Identifying cDNA Clones, Establishing Reading Frames, and. Deducing the Gene .... tides were added to the microtiter plate wells (50 plI per well), and the plate ...
Vol. 54, No. 1
JOURNAL OF VIROLOGY, Apr. 1985, p. 186-193 0022-538X/85/040186-08$02.00/0
Copyright © 1985, American Society for Microbiology
Use of Antibodies Directed Against Synthetic Peptides for Identifying cDNA Clones, Establishing Reading Frames, and Deducing the Gene Order of Measles Virus CHRISTOPHER D. RICHARDSON,* ALLA BERKOVICH, SHMUEL ROZENBLATT,t AND WILLIAM J. BELLINI Laboratory of Molecular Genetics, Intramural Research Program, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20205 Received 15 August 1984/Accepted 3 December 1984
A number of cDNA clones complementary to measles virus mRNA and 50S genome RNA have been generated. These clones have been mapped by restriction enzyme analysis and were subsequently sequenced by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). Computer analysis of these DNA sequences revealed open reading frames which potentially could code for a number of gene products. Portions of these putative polypeptides were synthesized, and rabbit antibodies directed against peptide-hemocyanin conjugates were produced. These antibodies were used to iinmunoprecipitate virus-specific polypeptides which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For each of the antisera tested, a unique protein was precipitated whose migration on polyacrylamide gels corresponded to standard gene products identified by monoclonal antibodies and antisera against measles virus. By using this method, we were able to assign the coding regions of cDNA clones to specific protein products and, subsequently, to order the genes of the 3'-terminal third of measles genome RNA.
as to unequivocally establish the gene order from the 3' terminus of measles 50S RNA. This gene sequence was determined to be N-P/C-M.
Measles virus is a paramyxovirus which encodes its genetic information in a single-stranded RNA (4.8 x 106 daltons) of negative polarity (4, 44). The genome is sequentially transcribed from the 3' terminus to yield possibly nine mRNA species (44). Six of these mRNAs specify the virion structural proteins: nucleocapsid protein (N), phosphoprotein (P), matrix or membrane protein (M), large or polymerase protein (L), a membrane glycoprotein (H) responsible for hemagglutination and attachment to the host cell, and another glycoprotein (F) which causes the membrane fusion that allows the virus to penetrate into the host cell (15). The other three or so mRNAs may specify nonstructural proteins but may also represent polycistronic readthrough mRNAs which have been described in other paramyxoviruses (20, 21, 46). The organization and content of the measles genome are not altogether clear. Indeed, the partial gene order of Sendai virus has only recently been more firmly established (11, 14) and shown to agree with that proposed for Newcastle disease virus (9). Sendai virus has been demonstrated to produce two related nonstructural proteins, C (molecular weight, 22,000) and C' (molecular weight, 24,000), both in vivo and in in vitro translation systems (10, 13, 14, 25). The functions of these proteins, which are produced from the same mRNA as the P polypeptide, are not known. Similar nonstructural proteins have been described for Newcastle disease virus (9), mumps virus (32, 33), canine distemper virus (19), and respiratory syncytial virus (22, 37) and have also been suggested for measles virus (32, 33). The significance and origin of these polypeptides are still presently unclear. In this study, we used antibodies prepared against selected peptides derived from the coding regions of the measles genomic RNA both to identify cDNA clones as well
MATERIALS AND METHODS Cells and virus. CV-1 and Vero cell lines derived from African green monkey kidney were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. The Edtnonston strain of measles virus (originally obtained from Erling Norrby) was grown in Vero or CV-1 cells in the presence of 0.1% bovine serum albumin (BSA). Virus was plaque purified and amplified by three successive passages in Vero cells at a multiplicity of infection of 0.1 PFU per cell at 37°C. The host cells were totally fused by 24 h postinfection, and virus was harvested from the cell supernatant at 36 to 48 h postinfection. A continuous cell line (MA160) which was persistently infected with the Mantooth strain of measles virus was obtained from Microbiological Associates, Walkersville, Md. Preparation of [35S]methionine-labeled