these two viral RNAs were mixed and analyzed by T1-oligonucleotide fingerprinting. These fingerprints were then compared to individual fingerprints of REV and REV-A (Figs. lA-D). By virtue of comigration in such mixing experiments, 19 oligonucleoticle spots were determined to be common to both viruses (Fig. 1D). This conclusion was supported by partial sequence determination by RNase A digestion of every numbered oligonucleotide in REV and REV-A as we previously reported (Breitman et al., 1980a). However, there were two excep- tions: REV spots #13 and #17 which were found to comigrate with REV-A spots #21 and #15b, respectively, have different sequences (Breitman et al., 1980a). The comigration of these spots was most likely fortuitous, since they were localized at different parts of the REV and REV-A genomes (see below). The remaining oligonucleotides which did not comigrate represent sequences unique to REV to REV-A. Thus REV and REV-A share only 17 out of 33 and 40 large T1-oligonucleotides, respectively.

Mapping of REV-Specific Oligonucleotides

To determine the genomic localization of the REV-specific oligonucleotides, and possibly the transformation-specific sequences, we first ordered these oligonucleotides on the REV 30 S RNA genome. The =P-labeled REV genomic RNA was par- tially degraded by treatment with mild alkali. The fragmented RNA containing poly(A) was selected by binding to an oligo(clT)-cellulose column, fractionated into several size classes by centrifugation through a sucrose gradient and then in- dividually analyzed by T,-oligonucleoticle fingerprinting. Figures 2A-D shows the


FIG. 1. Comparative analysis of Ti-oligonucleotides of REV and REV-A RNAs by two-dimensional polyacrylamide gel electrophoresis (“fingerprinting”). The mP-labeled RNAs of the REV (REV-A) released from the SOC6 cell line and REV-A were exhaustively digested with RNase Ti, and separated by two-dimensional polyacrylamide gel electrophoresis as described in Materials and Meth- ods. Migration was from left to right in the first dimension and from bottom to top in the second dimension. Numbering of spots is consistent with that published by Breitman et al. (199Oa). (A) REV(REV-A). (B) REV-A. (C) Equal mixture of REV(REV-A) and REV-A. (D) Schematic representation of (C): (0) T,-oligonucleotides specific for REV-A, (a) Ti-oligonucleotides specific for REV, (0) Ti-oligonucleotides common to both REV and REV-A. The relatively dark spot denoted by an arrow in (A) represents the poorly separated spots 2a and 2b of REV-A.

fingerprints of the poly(A)-containing RNA fragments of different size classes. Individual spots were ordered into several groups on the genome by noting the smallest poly(A)-containing size class that included them. Using Spirin’s formula of MW = 1550(S)2.’ for converting sedimentation value into molecular weight (Spirin, 1963), we have determined the approxi- mate map positions for all of the REV oligonucleotides (Fig. 3). It is seen that the REV oligonucleotides which are shared with REV-A (the oligonucleotides underlined with solid bars in Fig. 3) are scat- tered throughout the entire REV genome.

There is a relatively long contiguous stretch of REV-unique oligonucleotides close to the 3’-end. There are also several short stretches of REV-unique oligonucleotides in the middle of the REV genome.

Localization of REV-Spec@c and Presum- ably Oncogenic Sequences

Mapping of oligonucleotides on the REV genome showed that several regions of the REV genome contained oligonucleotides unique to REV (Fig. 3). However, some of these oligonucleotides might represent only minor base changes from the REV-A


FIG. 2. Ti-Oligonucleotide fingerprints of poly(A)-containing REV (REV-A) RNA fragments of different size classes. The =P-labeled poly(A)-containing REV (REV-A) RNA fragments of different size were digested with RNase Ti and separated by two-dimensional polyacrylamide gel electrophoresis. (A) 30 S, (B) 21-15 S, (C) 15-12 S, and (D) 12-4 S. The identity of the large Ti-oligonucleotides was determined by further sequence analysis with RNase A and by their location relative to neighboring spots. The oligonucleotides derived from the REV-A genome appear on the fingerprints as very faint spots. Only those spots of REV origin are numbered.

sequences. These oligonucleotides would the REV-specific sequences which are pre- then represent the sequences related to sumably responsible for the oncogenic po- the replication genes of REV-A, and not tential of the virus. The latter class of se-

5.7Kb 5Kb 4Kb 3Kb 2Kb 1 Kb

5’ ’ I I I I I PA

- 3’

( 25. 19. 2s. 1s )(x.30.42. 1.6. 12. 5.4. S.7)( 31. ~8,~~.~~~~~)~14,34x23.35.28.s1,s2~

