Cloned DNA copies of cowpea severe mosaic virus genomic RNAs: Infectious transcripts and complete nucleotide sequence of RNA 1

VIROLOGY

191, 607-618

(1992)

Cloned DNA Copies of Cowpea Severe Mosaic Virus Genomic RNAs: infectious Transcripts and Complete Nucleotide Sequence of RNA 1 XIAOJIANG CHEN AND GEORGE BRUENING’ Department

of Plant Parhology,

College of Agricultural

and Environmental

Received February

Sciences,

11, 1992; accepted August

University

of California,

Davis, California

956 16

13, 1992

Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member. Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter. Previously, similar clones of CPSMV RNA 2 had been obtained. A 5’-rUAUUWAAUUUU sequence is common to RNA 1 and RNA 2. From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences. Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions. The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831. The calculated molecular weight of the polyprotein is 208,000. Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5’-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites. Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24K/87K junction has a distinct aa pair in the CPSMV polyprotein. Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively. Published mutational analysis of the CPMV 24K proteinase and alignment of aa sequences from three comoviruses suggest that cysteine168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase. Results are discussed in the context of the resistance that some cowpea (Vigna unguiculara) lines exhibit against CPMV but not against CPSMV. 0 1992 Academic Press. Inc.

proteins needed for the cell-to-cell and long-distance movement, including the capsid proteins (Goldbach et a/., 1980; Wellink and Van Kammen, 1989). CPMV RNA 1 and RNA 2 direct the synthesis of polyproteins that are cleaved to generate mature proteins. This proteolytic processing is mediated by a proteinase, the 24K proteinase, that initially is a segment of the RNA l-encoded polyprotein 200K (Wellink et al., 1986, 1987). During translation in vitro, the 200K polyprotein is rapidly cleaved into an amino-terminal 32K protein and a carboxyl-terminal 170K protein (reviewed by Eggen and van Kammen, 1988). Within the 170K polyprotein, beginning at the amino-terminus, segments correspond to the mature proteins 58K (helicase motif protein; Gorbalenya et al., 1989a), VPg, 24K proteinase (Verver eta/., 1987), and 87K (polymerase motif protein; Argos, 1988). For CPMV, different orders of cleavage at the three sites of the 170K protein generate partial cleavage products 11 OK (24K-87K), 84K (58K-VPg-24K), and 60K (58K-VPg), in addition to the mature proteins (reviewed by Eggen and van Kammen, 1988). The nucleotide sequences of CPMV RNA 1 (Lomonossoff and Shanks, 1983) and the VPg-24K proteinase region of red clover mottle virus (RCMV) RNA 1 (Shanks

INTRODUCTION Cowpea severe mosaic virus (CPSMV) is a member of comovirus group (Bruening, 1978) of which the very well studied type member is cowpea mosaic virus (CPMV). CPSMV once was considered to be a strain of CPMV, but subsequently CPSMV was recognized as a distinct virus (F&on and Scott, 1979; de Jager, 1979). Comoviruses have two, separately encapsidated, single-stranded genomic RNAs of the messenger sense. The larger RNA 1 and the smaller RNA 2 are separately encapsidated in icosahedral capsids that are approximately 28 nm in diameter and composed of 60 copies each of the large coat protein, L, and the small coat protein, S (Wu and Bruening, 1971; Chen et al., 1989). The genomic RNAs bear a 5’-linked protein, VPg, and 3’ polyadenylate (reviewed by Eggen and van Kammen, 1988). Both genomic RNAs are necessary for systemic infection of the host plant. RNA 1 encodes functions required for virus replication, whereas RNA 2 specifies

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the Accession No. M83830. ’ To whom reprint requests should be addressed. 607

0042-6822/92

$5.00

Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

608

CHEN AND BRUENING

and Lomonossoff, 1990) are available. Our interest in CPSMV is derived from its ability to infect cowpea (Vigna unguiculata) lines that are highly resistant to CPMV (Beier et al., 1977, 1979). An inhibitor of the CPMV 24K proteinase may be a determinant of the resistance against CPMV that is exhibited by seedlings of the cowpea line Arlington (Ponz et al., 1988). We report construction of cDNA clones of CPSMV RNA 1 and tailoring of RNA 1 and RNA 2 clones to serve as templates for infectious transcripts. Similarities of the deduced amino acid residue (aa) sequences of the RNA 1 polyproteins of CPMV, CPSMV, and RCMV allow likely cleavage sites for the CPSMV RNA 1 polyprotein to be identified. MATERIALS Construction clones

AND METHODS

and amplification

of plasmid cDNA

CPSMV is the DG isolate studied in this laboratory and originally referred to as CPMV-DG (Beier et al., 1977, 1979). CPSMV was propagated, virions were purified and subjected to Metrizamide gradient centrifugation, and genomic RNAs were recovered, all as described by Chen and Bruening (1992). The methodology for cDNA cloning was as described by Alexander (1987) and applied by us (Chen and Bruening, 1992) using dT-tailed plasmid pCGN 1703 (O’Neill, 1989) as primer for cDNA synthesis catalyzed by ribonuclease H-less Maloney murine leukemia virus reverse transcriptase. To overcome a tendency of Escherichia co/i cells to lose plasmids that have CPSMV RNA 1 inserts, freshly inoculated cultures in 5 ml of LB medium (Sambrook et al., 1989) containing 150 pg/ml ampicillin were grown for 5 hr, and cells were collected by centrifugation. Cells were washed two or three times with fresh, ampicillin-containing LB medium and were suspended in 20 ml of the medium. After another 5 hr at 37”, the wash was repeated, and the cells were suspended in 50 to 100 ml of the ampicillin medium. The culture was incubated at 37” overnight. Analysis

of in vitro transcripts

for infectivity

Plasmid clones of CPSMV RNA 1 and RNA 2 were linearized at the 3’ end of the inserted sequence using restriction endonuclease Sal1 or Asp7 18, respectively. ln vitro transcription was in the presence or absence of the cap analogue m7G(5’)ppp(5’)G (Verver et al., 1987). Standard reactions of 100 ~1 contained 5 pg of linearized template DNA, 100 U bacteriophage T7 RNA polymerase and 50 U RNasin ribonuclease inhibitor (Pro-

mega, Madison, WI) in 40 mM Tris-HCI, 20 mn/r MgCI,, 10 ml\/l DlT, 100 pg/ml bovine serum albumen, 1 mM each rNTP. When m7G(5’)ppp(5’)G was included in the reaction mixture, it was supplied at 2 mlL1, and the rGTP concentration was reduced to 0.2 mM. A fraction of the transcription reaction mixture was analyzed by electrophoresis through 0.7% agarose gel to estimate the extent of synthesis and size distribution of the RNA products. Nucleic acids were precipitated with ethanol and dissolved in diethylpyrocarbonatetreated water to the desired stock solution concentration. Groups of 8-10 2-week-old seedlings of Chenopodium amaranticolor and cowpea (V. unguiculata) lines Blackeye 5 or Arlington were rubbed with solutions of nucleic acids from transcription reaction mixtures (30 pug/ml each RNA, singly or as RNA l-RNA 2 pairs), CPSMV virion RNAs (10 pg/ml) or buffer (0.05 M potassium phosphate, pH 7) alone on leaves that had been dusted with Carborundum powder. Plants were observed for a period of 3 weeks for development of symptoms. Infection by CPSMV was confirmed by immunodiffusion using antibody raised against purified virions and by transfer to Arlington cowpea seedlings and observation for characteristic CPSMV-induced symptoms. Nucleotide

sequence

determination

The 6-kbp cDNA insert of an RNA 1 cDNA clone was cut at the unique EcoRV site corresponding to a location 2 kbp from the 5’ end of RNA 1. The linearized plasmid then was digested either by Sstl at a unique site in the multiple cloning site (MCS; Fig. 1) or by Sal1 at a unique site to the 3’ side of the cloned RNA 1 sequence. The ends were blunted, by the action of bacteriophage T4 DNA polymerase and dNTPs, and ligated, giving two subclones. Controlled truncation of each end of the insert for each of the two subclones employed E. coliexonuclease III and nuclease Sl, and an ordered set of subclones was selected as described (Sambrook et al., 1989; Chen and Bruening, 1992). With few exceptions, each succeeding member of the ordered set differed in length from the previous member by about 100 bp. Partial nucleotide sequences of plasmid inserts were determined as described @anger et al., 1977; Chen and Bruening, 1992). The sequence of both strands in the region of the EcoRV site was determined using two chemically synthesized oligodeoxyribonucleotide primers, one with sequences corresponding to nt 201 1 through nt 2027 and the other complementary to nt 2226 through nt 2210, in the CPSMV RNA 1 sequence. Sequence data were assembled using the Microgenie program