lysates of infected cells. CV-1 cells (2 x 107) were infected with measles virus at a multiplicity of 1 PFU per cell. At 18 h postinfection, medium was removed and replaced with 10 ml of Eagle medium (without methionine) and 1 mCi of [35S]methionine (1,000 Ci/mniol). The infection was allowed to proceed until the cells completely fused (24 to 30 h postinfection). The cells were gently washed with 10 ml of cold phosphate-buffered saline (PBS) and lysed with 2 ml of RIPA buffer containing 0.1% BSA. Nuclei and DNA were sedimented by centrifugation at 30,000 x g for 15 min at 4°C. The radioactive supernatants were stored in 0.5-ml aliquots at -70°C, and the pelleted material was discarded. Peptide synthesis. Peptides were synthesized by the Merrifield solid-phase technique with a Beckman 990 automated peptide synthesizer (8). Amino acids were coupled to a 4-methyl-benzhydrylamnine (1% cross-linked) resin with diisopropylcarbodiimide as the coupling agent. All couplings were performed with a sixfold excess of t-butyloxycarbonyl-
* Corresponding author. t Permanent address: The Weizmann Institute of Science, Rehov-
ot, Israel. 186
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amino acids. For asparagine and glutamine, an equimolar amount of N-hydroxybenzotriazole dissolved in dimethylformamide was added to the protected amino acid. The protected peptide-resin (ca. 2 g) was dried under vacuum and treated with 20 ml of anhydrous HF at 4°C (under vacuum) in the presence of 2 ml of anisole and 200 ,ul of ethanedithiol for 1 h. The HF was evaporated with a stream of N2, and then the red residue was mixed with anhydrous ether and stirred vigorously; the precipitated peptide and resin were collected by filtration through a sintered glass funnel and washed three times with ethyl ether. Resin and the cleaved peptide were dried under vacuum, and the peptide was subsequently extracted by washing the resin with 50% acetic acid in water. The extract was lyophilized, and the crude peptide was desalted on a Sephadex G-10 column (1.5 by 250 cm) eluted with 50% acetic acid. Ninhydrin-positive fractions were pooled, lyophilized, and purified by high-pressure liquid chromatography on a Whatman ODS3 magnum C18 preparative column when necessary. Conjugation of peptides to keyhole limpet hemocyanin carrier protein. Peptides were coupled to a protein carrier protein, keyhole limpet hemocyanin, by two methods. For the first method, glutaraldehyde was used as the coupling agent. Peptide (5 mg) and keyhole limpet hemocyanin (5 mg) were dissolved in PBS (5 ml), glutaraldehyde was added to a final concentration of 0.25% (voUvol), and the reaction was allowed to proceed for 1 h at room temperature, after which lysine was added to a final concentration of 0.2 M to stop the reaction. For the second method, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used as a coupling agent. Peptide (5 mg), keyhole limpet hemocyanin (5 mg), and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (5 mg) were dissolved in PBS at 4°C. The reaction was allowed to proceed overnight, and the peptide conjugate was dialyzed against PBS (two times, 1 liter each) to remove excess coupling agent. Immunization of rabbits. New Zealand White rabbits were initially injected subcutaneously along the back with peptidehemocyanin conjugate in Freund complete adjuvant (1:1). Approximately 0.2 mg of peptide conjugate was injected at each of five sites. At 3 weeks, the rabbits were again injected along the back in a similar fashion with peptide conjugate in Freund incomplete adjuvant (1:1). At 5 weeks, the rabbits were injected subcutaneously along the back with peptide conjugate, again in Freund incomplete adjuvant, and intravenously with 2 mg of free peptide (dissolved in 1 ml of PBS) into the ear. The rabbits were subsequently boosted at 3-week intervals by injection of 2 mg of unconjugated peptide intravenously into the ear. Rabbits were first bled at 7 to 8 weeks after the initial injection. ELISAs. Enzyme-linked immunoadsorption assays (ELISAs) were used to test all antisera for their ability to react with the peptides used in immunization. Peptide antigen was bound to 96-well polyvinyl chloride microtiter plates (Dynatech, Alexandria, Va.). Briefly, 100 pul of 0.2% glutaraldehyde in PBS was added to each well, allowed to remain for 2 h, and removed by vigorously inverting the plate several times. Peptide (10 ,ug dissolved in 100 ,ul of PBS containing 0.05% sodium azide) was added to each well, and the plate was incubated overnight at room temperature. Excess peptide solution was removed by inverting the plate several times, and the wells were washed one time with PBS. Nonspecific binding sites in the wells were then blocked by adding 200 pul of a solution of PBS containing 10% goat serum, ovalbumin (30 mg/ml), and 0.05% sodium