FIG. 3. Ti-Oligonucleotide map of REV showing sequence relationship to REV-A. Individual oligonucleotide spots were ordered into five brackets by noting the smallest poly(A)-containing RNA fragment which included them as shown in Fig. 2. The relative order within a bracket is less certain. Sedimentation values (S) of poly(A)-containing RNA fragments were converted into kilobases (kb) by the formula kb = 1550(S)21/3 X 105 (Modified from Spirin, 1963). The circled oligonucleotides are those specific for REV (see text). The oligonucleotides underlined with a solid bar are those REV oligonucleotides which have identical counterparts in REV-A. The oligonucleotides underlined with a single line are those which have related but not identical sequences in REV-A (see Fig. 4). The oligonucleotides underlined with a jagged line are those unique to REV.


quences might comprise only a subset of these REV-unique regions. To identify the genetic region which represents the truly REV-specific, and presumably oncogenic sequences, two approaches were taken. The first approach was to identify the REV oligonucleotides not identical, but highly related, to REV-A sequences. These oligonucleotide spots were determined by hybridizing 32P-labeled REV RNA to an excess of cDNA complementary to REV- A (cDNAszvJ under conditions of moderate stringency. The hybrids were then digested with either RNase T1 plus RNase A or RNase T1 alone. The 32P-labeled REV RNA still hybridized to REV-A cDNA was recovered in the void volume of a Sephadex G-100 column, and following denaturation, was digested with RNase T1 and fingerprinted. As shown in Fig. 4, the fingerprints of the hybrids that had been digested with RNase T1 alone contain all of the REV spots which had been determined to have identical REV-A counterparts (Fig. 1). In addition, these fingerprints also contain seven other REV spots (X, 1,4, 5, 8, 10, 23) which do not have counterparts in REV-A. It suggests that these addi- tional spots are related to, but not identical to REV-A sequences. These oligonucleotide sequences are located mainly at the 5’-half but also include a short region close to the 3’-end (the regions singly underlined in Fig. 3). Thus, the 5’-half and the extreme 3’-end of the REV genome consist entirely of the sequences either identical or related to the REV-A replication sequences. It is deduced from these data that only the region between approximately 1 and 2.7 kb contains the truly REV-specific and presumably oncogenic sequences. It is interesting to note that when the hybrids were digested with both RNase T1 plus RNase A, all these related but not identical oligonucleotides disap- peared (data not shown). Therefore these oligonucleotides represent related sequences which differ by at least one C or U residue from their counterparts in REV-A.

The second approach is to identify directly the REV-specific oligonucleotides by a two-step hybridization experiment. In

FIG 4. Oligonucleotide fingerprint of REV (REV- A)/REV-A hybrid molecules. saF’-labeled REV (REV- A) from the SOC6 cell line was hybridized to an excess of cDNAssv.* under conditions of moderate stringency. Nonhybridized RNA was removed by digestion with RNase T1 followed by passage through a Sephadex G-166 column. The S2P-labeled RNA in the hybrid was heat-denatured, digested with RNase T1, and separated by two-dimensional polyacrylamide gel electrophoresis. The identity of oligonucleotides was determined as described in the legend to Fig. 2. Only those oligonucleotides derived from REV are numbered.

the first step, cDNA complementary to REV RNA was hybridized to an excess of cold REV-A RNA. 32P-labeled REV RNA was subsequently added to this hybridization mixture and the second step of hybridization was carried out. Since the cDNA sequences which are related to REV-A were not available for hybridization in this second step, only REV-specific cDNA sequences were left to hybridize to 32P-labeled RNA. The final hybrids were digested with RNase T1 plus RNase A or with RNase T1 alone and the undigested RNA excluded from a Sephadex G-100 column was fingerprinted as described above. As shown in Fig. 5A, all the REV spots determined in the first approach to be unique by virtue of their absence in the fingerprints of the REV-A-related sequences (Figs. 3 and 4), #9, 11, 13, 17, 20, 21, 24, 26 and 27, are present in this fingerprint. All these REV-specific oligonucleotides were localized in the region between 1 and 2.7 kb from the 3’-end (the


FIG. 5. Oligonucleotide fingerprints of REV-specific sequences. In an initial hybridization step REV (REV-A) cDNA was hybridized to an excess of REV-A RNA (A), or to an excess of RNA mixtures of REV-A, CSV, SNV, and DIAV (B). In a second hybridization step, =P-labeled REV (REV-A) RNA was added to this hybridization mixture and further incubation was performed. The hybrids were recovered by Sephadex G-100 column chromatography after digestion of the non- hybridizable RNA with RNase Ti. The =P-labeled REV RNA present in the hybrids were heat- denatured, digested with RNase Ti, and separated by two-dimensional polyacrylamide gel electrophoresis. These oligonucleotides represent REV-specific sequences (see text). The identity of oligonucleotides was determined as described in the legend to Fig. 2.

