Purification and chemical and immunological characterization of avian reticuloendotheliosis virus gag-gene-encoded structural proteins

VIROLOGY

140, 289-312

(1985)

Purification and Chemical and Immunological Characterization of Avian Reticuloendotheliosis Virus gag-Gene-Encoded Structural Proteins’ WEN-PO Litton

Bimetics, National

TSAI, TERRY

D. COPELAND,

Inc.-Basic Research Program, Laboratory Cancer Institute, Frederick Cancer Research Received

July

27, 198.& accepted

AND

STEPHEN

OROSZLAN’

of Molecular Virology and Carcinogenesis, Facility, Frederick, Maq&zd 21701 September

27, 1984

Five gag-gene-encoded structural proteins, designated ~12, pp18, pp20, ~30, and p10 were purified from replication-competent avian reticuloendotheliosis-associated virus (REV-A) by high-performance liquid chromatography complemented with chloroformmethanol extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on amino acid composition and NHz- and COOH-terminal sequence analysis ~12, pp18, ~30, and p10 are distinct from one another, whereas pp20 is likely identical to pp18 in primary structure. The p12 was resistant to Edman degradation and was found to be myristylated at the NHz-terminal amino group. Sequence comparisons among the retrovirus family show that pp18/pp20 and p10 are, respectively, homologs of phosphoproteins and nucleic acid-binding proteins. A comparison of terminal sequences with the nucleotide sequence of spleen necrosis virus (SNV) revealed that the gag genes of SNV and REV-A are highly conserved; together with the identification of REV-A gagprecursor polyprotein, PrGOB’ in immunoprecipitates of radiolabeled cell lysates, this comparison also led to the establishment of the organization of Pr60B”8, viz., NHz-p12pp18-p30-plO-OH. Sequence comparisons show that REV-A/SNV is related to mammalian type C viruses: the pp18-p30 region is most homologous to the macaque/colobus group and least to simian sarcoma virus (SSV), whereas both the 5’- and 3’-gag regions (i.e., p12 and ~10) are clostest to SSV. Immunological studies using monospecific antisera and Western-blot analysis showed that antigenic determinants of REV-A p30 are conserved in most of mammalian type C and type D viruses, but those of REV-A p12 are shared only with simian sarcoma-associated virus (SSAV) and endogenous viruses of macaques. @ 1985 Academic Press, Inc.

duck infectious anemia virus (DIAV), duck spleen necrosis virus (SNV), and chick syncytial virus (CSV) (Purchase et al, 1973; Maldonado and Bose, 1976). The fifth member of the REV group, a replicationdefective transforming virus (REV-T), has also been characterized (Hoelzer et ak, 1979) and the complete sequence of v-rel oncogene has been determined (Stephens et aL, 1983). Serological studies indicate that REV infection is sporadic to frequent among commercial chickens in the United States (Aulisio and Shelokov, 1969; Witter et aL, 1982) as well as in several other countries (Bagust and Dennet, 1977; Wakabayashi and Kawamura, 1977; Yamada et aL, 1977; Ianconescu and Aharonovici, 1978; Neumann et aL, 1981; Howell et aL,

INTRODUCTION

Reticuloendotheliosis viruses (REVS) are a group of type-C retroviruses, some of which were shown to cause neoplasms in avian species from which the viruses were originally isolated (Robinson and Twiehaus, 1974). This virus group consists of at least four replication-competent antigenically related members, viz., reticuloendotheliosis-associated virus (REV-A), r The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. * Author to whom requests for reprints should be addressed. 289

0042-6822185 Copyright All rights

$3.00

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

290

TSAI,

COPELAND,

1982). Biochemical and immunological studies indicated that the reverse transcriptase (RT) of REVS may be more similar and antigenically more closely related to RT of mammalian type-C viruses than to RT of avian retroviruses (Mitzutani and Temin, 1973; Moelling et a& 1975; Bauer and Temin, 1979,198O). REV-A p30 has also been found to be related in protein structure and antigenicity (Hunter et aL, 1978; Charman et al, 1979; Barbacid et aL, 1979) to ~30s of mammalian type-C retroviruses and not to those of the avian leukosis-sarcoma virus complex. On the basis of their p30 NHz-terminal sequences and antigenic relatedness mammalian type-C viruses have been subdivided into four subgroups: mouse, rat, and cat viruses in subgroup I; baboon endogenous virus and the highly related RD-114 (a feline endogenous virus) in subgroup II; gibbon ape and woolly monkey viruses in subgroup III; and viruses of Old World monkeys such as macaques and colobus in subgroup IV (Oroszlan and Gilden, 1980). By this criterion, REV-A has been shown to fall into the group of macaque and colobus viruses (Oroszlan et al, 1981a,b). The question is thus raised as to the origin of REV-A since it is not endogenous in normal avian cell DNA. Our aim has been to structurally and immunologically characterize the gag-gene products of the replication-competent helper virus REV-A, in order to obtain further insights into the genetic relationship between this virus and mammalian type-C viruses. Previously, only the p30 of REV-A had been studied (Hunter et al, 1978; Oroszlan et al, 1981a). For the present study, we purified and characterized the gag-gene-encoded internal proteins of REV-A, grown in chicken cells. The NHz- and COOH-terminal amino acid sequences of each purified protein were determined and compared to known sequences of mammalian type-C virus proteins. A comparison of NHz- and COOHterminal amino acid sequences of the internal structural proteins of REV-A with the partial nucleotide sequence of SNV RNA (O’Rear and Temin, 1982) revealed that these two members of the REV group

AND

OROSZLAN

are highly related, and permitted the determination of the organization of the gag gene in REVS. Using antisera raised against the purified proteins for immunoprecipitation of lysates from radiolabeled virus-producing cells in combination with pulse-chase experiments, we identified a GOK-Da polyprotein as the precursor of the gag-gene-encoded structural proteins. In addition, the antibodies were used to detect cross-reactions between REV-A and other retroviruses. MATERIALS

AND

METHODS

Wraa REV-A was grown in REV/ cBMC, a continuous line of transformed chicken bone marrow cells (Franklin et al., 1974). It was purified by standard sucrose density gradient centrifugation methods (Benton et aL, 1978) and obtained from the Biological Products Laboratory, NCI-FCRF. Protein pur$ication. The gag-gene-encoded proteins of REV-A were purified by reverse-phase (RP) high-performance liquid chromatography (HPLC) by previously described procedures (Henderson et aL, 1981b) with slight modifications. For most proteins RP-HPLC was combined with size separation effected by sodium dodecyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). To recover proteins from gels, the protein bands were first visualized by KC1 staining (Hager and Burgess, 1980), then sliced and extracted with 5% acetic acid. For the phosphoproteins a previously described solvent extraction method using neutral chloroform-methanol (Olpin and Oroszlan, 1980) was also included. SDS-PAGE was also used to determine purity and approximate molecular weights of the proteins. Reduction and carboxamidomethylation. Protein was reduced in a solution containing 6 M guanidine-HCI, 0.17 M dithiothreitol (DTT), and 0.1 M NaHC03, pH 8.5. Samples were flushed with Nz and incubated for 3 hr at room temperature. Reduced proteins were then alkylated with 0.4 M iodoacetamide in the dark at room temperature overnight (Henderson et aL, 1981a). The modified proteins were then dialyzed and recovered by lyophilization.

AVIAN

RETICULOENDOTHELIOSIS

Digestian with endoproteinase Lys-C. The carboxamidomethylated proteins were subjected to digestion by Lys-C (Henderson et al, 1981a). The generated peptides were subsequently isolated by RP-HPLC. Amino acid analysis. The composition and molecular weight of the purified proteins or peptides were determined with a Durrum D500 amino acid analyzer as previously described (Henderson et al., 19’78). The data were analyzed by a computer program which selects a minimum molecular weight for a peptide chain in such a way as to minimize the departure from integral values for all residues (Boyer et al., 1973). NH&rminal sequence analysis. Semiautomated amino-terminal Edman degradations (Edman and Begg, 196’7) were performed in a spinning-cup liquid-phase system (Oroszlan et al., 1978) in the presence of Polybrene (Tarr et aL, 1978) with a Beckman 890C sequencer. Phenylthiohydantoin (PTH) derivatives of amino acid were then identified and quantitated by RP-HPLC on a Waters phenylalkyl column (Henderson et al+, 1980).