MEASLES VIRUS GENE ORDER
187
azide for a period of 5 h at room temperature. Blocking solution was removed by inverting the plate several times and washing the wells three times with PBS. Five- and 10-fold dilutions of rabbit antisera directed against the peptides were added to the microtiter plate wells (50 plI per well), and the plate was incubated at room temperature for 2 h. Preimmune sera were included as controls. The diluted antisera were then removed, and the plates were washed three times with PBS. Horseradish peroxidase conjugated to goat anti-rabbit antibodies (Miles-Yeda Ltd., Rehovot, Israel) was diluted 1:500 in PBS containing 0.02% Tween 20; 150 pul of this mixture was added to each well, and the plate was incubated for 2 h at room temperature. Peroxidase-conjugated antisera were removed, and the microtiter plate was washed three times with PBS and then three times with distilled water. Enzyme substrate, which consisted of 5-aminosalycylic acid (1 mg/ml) and 0.01% hydrogen peroxide (added just before the assay), was added to each well (150 pul per well) and incubated at room temperature for 1 h. A brown color developed in positive wells which could be quantitated at 450 nm in a spectrophotometer. The antibody titer was taken as the reciprocal of the dilution giving 50% of the maximum color development. Antibodv purification by affinity chromatography. Peptide (40 mg) was coupled to 3 ml of AffiGel-10 (Bio-Rad, Richmond, Calif.) in 0.1 M NaHCO3 (pH 8.0) with gentle agitation for ca. 11 h at 4°C. Unreacted coupling sites were then blocked by the addition of 0.1 ml of 1 M ethanolamine (pH 8) for 2 h at room temperature. The peptide-Sepharose column was sequentially washed with (i) 0.1 M citrate buffer (pH 2.5) containing 1 M NaCl, (ii) 6 M guanidine hydrochloride in 0.1 M Tris (pH 7.4), and, finally (iii) PBS containing 0.05% sodium azide before use in antibody purification. Antibodies were first selected by passing 30 ml of rabbit antisera over a 5-ml staphylococcus A protein-CL4B Sepharose column (Pharmacia, Sweden) which was equilibrated in PBS. The column was washed with PBS (20 ml), and the bound antibody was eluted with 0.1 M citrate buffer (pH 2.5) containing 1 M NaCl and then dialyzed against PBS (two times, 1 liter each) at 4°C for 12 h. The protein A column was then washed with 4 M guanidine hydrochloride in 0.1 M Tris (20 ml; pH 7.4) to remove tightly adsorbed antibodies, equilibrated in PBS containing 0.05% (wt/vol) sodium azide, and stored at 4°C for future use. The antibodies eluted from the protein A column were next subjected to affinity chromatography over the peptideconjugated Sepharose columns. Antibodies in ca. 5 ml of PBS were passed slowly over the peptide-Sepharose column, and the column was washed with 15 ml of PBS and eluted stepwise with (i) 0.1 M citrate buffer (pH 6), (ii) 0.1 M citrate buffer (pH 4.5), (iii) 0.1 M citrate buffer (pH 2.5) containing 1 M NaCl, (iv) 3 M NaSCN in 0.1 M Tris (pH 7.4), and, finally (v) 6 M guanidine hydrochloride in 0.1 M Tris (pH 7.4). The A280 of the protein eluted was monitored simultaneously. The various fractions were then dialyzed against PBS at 4°C and used in immune precipitation reactions. Routinely, the fractions eluted with 0.1 M citrate (pH 2.5, 1 M NaCl) contained antibodies of the highest titer. These antibodies were further concentrated by ultrafiltration centrifugation with Centricon 30 membranes (Amicon;
Danver, Mass.). Preparation of mRNA. Tissue culture flasks (150 cm2) containing Vero cells were infected with measles virus at a multiplicity of 1 PFU per cell. When the cells were 80 to 90% fused, they were washed with TNE buffer (0.14 M NaCl, 10 mM Tris [pH 8.8], 2 mM EDTA) and then lysed with the
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RICHARDSON ET AL. 2;|
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5 4 FIG. 1. Array of overlapping clones spanning the 3' portion of the measles 50S genome RNA. These clones have been identified through hybrid arrested translation, hybrid selection-translation, restriction endonuclease mapping, and finally through use of antibodies directed against synthetic peptides encoded by specific regions of the DNA (arrows). The positions of the boundaries between the genes were deduced from the nucleotide sequence as described in the text.
