VIROLOGY 146. 282-291 (1985) Nucleotide Sequence at the 3’ Terminus of Pepper Mottle Virus Genomic RNA: Evidence for an Alternative Mode of Potyvirus Capsid Protein Gene Organization’ WILLIAM G. DOUGHERTY,**’ RICHARD F. ALLISON,* T. DAWN PARKS,? ROBERT E. JOHNSTON, MARK J. FEILD,$ AND FRANK B. ARMSTRONG+ Departments of *Plant Pathology, fCrop Science, ~Microbiology and Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7616 Received December 26, 1984; accepted April 20, 1985 The sequence of the 3’-terminal 1481 nucleotides of the pepper mottle virus (PeMV) genome has been determined. The sequence was determined by dideoxy nucleotide sequencing of complementary DNA which had been inserted into Ml3 bacteriophage cloning vectors and was confirmed by sequencing selected regions of PeMV RNA. A discrete open reading frame of 993 nucleotides, ending 333 nucleotides from the 3’- terminal polyadenylate tract, was identified that potentially encoded a 3’7,669-MW protein. The amino acids predicted at positions 64 through 84 of this putative polypeptide were identical to the amino-terminal 21 amino acids of the PeMV capsid protein ascertained by chemical sequencing. These combined nucleotide and amino acid sequence data suggest that the PeMV capsid protein is encoded by the 3’-most cistron on the genomic RNA and that it may be expressed as a precursor that is proteolytically processed to produce the mature capsid protein. 0 1985 Academic PESS. IK INTRODUCTION Pepper mottle virus (PeMV) is a mem- ber of the potato virus Y group and ap- pears to be related to potato virus Y and tobacco etch virus (TEV) (Edwardson, 1974; Purcifull et al., 1975). These viruses have overlapping host ranges and the capsid and cylindrical inclusion proteins of all three viruses are serologically re- lated (Purcifull et al, 1973, 1975; Nelson and Wheeler, 1978). Although PeMV and TEV are related, their capsid proteins apparently are formed by two distinct mechanisms in the rabbit reticulocyte cell-free translation ‘Paper No. 9766 of The Journal Series of the North Carolina Agricultural Research Service, Ra- leigh, N. C. 2’7695-7601. Mention of trade or company names does not imply endorsement by the North Carolina Agricultural Research Service of the prod- ucts names nor criticism of similar ones not men- tioned. *Author to whom requests for reprints should be addressed. system (Dougherty and Hiebert, 198Oc). Cell-free translation of TEV RNA results in the synthesis of TEV capsid protein of apparent molecular weight 30,000 as well as the synthesis of several higher molec- ular weight products (-85,000). Some of these proteins are immunoreactive with monospecific antisera to TEV capsid and nuclear inclusion proteins. We have de- termined that a single open reading frame (ORF) is present on TEV genomic RNA which encodes a high-molecular-weight protein containing the capsid protein se- quence (Allison et al, 1985). Therefore, the TEV genome apparently expresses the large nuclear inclusion and capsid protein genes as a polyprotein which subsequently is processed. In contrast, cell-free trans- lation of PeMV RNA from purified virions results in the synthesis of discrete prod- ucts with little or no synthesis of poly- proteins (Dougherty and Hiebert, 198Oc). Because of these differences in cell-free translation products, we compared the genomic organization of PeMV and TEV 0042-6822/85 $3.00 Copyright Q 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. 282
Transcript
VIROLOGY 146. 282-291 (1985)
Nucleotide Sequence at the 3’ Terminus of Pepper Mottle Virus
Genomic RNA: Evidence for an Alternative Mode of Potyvirus
Capsid Protein Gene Organization’
WILLIAM G. DOUGHERTY,**’ RICHARD F. ALLISON,* T. DAWN PARKS,? ROBERT E. JOHNSTON, MARK J. FEILD,$ AND FRANK B. ARMSTRONG+
Departments of *Plant Pathology, fCrop Science, ~Microbiology and Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7616
Received December 26, 1984; accepted April 20, 1985
The sequence of the 3’-terminal 1481 nucleotides of the pepper mottle virus (PeMV) genome has been determined. The sequence was determined by dideoxy nucleotide sequencing of complementary DNA which had been inserted into Ml3 bacteriophage cloning vectors and was confirmed by sequencing selected regions of PeMV RNA. A discrete open reading frame of 993 nucleotides, ending 333 nucleotides from the 3’- terminal polyadenylate tract, was identified that potentially encoded a 3’7,669-MW protein. The amino acids predicted at positions 64 through 84 of this putative polypeptide were identical to the amino-terminal 21 amino acids of the PeMV capsid protein ascertained by chemical sequencing. These combined nucleotide and amino acid sequence data suggest that the PeMV capsid protein is encoded by the 3’-most cistron on the genomic RNA and that it may be expressed as a precursor that is proteolytically processed to produce the mature capsid protein. 0 1985 Academic PESS. IK
INTRODUCTION
Pepper mottle virus (PeMV) is a mem- ber of the potato virus Y group and ap- pears to be related to potato virus Y and tobacco etch virus (TEV) (Edwardson, 1974; Purcifull et al., 1975). These viruses have overlapping host ranges and the capsid and cylindrical inclusion proteins of all three viruses are serologically re- lated (Purcifull et al, 1973, 1975; Nelson and Wheeler, 1978).