COOH-terminal

sequence

analysis.

COOH-terminal amino acid sequences were determined by digestion with carboxypeptidase A as described (Oroszlan et al, 1978). Statistical analysis. The homology between two protein sequences was determined by the computer program “ALIGN” (Dayhoff, 1976). The program is designed to compute a numerical value for any given alignment of two sequences based on the mutational data-scoring matrix. For each break introduced in order to obtain the best alignment, a penalty is assigned. The final alignment score is the number of standard deviations (SD) by which the maximum score for the real sequences exceeds the average score for the random permutations. Three hundred random runs were performed to determine alignment scores. Antisera. Antisera were produced in rabbits. Two methods were used for the preparation of protein inocula. The lyophilized purified protein was dissolved in 0.5 ml phosphate-buffered saline (PBS) and

VIRUS

291

the solution was then mixed with an equal volume of complete Freund’s adjuvant for the initial injection, or with incomplete adjuvant for subsequent injections. Alternatively partially purified proteins were electrophoresed in SDS-PAGE, and then stained with KCl. The appropriate protein band was then excised and minced with an equal volume of PBS. The resulting suspension was ready for injections. Freund’s adjuvants were used with REVA p10 inoculum, and for initial REV-A pp18 injections. Protein-gel suspensions were used for REV-A p30 and ~12 and later injections of pp18. An amount of 100 pg of protein was used for the initial injections and 50 pg for each subsequent injection. The administration routes, immunization, and bleeding schedules were similar to those described by Morgan et al. (1983). Iodinated lz51-protein A. ?-Protein A (IPA) was routinely prepared by use of IODO-GEN (1,3,4,6-tetrachloro-3a,6a-diphenylglycouril) according to the method previously described (Chevez and Scheraga, 1977; Fraker and Speck, 1978). Iodination was performed at 4” for 30 min in a mixture of 200 j~l containing 0.2 mg of IODO-GEN and 25 rg protein A in 50 ~1 of reaction buffer (0.4 M Tris, 4.0 mM EDTA, pH 7.4), and 1 mCi of Nalz51. The reaction mixture was then applied to a Sephadex G-25 (medium) column equilibrated in PBS for separation of free isotope from lz51-protein A.

Electroblotting and immunoautwadiography. The specificity, titer, and crossreactivity of the antisera were determined by electroblotting and immunoautoradiography (Renart et aL, 1979; Symington et al, 1981) as described by Morgan et al (1983). Viral proteins were first resolved on 8 to 20% SDS-polyacrylamide slab gels 1.5 mm in thickness. Routinely, a sample of about 75 pg viral protein was loaded in a single slot about 0.5 cm wide. Immediately after electrophoresis, the gel was equilibrated with transfer buffer (0.04 M sodium phosphate, pH 6.4) at room temperature for 1 hr. Proteins in the gel were then transferred electrophoretically to diazo paper in a Trans-Blot cell (Bio-Rad

292

TSAI,

COPELAND,

Laboratories) filled with transfer buffer. Electrophoresis was performed at 1.5 A for 2 hr with constant cooling. After transfer, the diazo paper was incubated with 1% bovine serum albumin (BSA) in transfer buffer for 2 hr at 37” and then rinsed with distilled water. Usually strips with an average width of 0.5 cm or wider were cut and treated with appropriate antisera. If not used immediately, the strips were air-dried and stored at -20”. For detection of antigens, the antiserum was diluted with TENG-N (0.05 M TrisHCl, pH 7.4, 0.005 M EDTA, 0.15 M NaCl, 0.25% Knox gelatine [gelatin], 0.05% Nonidet-P40 [NP-40]), wherein the proteintransferred paper was incubated at 37” overnight. The paper was then rinsed with water and washed in TENG-N at 37” for 2 hr, and then reacted with lz51-labeled protein A (IPA) at 50,000 cpm/ml in TENG-N at 37’ for 2 hr. The unbound isotope was washed off at 37” for 2 hr with TENG-S (0.05 M Tris-HCl, pH 7.4, 0.005 M EDTA, 1.0 M NaCl, 0.25% Knox gelatine, 0.4% sodium-N-lauroyl sarcosinate). After being rinsed in Hz0 and airdried, the paper strips were exposed generally overnight to a flashed X-ray film with an intensifying screen. Carbon 14labeled protein standards were included in the entire procedure to estimate molecular weights. Radiolabeling of cell cultures. In order to study the precursor polyprotein of REVA gag-gene products, [35S]methionine (40 pCi/ml, 1174.9 CYmmol) was used to metabolically label REV/cBMC and the virus produced as previously described (Schultz et ah, 1979). In brief, the infected cells were washed with warm PBS and labeled with the isotope in methionine-free Eagle’s minimum essential medium (EMEM) for a short pulse period. After being washed with cold PBS, the labeled cells were incubated with the regular medium. At different time periods from 0 to 22 hr the cells and the released viruses were harvested. Radioimmunoprecipitation. After labeling with [35S]methionine, cells were washed with cold PBS and disrupted with lysis buffer (0.02 M Tris-HCl, pH 7.5, 0.05

AND

OROSZLAN

M NaCl, 0.5% sodium deoxycholate, 0.5% NP-40). The pelleted virus was also solubilized in the same buffer. Immunoprecipitation of the cell lysates and pelleted virus with protein A-Sepharose and subsequent SDS-PAGE of the recovered immunoprecipitates were performed as previously described (Schultz et al., 1979). Radioactive bands were located by scintillation autoradiography (Laskey and Mills, 1975). RESULTS

Reverse-Phase HPLC (RP-HPLC) SDS-PAGE Analysis of REV-A Gene-Encoded Proteins

and gag-

REV-A proteins (450 pg) solubilized in guanidine-HCl at acidic pH were chromatographed on a PBondapak phenylalkyl column and fractions analyzed by SDSPAGE. The RP-HPLC elution profile is shown in Fig. 1 and the corresponding electrophoresis pattern in Fig. 2. From these figures, it is seen that RP-HPLC under the conditions employed (see legend to Fig. 1) separated fairly well, but not completely, five major viral proteins designated ~10, pp18/20, ~30, ~12, and ~20”“’ eluting in that order. The smallest hydrophilic protein, ~10, eluted at 28% acetonitrile (Figs. 1 and 2, fraction 13). pp18/ 20 was eluted at 39% acetonitrile (Fig. 1) and appeared as a doublet by SDS-PAGE (Fig. 2, fraction 17). The major peak which appeared at 49-50% acetonitrile (Fig. 1, fraction 24) contained primarily p30 (Fig. 2, fraction 24) and also several minor components of both larger and smaller molecular weights. This peak was followed by a substantial trailing extending to fraction 29 (58% acetonitrile) and thus overlapping another major viral protein peak eluting at 54% acetonitrile and containing p12 (fraction 27 of Figs. 1 and 2). Besides the peaks which contain these major proteins, an earlier peak, fraction 10, was void of proteins as analyzed with SDS-PAGE followed by Coomassie brilliant blue staining. When much larger quantities of viral proteins (~5 mg) were chromatographed, the chromatographic profile was comparable to the run shown

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RETICULOENDOTHELIOSIS

VIRUS

293

Minutes

1. RP-HPLC profile of REV-A proteins. Purified REV-A (total protein 450 pg) resuspended in TNE (0.01 M Tris-HCl, pH 7.2, 0.1 M NaCl, 0.001 A4 EDTA) was solubilized in 4 M guanidineHCl and then reduced in presence of 2-mercaptoethanol (2% v/v) buffered with NaHC03 to pH 9. It was then acidified (pH 2) with 10% trifluroacetic acid (TFA final concentration 0.05%) and applied to a pBondapak phenylalkyl column (0.25 in X30 cm). Proteins were eluted with a O-60% acetonitrile gradient in 0.05% TFA at a flow rate of 1 ml/min. Fractions of nonidentical volumes as shown were collected manually. FIG.