1
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same buffer containing 0.5% Nonidet P-40, 0.1% sodium deoxycholate, 1% P-mercaptoethanol, and RNasin (100 U/ml, 2 ml per flask). Nuclei were removed by centrifugation at 1,800 x g for 5 min, and the supernatant was adjusted to 0.1% sodium dodecyl sulfate (SDS) and 0.3 M LiCI. Polyadenylated mRNA was selected by passing the supernatant directly over oligodeoxythymidylate-cellulose columns. Reticulocyte translation of measles mRNA. Rabbit reticulocyte lysate which was treated with micrococcal nuclease to remove endogenous mRNA was obtained from Promega Biotec (Madison, Wis.). No adjustment of Mg or K ion concentrations was found to be necessary. Each assay mixture consisted of 15 ,ul of lysate, 2.5 ,ug of mRNA, 1 ,ul (500 U) of RNasin, 1 p.l of 10 mM amino acids without of [35S]methionine (50 ,uCi; 1,000 methionine, and S Ci/mmol). Incubations were for 1 h at 30°C, after which the reactions were terminated by the addition of either SDS electrophoresis sample buffer (50 ,u1) or RIPA buffer (200 p.). Immunoprecipitation and SDS-polyacrylamide gel electrophoresis. RIPA buffer containing 0.1% BSA was added to infected cell lysate (50 ,ul) or reticulocyte translation cocktails (27 ,ul) which had previously been labeled with [35S]methionine. Antisera (10 p.l) or affinity-purified antibodies (10 to 20 p.g of protein) were added to the diluted lysates and incubated for 1 h at 4°C. Staphylococcus A protein conjugated to Sepharose Cl-4B (50 p.l of a suspension of 1.5 g of Sepharose in 5 ml of RIPA) was added to each assay and incubated for another hour with agitation every 15 min. Immunoglobulin, antigen, and protein A-Sepharose beads were then sedimented by centrifugation for 10 s in an Eppendorf microfuge. The bead complex was then washed by briefly vortexing the beads with 1 ml of RIPA buffer (containing 0.1% BSA), sedimenting them by centrifugation for 10 s, and aspirating off the supernatant. This process was repeated four additional times (the final wash was performed in RIPA buffer without BSA). The beads were finally suspended in SDS-gel electrophoresis sample buffer (0.06 M Tris [pH 6.8], 4% SDS, 40% glycerol, 3% dithiothreitol, 0.005% bromphenol blue) and heated for 5 min at 100°C before electrophoresis. Radioactive proteins were subjected to electrophoresis on SDS-polyacrylamide gels which were either 15% acrylamide (acrylamide/bisacrylamide weight ratio, 173.1:1) or 8% acrylamide (acrylamide/bisacrylamide weight ratio of 37.5:1).