COWPEA SEVERE MOSAIC

and computer board (Beckman, Palo Alto, CA). Sequence comparisons and related analyses were performed with the software packages of the University of Wisconsin Genetics Computer Group (GCG), version 7.0. Deleting nonviral of the cDNA

sequences

at the 5’ end

As the first step in eliminating sequences between the cDNA 5’ end and the bacteriophage T7 promoter, plasmids bearing CPSMV RNA 1 inserts were digested with Apal and Sall, and those bearing RNA 2 inserts

A

T7 pcomcmr

CPSMV sequence

FIG. 1. Methodology for precisely deleting from pCGN1703-derived plasmids the sequences located between the bacteriophage T7 promoter and the cloned insert of cDNA transcribed from cowpea severe mosaic virus (CPSMV) RNA 1 or RNA 2. The top strand corresponds to the encapsidated polarity of CPSMV RNAs. CPSMV cDNA sequences are in bold font, beginning with the dTAlTAAAATTTT sequence that here was shown to be common to the 5’end of both CPSMV genomic RNAs. The remainder of the CPSMV RNA sequence is presented as X:Y base pairs and the poly(dA):poly(dT) 3 tail. (A) In the starting plasmid, the bacteriophage T7 promoter is separated from the CPSMV RNA sequence by(i) a restriction endonuclease Apal site, (ii) the remaining multiple restriction endonuclease sites (MCS:MCS’) derived from the adaptor during cloning, and (iii) oligo(dC):oligo(dG) introduced in the process of dG tailing the first strand cDNA. The plamid was linearized by the action of Apal and denatured. DNA synthesis, with the strand complementary to CPSMV genomic RNA as template, was initiated with 27-mer primers, dTAlTAAAATf , designed to correspond to the 5’ terminal sequence of RNA 1 or of RNA 2. (B) The result of extension of the primer to the end of the template strand will be a molecule that is single-stranded only for a 57 nt, 3’ extension, which was removed by the action of bacteriophage T4 DNA polymerase in the presence of all four dNTPs. (C) Ligation of the product connected the bacteriophage T7 promoter to the CPSMV RNA sequence such that bacteriophage T7 RNA polymerase will generate CPSMV RNA preceded by only the single guanylate residue that is necessary for efficient initiatlon of transcription (arrow).

609

VIRUS RNA 1

-r.L-

-----

---4-+----d ,000

2000

-

---e----e --------

4000

3000

6000

5000

1

I

2

2

3

3 I,,,,,,, _ _-----

--

/ ... ..

I 1000 -

2000 -----------cc--cc--

3000 --

&

4000 ---

6000

5000 ---

-

FIG. 2. The three possible open reading frame (ORF) registers of the encapsidated polarity of CPSMV RNA 1. The locations of stop codons (long vertical lines) and AUG start codons (short vertical lines) are indicated within each of three long rectangles that represent the three reading frame registers. Above and below the set of three rectangles is a scale that shows the nucleotide sequence numbering. Arrows locate an overlapping subset of all of the sequences, including a portion of the 3’ polyadenylate, that were determined from various clones.

(Chen and Bruening, 1992) were digested with Apal and Kpnl. The inserts were transferred to similarly digested, modified plasmid pTZ18R. Figure 1 summarizes the subsequent steps. The insert-bearing plasmid was linearized by digestion with Apal and was extracted with phenol and chloroform. Extension of a primer (Fig. 1 B), with the 5’ terminal sequence of the corresponding CPSMV RNA, was in reaction mixtures of 100 ~1 containing 5 U of Taq DNA polymerase (Perkin Elmer Cetus Corp., Norwalk, CT), approximately 1 pg of plasmid, 0.7 pg of primer, 250 pl\/I of each dNTP, 10 mM Tris-HCI, 50 mM KCI, 1.5 m/l/l MgCI,. Incubation was at 95” for 6 min, 49” for 1 min, and 72” for 30 min. The product was incubated with bacteriophage T4 DNA polymerase (1 U/pg of DNA) in the presence of 250 PM of each dNTP at 12” for 30 min (Sambrook er a/., 1989) to remove the 57 nt 3’overhang (Fig. 1 B). The blunt-ended DNA subsequently was ligated by the action of bacteriophage T4 DNA ligase (1 Weiss U/pg DNA) overnight at 23” to complete the construction (Fig. 1C). To select clones with the guanylate residue of a bacteriophage T7 promoter fused directly to the 5’ nucleotide sequence of CPSMV RNA, two oligodeoxyribonucleotide primers were prepared, one corresponding to 21 nt of the promoter and the other complementary to nt 118 through 99 of both CPSMV RNA 1 and CPSMV RNA 2. To recover possible RNA 1 clones, small, 0.5- to 1-mm-diameter colonies were selected; colonies of any size were analyzed as possible RNA 2 clones. A 20-~1 suspension of cells was heated to 100” for 15 min in polymerase chain reaction (PCR; Sambrook et al., 1989) buffer and dNTPs. The suspension was cooled to room temperature and centrifuged. Taq DNA polymerase, 0.5 U, was added and 25 cycles of PCR were performed using a program of 1 min each at 92, 49, and 72”. The PCR products were resolved by

610

1 194 13 293 46 392

QRHRRLLTLSYGAMCFQFSNSVGDEGIEVDDDE CAGAGGCACCGACGGTTGTTGACACTGTCATATGGTGCTATGTG~C~~CTC~~CAGTAGGGGATGAGGGCATAG~GTGGATGATGATGAG

79 491

LMFEIFDALLRTKISNSKGMTHLYSWMRGVYLS TTGATGTTTGAAATATTTGATGCATTGCTGCGCACAAAGA~C~CTCA~GGGCATGACGCAC~ATACAGCTGGATGCGTGGAG~ATCTCAGC

112 590

TFKVEVQCDDYNSNLLEKDLAGEAQGLSQFVSG ACATTCAMGTAGAGGTGCAGTGTCATGATGA~ACMCTC~TCTGCTGGAGMGGA~GGCTGGAGAAGCTCAGGGTC~CACAGTTCG~CAGGA

145 689

LADWIPSRVKTLAGYAAEGIIEAFKKHFDKLLV CTTGCTGACTGGATTCCCAGTCGTGTAAAGACCTTGGCAGGATATGCCGCTGAGGGCATAA~GAGGCC~MG~GCAC~CGACAM~GC~G~

178 788

EYCPMAVAACSWITTVWTTIKEWVQSAMDAMSW GAGTACTGTCCTATGGCCGTGGCTGCGTGTAGTTGGATMCTACAGTATGGACCACTATCMAGAGTGGGTCCMTCTGCGATGCATGCTATGTCGTGG

211 887

IMAGCTELISWGMCVIAGSCALSLLEKALVAMG A~ATGGCTGGATGCACTGMCTCA~C~GGGGMTGTGTGTCA~GCAGG~CATGTGC~ATCG~A~GGA~GGCA~GGTAGCTATGGGT

244 986 277A E L L Q L V S L AV G A V S S AT 1085 GCTGAGTTGTTACMCTAG~CA~GGCCGTGGGAGCAGmCACAGATGTGAGC

5

5

C

F

QS

P

V

G

QAT

DV

5

318 1184

A E 5 Q+S G G V E M L E S LA K N L T N F C 0 G T L V S I G K GCTGAGAGTCMTCAGGTGGAGTTCAGATGCTAGAGAGTCAC~GCTMGMC~GACCM~CTGTGATGGCACTCTGGTATCTATCGG~GAC~GC

T

C

343 1283

N A V N S I N TAA G T I K N L VG R L L S M L S N F A MCGCTGT~~CGATCMTACGGCTGCAGGTACCATC~TCTGGTAGGTAGACTC~GTCTATGCTMGTM~GCATAC~CTACTAGGT