Although PeMV and TEV are related, their capsid proteins apparently are formed by two distinct mechanisms in the rabbit reticulocyte cell-free translation
‘Paper No. 9766 of The Journal Series of the North Carolina Agricultural Research Service, Ra- leigh, N. C. 2’7695-7601. Mention of trade or company names does not imply endorsement by the North Carolina Agricultural Research Service of the prod- ucts names nor criticism of similar ones not men- tioned.
*Author to whom requests for reprints should be
addressed.
system (Dougherty and Hiebert, 198Oc). Cell-free translation of TEV RNA results in the synthesis of TEV capsid protein of apparent molecular weight 30,000 as well as the synthesis of several higher molec- ular weight products (-85,000). Some of these proteins are immunoreactive with monospecific antisera to TEV capsid and nuclear inclusion proteins. We have de- termined that a single open reading frame (ORF) is present on TEV genomic RNA which encodes a high-molecular-weight protein containing the capsid protein se- quence (Allison et al, 1985). Therefore, the TEV genome apparently expresses the large nuclear inclusion and capsid protein genes as a polyprotein which subsequently is processed. In contrast, cell-free trans- lation of PeMV RNA from purified virions results in the synthesis of discrete prod- ucts with little or no synthesis of poly- proteins (Dougherty and Hiebert, 198Oc).
Because of these differences in cell-free translation products, we compared the genomic organization of PeMV and TEV
0042-6822/85 $3.00 Copyright Q 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
282
PeMV CAPSID PROTEIN GENE AND ITS PRODUCT 283
RNA. We report here the nucleotide se- quence of the 3’-terminal1481 nucleotides of the PeMV RNA genome within which a discrete ORF has been identified. The amino acid sequence predicted from the nucleotide sequence has been compared to the chemically determined amino acid composition and amino-terminal amino acid sequence of PeMV capsid protein. This comparison suggests that the capsid protein is expressed as a precursor which is processed subsequently by proteolytic cleavage to the capsid protein associated with purified virions.
MATERIALS AND METHODS
Virus and RNA purificaticm. PeMV was purified from Nicotiana tabacum (Burley 21) 20 to 30 days after inoculation. Puri- fication of virus and RNA isolation have been described previously (Dougherty and Hiebert, 1980a).
Synthesis of complementary DNA. The complementary DNA (cDNA) synthesis protocol was drawn from a number of published reports (Buell et aL, 1978; Retzel et ah, 1980). TEV RNA (20 pg) was incu- bated in a final reaction volume of 50 ~1 containing 50 mM Tris-HCl (pH 8.3), 8 mM MgClz, 25 mM NaCl, actinomycin D (50 ng/pl), 4 pg of oligodeoxythymidyl- atelzmls [oligo(dT12-18)], 400 PM each of dATP, dGTP, dCTP, and dTTP, 50 &i of [a-3H]dCTP and 36 units of avian myelo- blastosis virus (AMV) reverse transcrip- tase (Life Sciences Inc., St. Petersburg, Fla.). After cDNA synthesis at 42” for 90 min, NaOH was added to 0.4 M and the mixture was incubated at 56” for 1 hr. The cDNA was separated from unin- corporated nucleotides by gel filtration through Sephadex G-100 buffered in 20 mM HEPES (pH 7.5), 10 mM NazEDTA, and 1.0 M NaCl. Fractions containing the cDNA were pooled, and the cDNA was precipitated overnight at -70” by adding tRNA to a final concentration of 10 pg/ ml and 2 vol of 95% ETOH. The cDNA was pelleted by centrifugation for 30 min at 12,000 g, washed twice with cold 70% ETOH, dried under vacuum, and resus- pended in 50 ~1 of H20.
Synthesis of double-stranded cDNA. Homopolymeric tracts of deoxycytidine were added onto the 3’ termini of the cDNA molecules in a reaction catalyzed by terminal transferase (Deng and Wu, 1981). The cDNA was then made double- stranded by adding 32 units of AMV re- verse transcriptase and oligo(dG1& as a primer to a loo-p1 reaction mix whose final concentrations were 50 mM Tris- HCl (pH 8.3), 8 mM MgCla, 400 PM each of dCTP, dTTP, dGTP, and dATP, 50 &i of [a-32P]dCTP and 10 mM DTT. The re- action was incubated at 42” for 60 min and the double-stranded cDNA was sep- arated from unincorporated nucleotides by Sephadex G-100 gel filtration and ethanol precipitation as described above. The double-stranded cDNA was collected by centrifugation, resuspended in a lOO- ~1 reaction volume, and incubated for 15 min at room temperature with mungbean nuclease according to the manufacturer’s instructions (Pharmacia P-L Biochemicals, Piscataway, N. J.) to generate blunt ends. The reaction was terminated by phenol: chloroform extraction and the sample was extracted with ether, and precipitated with ethanol at -70”. The double-stranded cDNA was collected by centrifugation and resuspended in water.