in Fig. 1. All viral proteins used in this study (~10, plW20, ~12, and ~30) were further purified as described below. The final purification and structural analysis of p20env, eluting only with 1-propanol (Fig. 1, fraction 37) will be reported elsewhere. Puti$catim

of REV-A

p10

As shown in Figs. 1 and 2, REV-A p10 was obtained in a high state of purity by a single RP-HPLC run. This protein component was eluted from the phenylalkyl column as an apparently homogenous band even when larger amounts of viral proteins were chromatographed. The resulting purified protein was used for sequence analysis. It has been shown previously that the ~10 homologs of type-C viruses have a relatively high cysteine content (Henderson et aL, 1981a). Since cysteine residues cannot be accurately determined by amino acid analysis nor identified by Edman degradation because of their instability, it was necessary to

chemically modify the cysteines of REVA p10 and purify the modified protein for more complete structural studies. RPHPLC-purified p10 was reduced, carboxamidomethylated, and then reisolated by RP-HPLC on a PBondapak Cl8 column. The modified p10 was recovered in fraction 11 eluting at 28% acetonitrile (Fig. 3A). In SDS-PAGE analysis (Fig. 3B, fraction ll), modified p10 appeared as a single sharp band using either 5.5 or 1.8 pg of the protein and showed no tapering front edge as detected in a previous RP-HPLC run (Fig. 2, lanes 13 and 14). It should be noted that carboxamidomethylated p10 migrated in SDS-PAGE somewhat faster than unmodified p10 and at a rate similar to that of aprotinin (6.5K). Purification

of REV-A

pp18 and pp20

As shown in Figs. 1 and 2, pp18 and pp20 were coeluted in a single peak from a phenylalkyl column. To separate pp18 and pp20 from each other, the peak pool of REV-A pplW20 obtained from RP-

TSAI,

294

COPELAND,

AND

Fraction

t

OROSZLAN

Number 19

4

200K + 1163K+ 925K-b 662Ka

3lK-b

21 SK144K-b

Fraction S

V

23

Number

31 24 25 26 2? 26 29 30 32

33 34 S

2OON-e 116.3K-b 92 SK662K+

31K-O

ame

14 OK-+

c

6 5U-W

FIG. 2. SDS-PAGE analysis of fractions in the RP-HPLC fractions 4 to 34 were electrophoresed and stained with labeled with fraction number, corresponding to fractions in standards for molecular weights. The protein markers (116.3K), phosphorylase B (92.5K), bovine serum albumin anhydrase (31K), soybean trypsin inhibitor (21.5K), lysozyme lane contains whole disrupted REV-A. In each of these lanes

HPLC was first lyophilized and run on SDS-PAGE. The proteins were then visualized by KC1 staining (Hager and Burgess, 1980). REV-A pp18 and pp20 bands were individually sliced out from the gel, extracted, and rechromatographed on a Cl8 column. The chromatographs for REVA pp20 and pp18 are shown in Figs. 4A

as shown in Fig. 1. Entire samples of Coomassie brilliant blue. Lanes are Fig. 1. The “S” lane contains protein are: Myosin (29OK), @galactosidase (66.2K), ovalbumin (45K), carbonic (14.4K), aprotinin (6.5K). The “V” a total of 40 pg protein was applied.

and B, respectively. It shows that pp20 was eluted at 35% acetonitrile (Fig. 4A, fraction 19), whereas pp18 eluted at 33.5% (Fig. 4B, fractions 18, 19). Subsequent SDS-PAGE analysis (Fig. 4C) showed both the pp18 and the pp20 peaks to contain single bands, indicating a high state of purity.

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VIRUS

295

contaminated primarily with ~30. When the pool of plBcontaining fractions was rechromatographed on a Cl8 column, ~30 was eluted at 49% acetonitrile (fraction 10, Fig. 5A) whereas ~12 was eluted at 60% (fraction 15, Fig. 5A). In SDS-PAGE analysis, as shown in Fig. 5B, fraction 15 was found to contain ~12 and a minute amount of ~30. ~12 was further purified by SDS-PAGE and rechromatographed on a Cl8 column, after which pl2-containing fractions had no detectable ~30 by SDS-PAGE analysis.

,:;1I ;. :‘-11 Fractmn

0

B

10

20

30

Number

40 Minutes

s

50

” 1 -a _

60

70

0

80

F11 .

.,

92.5K-w 55.2K-W

45K+

21K-m

21.5K-. 14.4K-W &5K-. 5.5vg

1.8l.g

FIG. 3. RP-HPLC profile and SDS-PAGE analysis of reduced and alkylated REV-A ~10. (A) RP-HPLC profile on a pBondapak Cl8 (0.25 in X30 cm) column. Flow rate was 1 ml/min. Fractions of nonidentical volumes were collected manually. (B) SDS-PAGE analysis of fraction 11 of Fig. 3A. In the lanes shown under the bar, samples containing 5.5 pg (left lane) and 1.8 pg (right lane) of p10 were applied. Lane S, protein standards for molecular weights. The protein markers are same as in Fig. 2 excluding myosin and P-galactosidase. Lane V, REV-A; a total of 40 pg of protein was applied.

Pur$cation

of REV-A ~1.2

As shown in Figs. 1 and 2, in chromatographing REV-A proteins on the phenylalkyl column the REV-A p12 peak was

PurQkation

of REV-A

p30

Purification of REV-A ~30 by conventional methods has previously been described (Hunter et aL, 1978; Charman et aL, 11979). The amino acid composition as well as the partial primary sequence of this protein has also been reported (Hunter et aL, 1978; Oroszlan et aL, 1981a). In the present study, REV-A ~30 was purified by RP-HPLC followed by SDSPAGE. As shown in Fig. 1~30 was eluted in a single RP-HPLC run mainly in a sharp peak, which, however, also contained other proteins (Fig. 2). In a large scale (~5 mg total protein) HPLC separation, REV-A ~30 was eluted in a similar fashion. To further purify ~30, the peak fractions containing this component were lyophilized and then subjected to SDSPAGE separation, and rechromatographed on a Cl8 column as for ~12. ~30 appeared as a single band in subsequent SDSPAGE analysis indicating a high state of purity.

Amino Acid Composition teins

of REV-A

Pre

The amino acid composition and the calculated molecular weight of REV-A ~12, pp18, pp20, ~30, and ~10 are shown in Table 1. The data are based on a single 24-hr hydrolysis. The composition of pp20 was found to be identical to that of pp18, otherwise each protein had a distinctive composition. REV-A pp18/pp20 is exceptionally rich in proline (22.5%) as compared with other proteins (6.6% in ~12, 7.5% in ~30, and 12.2% in ~10). REV-A

296

TSAI,

COPELAND,

AND

OROSZLAN

FIG. 4. RP-HPLC profiles of REV-A pp20 and pp18 and SDS-PAGE analysis of their RP-HPLC fractions. (A) REV-A pp20 profile; (B) REV-A pp18 profile. A PBondapak Cl8 column (0.25 in X30 cm) was used. Flow rate was 1 ml/min. Fractions of nonidentical volumes were collected manually; (C) SDS-PAGE analysis of REV-A pp20 and pp18 fractions from RP-HPLC. Lane A19, a sample from F19 of Fig. 4A, lane B, 18-19, a sample from F18-19 of Fig. 4B; lane 0, pp18/pp20 prior to separation. Lane S, protein standards for molecular weights. The protein markers are same as in Fig. 2 excluding myosin and j+galactosidase. Lane V, REV-A; a total of 40 ng of protein was applied.

p12 has a relatively high glycine content (13.2%) as compared with other proteins (7.1% in pp18, 4.5% in ~30, and 12.2% in plO), but lacks methionine. REV-A p30 is relatively rich in dicarboxylic acids and their amides, glutamic acid/glutamine plus aspartic acid/asparagine (28.4%, altogether) as compared with other proteins (17.6% in ~12, 18.8% in pp18, and 16.3% in ~10). In contrast, REV-A p10 is relatively rich in the basic amino acids, lysine and arginine (20.4%, altogether) as compared with the other gag proteins (14.3% in ~12, 9.4% in pp18, and 14.6% in p30), but lacks methionine, isoleucine, and phenylalanine. In order to further characterize REVA gag-gene products, the amino acid composition of each protein was analyzed by grouping the residues into three classes: polar, intermediate, and nonpolar (Capaldi and Vanderkooi, 1972) as shown in Table 2. Among the gag-gene products, p30 appears to have the highest percentage of polar amino acids (42.9%), of which glutamine/glutamic acid constitute 42.6%; p10 is the next most polar protein, in which lysine (8 residues) and arginine (2 residues) make up more than half of its polar residues (18 residues); p12 and pp18 are

much less polar and have relatively higher contents of nonpolar residues than p30 and ~10. The overall distribution of polar and nonpolar amino acid residues thus appear to be similar to that found in other mammalian type C virus gag proteins (Oroszlan and Gilden, 1980). Amino-Terminal Sequences of REV-A p1.2, pp18, ppZ0, and p10 ~12. Four nanomoles of protein were subjected to high-sensitivity microsequence analysis by semiautomated Edman degradation. No PTH amino acids were detected for the first 10 cycles. The resistance of this protein to Edman degradation indicates that its NH2 terminus is blocked. In an independent study using dipeptidyl carboxypeptidase digestion followed by gas chromatography-mass spectral analysis, a six-residue peptide derived from purified REV-A p12 was found to have a sequence of Gly-Gln-Ala-Gly-SerLys and shown to be myristylated at the amino-terminal glycine (unpublished results obtained in collaboration with L. E. Henderson and H. C. Krutzsch). pp18 and pp20. Eight nanomoles of pp18 protein were degraded in a single mi-