Y

K

L

L

G

376 1382

L E 5 T F L R 0 A 5 VV F 5 E TTAGAATCCACArrCTTGCGAGACGCTTCCGTGGTTTmCTGCC

D

Q

F

LA

409 1481

KAY IN Q D E L M V L R S L IT R G E VMQ R E M I M G G ~GGCATACATCAACCAGGATGMC~ATGGTGTTGCGATCTCTCA~ACCAGAGGTGMG~ATGC~GGGAGATGATCATGGGTGGTATG~G~

M

KV

442 1580

S P T V C G L I N KG CT D L A K L M A G A V M H G T S G TCACCTACAG~GTGG~GATCMT~GGATGCACGGATCTCGCCMATTMTGGCAGGGGCTGTGATGCATGGMCGAGTGGTACTCGG~TA

T

R

K

I

475 1679

P F V VY A H G A S R V G K TMV CCA~GTTG~ATGCACATGGGGCTTCTAGAGTTGGTAAAAGGAGAG

E

L

G

E

508 1778

0 C V Y P R N V V D D Y W 5 G Y K R Q P I V V I D 0 F GA V GACTGTGTGTATCCACGAAATGTGGTAGATGACTACTGGAGTGGGTAC~GACMCCTA~G~GTCA~GATGA~GGTGCTGTGTC~CAGAT

S

S

0

541P S A E A Q L I P L I S 5 A P Y P L NM AD L S E KG M H F 1877 CCTTCTGCAGAAGCTCMTT~~CCATTGATCTCTAGTGCTCCCTATCCCC~~CATGGCTGATCTCTCTGAGMGGGAATGCAC~GA~CAGCT

D

S

A

NV

D

I

G

N

W

R

L

L

KQ

I

I

E

D

SW

F

C

R

K

Q

E

L

574 I VM C 5 S N F I E C 5 P E S KV R D EM A F R N R R H V L F TV 1976 ATCGTCATGTGCTCATCCMmCATTGAGTGTTCACCAGAGTGTTCACCAG~GCMGGTGCGTGACGMATGGCA~CAG~CAGACGACATGTGCTC~CACTGTC 607 S L D P N I P Y D G D D I T K N Q I Y E I K T W F H D S 2075 TCACTTGACCCTMTATACCATATGATGGTGATGATATCACMACMTC~TATATG~ATC~AC~GG~CATGA~CGTATCATG~GMGCA

H

V

E

A

640 T F T S Y G D L L A Y C K N K W V E H NT E Q E A N L K Q L 2174 ACmCACATCATATGGGGACTGCTGGCATATTGCMMACAAGTGGGTGGAGCACMTACTGAGCMGAGGCCMC~GMGCMC~GGAGTT~A

G

V

K

673 2273

K E S VA F Q Q F R S I L D AAGGAGAGCGTTGCA~CAGCAGmCGTTCCATTCTTGAmGG

L

E

T

P

706 2372

D G R C H F V 5 C Y D K 5 G I L R H Y T I DA T G D V Q E M GATGGTAGGTGCCAC~GTGTCATGTTATGATAAGAGTGGTATACTCAGGCACTATACTA~GATGCMCTGGAGATGTGCAAG~TGG~GG~

E

K

V

739 2471

D S 5 L D D I L L E K T N KM V L A A Y KM I KY H K D GA~CCTCTCTAGATGACATCCTA~GGAM~CC~C~~TGGTC~AGCTGCATATAAGATGA~MGTACCAC~GGACACCMTCTGGTCA~

T

N

L

V

I

772 2570

K T Q LA 0 L V D P T K Y T A 0 F Q F D G V I G 5 P L F S AAAACCCAGCTTGCAGAmGGTGGATCCCACMAGTATACTGCAGA~CCAG~GACGGTG~ATAGGATCACCAC~CAGCAGCCMGT~TG

5

Q

V

M

805 2669

P S V KA L P L WQ R MV L Y T V G Q N L G RT H S S W Y E G I K CCMGTGTCAAGGCATTACCACTGTGGCAAAGCATGGATGGTACTGTACACTGTTGGGCAG~TCTGGGAAGMCTCA~CTAG~GGTATGAGGGCATCMG

838 2768

D K C M L A L S KAY S T E I K DW P V A L K I V V G V I LA GACMGTGCATGCTTGCACTATCAAAAGCATACTCMCTGAGATC~GGA~GGCCTGTAGCACTC~~~G~G~GGAGTGATACTGGCTACTGTA

T

V

87l.A 2867

G KA F W R F Y A S MA 0 A G N G G H F V G AVA S A FAG S GCAGGTMGGCA~GGAGGTTCTATGCCTATGCCTCAATGGCAGATGCAGGC~TGGTGGACAC~GTGGGAGCCG~GC~CCGCA~GCAGGAAGTCM

Q

LA

V

F

V

N

Q

D

A

E

N

F

K

Q

Y

R

sequence of the insert of plasmid pDGl-2, corresponding to CPSMV RNA 1. In the lines above the mucleotide sequence is FIG. 3. Nucleotide displayed the aa sequence deduced for the long ORF. Cleavage sites proposed on the basis of comparison with the cleavage sites for the CPMV RNA 1 polyprotein (Lomonossoff and Shanks, 1983) are glutamine-serine. +; glutamine-methionine, 0; glutamine-alanine, 7

COWPEA SEVERE MOSAIC

611

VIRUS RNA 1

904AVVAQeS RK P NR F DVAQY RY RN I P L RKR NWA E 2966 GCGGTTGTTGCACAGAGTAGGMGCCCAACAGGmGATGTGTGGCTCAGTACAGGTACCG~CATACCTCTMGGMGAG~~GGGCAGMGGGCM

GQO

937MS L D Q S TM L I M E KC K A N F V F 5 N I S C Q I VM L 3065 ATGAGTCTGGATCAGTCCACAATCCTCATMTGGAAAAGTGC~GGCCM~CGTC~AGC~CATTAGCTGTCAGATAG~ATG~GCCTGGGCGA

P

G

R

970Q F L C Y K H V F A S L N S PM Y V D I Y T A N K K Y K L Y Y 3164 CAATTCTTGTGCTACAAACATGTGmGCTAGTCTAGTCTCMTAGTCCMTGTATGTGGATA~ATACTGCCMCMGMGTATMACTCTA~ACAMCCT