Homopolymeric tracts of deoxycytidy- late were added in a reaction catalyzed by terminal transferase (Deng and Wu, 1981). The sample was extracted with phenol and ether, and precipitated with ethanol. The double-stranded cDNA was resuspended in annealing buffer [lo mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl] (Gordon et al, 1978) and annealed with an equimolar amount of PstI digested pBR322 plasmid DNA that had been mod- ified with homopolymeric tracts of deoxy- guanylate (Deng and Wu, 1981).
Transformation and identification of re- combinant DNA clones. Escherichia coli, strain LE 392, was subjected to calcium shock and transformed with the annealed plasmid-insert mix, as described by Dagert and Ehrlich (1979). Transformed bacteria were plated on L agar containing tetra- cycline (5 pg/ml) and incubated for 24 hr at 37”. Replicas of bacterial colonies were
284 DOUGHERTY ET AL.
plated onto an L-agar plate containing ampicillin (100 pg/ml) and an L-agar plate containing tetracycline (5 pg/ml). Colonies that were tetracycline resistant and am- picillin sensitive were screened by colony hybridization with a 32P-labeled randomly primed cDNA probe as described by Han- ahan and Meselson (1980).
Synthesis of oligo(dT) and randomly primed PeMV cDNA for hybridization- studies. Randomly primed and oligo- (dTr,-,,) primed cDNA molecules, labeled with [a-32P]dCTP, were synthesized as de- scribed above with two modifications. Un- labeled dCTP was omitted from the re- action and [a-3H]dCTP was replaced with [a-32P]dCTP. For randomly primed cDNA synthesis, the oligonucleotide primer was made as described by Taylor et al. (19’76), and 4 pg of the randomly cleaved DNA was substituted for oligo(dT12-18) in the reaction. The remaining reaction condi- tions and purification by gel filtration were as described above.
Electrophoresis of DNA and gel hybrid- ization experiments. DNA was analyzed by agarose gel electrophoresis through 1.0% agarose gels in TAE buffer [40 mM Tris-acetate (pH 8.0), 20 mM sodium ac- etate, 20 mM sodium chloride, 2 mM EDTA]. Electrophoresis was performed with a water-cooled raft horizontal gel apparatus at 15 V/cm for 4 hr.
DNA was transferred to nitrocellulose and hybridization experiments were car- ried out as described by Wahl et al. (1979) except that dextran sulfate and sodium dodecyl sulfate were omitted.
cDNA plasmid inserts to be used in sequence determination or hybridization studies were isolated by electrophoresis onto NA 45 membrane according to the manufacturer’s instructions (Schleicher and Schuell, Keene, N. H.).
DNA sequence determination. Purified PstI excised cDNA inserts were digested with XhoI, TaqI, or Sau3A1, and DNA fragments were ligated into the PstI-SalI, AccI, or BamHI sites of the replicative form of the bacteriophage M13mp9 or -mplO (Messing, 1983). Transfection was performed, and colorless plaques were se- lected. Bacteriophage were screened by
dot hybridization with 32P-labeled PeMV cDNA or cDNA insert probes, labeled with [a-32P]dATP by nick translation (Rigby et ah, 1977). The DNA nucleotide sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) using a 17-nucleotide universal primer purchased from Pharmacia P-L Biochemicals.
RNA sequence determination. The length of the polyadenylate tract of PeMV RNA was determined directly from the RNA as described by Ahlquist and Kaes- berg (1979).
Two regions of the sequence reported in this paper were determined using RNA in a sequencing reaction described previ- ously by Zimmern and Kaesberg (1978). The oligonucleotide primers used in these studies were 5’-T-T-T-T-T-T-T-T-T-G-3’ (Pharmacia P-L Biochemicals) and 5’-C- C-T-T-C-C-T-G-C-G-C-T-A-T-T-G-A-3’ (synthesized by R. Sederoff and B. Gwynne).
Protein analysis. Amino acid composi- tion was determined as described previ- ously (Lipscomb et al, 1974) except that hydrolysis was performed for 24, 48, and 60 hr.
Amino acid sequencing was carried out in an Applied Biosystem gas-phase se- quencer by the aqueous TFA conversion method (Hewick et al, 1981).
Computer analysis. Nucleic acid and protein data were analyzed with a 64K, CP/M based microcomputer using soft- ware developed by one of us (R.E.J.) (manuscript in preparation).