jagged line and circled numbers in Fig. 4). This result is consistent with the conclusion drawn from the first approach. There- fore, we conclude that this region probably represents REV-specific and presumably oncogenic sequences. Assuming an AL- W-like gene order for RE viruses, this region will correspond to the env gene of the REV-A genome. Unexpectedly, the fingerprint of this hybrid contains two ad- ditional spots, #10 and #14, which also appeared in fingerprints of REV-A-related sequences (Fig. 4). Since spot #14 is known to be identical to REV-A spot #16, both by base composition (Breitman et al., 1980a) and by comigration on 2-D gels (Fig. l), its presence here is probably an artifact of hybridization. Moreover, since spot #14 lies adjacent to the REV-specific region on the REV genome (Fig. 3), it is likely that the REV-A RNA hybridized to it in the first hybridization step was subject to strand displacement by 32P-labeled REV RNA in the second hybridization step. This conclusion is supported by the

fact that its intensity varied from experiment to experiment (compare Figs. 5A and B). The presence of REV spot #lO in these fingerprints is more difficult to in- terpret. As shown in Fig. 3, spot #10 is surrounded by REV-specific sequences. It is possible that REV-unique sequences do indeed map as two distinct regions interrupted by a short stretch of REV-A-related sequences. Such a short stretch of sequences would be unable to form a stable hybrid with REV-A RNA and occasionally be subject to strand displacement in the hybrids. Alternatively, spot #lO could represent REV-unique sequences, with its ap- pearance in fingerprints of REV-A-related sequences (Fig. 4) being fortuitous. For example, sequences related to the REV spot #10 may be present in an otherwise unrelated region of the REV-A genome. These sequences could hybridize to it under some hybridization conditions.

From the results of these two hybridization experiments, we conclude that the REV-specific, and possibly transforma-

680 COHEN, WONG. AND LA1

tion-specific, sequences are localized as a contiguous stretch from 1.0 to 2.7 kb from the 3’-end, possibly interrupted by a short stretch of sequences related to REV-A. In contrast, the REV-A-related sequences map from 0 to 1 kb and from 2.7 to 5.7 kb in a 3’ to 5’ direction (Fig. 3).

Lack of Tran#brmation-Related Se- quences in SNV, CSV, and DIAV

Since the other RE viruses, SNV, CSV, and DIAV, are closely related to REV-T (Kang and Temin, 1973; Purchase et al., 1973), and the original stock of SNV has been shown to cause reticuloendotheliosis in viva (Trager, 1959; Purchase and Wit- ter, 1975), it is interesting to determine whether these RE viruses contain any sequences identical or related to the transformation-specific sequences of REV. To this purpose, the two-step hybridization/ fingerprinting experiment described as the second approach for REV-specific sequences was employed, except that, in the first hybridization step, cDNA complementary to the REV RNA was hybridized to an excess of RNA mixtures of SNV, CSV, DIAV, as well as REV-A. The =P- labeled REV RNA was subsequently added and further hybridization was carried out. The hybrid was analyzed by T1-oligonucleotide fingerprinting. If any of SNV, CSV, or DIAV contains transformation- specific sequences related to those of REV, then some of the REV-specific oligonucleotides as defined in Fig. 4 would have dis- appeared from such fingerprints. As shown in Fig. 5B, this fingerprint is identical to that obtained by hybridization with REV- A alone (Fig. 5A). This result suggests that SNV, CSV, or DIAV do not contain appreciable amounts of the transformation-related sequences found in REV. This conclusion is consistent with that obtained with RNA. DNA hybridization (Wong and Lai, 1981).