K

P

1003QNRVYFETtlSEIMLYKDASLEDIPASCWDLFCF 3263 CAGMTAGGGTATAC~GAGACTGATAGTGAGATCATGCTATACMGGATGCCAG~GGMGACATACCTGCCAGCTGCTGGGATC~G~T l036DAEKSLPRGSFPAEILSCKLDRTTNQHIPEWAD 3362 GATGCGGAAA~G~GCCACGAGGTAGCAGAAATCAACATATCCCGGAATGGGCCGAC l069ISARTVNQKLDVEFGEYQTIFYSYLQYDVSTKA 3461 ATCTCAGCTCGTACTGTCAATCAAAAACTGGACGTGG~~GGGGAGTACCAAACCATC~A~CCTATCTCCAGTATGATGTATCCACMAAGCT ll02EDCGSLIIATIDGRKKIIGIHTAGRANRSGFAS 3566 GMGATTGTGGTTCCCTPTMTAGCAACCATTGATGGTAGG~MGATMTAGGGATCCACACTGCTGGACGGGCMATAGGAGTGG~GCMGT ll35YMPQVEIPVQAQ?AAEKFFDFLEKEQHVTEGIGK 3659 TATATGCCGCAGGTAGAAATACCAG~CMGCACMGCAGCGGMMG~C~GAT~CTTGAG~GMCAACATG~ACTGAGGGCA~GGAMG ll68VGNLKKGVWVPLPTKTNLVETPKEWHLGTEKTK 3758 GTGGGAMTCTCMGAMGGAGTCTGGGTTCCA~ACCCACTMGACCMTC~GTGG~CACCAMAGAGTGGCATCTGGGCACTGAG~CAMA ~~~~EPSILSSTDLRLGDKQYDPFVGGIQKYAEPMGI 3857 GMCCAAGTATTCTCAGCAGTACGGAmAAGGCTCGGCTCGGTGATMGCAGTATGATCCC~G~GGAGGMTACAGMGTACGCCGAACCMTGGGAA~ l234LDDEVLRHVATDIVEEWFDCVDPQEDTFEEVDL 3956 CTAGATGATGAGGTGCTCCGGCACGTGGCMCATAGGAGGAAGTTGACCTG l267QVAINGLEGMEYMERVPMATSEGFPHILTRKSG 4055 CAGGTTGCTATCMTGGTCTGAAGGAATGGAATACATGGMAGAGTTCCTATGGCMCATCTGMGGC~CCCACACAT~GACAAGGAAAAGTGGG l300EKGKGRFVYGDGEIFDLIPGTSVHEAYLTLEET 4154 GMAAAGGCMAGGTAGGmGTATATGGGGATGGAGAkATGAGGCATATCTGACACTGGAAGAGACT l333CADTVPALVGIECPKDEKLPLRKIYEKPKTRCF 4253 TGTGCGGACACTGTTCCAGCCCTGGTTGGGATTGAATGTCCAAAAGATGM~C~CC~GCGGAAGATCTATGAGAAGCCTMAACAAGATG~C l366TVLPMEYNLVVRRKFLKFVVFIMKNRHRLSCQV 4352 ACTGTACTTCCTATGGMTATMTCTTGTAGTTAGGAGGAM~CTCAAG~GTGGTGT~A~ATGMGAATCGGCACAGA~ATCCTGCCAGGTG l399GINPYGMEWSRLAMSLLEKGNNILCCDYSSFDG 4451 GGCATCMTCCATATGGCATGGMTGGAGTCGCCTGGCAATGAGC~GC~GAGMAGGAAACAACA~~ATG~GTGA~ACAGTTCATTTGATGGG l432LLTKQVMHLMSEMINELCGGSSRLKQQRTNLLM 4550 TTGTTGAC~GCAAGTTGCATCTCATGAGTGAMTGATCMTGAACTGTGTGGGGGATC~CGCGCCTAMGCAACAGCGCACCM~TGTTAATG l465ACCSRYALCKGEVWRVECGIPSGFPLTVICNSI 4649 GCATGTTGTTCCAGGTATGCA~ATGCMGGGAGAAGTGTGGCGCGTTGMTGTGGMTTCCCTCTGGA~CCA~GACTGTCA~TGCAACAGCA~ l498FNELLVRYSYIKICQQARVPATITYGFSTFVKM 4748 TTCMTGAGCTATTGGTTPGATACAGCTACA~AAGA~GCCAACAAGCGCGTGTGCCAGCCACCATAACATACGG~AGTACC~TGTAAAGATG l531VTYGDDNLLSVQSAITHVFDGTKLKEFLKLNGI 4847 GTGACTTATGGTGATGATMCTTACTGAGCGTTCAGTCTGCAATCACTCACGTG~GATGGMCCAAGCTC~GGAG~CTGMACTCAATGGCATC l564TITDGKDKTSPVLNFRNLEDCDFLKRGFKKESD 4946 ACCATCACTGACGGCAAAGACAAAACTTCCCCTGTGC~AA~CGGAA~GGAAGA~GTGAC~CTC~AGGGGC~MGMAGAAAGTGAT l597VVWVGPEEKESLWAQLHYVTTNNLEKHEAYLVN 5045 GTAGTGTGGGTTGGACCAGAGGMMGGMTCGCTGTGGGCACAACTCCATTATGTCACGACCMCMTC~GA~GCATGMGC~A~TGGTGMC l630VVNVIRELYLHDPREAAELRRKAIQNVDFLKEN 5144 G~GTAAACGTGATCAGGGAGTACCTACATGATCCMGAGAGGCTGCTGAG~GCGTCGCMGGCMTACA~TGTAGAC~C~GAAGGAAMT l663PKDLPTMAAIKEFYNMQRQQQFVDSNDNLDSLL 5243 CCTA~GA~GCCCACTATGGCTGCTATCMGGAGTTCGTCTTCTG l696NPDFLFVAPHRKMHEAEMELVPKWYLRDLGKAP 5342 AATCCAGATTT~G~G~GCCCCACATAGMAGATGCATGAGGCCGAGATGG~~AGTGCC~AGTGGTATCTGAGAGATC~GGCMAGCTCCA l729INVLTGEADRICVLVNASIPDHLLPEKVVNISW 5441 ATAMTGTTCTMCTGGAGMGCTGATCGMT~GTGTTCTATATCATGG 1762PYGPGRGGLPTHGWAQANLYNPNSAVVKKLRTL 5540 CCTTATGGACCCGGMGGGGAGGACTGCCGACTCATGG1T 1795VNQNPDDRVDICFRHDAVPVAIATIIFLVHLGK 5639 GTGMTCAAAATCCTGATGATCGAGTGGATATATGTmAGGCA~AGGAAAG l828VKGRSANEYLTKIIDSAKSLKFLPKECDIIF. 5738 G~~GGGAGMGCGCTAATGMTAT~GACTAAAAT~CTGAGTG 5837 5936

TA~GAG~GTGTGTCTCTTTTC~ACTCTCAGTCTGMTAACTGGCA~~CGCCAA~ATMM~ATAATGTGTGA~G~GTGTGTGATTTC TACAGTACATGlTAlTACTTTTAn

FIG. 3-Continued

CHEN AND BRUENING

612 58K helkase

32K 257 CPSMV +

313

t I

595

5957

41% (62%)

cf’hJ’4

207 •(

326

t 1

67K polymerase

24K VPg pmlease

66% (6%) to 1I

593

45% (66%) P 206 1

5605 711

5889

75% 55% (69%) (74%)

ti

206

64% (6%)

44% (64%)

RCMV

4 QIS

?

O/G

t WA

OQ/M

FIG. 4. Comparison of the locations, sizes, and amino acid sequence relationships of the proteins deduced to be the cleavage products of the polyprotein or polyprotein fragment encoded by the long ORF of RNA 1 of CPSMV (Fig. 3) cowpea mosaic virus (CPMV; Lomonossoff and Shanks, 1983) and red clover mottle virus (RCMV; Shanks and Lomonossoff, 1990). Numbers just above the rectangles identify the positions of the first residue of the start codon and of the stop codon of each long ORF. Numbers inside the segmented rectangles indicate the length, in aa, of the determined and deduced polyprotein cleavage products. Numbers below the CPSMV diagram show the percentage of identical and, in parentheses, similar aa, when CPSMV and CPMV were compared. Percentages below the CPMV diagram relate CPMV and RCMV proteins, and the numbers below the RCMV diagram relate RCMV and CPSMV proteins. The key at the bottom of the figure identifies aa pairs at the sites at which cleavage has been determined or postulated to occur.

electrophoresis through 9% polyacrylamide gel (Sambrook eta/., 1989). A 139-bp product was diagnostic of the expected clone. RESULTS AND DISCUSSION Selection of possible CPSMV RNA 1

full-length

cDNA clones of

As reported (Chen and Bruening, 1992) four zones were visible by their turbidity after Metrizamide gradient centrifugation of CPSMV virion preparations. Virions were recovered from the zone of greatest density and were subjected to another cycle of gradient centrifugation, giving two major zones. Using as templates pooled RNA derived from the two zones, with an RNA 1-to-RNA 2 ratio of about 20: 1, transcripts with about equal amounts of the RNA 1- and RNA 2-directed products were obtained, as estimated by autoradiography after electrophoresis through 0.7% alkaline agarose gels (Sambrook et a/., 1989; results not shown). This first strand cDNA preparation was used in subsequent steps as described (Alexander, 1987; Chen and Bruening, 1992) to produce cloned plasmids bearing CPSMV genomic RNA sequences.