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RETICULOENDOTHELIOSIS

0

Fraction

I s

0

2

3

4

6

10

13

VIRUS

Number 14

15

297

0 17

18

19

21

22

92.5K 66.2K 45K

21.5K

-b

14.4K

-D

6.5K

-m

FIG. 5. Separation of REV-p12 and p30 by RP-HPLC. (A) RP-HPLC profile of REV-A p12 and REV-A p30 on a Cl8 PBondapak column (0.25 in X30 cm). Flow rate was 1 ml/min. Fractions of nonidentical volumes were collected manually. (B) SDS-PAGE analysis of samples from fractions in Fig. 5A. Lane S, protein standards for molecular weights. The protein markers are same as in Fig. 2 excluding myosin and /3-galactosidase. Lane V, REV-A; a total of 40 pg of protein was applied.

crosequence analysis; the NHz-terminal amino acid sequence and the yield in nanomoles of the various amino acids at each cycle are given in Fig. 6. To determine the sequence of REV-A pp20, 4 nmol of protein were subjected to similar analysis. This sequence, as shown in Fig. 6, is identical to the first five residues of pp18, consistent with the compositional identity between pp18 and pp20, and indicating

that these two proteins are likely to be identical in primary structure. ~10. The NHz-terminal sequence of REV-A p10 (initial 3’7 residues) is shown also in Fig. 6. The quantitative data from the analysis of intact unmodified protein is shown under the sequence (the first row) up to residue 30 but with the exception of residue 28. The amino acid in this position and residues 31 and 32 were iden-

298

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AND

TABLE AMINO

ACID

COMPOSITION

1

OF REV-A

Number

OROSZLAN

gcf@GENE

of residues

per

PROTEINS mole”

Amino acid

P12

PPl8

PP20

P30

Asp Thr Ser Glu Pro GUY Ala Val Met Ile Leu Tyr Phe His LYS Arg CYS

7.8 (8) 5.5 (6) 4.1 (4) 8.0 (8) 5.9 (6) 12.2 (12) 4.1 (4) 6.3 (6) 0.3 (0) 4.6 (5) 9.4 (9) 2.8 (3) 4.6 (5) 2.1 (2) 9.0 (9) 4.0 (4) ND

3.2 (3) 2.8 (3) 6.5 (7) 12.9 (13) 19.0 (19) 6.1 (6) 8.9 (9) 3.8 (4) 1.9 (2) 1.0 (1) 4.9 (5) 2.1 (2) 2.2 (2) 1.1 (1) 1.1 (1) 7.4 (7) ND

3.1 (3) 2.8 (3) 6.4 (6) 12.9 (13) 19.0 (19) 6.3 (6) 8.9 (9) 3.7 (4) 1.9 (2) 1.1 (1) 4.9 (5) 2.0 (2) 1.7 (2) 1.1 (1) 1.1 (1) 7.1 (7) ND

26.8 (27) 20.2 (20) 14.3 (14) 49.2 (49) 20.2 (20) 12.1 (12) 18.9 (19) 14.9 (15) 3.1 (3) 10.8 (11) 19.0 (19) 7.9 (8) 8.7 (9) 2.6 (3) 12.7 (13) 26.3 (26) ND

Total* MW

91 9924

85 9089

84 9002

268 30,681

“Values based on one sample the parenthesis. *Minus cysteine and tryptophan ‘Not determined.

hydrolyzed except

for for

24 hr and ~10 which

tified and quantitated by degrading the cysteine-containing peptide (data shown in second row) generated from carboxTABLE POLARITIES

2

OF REV-A

gag-GENE

Percentage

of total

Protein

Polar*

P12 ~~18 P30 PlO

31.9 28.2 42.9 36.7

Intermediate” 29.7 22.4 21.3 26.5

PROTEINS’ amino

rounded is minus

off to the tryptophan

nearest

PlO 2.1 1.9 2.7 6.0 6.0 6.1 3.9 1.9 0.0 0.0 3.9 0.9 0.0 1.0 7.7 3.0 2.7

(2) (2) (3) (6) (6) (6) (4) (2) (0) (0) (4) (1) (0) (1) (8) (3) (3)

51 5442 integers

as shown

in

only.

amidomethylated p10 (see above) by LysC endoproteinase and purified by RPHPLC. The assignment of amino acids beyond residue 32 is based only on qualitative data (appearance or disappearance of the peaks of newly arising amino acids) from the analysis of intact ~10.

acids Nonpolard 38.5 49.4 35.8 36.8

a Amino acids divided into three classes according to Capaldi and Vanderkooi (1972). *Polar: Lys, Arg, Asp, Asn, Glu, Gln (the number of amides was not separately determined). ’ Intermediate: His, Ser, Thr, Gly, Tyr. d Nonpolar: Ala, Val, Leu, Ile, Met, Pro, Phe, Cys, Trp.

COOH-Teminal

Sequence Analysis

The carboxyl-terminal sequences of REV-A gag proteins were determined by digestion for various time periods with carboxypeptidase A and analysis of amino acids released. The data are summarized in Table 3. On the basis of this kinetic data the carboxyl-terminal sequence of REV-A p12 is -Ser-Lys-Val-Leu-OH. When REV-A pp18 and pp20 were analyzed, tyrosine was the only amino acid liberated even after a 120-min digestion

AVIAN

RETICULOENDOTHELIOSIS

VIRUS

299

Compatim of RE V-A and SNV Sequences and Their Organization in the gag Gene A comparison of the partial amino acid sequences deduced from the nucleotide sequences of SNV (O’Rear and Temin, 1982), with the NHz-terminal and COOHterminal sequences of the structural proteins of REV-A as determined in this study was done to assess the genetic relatedness of the two viruses. The alignment of these sequences, shown in Fig. 7, permitted the determination of the precise organization of REV-A and SNV in their gag genes. It is seen that the six-residue NH2terminal sequence of REV-A p12 matches perfectly the two- to seven-residue segment of SNV (when numbering starts with the initiator methionine). The fourresidue COOH-terminal sequence, SerLys-Val-Leu-OH, of REV-A p12 is also identical to the llO- to 113-residue seg-

P!!

FIG. 6. NH,-terminal amino acid sequence A pp18, pp20, ~10, and ~12. Numbers below are yields in nanomoles of PTH amino acid tives as obtained by degrading the unmodified teins and a p10 fragment. Inputs for Edman dation were as follows: pp18, 8 nmol; pp20, ~10, 10 nmol for intact protein, 3 nmol fragment.

of REVresidues derivaprodegra4 nmol; for the

period from either. Similarly only a single amino acid, leucine, was released by carboxypeptidase A from REV-A ~10. TABLE CARBOXYL-TERMINAL

AMINO ACID ANALYSIS OF REV-A BY DIGESTION WITH CARBOXYPEPTIDASE Moles

Time (min)

Protein Pl2

5 20 60 120

~~16

PP20

PlO

a Stock

of amino

acid

Leu

Val

LYS

Ser

0.64 0.70 0.69 1.06

0.56 0.64 0.64 1.00

0.05 0.11 0.32

0.06

ga&GmE A”

released/mole

PROTEINS

of protein COOH-terminal sequence

5r

-Ser-Lys-Val-Leu-OH

5 20 60 120

0.05 0.12 0.40 0.42

-Tyr-OH

5 20 60 120

0.05 0.34 0.62 0.71

-Tyr-OH

1 5 10 20 120 solution

3

0.92 1.09 1.05 1.02 1.01

of carboxypeptidase

-Leu-OH A of 25 mg/ml,

specificity

of 76 Wmg,

diluted

at 1:5000

was

used.