Relatedness of Genomic Sequences of all RE Viruses

Although CSV, SNV, and DIAV do not contain any REV-specific sequences, all of

them cause various diseases in fowls (Pur- chase and Witter, 1975). In an attempt to possibly identify the genetic basis for their varied pathogenicities in viva, we studied the sequence relatedness of these viruses by oligonucleotide fingerprinting. All the RE viruses contain a 35 S RNA genome of the same size as determined by comigration on polyacrylamide gels (data not shown). None of them contain a small RNA species similar to that of REV. Fin- gerprinting analysis of these viruses showed that all of them had very similar T1-oligonucleotide fingerprints, but each virus also contained some unique spots (Figs. 6 and 7). The exact identity of each oligonucleotide was determined by comparing with fingerprints of the different RNA mixtures (Figs. 7A-F) and also by mapping of their individual RNA genomes (see below). The spots which comigrated in the fingerprints of RNA mixtures and were located at the same positions of their RNA genomes are considered to have identical sequences. In some cases, the identity of the oligonucleotides was further confirmed by partial sequence analysis with RNase A digestion. The mapping of large T1-oligonucleotides on the RNA genome for all the RE viruses was performed by fingerprinting poly(A)-containing RNA fragments of different sizes as described for REV (data not shown). Figure 8 sum- marizes the map orders of all the large oligonucleotides in all of the RE viruses and their interrelationship. Several con- clusions can be drawn from these data: (1) SNV and DIAV are most closely related. They share more than 35 large T1- oligonucleotides while either virus contains only two to three specific oligonucleotides. This is consistent with their evo- lutionary history, since both SNV and DIAV were originally isolated from ducks (Trager, 1959; Ludford et al., 1969). (2) REV-A is more closely related to CSV than to SNV/DIAV. (3) SNV, DIAV, and CSV contain an oligonucleotide spot (#2 in SNV and DIAV, and #3 in CSV) which is shared with spot #l of REV but not present in the genome of REV-A. The identity of this spot in different viruses has been confirmed by partial sequence


FIG. 6. Oligonucleotide fingerprints of 70 S RNA from different viral strains belonging to the reticuloendotheliosis group. (A) REV-A. (B) CSV. (C) SNV. (D) DIAV. Numbering of spots for CSV, SNV, and DIAV is arbitrary.

analysis (data not shown). This spot has a related, but not identical, counterpart in REV-A. (4) A stretch of sequences localized at 2.0-2.5 kb from the 3’-end contains most of the unique oligonucleotides in all of the RE viruses. This region corresponds to the 5’-half of the transformation-specific sequences in the REV genome. (5) All the RE viruses contain identical 3’-end sequences (spots Sl and S2 in Figs. 1, 6, and 8). This region might be analogous to the extreme 3’-end of the C region in the AL-D complex. (6) By comparing with the oligonucleotide map of REV which has a 5.7 kb genome (Fig. 8), it can be seen that a nearly 3 kb stretch of sequences close to the 5’-end of the REV-A genome is deleted in the REV genome. This dele- tion is localized in a region corresponding

to the gag-pol gene of the AL-SV complex. Furthermore, the REV genome also de- letes approximately 1.5 kb of REV-A sequences in a region corresponding to the env gene. These deletions could account for the genetic defectiveness of REV and suggest that the REV-specific sequences represent a substitution and not an addition of sequences.

DISCUSSION

Characteristics of REV-Specific Sequences Our results have shown that the REV-

specific sequences are localized as a contiguous stretch, possibly interrupted by a short stretch of REV-A-related sequences, approximately 1 kb to 2.7 kb from the 3’- end of the REV genome. These sequences are bordered by approximately 3 kb of


REV-A-related sequences on the 5’ side and by 1 kb on the 3’ side. This result is consistent with the recent observations obtained with electron microscopic heteroduplex studies, which showed that the REV-specific transformation sequences were 1.6-1.9 kb in length and were localized in the env region of REV-A (Hu et al., 1981). Our oligonucleotide fingerprinting analysis further showed that REV-T was the only reticuloendotheliosis virus which contained these transformation-specific sequences. Analogous to all the known sarcoma- and leukemia-specific sequences, we, erb, myb, and mat (Spector et al., 19’78; Roussel et al., 1979; Wong et al., 1981), these REV-related transformation sequences (“rel”) also have cellular counterparts in normal avian cells (Wong and Lai, 1981). Thus, it suggests that REV was probably derived from recombination between an RE virus and cellular “ret” sequences. In this connection, it is interesting to note that the specific sequences of REV map adjacent to the 3’ side of REV spots #31 and #18 (Figs. 3 and 8). These two oligonucleotides, which are also pres-

ent in the genomes of REV-A and CSV, map adjacent to the 5’ side of a very variable region where most of the unique spots of CSV, REV-A, and SNV/DIAV map (Fig. 8). Hence, it is possible that REV spots #31 and #18 represent the boundary of a region where frequent recombinations and mutation take place. These mutations and recombinations may have resulted in di- vergence of different RE strains and, in the case of REV, the acquisition of a cellular oncogene. It is interesting that this region, presumably corresponding to the 5’-half of the env gene, is also a variable region in the avian leukosis-sarcoma virus complex (Hu et a,?., 1978) which is related to the host range of the viruses. Our oligonucleotide mapping (Fig. 8) has also shown that the genome of the oncogenic REV has about 3 kb of sequences deleted in a separate region at the 5’-half of the genome. This genetic structure of REV is thus distinct from that of other avian acute leukemia viruses, which contain transformation-specific sequences linked to the gap-pol gene (Lai et al., 1979; Hu et al., 1979; Mellon et al., 1978). In this re-