RESULTS
Recowhinant DNA Molecules Containing PeMV Nucleotide Sequence
Twenty-eight recombinant DNA plas- mids containing cDNA inserts derived from PeMV RNA were identified. The cDNA inserts of these plasmids ranged in size from 300 to 1500 base pairs (bp) and hybridized with randomly primed or oligo(dT12-18) primed 32P-labeled PeMV cDNA. PeMV cDNA inserts did not hy- bridize with oligo(dT) primed cDNA of TEV RNA under the hybridization con-
ditions used in this study (data not The 3’-terminal polyadenylate region shown). Two of these recombinant DNA was variable in length, ranging from 20 molecules, pPMV-6 (-900-bp insert) and to 140 adenosines, with most from 30 to pPMV-4 (- 1400-bp insert) were selected 50 adenosines long (data not shown). The for sequence analysis. sequence of the 3’-terminal 1481 nucleo-
tides adjacent to the polyadenylate region
Nucleotide Sequence of PeMV 3’-Terminal is presented in Fig. 2.
1481 Nucleotides
The sequence of 1481 nucleotides adja- Open Reading Frames
cent to the 3’-polyadenylate region of Computer analysis of the sequence re- PeMV genomic RNA was determined. The vealed a single large ORF of 990 nucleo- relationship of the cDNA inserts to each tides on the (+) sense (virion polarity) other and to the PeMV genomic RNA is RNA (Fig. 3). The large ORF began with presented in Fig. 1, along with the strategy an AUG codon and was terminated by a used to determine the nucleotide sequence. single UGA codon located 333 nucleotides The nucleotide sequence adjacent to the from the 3’-terminal polyadenylate region 3’-polyadenylate tract and the internal (Figs. 2 and 3). This ORF would encode region indicated also was determined by a 37,669-MW protein. Previous genome analysis of direct transcripts of the ge- mapping studies have assigned the gene nomic RNA. The sequence obtained from coding for the capsid protein to this region PeMV RNA agreed with the nucleotide (Dougherty and Hiebert, 198Oc). sequence derived from cDNA recombinant Another ORF could be located 12 nu- molecules. cleotides upstream from the first AUG
codon (position-1326) of the 990-nucleotide ORF. This putative ORF and the 990-
PEMV RNA nucleotide ORF were both in reading frame 2 (Fig. 3). Our previous genome
+%“A
/- I mapping studies suggested that the large-
/-- I nuclear-inclusion-like protein gene was
rc ! PoL” A v -
located adjacent to the capsid protein i z
pPMV-4 y 55 i gene (Dougherty and Hiebert, 198Oc) so - --
---- =- TAQ I this sequence could encode the carboxy-
-
-- - terminal amino acids of the large-nuclear- - = RSA I zz inclusion-like protein. However, we believe -- - PST l-Xl+0 I that the large-nuclear-inclusion-like pro-
tein is more likely to be encoded in reading pp,v-,j r frame 3 (Fig. 3) which overlaps 73 nucleo-
- TAOI tides of the 990-nucleotide ORF. This is - PST I suggested because the predicted amino
acid sequence translated from this ORF _ 100 rwcleof\de, displayed 59% homology with the pre-
FIG. 1. Nueleotide sequencing strategy. The rela- dicted amino acid sequence of the large- tionship between PeMV genomic RNA and the two nuclear-inclusion-protein gene sequence of complementary DNA (cDNA) recombinant molecules TEV (Fig. 4). No amino acid homology (pPMV-4 and pPMV-6) is presented. The heavy lines (above schematic of pPMV-4) indicate those regions
was detected between the TEV large-nu-
of the sequence determined from the RNA directly. clear-inclusion gene product and the pre-
The light lines presented under the two cDNA dicted gene product of reading frame 2.
inserts represent sequences derived from subcloning A number of short ORFs were identified TuqI, Z&XI, or XhoI-PstI cDNA fragments into the in the sequence of the (+) sense and (-) bacteriophage Ml3 and determining the nueleotide sense RNA that could code for proteins sequence using the di-deoxynucleotide sequencing with molecular weights less than 8000 reaction. (Fig. 3).
PeMV CAPSID PROTEIN GENE AND ITS PRODUCT 285
286 DOUGHERTY ET AL.
*UC CA” AC” NJ* C”” CGA 0°C AAG MC MC WA ““G”ocu(jccucucuccoo~*“*“*“~“~““*~”*~G”~”~””””~~““““~~“G”*~”~ 2: ““““*““G”McuAG”u”c4G”““GAA”*““*“”cA”MA”AGAGG”~ uo*uuucG”cIuuouooucI”~*~~~““~G”~*””~G””~~”~~ -1.1 n%G”cG”“G”“G”“G”~~~“*“~*““~~~G~“”~G” ~~~“M)G~““~~““~-“~“G”“*~*-G”~~~- -22 G”AAAc”“CM”C*GG*G*C*poly *zo-,*o -I
FIG. 2. The nucleotide sequence and possible organization of the 3’-terminal 1481-nucleotide of PeMV RNA. The RNA sequence and major translation product of reading frame 2 is presented. Above the nucleotide sequence is the single letter abbreviation for the amino acid encoded for by the codon. Nucleotides are numbered from the 3’ terminus. Amino acids are numbered from the first methionine of the large open reading frame. The four possible initiation triplets are underscored. The arrow indicates the putative cleavage site resulting in alanine as the amino- terminal amino acid of the capsid protein.