300

TSAI,

COPELAND,

FIG. 7. The order of gag-gene products in REV-A gag gene determined by alignment of partial sequences of REV-A ~12, pp18, and p30 with SNV homologs deduced from nucleic acid sequences (from O’Rear and Temin, 1982). Differences between SNV and REV-A are underlined. The Arabic numbers over the sequence indicate the residue numbers of SNV beginning with initiator methionine as residue 1. The REV-A p30 NHz-terminal sequence is taken from Oroszlan et al. (1981a). The one-letter code for amino acids is: A, alanine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; X, unidentified; Y, tyrosine.

ment of SNV. Thus REV-A p12 is determined to be encoded by the NH2 end of the gag gene. In addition, similarity in the amino acid composition between p12 of REV-A (as determined in this study) and that of SNV (as deduced from the nucleotide sequence), and the demonstration of a “blocked” myristylated NH2 terminus of REV-A p12 are consistent with such a position for this protein. Aligning the first residue of REV-A pp18 protein sequence with residue 114 of SNV immediately adjacent to the carboxyl terminus of SNV ~12, the first 28 residues of REV-A pp18 (except three positions, underlined in Fig. 7) match the SNV sequence running from residues 114 to 141. The COOH-terminal tyrosine of REV-A pp18 is aligned with residue 199 in SNV. This alignment gives an 86-residue polypeptide for both SNV and REV-A pp18. This is in good agreement with the total residue number of REV-ppl8 as determined by amino acid analysis (Table 1). Thus pp18 is adjacent to and continuous with the COOH terminus of ~12. The first p30 NHz-terminal residues of REV-A p30 (Oroszlan et aL, 1981a) are identical with the SNV sequence running from resi-

AND

OROSZLAN

dues 200 to 229, excepting an exchange of Thr - Gly (underlined). It is clearly shown that REV-A p30 is contiguous with the COOH terminus of REV-A pp18. Thus, the three REV-A structural proteins discussed above occur in the order of NH2p12-pp18-p30-. The position of REV-A p10 was determined by analogy to MuLVs. The structural results reported in this study show that REV-A ~12, pp18, ~30, and p10 are the homologs (see below), respectively, of MuLV ~15, ~12, ~30, and p10 which are aligned in the order NHz-p15-p12-p30p10 in Pr65g”g (Barbacid et aZ., 1976a; Oroszlan et aL, 1978; Schinnick et aL, 1981). Accordingly, REV-A p10 is placed next to the carboxyl terminus of ~30. The organization of REV-A in the gag gene is thus established as NH,-p12-pp18-p30plO-OH. Characterization of REVA gag-Gene-Encoded Precursor Polyprotein In pulse-chase experiments designed to identify gag precursors, cells producing REV-A were pulse-labeled with r5S]methionine for 20 min and chased for 0,0.25, 0.5, 1, 2, 4, and 22 hr. Virus released from the labeled cells was also harvested at 4 and 22 hr of the chase. The proteins precipitated by anti-REV-A p30 from cell lysate and virus were electrophoresed in SDS-PAGE and detected by autoradiography. The resulting protein patterns are shown in Fig. 8. The major product evident at the end of the pulse (O-time chase) was a 60K species, designated Pr60gag (lane 1). This molecule was present in the cells for a short period during the chase (15 min as shown in lane 2) and gradually disappeared thereafter (lane 3). As judged from the intensity of the protein bands, more than half of these molecules were removed by the end of the 60-min chase (lane 4). In another hour, the protein had decreased by about two-thirds (lane 5) and it was nearly completely removed from the cells at the end of 4 hr (lane 6a). No trace was found after a 22-hr chase (lane 7a). This molecule was not found in extracellular virus collected at the end of the 4 or 22-

AVIAN

s

1

234

RETICULOENDOTHELIOSIS

56a6b6c7a7b7c

S

FIG. 8. Analysis of REV-A gag-gene-encoded precursor polyproteins in pulse-chase REV/cBMC and REV-A viruses by immunoprecipitating. REV/cBMC was labeled with pS]methionine (40 pCi/ml, 1174.9 Ci/mmol) for 20 min and chased for seven different times and viruses were harvested at two different times. The cells were then lysed and then the lysates clarified. Immunoprecipitation was then performed in 0.5 ml of mixture containing a sample of cell lysate or solubilized viruses, 5 ~1 of antiserum to REV-A ~30, and 40 ~1 of protein A Sepharose CL4B suspension. The precipitates were then subjected to SDS-PAGE analysis. Samples of cell lysate with 2 X lo6 cpm or of solubilized viruses with 14 X lo* cpm were used for each precipitation. The immunoprecipitates of cell lysates from cells pulse-chased for different times were applied in: lane 1, 0 time; lane 2, 15 min; lane 3, 30 min; lane 4, 60 min; lane 5, 2 hr; lane 6a, 4 hr; lane ?‘a, 22 hr. The viruses pulse-chased for two different times were applied in: lane 6b, 4 hr; lane 7b, 22 hr. The viruses without immunoprecipitation were applied in: lane 6c, 4 hr; lane ?c, 22 hr. Lane S, protein standard for molecular weights. Protein markers are phosphorylase B (92.5K), BSA (69K), ovalbumin (46K), carbonic anhydrase (30K), lactogobulin A (18.4K), cytoehrome (12.3K).

hr chase (lanes 6b and 7b). REV-A ~30, the major product detected in extracellular viruses (lanes 6b and 7b), appeared concomitantly with the disappearance of intracellular Pr6Wag during the extended chase period (lanes 6a and 7a). The corresponding whole virus electrophoretic patterns (lanes 6c and 7c) show existence of ~30, but not Pr6Wag in any appreciable amounts, indicating nearly complete processing of the polyprotein. Other minor products, 160K, llOK, and 46K proteins were also evident intracel-

VIRUS

301

lularly at the end of the pulse. The 160K species was probably the gag-PO1 precursor as found previously in REV/c BMC (Hoelzer et al, 1980). The origin of the 1lOK species found in cells is unknown. It may be a highly glycosylated Pr6Wag similar to gP94gag found in R-MuLV-infected cells (Schultz et aZ., 1979). Alternatively, it may not be virally coded. In fact, the 46K protein is thought to be actin which is nonspecifically bound to the immunoglobulin.

Protein Sequence Relutimships between REV-A and Other Type-C Viruses It has been shown previously that the REV-A p30 NHz-terminal sequence is highly related to mammalian type-C viruses and is most related to the macaquecolobus group (Oroszlan et al, 1981b). In the present study, the newly determined NH,-terminal sequences of REV pp18 and p10 are compared, respectively, with their structural homologs from other type-C viruses. Optimal pairwise alignments of REV-A pp18 with CPC-1 (endogenous virus of Colobus polgkmos) pp18, BaEV (baboon endogenous virus) pp18, R-MuLV pp12, and SW (simian sarcoma virus) pp12 were made using the computer program ALIGN of Dayhoff (Fig. 9), and the corresponding alignment scores were also obtained. Although, in these four alignments, the scores are all less than 3.0 SD (a value considered statistically not significant). These results are in line with previous findings indicating that the gaggene regions coding for the phosphoproteins of various mammalian retroviruses are highly divergent (Oroszlan and Gilden, 1980). As already described (see text and Fig. 7), 24 out of 27 residues of the NHzterminal sequence of REV-A pp18 and SNV pp18 were found to be identical. To obtain an optimal assessment of the genetic relationships between the 5’ region of the gag gene of REVS and that of other mammalian type-C viruses, we took advantage of the complete nucleotide sequences available for SNV p12 and pp18 (O’Rear and Temin, 1982) and compared

302

TSAI,

0.