3
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RESULTS Identification of cDNA clones spanning the 3' portion of the measles 50S genome RNA. Clones complementary to both 50S genome RNA (5, 6, 6a) and mRNA (36) have previously been generated. The clones derived from mRNA were identified by hybrid selection of mRNA from infected cells and then protein translation in reticulocyte lysates. However, proteins are not glycosylated in this in vitro system and the nonglycosylated proteins often run aberrantly on SDSpolyacrylamide gels when compared with their in vivo counterparts from infected cells. For example, the measles hemagglutinin H migrates with an apparent molecular weight of 68,000 when synthesized in vitro, which is close to that of the phosphoprotein P. This same glycoprotein migrates at 78,000 daltons when synthesized in vivo (unpublished data). An assay system independent of the vagaries of in vitro translation was required which could be applied to the gene products synthesized in infected cells (in vivo). Since peptides derived from the cDNA clones of hepatitis B, influenza, and foot and mouth disease viruses (26, 27) have shown promise in the production of potential antisera vaccines, we felt that antisera against peptides corresponding to portions of amino acid sequence deduced from the cDNA could be used to immunoprecipitate measles protein. This strategy sucessfully demonstrated that the Cl-G clone, presumed to be a hemagglutinin clone, actually contained coding regions of the measles P protein gene (5, 6). A similar approach was applied to the remaining cDNA clones spanning the 3' portion of the measles 50S genome. The clones were ordered by restriction endonuclease mapping and sequenced by the method of Maxam and Gilbert (30; Bellini et al., in press). The array of overlapping clones used in the present study are diagramed in Fig. 1. Starting with Cl-G, we identified overlapping clones from both measles genomic and mRNA-derived libraries. We "walked along" the measles genome in both directions until we reached Cl-15, a clone which overlaps by 400 bases (35a) the 3' end clone described by Billiter et al. (7). In the other direction, the array of clones stretched to clone Cl-M and lOE11, putative M clones. The total representation of measles genetic information in the array is ca. 4.8 kilobases.
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TABLE 1. Nucleotide sequence of intercistronic boundary regions Sequence
Gene boundary
Virus
Measles
Vesicular stomatitis
Sendai a Determined by Bellini et al. (6a). b
N-P, P-M N-NS, NS-M, M-G, G-L
5'... UU A U(A)6C U UA GGA ... 3'a 5'... UA U G(A) C U AA C AG...3b
N-P, P-M
5'... UA A G(A)5 C U UA GGG 3"
Determined by Rose (35), Schubert et al., (38), and McGeoch (31). C Determined by Gupta and Kingsbury (18), Giorgi et al. (14), and Shioda et al. (41).
Both strands of all clones shown in Fig. 1 have been sequenced in their entirety by Maxam and Gilbert chemical sequencing. These sequences and the characteristics of the genes and of the proteins they code will be reported in detail elsewhere (6a, 35a). Since some of the clones were obtained by oligodeoxythymidylate priming on the polyadenylate tail of measles mRNA, they represented the very 3' end of the respective mRNAs. In the case of Cl-G and Cl-15, this conclusion was also supported by the sequencing data, which showed that the clones terminated with a sequence very similar to that found at the end of mRNAs from other negative-strand RNA viruses (14, 18, 31, 35, 38). Furthermore, clones made from genomic RNA covered these regions and thus spanned two adjacent genes. Sequence analyses of these clones revealed not only the conserved sequences described above, but also sequences that were very homologous to the polyadenylation site and intercistronic boundaries found in other negative-strand RNA viruses (14, 18, 31, 34, 35, 38). Table 1 lists the sequences that appear in the measles virus genome together with the cognate sequences from other viruses. For the purpose of Fig. 1, we assigned the boundary between the N, P, and M genes to the position between the run of six adenylates which correspond to the first six adenines in the mRNAs and the cytosine that follows them. By analogy to the Sendai system, the measles mRNA most likely starts and the intervening with the sequence, GpppAGGA. trinucleotide CUU may not be transcribed at all. Nonetheless, we have arbitrarily assigned it to the downstream gene. With these positions designated as the points of demarcation, the genes for N, P, and M are 1,691, 1,657, and 1,472 nucleotides long, respectively. Preparation and purification of antipeptide antibodies. To further support these assignments and to help define the actual translation starts for the various proteins, we synthesized peptides specified by the sequences in the indicated regions of Fig. 1. Peptides were constructed from potential coding regions near the anticipated NH2 and COOH termini of the proteins and antibodies were produced against these .