Comparison of the Large ORF with PeMV Capsid Protein Amino Acid Sequence
The amino acid sequence of the pre- dicted translation product of the 990 nu- cleotide ORF was compared with amino acid sequence data obtained directly from PeMV capsid protein. The protein product predicted from the nucleotide sequence data should have a molecular weight of
-1483 -loo0 -500 -1
t-1 51’; 14i43 lo&l do :
FIG. 3. Distribution of open reading frames (ORF) contained within the 3’-terminal 1481 nucleotides of PeMV RNA. White blocks represent ORFs which encode proteins greater than 20 amino acids in length. Results of the analysis are presented for the three reading frames for both (+) sense [virion polarity] and (-) sense RNA.
37,669 and an amino-terminal methionine. In contrast, PeMV capsid protein, isolated from PeMV-infected tobacco tissue or from purified virions, has a molecular weight of 32,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis (SDS-PAGE) (Allison et al., 1984). The primary sequence of amino acids at the amino-terminus of PeMV capsid protein was determined using two different purified PeMV capsid protein preparations. The sequence of the first 21 residues of the amino-terminus was iden- tical to the nucleotide predicted sequence of amino acids 64 through 84 of the large ORF (Fig. 5). A protein containing the primary sequence of amino acids 64 through 330 of the large ORF had a cal- culated molecular weight of 30,120, a value in reasonable agreement with that ob- tained by SDS-PAGE of the capsid pro- tein.
The amino acid composition of purified PeMV capsid protein was determined chemically and compared to the amino
TIN-NAT sr Y-L-K-V-L-Y-D-Y-D-I-P-T-T-E-T-L-Y-F-Q- capsld protein
FIG. 4. A comparison of the nucleotide predicted amino acid sequence of the large-nuclear- inclusion protein of the not-aphid-transmitted isolate of tobacco etch virus (TEV-NAT) and the predicted amino acid sequence of PeMV reading frame 3. The carboxy-terminal amino acids of the putative TEV nuclear-inclusion protein are presented. The arrow indicates the putative cleavage of the TEV polyprotein which results in the synthesis of the large-nuclear-inclusion and capsid proteins. The carboxy terminus of the predicted protein of PeMV reading frame 3 is noted.
acid composition predicted from nucleotide sequence data (Table 1). The amino acid composition of purified PeMV capsid pro- tein was in close agreement with the predicted composition of a protein begin- ning at amino acid 64 and ending at amino acid 330 of the large ORF.
$-Terminal Untranslated Region
The 3’-untranslated region had a num- ber of unique characteristics. This 330- nucleotide region was 66% adenosine and uridine and had three regions, 35 to 40 nucleotides in length, which displayed considerable homology. Stable secondary structures could be generated only for two of the three regions. Additionally, direct repeats of a 9- and a 13-nucleotide se-
quence were present in the 3’-untranslated region. Some of these features are pre- sented in Fig. 6.
DISCUSSION
Dougherty and Hiebert (198Oc) have proposed that the capsid protein gene maps proximal to the 3’ terminus. The proposed genetic order for PeMV genomic RNA is: 5’ terminus-78,000 protein-49,000 nuclear-inclusion-like protein-41,000 pro- tein-68,000 cytoplasmic-inclusion protein- 56,000 nuclear-inclusion-like protein- 32,000 capsid protein-3’ terminus. The nu- cleotide and amino a&d sequence data presented here definitively locate the PeMV capsid protein gene proximal to the 3’ terminus of the genomic RNA.
Redkted horn mJckosde **qwm*
Chemically delwmlned
1 A-H-D-T-X'%-T-GGN-S
2 A-,+C-T-,-D-T-G-G-N-S-K-K-D-V-K-P-E-Q-G-S
FIG. 5. Comparison of the nucleotide predicted and chemically determined sequence of the amino-terminal amino acids of PeMV capsid protein. The nucleotide predicted sequence was generated by computer translation of the nucleotide sequence of the 99Cknucleotide open reading frame. The chemically determined sequences were generated by amino acid sequence analysis of PeMV capsid protein using a gas-phase protein sequencer. Chemical sequence determination was carried out on two different capsid protein preparations.
288 DOUGHERTY ET AL.
TABLE 1
AMINO ACID COMPOSITION OF THE PeMV
CAPSID PROTEIN
Number of amino acid residues
Amino acids
Ala (A)
Arg W Asx (D & N)
CYS (Cl Glx (E & Q)
GUY (G) His (H) Ile (I) Leu (L)
LYS W) Met (M) Phe (F) Pro (P) Ser (S) Thr (T)
Trp W) Tyr 0’) Val (V)
Predicted from
nucleotide sequence
23 15 28
1 30 17
6 13 15 18 13
5 14 18 20
3 9
19 267
Determined chemically”
24 17 34
1 31 17
6 12 18 20
8 8
14 16 23
1 8
20 278
a Composition derived from acid hydrolysis of pro- tein carried out for 24, 48, and 60 hr. All amino acids were detected by post-column derivatization except for histidine. Cysteine was determined as cysteic acid (performic acid oxidation). Tryptophan was determined using 2% thioglycolic acid in the hydrolysis.