FIG. ‘7. Comparative fingerprinting analysis of 70 S RNAs from REV-A, CSV, SNV, and DIAV. (A) Equal amounts of REV-A and CSV RNA. (B) Equal amounts of REV-A and DIAV RNA. (C) Equal amounts of CSV and DIAV RNA. (D through F) Schematic representation of fingerprints A-C, respectively.

GENOMIC SEQUENCES OF REV

5.7Kb

FIG. 8. Oligonucleotide maps of genomic RNAs from different RE viruses. Ordering of the oligonucleotides followed the procedures described in Fig. 2. The identical oligonucleotides present in different viruses are linked by single lines. Circled oligonucleotides represent the oligonucleotides unique to a particular virus strain. The oligonucleotide map of REV is shown at the top of the diagram for comparison. REV sequences identical or related to REV-A are represented by the hatched bar under the REV oligonucleotides.

spect, REV resembles the defective murine sarcoma virus (Hu et al., 1977).

Relationship among werent RE Viruses

All the RE viruses are genetically related and could not be distinguished by the extent of their nucleic acid sequence homology (Kang and Temin, 1973). However, our oligonucleotide mapping studies re- vealed further details on their genetic relatedness. The oligonucleotide fingerprints of SNV and DIAV are most closely related (Fig. 8). This is consistent with the facts that both were originally isolated from ducks (Trager, 1959; Ludford et al., 1969) and that both viruses have very strong crossreacting neutralizing anti- body (Purchase et al., 1973). CSV and REV-A are also closely related in their oligonucleotide maps, although CSV was originally isolated from chicken and REV- A, or its parental virus (REV-T), was isolated from turkey (Robinson and Twie- haus, 1974; Sevoian et al., 1964). Our data further suggest that CSV is probably most closely related to the progenitor virus: (1) CSV shares 10 T,-oligonucleotides with

REV-A that are not shared by SNV or DIAV. (2) CSV shares 7 Ti-oligonucleotides with DIAV and SNV that are not shared by REV-A. (3) Only one T1-oligonucleotide is shared by REV-A, SNV, and DIAV that is not also shared by CSV. Al- though the number of shared oligonucleotides cannot be used unequivocally as a quantitative basis for comparing virus strains, these data nevertheless suggest that CSV is more closely related to the other RE viruses, particularly since these RE viruses have very similar overall sequences as determined by nucleic acid hybridization (Kang and Temin, 1973).

Despite the fact that the original stock of SNV has been associated with causing reticuloendotheliosis (Trager, 1959), the genome of SNV was not found to contain any REV-specific sequences (Fig. 5B, Wong and Lai, 1981). However, we have been unable to locate any stocks of SNV that are presently leukemogenic. It has long been known with REV-T that passage in fibroblasts results in attentuation of the incidence of reticuloendotheliosis when in- jected back into birds (Witter et al., 1970; Sevoian et al., 1964; Campbell et al., 1971).


Furthermore, it has recently been demonstrated that accompanying viral attenuation is a loss of the REV RNA component (Breitman et al., 1980b). Apparently, all present stocks of SNV have at one time or another been passaged in fibroblasts. It is possible that the original isolates of SNV, which caused both reticuloendotheliosis and spleen necrosis (Purchase and Witter, 19’75), contained an REV or an REV-like component which has subsequently been lost after repeated passage in fibroblasts.

All of the RE strains differ mainly in the region corresponding to the env region, although some minor variability also oc- curs in other regions of the genome. This is consistent with the finding that neutralizing reactions are weaker with het- erologous RE strains than with homologous RE strains (Purchase et cd., 1973). Whether the differences in this genetic region could account for the varied pathogenicities of these viruses is not clear. It is possible that differences in env proteins could change or expand the target cell do- main of the viruses and thus explain the different pathogenicities associated with the various RE viruses.

ACKNOWLEDGMENTS

This work was supported by United States Public Health Service Research Grant CA 16113, awarded by the National Cancer Institute, and Grant CA 24426 of the Clinical Cancer Education Program. Robert Cohen was the recipient of a NIH predoctoral training fellowship. Timothy Wong is a postdoctoral fellow’of the Leukemia Society of America.

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