Among the more than 100 colonies selected for small-scale preparation of plasmid DNA (Sambrook et a/., 1989) none gave a plasmid with a full-length insert. Restriction mapping revealed that the largest, 5.4-kbp insert has a 1 .l -kbpXbal fragment located at its 5’end. 32P-labeled probes were prepared by electrophoresis of theXbal fragment through 1.2% low-melting-temperature agarose gel and subsequent priming of DNA synthesis directly in a solution of melted agarose derived from the excised gel piece (Sambrook et al., 1989), using random hexadeoxyribonucleotide primers. Of the over 3000 colonies screened with Xbal-fragmentderived probe, 140 gave hybridization signals. Twelve of the colonies were sources of plasmids that had an insert of the expected 6-kbp size. Digestion of the plasmids with 11 restriction endonucleases revealed that 10 out of the 12 plasmids have identical insert maps. Blot hybridization of electrophoretically resolved CPSMV RNAs (Chen and Bruening, 1992) with probes derived from each of these 10 clones showed that each hybridized specifically to RNA 1 (data not shown). Preparations of the corresponding 10 plasmids were linearized with SalI, and transcripts were prepared and analyzed by in vitro translation, gel electrophoresis of the products and autoradiography (Chen and Bruening, 1992; Verver et al., 1987; Ponz et al., 1988; Laemmli, 1970). Six of the plasmids directed a pattern of translation products similar to that obtained by the in vitro translation of CPSMV virion RNA 1 (as also reported by Beier et a/., 1981). Prominent bands had mobilities corresponding to protein molecular weights 200,000, 170,000, 87,000/84,000, 60,000, and 32,000. The 4 other patterns were of apparently truncated, or no, translation products (results not shown). Of the six clones from which the expected translation products were derived, four had the sequence dTATTAAAATilT adjacent to a dC track (Fig. 1A), the two other clones having 5’ truncations of this sequence. The four apparently full-length clones of CPSMV RNA 1 were designated in the odd-numbered series pDGl-1 through pDGl-7. Four of the apparently full-length clones of RNA 2 already derived (Chen and Bruening, 1992) were designated pDG2-1 through pDG2-7. All eight plasmids were linearized and transcribed in vitro in the presence of the cap analogue m7G(5’)ppp(5’)G. The transcripts are expected to begin with a 42-nt MCS derived from the vector, followed by 14-l 6 rC residues. The RNA 1 and RNA 2 transcripts also are expected to bear 25 and 9 vector-derived nt, respectively, to the 3’ side of whatever portion of the polyadenylate was retained in the clones. The in vitro reaction produced approximately 10 molecules of transcript per molecule of DNA template. The transcripts

COWPEA SEVERE MOSAIC

did not prove to be infectious when applied to leaves of C. amaranticolor or primary leaves of Blackeye 5 cowpea, although plants inoculated with CPSMV genomic RNAs uniformly were infected. Modification of plasmid clones for production infectious transcripts

of

The removal of the nonviral sequences at the 5’ end of the cDNA inserts, except for the guanylate that is indispensable for transcription (Eggen et a/., 1989) was difficult because no restriction endonuclease site occurs only in the MCS and close to the 5’ end of the insert, and the dC:dG tracks resisted digestion by exonuclease much more strongly than did the 5’end of the insert. Partial digestions with HindIll did not release the expected fragment that could have been replaced with the appropriate PCR-derived fragment. The exonuclease activity of neither bacteriophage T4 DNA polymerase nor bacteriophage T7 gene 6 protein removed only the desired sequence, as judged by screening with an oligodeoxyribonucleotide specific for the desired sequence, although this approach was successful with other sequences (not shown). Ultimately, the noncDNA sequences were removed as described in Fig. 1. We obtained CPSMV RNA 1 clones pDGl-2 and DGl4, derived from pDGl-1 and pDGl-3, respectively, but none from pDGl-5 or pDGl-7. The method presented in Fig. 1 also generated four suitable RNA 2 clones, the even-numbered series pDG2-2 through pDG2-8, from the odd-numbered series pDG2-1 through pDG2-7. Transcription of all six of the even-numbered, tailored plasmids, without cap analogue, generated an average of only about two RNA molecules per template. C. amaranticolor and Blackeye 5 cowpea were inoculated with a mixture of all four RNA 2 transcripts combined with either the transcript of pDGl-2 or the transcript of pDGl-4. Both inocula produced symptoms, the pDGl-2 transcript mixture generating about 4~ the number of local lesions on C. amaranticolor as were induced by the pDGl-4 transcript mixture. Transcript of pDGl-2 was separately combined with each of the four RNA 2 transcripts, all transcripts being synthesized in the presence of m7G(5’)ppp(5’)G. Transcripts of the pDGl-2/pDG2-2 pair and the pDGl-2/ pDG2-4 pair, each RNA being supplied at a final concentration of 30 pg/ml, and CPSMV genomic RNAs supplied at 10 pg/ml, induced, respectively 24, 1.5, and 80 local lesions per leaf on C. amaranticolor. These inocula also infected, respectively, all, 550/o, and all of the cowpea seedlings, The 5’-capped transcripts of these plasmids were about 3~ as infectious as the corresponding uncapped transcripts. Normalizing for

VIRUS RNA 1

613

differences in concentration of RNA in the inoculum, in various experiments the capped transcripts were about 59/o as infectious as authentic CPSMV genomic RNAs. No symptoms were observed on plants rubbed with buffer, with the transcripts of individual clones, or with the combinations of transcripts of pDGl-2 and either pDG2-6 or pDG2-8. Thus, removing all of the 5’ extraneous nt, except the initially transcribed guanylate, converted apparently noninfectious transcripts of two each of the RNA 1 and RNA 2 clones to infectious transcripts. We conclude that plasmids pDGl-2 and pDG2-2 encode nucleotide sequences that correspond either to infectious CPSMV RNAs or to sequences that very readily are mutated to infectious RNAs. The conversion of noninfectious to infectious transcripts by removal of extraneous nt at the 5’ end of the insert is consistent with observations in other systems, in which extraneous 5’ nt were found to be more detrimental than extraneous 3’ nt (Dawson et al., 1986; Eggen et a/., 1989; Janda et al., 1987; Rice et al., 1987; Shaklee et al., 1988; van Der Werf et al., 1986; Vos et a/., 1988; Ziegler-Graff et a/., 1988). However, possibly the length of the 3’ polyadenylate track also influences the specific infectivity of transcripts. When combined with the same RNA 2 transcript, or the same mixture of RNA 2 transcripts, transcripts of RNA 1 clone pDGl-2, which has a 105~nt polyadenylate track, were more infectious than transcripts of pDGl-4, which has a 67 nt polyadenylate track (data not shown). The polyadenylate tracks of the RNA 2 transcripts were less disparate. The more infectious transcripts of pDG2-2 had an 89-nt polyadenylate track, whereas that of the less infectious pDG2-4 transcripts was 66 nt. As described above, only two RNA 1 clones that are sources of infectious transcripts were obtained from the more than 3000 transformants screened. Comparisons of the ratio of RNA 1 to RNA 2 in template mixtures and the products of the pCGN 1703-primed reverse transcription reaction suggest that RNA 2 is favored as a template by a factor of about 10 over RNA 1. In addition, inserts corresponding to the entire or nearly the entire CPSMV RNA 1 molecule apparently resulted in unstable plasmids with a deleterious effects on E. co/i. Full-length RNA 1 clones were obtained only from small bacterial colonies, and during attempts at removing the MCS and dC track, clones with about 5 kbp deleted from the central portion of the CPSMV RNA 1 sequence commonly were obtained. Colony counts after growing E. co/i bearing pDGl -2 or pDGl-4 overnight in LB culture containing 150 pg/ml ampicillin revealed that less than 2% of the viable cells had retained the ampicillin resistance character. Plasmid

614

CHEN AND BRUENING

A . . . . . . . . . . . . . . . . ..MK

FFAGQTVXDVIQHVSSPTTN

LRLLSYCNLKKEE.DCKML

:.I:lII.:I.:I...: “GLPEYPADSWLSQLTIE

FTPGMTVSSLLAQVTTNDF”

“FEIFDALLRTKISNSKG ::.

:.

::..

“LYSWMRGVYLSTFKVEVPC

:...

:..I.

LYKHYALFISNLVPRTLRFK

.1,1:.

:

:.

:I..:...:

:

VPMA”RvcSWLSQLWDKIV

I:

l

:.,:1::.1,

LQLVSLAVGAVSSATSSCFQ :. I:IIl...II...

SPVGQATDVSAESQ :.I

. .