The structural organization of the cap- sid protein gene is consistent with data obtained from cell-free translation of PeMV RNA (Dougherty and Hiebert, 198Oc). Cell-free translation of PeMV RNA in a reticulocyte lysate results in the synthesis of a discrete protein which co- migrates with PeMV capsid protein and is immunoreactive with antiserum to PeMV capsid protein (Dougherty and Hie- bert, 198Oc). High-molecular-weight poly- proteins which are immunoreactive with PeMV capsid protein antiserum are not detected. The capsid protein gene of PeMV is organized as a distinct open reading
frame beginning with an AUG initiator methionine codon and ending with a single UGA termination codon. Such a genomic organization would limit production of readthrough products and preclude poly- protein synthesis.
These observations are in contrast to the genome organization and cell-free translation results with TEV RNA. Trans- lation of TEV RNA results in the synthesis of large quantities of high-molecular- weight polyproteins (-85,000), in addition to a protein which comigrates with TEV capsid protein (30,000). Both the polypro- teins and capsid protein are immunopre- cipitated by antiserum to TEV capsid protein. Moreover, the coding region for TEV capsid protein is included within a large ORF of over 2100 nucleotides that presumably encodes for a polyprotein (Al- lison et ah, 1985).
The nucleotide and amino acid sequence data indicate that the PeMV capsid pro- tein gene may be translated initially into a higher molecular weight precursor pro- tein. However, the initiation codon for this precursor protein has not been iden- tified. Four AUG triplets are found in the large ORF prior to codons which are translated to give mature capsid protein. Presumably, any one of these initiation codons could be used. However, the AUG triplet beginning at -1192 conforms most closely with the consensus sequence for eucaryotic translation initiation sites (Kozak, 1984). Depending on the triplet codon used for initiation, 63, 34, 19, or 18 NHz-terminal amino acids may be re- moved by the apparent cleavage of a glu- tamine-alanine peptide bond resulting in a polypeptide with a molecular weight of 30,120. The glutamine-alanine dipeptide is present only once in the 37,669-MW protein. Proteolytic processing on the car- bony1 side of a glutaminyl residue has been reported for cleavage events of po- liovirus (Kitamura et al, 1981), encepha- lomyocarditis virus (EMC) (Palmenberg et aL, 1984), and cowpea mosaic virus (CPMV) (van Wezenbeek et aL, 1983). Cleavage required for TEV capsid protein production is at a glutamine-glycine di- peptide (Allison et aL, 1985). The possible
PeMV CAPSID PROTEIN GENE AND ITS PRODUCT 289
b
FIG. 6. Analysis of the 3’-terminal untranslated region of reading frame two of PeMV RNA. (A) The 3’-terminal untranslated sequence is presented. Regions showing homology are indicated as sequence 1, 2, and 3. The repeated I)-nucieotide (a) or l&nucleotide sequence (b) also are indicated. (B) A potential base-pairing scheme for two regions in the 3’-untranslated region is presented. The AG values for the secondary structures of sequences 1 and 2 were calculated to be - -6.0 and -8.4 kcal, respectively. The guidelines of Tinoco et al (1973) were used to esti- mate AG.
function of the amino-terminal amino ac- ids removed from the precursor removed is unknown; but perhaps processing is required for transport of the capsid pro- tein to the assembly site and/or in the regulation of virion assembly.
The nucleotide sequence and predicted secondary structures of the 3’-terminal untranslated region of reading frame 2 are unique to PeMV RNA (Fig. 6) and are not present in the TEV sequence (Allison et al, 1985). The high adenylate and uri- dylate composition of this untranslated region is similar to those reported for TMV RNA and for alphaviridae RNA (Goelet et al, 1982; Strauss et cd, 1984). Of particular interest is that PeMV has three homologous regions in the 3’-un- translated region. Repeated sequences have been demonstrated for the alphavi- ruses (Ou et aL, 1981, 1982), although the sequences in PeMV display no homology with alphavirus sequences. Ou and co- workers (1982) have hypothesized that such sequences may be involved in repli- case or transcriptase recognition and binding. Comparison of the untranslated region of PeMV with untranslated regions
of a number of plant and animal viruses did not reveal extensive nucleotide ho- mology. Viruses analyzed included tobacco mosaic virus (TMV) (Goelet et al, 1982), cowpea mosaic virus (Lomonossoff and Shanks, 1983; van Wezenbeek et al, 1983), brome mosaic virus (Ahlquist et cd, 1984), poliovirus (Kitamura et a& 1981), and Sindbis virus (Ou et cd, 1981, 1982).