1

5

10

REY-App18

iasar”NPnnP”cPsaPE~x~~~~~~~~

SSY

PnlooLL”“LLstPpP”PAALPPPLA

pp12

Ii

20

COPELAND,

AND

OROSZLAN

these statistical analyses are summarized in Table 4. As previously found with the fragments (Fig. 9), the scores from the comparison of the complete pp18 sequence are low (Table 4). While the value of 4.06 for the SNV ppl8/BaEV pp15 pair appears to be statistically significant, no significant relatedness was found between SSV pp12 and SNV pp18 (alignment score 0.38 SD), although the respective adjacent proteins, SSV p15 and SNV ~12, are highly related (see below). In this regard, it is also interesting to note that a similar comparison of the initial 46-residue segment of SNV pp18 (-50% of the complete sequence) with the NHz-terminal fragment (37 residues are known) of CPC-1 pp18 showed 16 identities and gave a corresponding alignment score of 4.11 SD (data not shown). This is particularly intriguing in the light of our previous findings indicating a closer relationship of REV-A p30 to CPC-1 p30 than to ~30s of other retroviruses (Oroszlan et al, 1981a, b; see also Table 4).

25

FIG. 9. Alignments of NHa-terminal sequence of REV-A pp18 (residues l-28) with sequences of phosphoproteins of four prototype mammalian type-C viruses, using computer program ALIGN of Dayhoff (19’76). The common residues are underlined. CPC-1 pp18r (A) is taken from Bess (1982); BaEV p15 (B), from Tamura (1983); R-MuLV p15 (C), from Versteegen et al. (1982); and SSV pl2 (D), from Devare et al. (1983).

the corresponding complete amino acid sequences with protein homologs of MMuLV, BaEV, and SSV. The results of TABLE

4

SUMMARYOF STATISTICAL.ANALYSESOFAMINOACID SEQLJENCERELATEDNESSBETWEENREV~~~PROTEINS AND RESPECTIVE HOMOLOGSOF VARIOUS RETROVIRUSES Identities/ possible matches

Number breaks

/M-MuLV ~15’ /BaEV ~12” /ssv p15d

39/108 34/106 43/108

4 4 4

9.60 7.59 11.13

/R-ML&V pp12” /BaEV ppl5’ /ssv ppl2d

22/80 28/85 12/68

5 8 4

3.15 4.06 0.38

p30

/R-MuLV p30 /BaEV p30 /SSAV/SSV /CPC-1 p30

13/22 13/21 13/27 18/30

1 2 3 0

6.5f 6.5f 6.4* 8.7’

~10 (l-37)

/R-M~Lv(1-35)~ /BaEV plO(l-35)’ /SSV plO(l-46)d

15/35 15/35 19/37

1 0 2

8.17 9.30 10.85

Sequences Complete sequences SNV ~12”

SNV

Fragments REV-A

REV-A

pp18”

Source. Taken (1983); “Versteegen

aligned

from “O’Rear and Temin et al (1982); fOroszlan

of

(1982); bHenderson et al. (1983); “Tamura (1983); et al (1981a); BOroszlan et al. (1981b); ‘Henderson

Alignment score (SD)

dDevare et al. et al. (1981a).

AVIAN

RETICULOENDOTHELIOSIS

The scores obtained for the extreme 5’ polypeptide p12 are high. Surprisingly, REV-A p12 shares a closer relationship to its homologs in SSV, BaEV, and MuLV than those obtained for ~30s (also shown in Table 4). It is also important to point out that based on these results, REV p12 appears to be more highly related to SSV p15 than to M-MuLV p15 or BaEV ~12. In contrast to the highly variable phosphoproteins of mammalian type-C viruses, the nonphosphorylated nucleic acid-binding proteins (NBPs) are highly conserved. These NBPs are characterized by homologous sequences containing three periodically placed cysteine residues are thought to be the putative nucleic acid-binding site(s) (Henderson et al, 1981a). Such regions have been identified in complete structures of MuLV p10 (Henderson et aL, 1981a), FeLV p10 (Copeland et uL, 1984) BLV p12 (Copeland et aL, 1983a), and HTLV p15 (Copeland et czL, 198313) as determined by direct protein sequencing, and also in the structure of BaEV p10 (Tamura, 1983) and SSV p10 (Devare et aL, 1983) as deduced from nucleotide sequences. The alignment of the initial 37residue segment of REV-A p10 (-66% of the molecule) with R-MuLV ~10, BaEV ~10, and SSV p10 is shown in Fig. 10. The occurrence of the typical cysteine-possessing conserved region in REV-A p10 is apparent. The high overall homology of REV-A p10 to the other NBPs is indicated by the alignment scores (in the range of 8 to 11 SD) shown in Table 4.

8.

1

5

10

15

303

VIRUS

Specificity of the Antisera tural Proteins

to REV-A Struc-

Specificity and titer of the antisera to ~12, pp18, ~30, and p10 raised in rabbits were determined by electroblotting immunoautoradiography. For this study, purified REV-A was disrupted and electrophoresed, and the viral proteins were transferred to DBM paper. To determine the specificity, the transfer strips were reacted with the antisera at 1:800 dilution and then with IPA as described under Materials and Methods. The results are shown as autoradiograms in Fig. 11. Individual antisera reacted strongly with the homologous proteins (Fig. 11, lanes lb, 2b, 3b, 4b) and a mixture of antisera to ~12, pp18, ~30, and p10 did not react more strongly with any of these viral components (Fig. 11, lane 5). All these antisera also recognized some additional protein bands, which were the precursor proteins, intermediary cleavage products, or fragments of primary products. Two intermediary proteins designated 42K and 33K were detected by both anti-ppl8 and anti-p30 antisera (Fig. 11, lanes 2b and 3b) and also by the mixture of the antisera (Fig. 11, lane 5). The intensity of these protein bands stained by the mixture is, however, stronger than that obtained with either anti-REV-A pp18 or anti-REV-A p30 alone. This indicates that these proteins, as a consequence of the possession of both pp18 and p30 antigenic sites, bound more antibodies in the presence of both

20

25

30

35

30

35

REV-Rp10 LRaEsnRIncsKKTPPcnGnPPLGKNpCnVCKrrGHW BdEYPI0 --Rn”“ry~RncXscFTnnnPKYDK09C*YCKERGHY c.

1 REV-RPI0 SS” PI0

5

10

15

20

25

10

45

***LRpEsnaEn~*‘**csK’KTPqCncnPPLGKNgCnvC~~~~”” nn”“sEGGsconnrGNLgNnRnKTqnoaapPcoqogcalZ~~~~~~

FIG. 10. Alignments of NHc-terminal sequence of REV-A p10 (residues l-37) with homolog sequences of three prototype mammalian type-C viruses, using computer program ALIGN of Dayhoff (1976). The common residues are underlined. R-MuLV p10 (A) is taken from Henderson et al (1981a); BaEV ~10, from Tamura (1983); and SSV ~10, from Devare et al (1983).

304

TSAI,

COPELAND,

FIG. 11. Specificity of rabbit antisera to REV-A ~12, pp18, ~30, and p10 as demonstrated by electroblotting immunoautoradiography. Proteins of purified REV-A separated by SDS-PAGE were electrophoretically transferred to DBM paper. The proteinbound DBM papers were then reacted with antisera followed by reaction with ‘l-protein A. Antisera at 1:800 dilution and preimmune sera at 1:200 dilution were used. The lanes and the corresponding antisera are lane lb, anti-REV-A ~12 serum; lane 2b, antiREV-A pp18 serum; lane 3b, anti-REV-A p30 serum; lane 4b, anti-REV-A p10 serum; lane 5, mixture of the antisera above. Lanes la, 2a, 3a, and 4a reacted with the respective preimmune sera. Lane S, protein standards for molecular weights. The protein markers are the same as in Fig. 8.

anti-ppl8 and anti-p30. Since pp18 and p30 have been shown to be adjacent in the precursor polyprotein, the 42K band may represent an uncleaved pp18-~30, and the 33K protein may be a truncated form of this molecule. Similarly the 28K-29K bands recognized by both anti-p12 and anti-ppl8 (Fig. 11, lanes lb and 2b) are probably uncleaved p12-pp18 or a portion of it. The identity of an approximately 68-kDa protein detected only by the antiREV-A p10 serum is not known. None of the preimmune sera stained any protein bands (Fig. 11, lanes la, 2a, 3a, 4a). The titer for each of the four sera (anti-p12, -pp18, -p30, and -plO) was found to be greater than 12,800.