CFQFSNSVGDEGIEWDDEL

I..:,

SAIEFFAAEFAVDIEGVHYN

::..I1 :I Ay”GQIRKNPSLLRISVVAY

DDYNSNLLEKDLAGFAQGLS

QFVSGLADWIPSRVKTLAGY

. ...:

:

.:

,,I:11

::

SWGMCVIAGSCALSLLEKAL :I:

.l,:..:l.::,,,l:ll

WISQASETMGWFLDGCRDLP

MALLSLAANCVSTVIVGGFF

AIKEQ..RHRRLLTLSYGAN

::I

APELEQYLQVZGDAVAQGVS

WVQSAMDAX9WINAGCTELI

:I..1

:..,

.

ELLLFCKCQFLEKMQASIVW

CP”AVAACSWITTVNTTIKE .III

:

l

TWGIATLATCSUSLVEKLL

313

CPSNV

326

CP”V

:..I:,.

I:.I:

.I

I:

1.:.

I:

AF”VSD”VA.ETNSYDVYP.F

I:.

99

AAFXXIFAFKKHFDKLLVEY .::.

.::,:

II

SVDAILVSFRKHFEKMVQEY

VAWGLISSSFDLAGIFVRSA

WGAXLTWNKRSRNCAEL

..,:1.111:,.:

179

.l:III:l::

QLLYKMbTWVPTFVRGAvDW

,111::

79 I

,I:1

I..

. . .

199 279

I:.

II:

VANGFLVEPFGLSGIFLRTG

WAAACYNYGTN.SKGFAEM

298

LGLFSTFLRDASWFSENVD

GWLKQISWCQCQFLAKAYIN

100

ll:I.

lllI.II

32K

:..:

PGEKDNAQ......

B SGGVFMLESLAKNLTNFCDG

TLVSIGKTCNAVNSINTAAG

TIKNLVGRLLSMLSNFAYKL

I:.1

IIII:IlII.III.I.I..l

.: I.I~.lI:l:II.:I

TLVSVGKTCTAWAISTCCG

NLKALAGRILGHLRDFIWKT

LGFETRFLADASLLFGEDVD

CTDLAKLNAGAYMHGTSGTR

r KIPFWYAH&SRV%4VI

:ll:II

.:

III:.

SSPVILLEGLA-NFCET QDEL”VLRSLITRGEVMQR. Ill:1

EMIWZGNKVSPTVCGLINKG

I:.:1

:

,.:

::

QDP.NMQILVLLEKGRQHP.KS

.II

:II.:..II

II

GLSKGG..ISPAIINLILKG

RNVXODYWSGYKRQPIWID

DFGAVSSDPSAFAQLIPLIS II:11 .:IIIIIl:I DFlUWTEPSAFAQj4INLIS

:lI:.::..Il,I.I

.I

.

I:1

NIGSMPKWnRAILFGIGVLL WAQ

....

595

CPSMV

593

CPMV

LLEKTN :

:I

I:

. . ..II..

:1..11,

II

.:

TRSQQHAEMEKNLAW”...

.:

I

GR.THSSWYEGIKDKC”LAL I

III

. . . .

GKEKEKTWYQVQVMQAL

.

KNVLAAYKMIKYHK

198

&!AFRN&FTVSLDPNI

299

: Il:IIl11:. II II. DFAFKNRRNVIVQVSNDPAK P

299

NQDAENF......KQRLETP

392

:i:1::::

:.

DTNLVIKTQIADLVDPTKYT :

l:II..:l

::I

SKAYSTEIKDWPVALKIWG

VILATvAGK,@WRFYASHAD

. ..I

I . SMISAGFDIIRPEKLPSEAK

I.. 395

ADFOFDGVIGSPLFSSWPlP I

SLNwIQA”LKEWVCPHHYD

I..),:I,,:::,:..,

199

AIVNCSSNFIECSPES!!

LSKYSEKWLLGAYEFLLCSE

II

NRLIEDFRXELELGEDCVYP

.:I:.l.Il:I.IIl.IIII QFFVFVSTNFLEVSPFAKVXD

HKSATFESHFKSLVEVLELG

.:,,:,:,I.::

100

SQVTKDFQDHYGLGGETVYS

..1..1:

IDDWYNVTCWFGECVGNPM

:I:.:1

IIIlIIlll:l.III: SAPYPINNAGLEEKGICFDS

GWLKAISDLRWFIAKSYCS

..:..II....:II::.II.

KHPFTIFFQGFRTGKSLIM

KESVAFQ.QFRSILDLAVFV

II..

.:.

Il.11.:::

.:.

IDATGDVQENEKVDSSLDDI

.:

:,

.III:Il.l..

1

NXW-.%“NTEQEANLKQLGVK

..:..,::,:/I

:,.:

SVKALPLWQRMVLYTVGQNL

,:,I.::

III::l:I:II

:.,:.

DGRCHNSCYDKSGILRHYT EKRVLYSIPYN..GEYCNAI

::(..(.I

II

DSYHVEATFTSYGDLIAYCK GRYNTVCVIEDYDELVAYLL

,.:

:.

SAPYPLNNADLSEKGMHFDS III

AYDAANFMNQIYTILAWKD

.I:

..lI..l

INDLEQLNRSCSVCGVRGVR

II..I:IllII:III:l::I RNPCDQYWSGYRRQPFVLWD PYDGDDITKNQIYEIKTWFH

:I

::II.::I.I,I:.:.

:.:

YDMYTKEIRDWPWIRVTCG

IVLAAIGGSAFWKVFCQLVG

ETPKEWHLGTEKTKEPSILS

STDLRLGDKQ..YDPFVGGI

::.I

.I.

I

I,...:

492 .I::.

KELNFIGKIGETYYHN(WVS

493

AGNGGHFVGAVASAFAGSQA

591

.iIl.

::1..1:11.:...

SGNGPVLHGVAAGAFSAEPQ

593

58K HELICASE

C AAEKFFDFLEKMHVTEGIG

F,VGNLKKGVWVPLPTKTNLV

:II.:IIlI.

.I:.II.Il::IlIIII.II

III.IIII:I.

GAEEEYFDFLPAEENVSSGVA

MVAGLKQGVYIPLPTKTALV

ETPSEWHLDTPCDKVPSILV

PTDPRIPAQHEGYDPAKSGV

SKYSQPMSALDPELLG

100

DIVEEWFDCMPQEDTFEEV

DLQVAINGLEGNEYMERVPM

ATSEGFPHILTRKSGEKGKG

RFVYGDGEIFDLIPGTSVHE

AYLTLEETCADTVPALVGIE

198

I::I I II. . :I I:II DVLELWHDCAMWDD.FGEV

.I: I:lI II:IIIIl:I: SLEEALNGCEGVEYMERIPL

IIIIIIIIII.I.: III1 ATSEGFPHILSRNGKEKGKR

RFVQZDDCVVSLIPGTTVAX

CPKDEKLPLRKIYEKPKTRC

FTVLPMEYNLVVRRKFLKN

VFI”KNRHRLSCQVGINPYG

“EWSRLANSLLEKGNNI-LTKQVMHLMSEN

IIIlIIII:II:::IIIIII CPKDEKLPHRWFDKPKTRC

II:IIIIlIIIIIIIII.II FTILPPlEYNLVVRRKFLNFV

III IlIIIIlIII.IlI: RFIbLUJR”RLSCQVGTNPYS

IIIIIII .: lIIl::III MEWSR LAARMKEKGNDVLCC

l::I..I::

.I

.I1

III1

Ill

I:...:

II:

QKYAEPMGILDDEVLRHVAT

III

:I:

II.:Il:

:..IIIII.l

II

INELCGGSSRLKQQRTNLLM

ACCSRYALCKGEVWR&I

PSGFPLTVICNSIFNELLVk

YSYIKIC-VPATITYGF

IIIIIII...II..I III1 INELCGGEDQLKNARRNLLH

lIIIl.I:II..IlIlIIII ACCSRLAICKNTVWRVECGI

IIIIl:lII.IiIIil:I:I PSGFPMTVIVNSIFNEILIR

.:...I. :. I I I: YHYKKLMRECQAPELMTQSF

ITDGKDKTSPVLNFRNLEDC~KKESDWWJGPEE

.I:1 :I. lIl.III: I VTPYFDGKKLKQSLAQGGVT

IlIlllIII

I

II.Il:l

AYEELFASAHRFVPALVGIE

ITDGKDKTSLELPFRRLEEC

.