Peptide
Amino Acid Sequence
(NH2) N15 PN 13 PC20 C1
C2 MN14
MC14
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peptides. The amino acid sequences of these various peptides are listed in Fig. 2. Antisera recovered from immunized rabbits were monitored by ELISAs at 0, 5, 10, and 16 weeks after the first injection. The immune titer was defined as the reciprocal of the antibody dilution which yielded 50% of the maximum color development by horseradish peroxidase. A titer of 50 for preimmune sera (0 weeks) was routinely determined to be the background level due to nonspecific binding of antisera to microtiter wells (Table 2). A dramatic increase in antibody titer between 5 and 16 weeks postimmunization was clearly evident (Fig. 2). Titers reached a maximum level of 100,000 to 200,000 at 4 to 5 months after immunization, depending upon the immunogenicity of the peptide. Immune titers of 100,000 usually indicated that the antisera could be used in immune precipitation reactions without further concentration or purification of antibodies. Antibodies which were purified by affinity chromatography were routinely more satisfactory for use in immunoprecipitation reactions from infected cell lysates, since they yielded SDS-gel autoradiographs with fewer contaminating radioactive bands. Antisera were first subjected to chromatography on staphylococcus A protein conjugated to Sepharose Cl-4B and finally purified over columns which consisted of specific peptides coupled to AffiGel 10. The elution profiles for the purification of antibodies against Cl are shown in Fig. 3 and are typical for the purification of antibodies against the remaining peptides. Immune precipitation of in vivo- and in vitro-synthesized measles proteins. Messenger RNA was purified from measles infected cells by oligodeoxythymidylate-cellulose chromatography and added to a reticulocyte in vitro translation system. The proteins P, N, F, and M and a 21,000-dalton protein (a probable candidate for C) were clearly resolved above the background of nonviral cellular translation products. These proteins were identified with monoclonal antibodies (Fig. 4). Antisera against the peptides PN13 and PC20 immunoprecipitated the phosphoprotein P, as one might expect. Clearly, antigenic sites at both the amino and
(COOH)
Gly Val Gly Val Glu Leu Glu Asn Ser Met Gly Gly Leu Asn Phe Ala Glu Glu Gin Ala Arg His Val Lys Asn Gly Leu Glu lie Lys Gly Ala Asn Asp Leu Ala Lys Phe His Gin Met Leu Met Lys lie lie Met Lys Asn Ala Ser Gly Leu Ser Arg Pro Ser Pro Ser Ala His Glu Ser Pro Gin Glu lie Ser Lys His Gin Ala Leu Gly Val Arg Val lie Asp Pro Ser Leu Gly Asp Arg Lys Asp Glu Met Ser Lys Thr Leu His Ala Gin Leu Gly Phe Lys Lys Thr
FIG. 2. Amino acid sequences of peptides from which antisera were prepared.
RICHARDSON ET AL.
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TABLE 2. Titers of antisera directed against specific peptides deduced from cDNA sequences derived from measles genome RNA Peptide
0 weeks
N15 PN13 PC20 C1 C2 MN14 MC14
50 50 50 50 50 50 50
Antibody titers' 5 10 weeks weeks
16 weeks
20,000 100,000 200,000 200,000 20,000 100,000 100,000
5,000 20,000 50,000 20,000 10,000 20,000 20,000
1,000 6,000 2,000 10,000 2,000 6,000 2,000
a The antibody titer was taken as the reciprocal of the dilution giving 50% of the maximum color development in ELISAs which were performed as outlined in the text.