Antigenic

Relationship

between

REV-A

and Other Retroviruses To determine the relationship between REV-A and other retroviruses, each of the antisera in this study was tested

AND

OROSZLAN

against a panel of retroviruses. The electroblotting-immunoautoradiography technique was used to detect cross-reactions. The panel of test viruses included 19 type-C, 3 type-D, and 1 type-B retroviruses. In testing anti-REV-A p12 and antiREV-A p30 against the different viruses, usually an average of 75 pg of total viral proteins were electrophoresed per each 5mm wide slot of the SDS-polyacrylamide slab gel. The results are shown in Fig. 12 and in Table 5. Anti-REV-A p12 reacted specifically with REV-A p12 (Fig. 12A, strip V), and homologous proteins of two endogenous viruses of macaques, MAC-l (strip 1) and MMC-1 (strip 2), and SSAV (strip 3), but not with other viruses tested (Table 5). In addition, antiserum to p12 detected again the 29K protein from REVA as previously shown. A protein of a similar size was also detected in a number of other viruses including SSAV, MAC-l, and MMC-1 (Fig. 12A). For anti-REV-A p30 the results are shown in Fig. 12B and Table 5. This antiserum reacted with homologous proteins from all the type D’s and the type C’s except BLV and RSV (Table 5). The autoradiogram in Fig. 12B shows the reaction between anti-REV-A p30 and each of 11 heterologous viruses, including two mouse viruses (strips 1 and 2) of subgroup I, RD-114, a feline endogenous virus (strip 3), and a baboon endogenous virus (strip 4) of subgroup II, two macaque endogenous viruses (strips 5 and 6) of subgroup IV, a gibbon ape virus (strip 7), and the woolly monkey virus (strip 8) of subgroup III, and three type-D viruses (strips 9,10, and 11). The apparent molecular weights of the detected homologous proteins appeared to differ slightly ranging from 28K to 30K. In general, this is in agreement with previous studies from various laboratories except for SMRV ~30, which was previously estimated to be -36K (Colcher et al, 1977; Hino et aZ., 1977). Evidently, REV-A p30 is broadly related to ~30’s of other retroviruses, including all the known primate viruses and most of the type C’s, but not to BLV or type-B virus. AntiREV-A pp18 and ~10, reacted only with

AVIAN

As

Y

1

RETICULOENDOTHELIOSIS

2

B

3

(19K-w

69K-+

46K-N

46K+

3OK-F

30K*

18.4K+

18.4K*

12.3Kt

12.3K-W

sv

VIRUS

12

345678

305

a 10 11

FIG. 12. Reactions of antisera to (A) REV-A p12 and (B) REV-A ~30, respectively, with various retroviruses as demonstrated by electroblotting immunoautoradiography. Proteins from various purified viruses were applied to SDS-gel in an average of 75 pg of protein/O.5 cm in width of application slot. Following electrophoresis, the proteins were transferred to DBM papers. The protein-bound DBM papers were then reacted with the antiserum followed by reaction with ‘%Iprotein A. Antiserum at 1:800 was used for reaction with REV-A and at 1:200 for the others. (A) Reactions of antiserum to REV-A p12 with the following viruses: lane V, REV-A; lane 1, MAC-l; lane 2, MMC-1; lane 3, SSAV. (B) Reactions of antiserum to REV-A p30 with the following viruses: lane V, REV-A; lane 1, R-MuLV, lane 2, M-MuLV, lane 3, RD-114; lane 4, BaEV, lane 5, MAC-l; lane 6, MMC-1; lane 7, GaLV; lane 8, SSAV; lane 9, SMRV, lane 10, MPMV; lane 11, PO-l-Lu. Lane S, protein standards for molecular weights. The protein markers are same as in Fig. 8.

REV-A, but not with any of the other viruses tested (Table 5). DISCUSSION

In the present study, five gag-gene-encoded structural proteins of REV-A, designated ~12, pp18, pp20, ~30, and ~10, were purified and monospecific antisera to them were produced. It was found that pp18 and pp20 are identical in amino acid composition, and NH2- and COOH-terminal sequences. In addition, antiserum raised against REV-A pp18 detected not only pp18 but also pp20 (Fig. 11, lane 2b and data not shown). Thus pp18 and pp20 are likely to be identical in primary amino acid sequence despite the difference between their apparent molecular weights, which may be due to post-translational modification (e.g., phosphorylation). On the basis of similar structural analyses, the other three proteins, ~12, ~30, and p10 were found to be different from one another and from ~~18120. Each of them has been shown to have a unique NH2terminal sequence. Moreover, each of the

antisera raised against these four proteins reacted specifically with its own antigen only, and did not cross-react with the other REV-A mature structural proteins (Fig. 11). The map order for REV-A was unambiguously determined to be NHBp12-pp18-p30-plO-OH in the gag-gene coding for the precursor polyprotein, Pr6Wag, identified in pulse-chase experiments. A comparison of REV-A proteins by amino acid composition and sequence with the known structural proteins of other type-C retroviruses readily identified each of them to be the homolog of one of the gag-gene-encoded structural proteins of mammalian type-C viruses. The NHz-terminal gag-protein p12 appears similar to and a homolog of MuLV p15 both in overall amino acid composition and in hydrophobicity, as indicated by its strong affinity for the hydrophobic surface on the RP-HPLC supports. A major contributor to the strong hydrophobicity is the covalently bound NHz-terminal myristyl group, which was shown to occur in MuLV ~15s (Henderson et d, 1983) and

306

TSAI, TABLE

COPELAND,

5

SUMMARY OF REACTIONS OF ANTISERA TO REV-A gag-GENE-ENCODED PROTEINS WITH VARIOUS RETROVIRUSES

Antiserum Virus

Type C REV-A R-MuLV M-MuLV AKR-MuLV MCF-247 292 A-MuLV HaLV MiLV FeLV RD-114

BaEV GaLV SSAV MAC-l MMC-1 CPC-1 OMC-1 BLV RSV

Pl2

PPl3

p3O

PlO

+ -

+ -

+ -

NT” NT NT NT NT + + + -

NT NT NT NT NT -

+ + + + + + + + + + + + + + + +

NT -

NT -

NT -

NT NT NT NT NT NT -

Type II MPMV SMRV PO-l-Lu

-

-

-

NT

NT

NT

Type B MMTV

-

-

-

“NT

= Not

-

tested.

also in REV-A p12 (see above). Previously, MuLV p15 was observed to be associated with membranes (Lezneva et al, 1976; Barbacid et al, 1978; Pepinsky and Vogt, 19’79), and the hydrophobic fatty acid moiety may have a major role in the membrane binding of MuLV p15 as well as of REV-A ~12. By comparison of NH2 and COOH termini as well as amino acid compositions, REV-A p12 and SNV p12 are very similar if not identical in primary structure. A comparison by Dayhoff’s ALIGN program of SNV p12 (O’Rear and Temin, 1982) and SSV p15 (Devare et cd, 1983) showed that these proteins share an identical lo-residue segment, Glu-Trp-

AND

OROSZLAN

Pro-Thr-Phe-Gly-Val-Gly-Trp-Pro, located in approximately the middle of the molecules. This sequence is also highly conserved in M-MuLV ~15, where the first Gly is substituted by Asn. In contrast, this segment is not conserved in BaEV p12 (Tamura, 1983). Apparently, it contributes greatly to the higher alignment scores obtained for SNV plB/SSV p12 and for SNV pl2/M-MuLV p15 pair than for SNV plB/BaEV p12 (Table 4). In studies of interviral cross-reactivities using anti-REV-A p12 hyperimmune serum and electroblotting immunoautoradiography, we detected a cross-reaction between REV-A p12 and SSAV ~15, but not with MuLV-pl5 or BaEV p12 (Table 5). Assuming that REV-A p12 and SNV p12 as well as SSAV and SSV ~15s all have in their sequence the above-described lo-residue segment or a substantial part of it, one is tempted to conclude that the structure of antigenic epitope shared between SNV ~12 and SSAV p15 is defined by the amino acid sequence of the above common segment. Because of the substantial divergence, no cross-reaction is expected with BaEV ~12. Moreover, it appears that the substitution of Gly to Asn as in MuLV ~15, is sufficient to change antigenic specificity and eliminate crossreactions. It remains to be seen whether similar sequences account for the observed cross-reactivities between REV-A p12 and MAC-l/MMC-1 counterparts (Table 5). Because of the negative reaction with CPC-1, we would expect at least one amino acid substitution in its corresponding gag protein as found for MuLV ~15. In previous studies, type- and group-specific antigenicity of MuLV p15 were demonstrated (Strand et al, 1974; Barbacid and Aaronson, 1978), but there is little evidence for interspecies-specific antigenic determinants. The results presented here indicate that anti-REV-A p12 possesses interspecific antigenic activity limited to the two primate subgroups of type-C viruses. The finding that REV-A p30 is antigenically related to that of all of the mammalian type-C viruses is consistent with the previous analyses of sequence homology (Oroszlan et al, 1981b). A seven-resi-