The nucleotide and amino acid sequence data presented here demonstrate that or- ganization and expression of the genome of PeMV and TEV are different. In the PeMV genome, the capsid protein coding sequence is present as a discrete ORF. Additional, preliminary evidence suggests the PeMV capsid protein gene and the adjacent gt ne for a PeMV protein similar to the TEV 54,000-MW nuclear inclusion protein are encoded in different reading frames. In contrast to the PeMV organi- zation, TEV capsid protein sequence is part of a large ORF which codes for at least two viral products (nuclear inclusion and capsid proteins) (Allison et uL, 1985). The genome structure described here for PeMV predicts that capsid protein expres- sion requires the participation of a sub-
290 DOUGHERTY ET AL.
genomic mRNA or internal initiation of translation at or near the beginning of the large ORF. However, PeMV and TEV capsid protein gene expression is similar in two respects. Both capsid proteins are expressed initially as higher molecular weight precursors and these precursors are processed by a co- or post-transla- tional proteolytic event into capsid pro- teins.
ACKNOWLEDGMENTS
We thank Ronnie Johnson and Mary E. Kelly for excellent technical assistance. This research was supported in part by the National Science Founda- tion, the U. S. Department of Agriculture Competitive Grants Program, and the North Carolina Agricul- tural Research Service.
REFERENCES
AHLQUIST, P., DASGUPTA, R., and KAESBERG, P. (1984). Nucleotide sequence of the brome mosaic virus genome and its implication for viral replication. J. Mol. BioL 172, 369-383.
AHLQUIST, P., and KAESBURG, P. (1979). Determina- tion of the length distribution of poly(A) at the 3’ terminus of the virion RNAs of EMC virus, polio- virus, rhinovirus, RAV-61 and CPMV and of mouseglobin mRNA. Nucleic Acids Res. 7, 1195- 1203.
ALLISON, R., JOHNSTON, R., ARMSTRONG, F., HORTON, R., and DOUGHERTY, W. G. (1984). A comparative study of the capsid proteins and the 3’ terminal nucleotide sequences of the potyviruses tobacco etch and pepper mottle. Phytopathdogg 74,859.
ALLISON, R. F., SORENSON, J. C., KELLY, M. E., ARMSTRONG, F. B., and DOUGHERTY, W. G. (1985). Sequence determination of the capsid protein gene and Banking regions of tobacco etch virus: Evidence for the synthesis and processing of a polyprotein in potyvirus genome expression. Proc. NatL Acd Sci. USA 82.3969-3972.
BUELL, G. N., WICKENS, M. P., PAYVAR, F., and SCHIMKE, R. T. (1978). Synthesis of full length cDNAs from four partially purified oviduct mRNAs. .I. BioL Churn. 253,2471-2482.
DAGERT, M., and EHRLICH, S. D. (1979). Prolonged incubation in calcium chloride improves the com- petence of Escherichiu wli cells. Gene 6, 28-28.
DENG, G.-r., and Wu, R. (1981). An improved proce- dure for utilizing terminal transferase to add homopolymers to the 3’ termini of DNA. Nucleic Acids Rex 9, 4173-4188.
DOUGHERTY, W. G., and HIEBERT, E. (1980a). Trans- lation of potyvirus RNA in a rabbit reticulocyte lysate: Reaction conditions and identification of
eapsid protein as one of the products of in vitro translation of tobacco etch and pepper mottle viral RNAs. ViroZogy 101, 466-474.
DOUGHERTY, W. G., and HIEBERT, E. (1980b). Trans- lation of potyvirus RNA in a rabbit reticulocyte lysate: Identification of nuclear inclusion proteins as products of tobacco etch virus RNA translation and cylindrical inclusion protein as a product of the potyvirus genome. ViroZogogy 104, 1’74-182.
DOUGHERTY, W. G., and HIEBERT, E. (198Oc). Trans- lation of potyvirus RNA in a rabbit reticulocyte lysate: Cell-free translation strategy and a genetic map of the potyviral genome. Virology 104, 183- 194.
EDWARDSON, J. R. (1974). Some properties of the potato virus Y-group. Fla, Agrk Exp. Stn Monogr. ser. 4.
GOELET, P., LOMONOSSOFF, G. P., BUTLER, P. J. G., AKAM, M. E., GAIT, M. J., and KARN, J. (1982). Nucleotide sequence of tobacco mosaic virus RNA. Proc. NatL Acad. Sci USA 79. 5818-5822.
GORDON, J. I., BURNS, A. T. H., CHRISTMANN, J. L., and DEELEY, R. G. (1978). Cloning of a double- stranded cDNA that codes for a portion of chicken preproalbumin. J BioL Chem 253,8629-8639.
HANAHAN, D., and MESELSON, M. (1980). Plasmid screening at high colony density. Gene 10, 63-67.