I99 298

IIIIIIIlI.IIIl.::..I DYSSFDGLLSKQVNDVIA 1

299

1

STFVXJP.‘TY%~NLLSVQSA :,

..::

398

:I,,,,,,,:II...

DKLIGLVTYGDDNLISWAV

399

3 AYL V,&“,NVIRELYLHDPRE

KESLWAQLHWTNNLEKHE

illII.I

.II.

IIIIIIII

.II...

2 IT”VFDGTKLKEFLKLNGIT

98

Il.l:I

I..:1

I.lII.IIIII..II

:II:

DFLKRTFVQRSSTIWDAPED

II.

498

III.IIIII:IlII:l.IiI

KASLWSQLHWNCNNCEKEV

AYLTNWNVLRELYMHSPRE

VAPHRKMHEAEM..ELV?KW

YLRDLGKAPINVLTGWRI

499

4 AAELRRKAIQNVDFLKENPK

DLPTMAAIKEFYN”QRQCQF

,.l:ll,.:..I.::...

IIIl:I.:.III:

VISNDNLDSLLNPDFL...F

IIII.

ATEFRRKVLKKVSWITSG..

DLPTLAQLQEFYEYQRQQGG

CVLVNASIPDHLLPEKVVNI

SWPYGPGRGGLPTHGWAQAN

1.1.

.I:

II:

.:

~FLFNTLYPQSSLPDGCHSV

VMENAKAFKFLPEEFNFAFS

l.llIIIII::I

[email protected]

IIDSAKSLKFLPKECDIIF. :::.II.:IIII.I

.I.

::

.

712

CPS”V

DV

711

CP”V

I

.I.II.

I

I

..I:1

:

I,:..:

1:I.I

I

.:.

593 :.,

ADNNDTCDLLTSVDLLGPPL

SFEKE+MHGCKVSEEIVTK.

. . . ..NLAYyDFKRKGEDEV

LYNPNSAVVKKLRTLVNQNP ,:..:l.:l:I,I

DDRVDICFRHDAVPVAIATI ..II.l..:,,,.,..::

IFLVHLGKVKGRSANEYLTK I: :.. .I.

KKRVIFCARDN~~V~VWIVAL

LCA~RJ~KLWPTAVSNATLVK

ISRKDSNINKIIRTAV..SS

. .

: 591 693 I.1 689

87K POLYMERASE

FIG. 5. Comparison of the deduced amino acid sequence of three genes of CPSMV RNA 1 and CPMV RNA 1. The alignment was performed by the program GAP of the GCG package using the algorithm of Needleman and Wunsch (1970) and a symbol comparison table based on the

COWPEA SEVERE MOSAIC

yields after overnight growth of bacteria at 37” were 2 to 8 pg/O.5 ml culture for RNA 2 clones, about 10 pg/ 100 ml for RNA 1 clones with the MCS and dC track, and about 5 pg/l for RNA 1 clones without the MCS and dC track. Washing the cells twice during growth of the E. co/i culture and suspending them in fresh, ampicillin-containing medium resulted in more than 80% of the cells retaining ampicillin resistance after a final, overnight incubation. The yield of plasmids pDGl-2 or pDGl-4 under these conditions was 150 to 400 pg/50 ml of culture. Presumably, if the ampicillin is not replenished by the wash procedure, the P-lactamase secreted by a small proportion of plasmid-bearing cells was sufficient to protect the bulk of cells that had lost the plasmid. The cause of the instability of CPSMV RNA 1 clones is unknown. Nucleotide sequence CPSMV RNA 1, as derived from clone pDGl-2, and probable locations of polyprotein cleavage sites Clone pDGl-2 was selected as the template for determination of the nucleotide sequence of RNA 1. The nucleotide sequence of pDG2-2 already is known, from that of its precursor pDG2-1 (Chen and Bruening, 1992). Completely overlapping sequences were obtained from subclones of pDGl-2 for both strands of the cDNA insert (Fig. 2). The sequences of the two strands were perfectly complementary. The length of the cDNA insert was determined to be 5957 nt, exclusive of the 3’-polyadenylate. Analyses for VPg of comovirus RNAs (Daubert and Bruening, 1979) and nt sequence determination of comovirus RNAs and cDNA clones (CPMV RNA 1, Lomonossoff and Shanks, 1983; CPMV RNA 2, van Wezenbeek et al., 1983; bean pod mottle virus RNA 2, Macfarlane et al., 1991; RCMV RNA 2, Shanks et a/., 1986; CPSMV RNA 2, Chen and Bruening, 1992) suggest that all comovirus RNAs may begin with the same VPg-UAUUAAAAU . structure. CPSMV RNA 1 and CPSMV RNA 2 have the first 12 nt in common and have identities of 88% for the first 150 nt and 66.5% for the entire 5’ noncoding sequence. The 3’ noncoding region of the two RNAs, however, have only 60% identity for the 3’-most 62 nt.

VIRUS RNA 1

615

A search of the six reading registers revealed a long open reading frame (ORF) only for one register of the genomic sense RNA (Fig. 2). The long ORF, which starts with an AUG at position 257 and ends with an UGA beginning at position 5831, is 1858 codons long (Fig. 3) encoding a polyprotein with a molecularweight of 208,000. The longest ORF in the negative polarity has only 102 codons. A comparison of the sequences of aa deduced for the polyprotein of CPSMV RNA 1 (Fig. 3) the polyprotein of CPMV RNA 1 (Lomonossoff and Shanks, 1983) and a RCMV RNA 1 polyprotein fragment (Shanks and Lomonossoff, 1990) suggests that the presumed polyprotein processing sites divide the RNA 1 polyproteins of these viruses into proteins of very similar sizes and similar sequences (Fig. 4). Of the deduced or determined cleavage sites in the RNA 1 and RNA 2 polyproteins of BPMV, CPMV, CPSMV, and RCMV, the three locations that have identical aa pairs defining the cleaved peptide bond all are located in the RNA 1 polyprotein, at 32W58K, 58KNPg, and VPg/ 24K. The fourth RNA 1 polyprotein site, at the 24W87K juncture, is Q/A for CPSMV and Q/G for CPMV and RCMV. No strong consensus sequence was observed for the cleavage sites of the RNA 1 polyproteins. For polyproteins of CPSMV, the six sites may be summarized as (A,V)X(A,G,L,S)Q/(A,M,S), and for CPMV they are (A,D)X(A,P)Q/(G,M,S). The corresponding representation for five RCMV cleavage sites is (A,G)X(A,P)Q/(G,M,S,T) and for two BPMV sites is (T,V)X(A,P)Q/ (MS). For the -4 position in 19 cleavage sites of BPMV, CPMV, CPSMV, and RCMV, the count is 13 A, 3 V, and 1 each for D, G, and T. The -2 position of BPMV, CPMV, and RCMV is occupied by A and P only, whereas occupation of this site is 3 A and 1 each of G, L, and S for CPSMV. The aa count at the +l sites for the four viruses is 8 S, 5 M, 4 G, and 1 each of A and T. No other pattern of conserved aa was obvious in the vicinity of the polyprotein cleavage sites. The alignments of the RNA 1 polyprotein aa sequences of CPMV, CPSMV, and RCMV show many patches of local identity (Figs. 5, 6). The alignment of the VPg for three viruses showed that each has 28 aa

evolutionary distance between the amino acids as presented by Gribskov and Burgess (1986). Residues that are identical in the two sequences are Indicated by vertical lines. Evolutionarily similar amino acid pairs are indicated by either a colon (more closely related) or a dot (less closely related). (A) Comparison of the sequence of the 32K protein gene. (6) Comparison of the sequence of the 58K putative helicase. Regions I and II, marked by brackets above the CPSMV sequence, show similarities to the 72K protein of tomato blackring nepovirus (Habili and Symons, 1989); to the Cl protein of tobacco vein mottling potyvirus, tobacco etch potyvirus, and plum pox potyvirus; and to the 2C protein of the picornaviruses polrovirus and foot-and-mouth disease virus (Gorbalenya ef al., 1989a; Habili and Symons, 1989). The rNTP-binding motif A of region I and motif B of region II are indicated by aa marked *. (C) Comparison of the sequence of the 87K putative polymerase. Regions 1, 2, 3, and 4 show similarity to the nepovirus 92K protein, potyvirus Nlb protein and pocornavirus 3D protein (Argos et a/., 1984; Habili and Symons, 1989). An identifiable regron 3 is present In many DNA and RNA polymerases, some of which use DNA and others use RNA as template (Argos, 1988). The “GDD” aa sequence, present in all RNA dependent RNA or DNA polymerases examined thus far (Argos, 1988) is marked with the symbol +++.