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due peptide Ser-Asp-Leu-Tyr-Asn-TrpLys encompassing residues 24 to 30 of REV-A p30 is shared completely by the ~30’s of R-MuLV/FeLV (subgroup I), BaEV (subgroup II), and MAC-l/CPC-1 (subgroup IV) (Oroszlan et al, 1981b). This peptide sequence without the Ser (i.e., six-residue DLYNWK) is also found in SSV p30 (Devare et aL, 1983) of subgroup III. This stretch of sequence may also be one of the antigenic sites common to REV-A and other mammalian type-C viruses as previously detected by competition radioimmunoassay (Barbacid et al, 1980a; Oroszlan et aL, 1981a). As already mentioned anti-REV-A p30 antiserum also reacted with other type C’s not studied previously, including HaLV, an endogenous hamster virus (Kelloff et aZ., 1970), MiLV, an isolate from mink cell line (Sherr et al, 1978), and three other murine viruses: AKR-MuLV (derived from AKR mice with high leukemia incidence), MCF-247, a recombinant dualtropic MuLV, (Hartley et aL, 1977) and 292-A-MuLV, an isolate from a steroid-treated BALB/c-Cy mouse infected with M-MuLV (Rosenberg and Baltimore, 1979) (Table 5). All early isolates of type-D retroviruses, MPMV, SMRV, and PO-l-Lu, which share a common interspecific antigen in their major internal proteins (Colcher et al, 1977,1978; Devare et al, 1978a; Hino et aL, 1977) were shown to react with anti-REV-A p30 in our studies. Thus, we demonstrated for the first time that the p30 of REV-A, a type-C virus, possesses antigenic determinants shared by both Old World and New World type-D viruses. Previously, DKV, an endogenous type C isolated from kidney cells of Columbian black-tailed deer (Aaronson et al, 1976), was shown to be antigenically related to SMRV, the sole New World type-D virus (Barbacid et CAL, 1980b). The Mason-Pfizer monkey virus (MPMV), horizontally transmitted among rhesus monkeys (Chopra and Mason, 1970; Devare et aL, 1978a) was shown to be genetically related to another Old World type D, PO-l-Lu, which was found to be endogenous in the spectacled langur (Presbytis obscures) (Benveniste and Todaro, 1977). Since rhesus monkeys, the

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host of MPMV as well as MMC-1, and some of the other primate virus hosts, such as stumptail monkeys and langurs, share similar natural habitats in Asia (Benveniste and Todaro, 1977), it is highly probable that type-D viruses are as closely related to REV-A as are type-C primate viruses. It would not be surprising to find that the newly isolated simian type-D retrovirus (Daniel et aL, 1984; Marx et aL, 1984; Stromberg et aL, 1984) is antigenitally related to REV-A ~30. REV-A pp18 appears similar to and a homolog of MuLV pp12 and the phosphoproteins of other mammalian type-C retroviruses in calculated molecular weight and in abundance of proline residues. The sequence of Pro-Pro-Pro-Tyr which is conserved in all of the known phosphoproteins of mammalian type-C viruses occurs also in REV-A pp18. This sequence may thus have functional significance. Another common feature found in the phosphoproteins is that their apparent molecular weights estimated with SDSPAGE are all about twice as large as the molecular weights calculated from amino acid compositions. This discrepancy may be caused by post-translational modification, but it is unlikely that phosphorylation alone is responsible. It should be emphasized that in interviral comparisons the alignments of partial or complete sequences of REV-A/SNV pp18 with a panel of retroviral homologs indicated a higher degree of relatedness to viruses of Old World monkeys (i.e., CPC-1) than to other mammalian retroviruses, as previously found with the ~30s (Oroszlan et aL, 1981b). The results that anti-REV-A pp18 did not detect any proteins in the heterologous retroviruses appear to correlate with the low degree of sequence homology between REV-A pp18 and other retroviral phosphoproteins and appear to be in agreement with previous studies which showed phosphoproteins to be type- and group-specific but not interspecies specific (Stephenson et aL, 1974). REV-A ~10, the smallest among the gag internal proteins, is rich in basic amino acids, a characteristic of the NBPs of type-C mammalian viruses. Its low affinity

308

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COPELAND,

for the hydrophobic RP support is consistent with the high polarity contributed mostly from basic amino acids. REV-A p10 is also homologous to other NBPs having the cysteine-containing putative nucleic acid-binding sites (Henderson et aZ., 1981a; Copeland et al, 1983a, b, 1984). In addition to the high homology within this binding-site sequence (Fig. lo), REVA p10 possesses a five-residue segment Gly-Arg-Pro-Pro-Leu which is identical to the sequence present only in SSV p10 (Fig. lOC), but not in MuLV (Fig. 10A) or BaEV ~10s (Fig. 10B). It has been previously shown that MuLV ~10s possess not only group-specific antigenic determinants (Long et al, 1977) but also react in interspecific fashion (Barbacid et al, 1976b; Schulein et ak, 1978). Thus it was rather unexpected that anti-REV-A p10 reacted only with REV-A and not with other viruses tested. While this appears to be a paradoxical finding, it should be emphasized that as already discussed for REVA p12 and as previously demonstrated for BLV and MuLV NBPs (Morgan et ah, 1983), a single amino acid exchange within an antigenic epitope can abrogate serological cross-reactions. The data presented in this report confirm and extend previous findings that the REV-A/SNV gag genes are significantly related to those of mammalian type-C viruses. Most interestingly, the limited sequence data show that in the phosphoprotein-p30 region REV-A/SNV is most homologous to Old World monkeys, particularly the macaque/colobus group, but least with SSAV/SSV, whereas in both gag 5’ and 3’ regions (i.e., p12 and ~10) REV-A/SNV is closest to SSAV/SSV. Nucleic acid studies showed that REV-A is homologous to CPC-1 in both gag and pal regions (Rice et aL, 1981) and that SNV is more homologous to the macaque/colobus group than to either R-MuLV/GaLV or BaEV/RD114 group in the 5’-terminal noncoding regions (Lovinger et al., 1981). Thus, based on previous studies and the results of protein sequence comparisons in this report it is tempting to speculate that REVS may have emerged from recombinations between progenitors of SSAV/SSV and CPC-1. Furthermore, im-

AND

OROSZLAN

munological studies showed that antibodies to purified REV-A ~20”“’ (transmembrane protein) cross-reacted with a panel of mammalian type-C and -D retroviruses (Tsai, 1983). Other previous studies also showed that primate type-C and -D retroviruses are linked in env-gene regions immunologically (Devare et al, 1978b; Barbacid et al, 1980b; Thiel et al., 1981) and genetically (Cohen et or, 1982; Chiu et aL, 1984). Thus, assuming that current retroviruses may have been derived from two progenitors, one for mammalian typeC viruses and another for type-A, -B, -D, and avian type-C groups on the basis of genetic relationship of the pol genes (Chiu et aL, 1984), multiple genetic interactions among many type-C and -D retroviruses, particularly primate viruses may have also occurred to generate the progenitor of REVS. In conclusion, these and other studies reflect on the complexity of the evolutionary history of this unique group of avian retroviruses. More detailed sequence information for primate viruses as well as REVS is needed to unravel the mammalian origin of REVS currently restricted to and spreading horizontally among birds. ACKNOWLEDGMENTS We thank Young Kim for assistance with protein sequencing, and Donald C. Fish and colleagues for assistance with immunizations. We are indebted to Nancy Rice and Alan Schultz for critical reading of the manuscript. Research sponsored in part by the National Cancer Institute, DHHS, under Contract NOl-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsements by the U. S. Government. REFERENCES AARONSON, S. A.,TRONICK, S. R., and STEPHENSON, J. R. (1976). Endogenous type C RNA virus of Odowileus hcmionus, a mammalian species of New World origin. Cell 9, 489-494. AULISIO, C. G., and SCHELOKOV, A. (1969). Prevalence of reticuloendotheliosis virus in chickens: Immunofluorescence studies. Prm Sot. Exp. Biol Med 130, 178-181.

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