HEWICK, R. M., HUNKAPILLER, M. W., HOOD, L. E., and DREYER, W. J. (1981). A gas-liquid solid phase peptide and protein sequenator. J. BioL Chem. 256, 7990-7997.
KITAMURA, N., SEMLER, B. L., ROTHBERG, P. G., LARSEN, G. R., ADLER, C. J., DORNER, A. J., EMINI, E. A., HANECAK, R., LEE, J. J., VAN DER WERF, S., ANDERSON, C. W., and WIMMER, E. (1981). Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature (London) 291, 547-553.
KOZAK, M. (1984). Point mutations close to the AUG initiation codon affect the efficiency of translation of rat preproinsulin in wivo. Nature (London) 308, 241-246.
LIPSCOMB, E. L., HORTON, H. R., and ARMSTRONG, F. B. (1974). Molecular weight, subunit structure, and amino acid composition of the branched chain amino acid amino transferase of Salmonella tvphi- murium. Biochemistry 13, 2070-2077.
LOMONOSSOFF, G. P., and SHANKS, M. (1983). The nucleotide sequence of cowpea mosaic virus B RNA. EMBO J. 2,2253-2258.
MESSING, J. (1983). New Ml3 Vectors for Cloning. In “Methods in Enzymology” (R. Wu, L. Grossman, and K. Moldave, eds.), Vol. 1OlC pp. 20-78. Aca- demic Press, New York.
NELSON, M. R., and WHEELER, R. E. (1978). Biological and serological characterization and separation of potyviruses that infect peppers. Phytopathdogy 68,979-984.
PeMV CAPSID PROTEIN GENE AND ITS PRODUCT 291
Ou, J.-S., STRAUSS, E. G., and STRAUSS, I. H. (1981). high specific activity in vitro by nick translation Comparative studies of the 3’-terminal sequences with DNA polymerase I. J. MOL Biol 113,237~251. of several alphavirus RNAs. virdogy 109, 281- SANGER, F., NICKLEN, S., and COULSON, A. R. (1977). 289. DNA sequencing with chain-terminating inhibi-
OLI, J.-S., TRENT, D. W., and STRAUSS, J. H. (1982). tors. Proc. Nat1 Ad sci. USA 74, 5463-5467.
The 3’-non coding regions of alphavirus RNAs STRAUSS, E., RICE, C. M., and STRAUSS, J. H. (1984). contain repeating sequences. J. Md Bid 156,719- Complete nucleotide sequence of the genomic RNA
730. of Sindbis virus. Virology 133, 92-110.
PALMENBERG, A. C., KIRBY, E. M., JANDA, M. R., TAYLOR, J. M., ILLMENSEE, R., and SUMMERS, J.
DRAKE, N. L., DUKE, G. M., PRTRATZ, K. F., AND (1976). Efficient transcription of RNA into DNA
COLLETT, M. S. (1984). The nucleotide and deduced of avian sarcoma virus polymerase. Biochim. Bie
amino acid sequences of the encephalomyocarditis phys. Acta 442, 324-330.
viral polyprotein coding region. Nucleic Acids Res. TINOCO, I., BORER, P. N., DENGLER, B., LEVINE,
12, 2969-2985. M. D., UHLENBECK, 0. C., CROTHERS, D. M., and
PURCIFULL, D. E., HIEBERT, E., and MC DONALD, GRALLA, J. (1973). Improved estimation of second-
J. G. (1973). Immunochemical specificity of cyto- ary structure in ribonucleic acids. Nature New
plasmic inclusions induced by viruses in the potato Biol. 246, 41-42.
Y group. Virology 55, 275-279. VAN WEZENBEEK, P., VERVER, J., HARMSEN, J., Vos,
P., and VAN KAMMEN, A. (1983). Primary structure PURCIFULL, D. E., ZITTER, T. A., and HIEBERT, E. and gene organization of the middle-component
(1975). Morphology, host range and serological RNA of cowpea mosaic virus. EMBO J. 2,941-946. relationships of pepper mottle virus. Phytopathol- w AHL, G. M., STERN, M., and STARK, G. R. (1979). ogy 65. 559-562. Efficient transfer of large DNA fragments from
RETZEL, E. F., COLLET~, M. S., and FARAS, A. J. agarose gels to diazobenzyloxymethol-paper and (1980). Enzymatic synthesis of deoxyribonucleic rapid hybridization by using dextran sulfate. Proc. acid by the avian retrovirus reverse transcriptase NatL Acad. Sci. USA 76,3683-3687. in vitro: Optium conditions required for transcrip- ZIMMERN, D., and KAESBERG, P. (1978). 3’-Terminal tion of large ribonucleic acid templates. Biochem- nucleotide sequence of encephalomyocarditis virus istry 19, 513-518. RNA determined by reverse transcriptase and
RIGBY, P. W. J., DIECKMANN, M., RHODES, C., and chain-terminating inhibitors. Proc. NatL Acad. BERG, P. (1977). Labeling deoxyribonucleic acid to Sci. USA 75,4257-4261.