616

CHEN AND SRUENING A

SRXPNRFDvaQYRYrNIPLrkRnWaegQ CPSMV SRKPNRFDm~YRY"NVPLkrRvWadaQ CPMV SRKPNFCFEvqQYRYkNVPLtrRsWgnaQ RCNV CO"S&UUS -#II QYNY N#PL XR W###Q 1 26

B n MsLoQStmlIMeKCkAnFVfsniscQIVMlPGRqFlcYkTivFaslnspMyVdIytankkY MSLDQSsvaIMsKCrAnLVfggt"lQIVPGRrFlaCkHfFthiktkLrVeI~dgrrY MSLDQStvsILnKChAkFIiasqhaQI~vPGRrFigYsHfFcnlkhp~VqIeta~tY NSLDQS## I# 1oc A # # QIVSXPGR PX HF c IVXI 1 40 44

XY 60

q

klyYkPqNrvyfetDSEimlYkdasLEDIpaSCWDLFCFDaEKsLPrgsF~E~lSCKld yhqFdPaNiydip.DSElvlYshpsLEDVshSCWLFCWDpDKeLPs.vFgADflSCKy" fhrYqPeMneyie.DSElcvYhsscLEDIshSCWDLFCWDpDKeLPk.kFsADfvSCKyn DSS# XY , IZD# .¶CNDLFC#D #K LP XPN PA##SfX# 77 95

120

0

rttnqhipeWAdIsartvnqkLdVefGeYqtifysylqYDv~kaeDCGSLIIatIdGrk kfggfyeaqYAdIkvrtkkecLtIqsG"YvnkvsryleYEapTipeDCGSLVIahIgGkh twtksveptWAnVdaevikedFtIcdGeYrntvstsirYEapTvmsDCGSMIItnVgGkt x!GS#XIX x #A## I: x G#Y XYX T 168

I Gu 180

*** KIIGIHtAGranrsGfAS*yM&eipvQAQ CPSMV KIVGVHvAGiqgkiGCASlLPplepiaQAQ CPNV KIVGIHvAGrdnkiGmASlLPpllpcaQAQ Fu2MV lD#G#H AG WGASXPX QAQ co"se"sus 210 FIG. 6. Multiple sequence alignment of the VPg and 24K proteinase of three comoviruses. The program “pileup” of the GCG package generated the alignments for aa sequences from CPSMV, CPMV, and RCMV. The derived consensus is shown below the aligned sequences. An aa that is identical among the three sequences is designated by its single-letter code in the consensus. # locates physiochemically conserved aa (Bordo and Agos, 1991). (A) The 28 aa VPg sequences of the three viruses are aligned. (El) Alignment of the 24K proteinase sequences shows a catalytic triad (0) identified by mutagenesis studies of Dessens and Lomonossoff (1991). The location of a loop, proposed (Gorbalenya ef al., 1989b) to connect two putative b-barrel domains of a “trypsin-like” proteinase, is indicated by a bracket under the sequence. The residues designated by Bazan and Fletterick (1988) as being part of the substrate binding pocket are indicated by asterisks. Numbering of aa is based on the 24K protein of CPSMV.

and that dissimilar aa occur at only four locations. Among the 24 aa showing similarity or identity, 6 are either lysine or arginine residues and only one is an aspar-tic or glutamic acid residue, giving the consensus molecule a substantial formal positive charge at neutral pH (Fig. 6A). Sequence comparisons of the VPg24K proteinase region suggest that CPMV and RCMV are more closely related to each other than either of them is to CPSMV. This conclusion previously was drawn from a comparison of the RNA 2 sequences of the three comoviruses (Chen and Bruening, 1992). Comovirus

24K proteinases

Sequence analyses and experiments have revealed three features that are common to the 24K comovirus

proteinases and picornavirus 3C proteinases: (i) a catalytic triad of cysteine, in place of the usual serine of a serine proteinase, histidine, and either aspartic acid or glutamic acid; (ii) a loop structure connecting the two putative six-stranded P-barrels that also are characteristic of chymotrypsin-like serine proteinases, and (iii) a presumed substrate-binding pocket (Kraut, 1977; Bazan and Fletterick, 1988, 1989; Gorbalenya et al., 198913; Shanks and Lomonossoff, 1990). In the 24K proteinase sequences of three comoviruses (Fig. 6B) cysteine residues are conserved at five locations, histidine residues at two, aspartic acid residues at six, and glutamic acid residues at two (Fig. 6B). The cited references are in agreement on the identity of the active site cysteine, corresponding to cysteine-168 in the CPSMV 24K proteinase. Disagreement about the identity of the histidine and acidic amino acid residues has been resolved for CPMV by analysis of mutant 24K proteinases (Dessens and Lomonossoff, 1991). The corresponding aa of CPSMV 24K proteinase are histidine-40 and glutamic acid-77. For the picornavirus 3C proteinases, evidence from site-directed mutagenesis also favors the glutamic acid-77 assignment (Hammerle et al., 199 1). The 7 aa assigned by Bazan and Fletterick (1988) to be part of the substrate-binding pocket were conserved between CPMV and RCMV (Shanks and Lomonossoff, 1990), but the sequence differs at two positions when comparisons with CPSMV are made (asterisks, Fig. 6B). Of the five proposed and confirmed polyprotein cleavage sites of both CPMV and RCMV, four have the cleaved peptide bond defined by the same aa pair. As is indicated above, of the six cleavage sites of CPSMV and CPMV, only three have the same aa pair. Thus it would be reasonable for the similarity between the postulated substrate-binding pockets of the CPMV and the RCMV 24K proteinases to be greater than the similarity between those of CPMV and CPSMV. The cleavage site with the most varied aa pair is that between the movement protein and the L coat protein, Q/S for CPSMV, Q/M for CPMV and BPMV, and Q/T for RCMV. In vitro analysis of cleavage at this site shows that neither the CPMV proteinase activity nor the CPSMV proteinase activity cleaves the reciprocal RNA 2 polyprotein (Goldbach and Krijt, 1982; Bruening et a/., 1987). It is this cleavage that was assayed in measurements of crude preparations of the inhibitor of polyprotein processing derived from the Arlington cowpea. The specificities and the differences in the putative proteinase binding pockets of the CPSMV and CPMV proteinases are consistent with the specificity of the inhibitor, which acted much more strongly against the proteinase activity of CPMV than that of CPSMVin vitro

COWPEA SEVERE MOSAIC

(Ponz et a/., 1988). Although the differences in the presumed substrate-binding pocket of the CPSMV and CPMV 24K proteinases may be sufficient to explain differences in substrate and inhibitor specificities for cleavage between the movement protein and the L coat protein in the RNA 2 polyprotein, the critical difference could instead or also reside in the 32K protein, which acts as a cofactor of the 24K proteinase in cleavage at this site (Vos et al., 1988). Of the 5 RNA 1-encoded proteins, 32K shows the greatest difference in aa composition between CPSMV and CPMV (Fig. 4). ACKNOWLEDGMENTS We are grateful to Danny C. Alexander and Christopher Glascock of Calgene, Inc., Davis, CA, for providing us with plasmid pCGN 1703 and the Apal adaptor and to Catherine A. Chay of this Department for tailing the plasmid with oligodeoxyribothymidylate. We appreciate advice and instruction on the use of the GCG package from personnel of University of California at Davis Computer Services. We thank Paul Feldstein and Steve Dauberi for valuable discussions and advice and an anonymous reviewer of the manuscript for pointing out the conservation of aa at position -2 of the polyprotein cleavage sites of comoviruses. This research was supported by the Division of Energy Biosciences of the U.S. Department of Energy under Award DE-FG-86ER13353 and by the Agricultural Experiment Station of the University of California. X. Chen was the recipient of a Regent’s Fellowship from University of California, Davis.

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