carboxy termini of this protein were exposed in the immunoprecipitation buffer. The DNA sequence of the P gene also predicted the existence of a second polypeptide which could be translated from a second open reading frame
through use of a second initiation codon (AUG) (6a). The molecular weight predicted from the DNA sequence for this putative polypeptide was 21,039. Antisera prepared against the two peptides, C1 and C2, specified by this reading frame immunoprecipitate a protein which migrated at a molecular weight of 21,000 to 22,000 on SDS-polyacrylamide gels. Antisera against MN14 and MC14 peptides also precipitated the matrix protein of measles virus (Fig. 4). These results clearly demonstrate that the clones BA7, M-5, M-15, and lOEll are, in fact, clones of the matrix protein gene. These results also show that both the amino and carboxy termini of the protein are exposed during the immunoprecipitation reaction. Another peptide, N15, has been shown to be highly conserved among the nucleocapsid proteins of Sendai, measles, and canine distemper viruses (35a, 41). Antibodies were prepared against this peptide and were found to immunoprecipitate the nucleocapsid protein of measles virus (Fig. 4). This result again confirmed that the clone Cl-15 contained a portion of the nucleocapsid gene. This antiserum has recently been shown to also precipitate the nucleocapsid protein of canine distemper virus (unpublished data).
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ELUTION VOLUME (MILLILITERS) FIG. 3. Purification of antibodies by affinity chromatography. Antisera from rabbits were first purified on columns of staphylococcus A protein conjugated to Sepharose Cl-4B as described in the text. The resultant antibody preparation was adsorbed to columns of peptide conjugate of AffiGel 10. The bound antibodies were sequentially eluted with citrate buffers at pH 6, 4.5, and 2.5 (containing 1 M NaCI) and then 3 M NaSCN, 1.5 M guanidine thiocyanate, and 6 M guanidine hydrochloride buffers as described in the text. A typical elution profile is shown. Antibodies eluted with citrate buffer (pH 2.5) containing 1 M NaCl were usually the most suitable for immunoprecipitation studies.
FIG.4 SDSpolyacrylamide gels of in vitro translation products immunoprecipitated with antipeptide antisera. Proteins were synthesized with [35S]methionine in a reticulocyte lysate, immunoprecipitated with the appropriate antisera, and electrophoresed on 15% polyacrylamide gels containing SDS. An autoradiogram of a dried gel is shown. Captions at the tops of the lanes refer to the imhmune reagent used to precipitate the labeled proteins; N, M, P, and F are previously characterized monoclonal antibodies. The right lane contains radioactive standard proteins.
VOL. 54, 1985
MEASLES VIRUS GENE ORDER '.....
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[35S]methionine-labeled cell lysates were prepared from measles-infected CV-1 cells and a cell line (MA160) that was persistently infected with measles virus. Immune precipitation of these with the antipeptide antibodies yielded identical results which were completely congruent with the above results (Fig. 5A and B). Antibodies directed against the peptides C1, C2, MN14, MC14, PN13, PC20, and N15 immunoprecipitated the appropriate protein (Fig. SA and B). Measles virus proteins were identified with antibodies against M, N, P, H, and F proteins. However, we could not clearly resolve the P and H proteins on a 15% acrylamide gel (Fig. 5A). An 8% acrylamide gel (Fig. 5B) was used to resolve the H and P proteins more efficiently, and one can clearly observe that antisera against PN13 and PC20 immunoprecipitated the P protein and not the H protein. A faint band which comigrated with H may represent a nonphosphorylated species of the P. Thus, with the aid of antibodies directed against synthetic peptides, we were able to unambiguously assign the order of the first three genes on the measles genome as N-P/C-M. Furthermore, these immune reagents allowed us to establish which reading frames are employed by the measles virus and to independently confirm that if translation is initiated with a methionine, then the first AUG in each of the open reading frames coding for P, C, and M is a translation initiator.
DISCUSSION of synthetic peptides to elicit antibodies of predetermined specificity and as potential vaccines has now gained universal appeal (1, 8, 27-29, 40, 42, 48). These antisera and peptides also have been used to probe the antigenic structure of a number of crystallized proteins and the results correlate well with the three-dimensional structure derived from X-ray diffraction data (2, 17). In addition, viral gene products have been identified and studied with antipeptide antisera against such viruses as influenza virus (3, 17), hepatitis B virus (12, 28), foot and mouth disease virus (8), polyomavirus (23), adenovirus (16), poliovirus (39) and retrovirus (43, 47). Studies with antipeptide antibodies were originally initiated by our laboratory as a means of firmly -identifying a cDNA clone (Cl-G) which was previously isolated (5, 6, 36). Hybrid selection-translation and hybrid-arrested translation could not allow conclusive assignment of this clone to either the P or the H gene of measles virus, since these proteins migrated at similar positions on SDS-polyacrylamide gels. Through the application of the technique we have described here in detail, we found that antisera directed against peptides specified by the open reading frame of the Cl-G clone immunoprecipitated the P protein (5, 6). Thus, we were able to firmly assign the Cl-G clone to the P protein gene of measles virus. The
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In this communication, we described the use of peptide specific antisera for the unambiguous assignment of other cDNA clones and to establish a partial gene order of the measles virus genome. We used antisera directed against synthetic peptides as a means for identifying and ordering cDNA clones which were derived from the 3'-terminal half of the measles RNA genome. The clones were determined to lie between highly conserved intercistronic sequences (shown in Table 1) which served to define the boundaries of the genes for measles virus. The P gene was found to contain a second open reading frame between its intergenic regions which could potentially code for another protein of 21,039 daltons. Two peptides were constructed from this second reading frame, and antibodies directed against these peptides were shown to immunoprecipitate this previously undefined gene product (which we called C) from both in vitro translation assays and infected cell lysates. Finally, our data established the gene order of the 3'-terminal portion of measles virus genome to be 3'-NP-(P+ C)-M-5'. Since the glycoproteins H and F are not efficiently translated in vitro, cDNA clones from these two regions of the genome are being characterized in a similar manner. The advantage of such an approach is twofold: it allows one to firmly identify cDNA clones and also supplies antisera for future study and purification of the viral proteins. The gene order of the 3'-proximal third of the Sendai virus genome has only recently been established through restriction analysis of random clones, dot blot analysis, and hybrid selection-translation techniques (11). We find the same gene order on the measles genome. The use of antisera directed against proteins specified by the coding regions of cDNA clones provides positive gene assignment and is more reliable than classical hybrid selection-translation or hybrid-arrested translation techniques. A similar approach is now being applied to the remaining two-thirds of the measles genome, which contains the F, H, and L protein genes. Due to the poor efficiency in translationl of these proteins in an in vitro reticulocyte translation system, antisera directed against peptides derived from open reading frames of DNA sequences seem to be the only way in which we can clearly assign cDNA to their corresponding genes. The benefits of antipeptide antibodies in identifying protein products of open reading frames, localizing intracellular gene products, following exon expression during splicing events, purifying protein gene products, or possibly acting as reagents for vaccines have previously been documented. In this communication, we provided a further use for antisera directed against peptides as a means of characterizing cDNA clones. ACKNOWLEDGMENTS We thank M. Brown, G. Knipfal, M. Janz, A. Nowry, H. Nutall, P. Frieda, and S. Kotsomitis for excellent technical assistance. The typing of this manuscript by Charlene French is also greatly appreciated. We are grateful for monoclonal and polyclonal antisera directed against measles virus proteins, which were obtained from E. Norrby, D. E. McFarlin, and W. Bohn. Finally, the advice, encouragement, and critical reading of this manuscript by Robert Lazzarini is gratefully acknowledged. LITERATURE CITED 1. Arnon, R. 1980. Chemically defined antiviral vaccines. Annu. Rev. Microbiol. 34:593-618. 2. Atassi, M. Z. 1975. Antigenic structure of myoglobin: the complete immunochemical anatomy of a protein and conclusions relating to antigenic structures of proteins. Immunochemistry 12:423-438.
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