Control and expression of 3′ open reading frames in clover yellow mosaic virus

179, 576-584

VIROLOGY

(1990)

Control and Expression of 3’ Open Reading Frames in Clover Yellow Mosaic Virus K. ANDREW Department

of Biochemistry,

WHITE

University

Received

May

AND

of Western 14, 1990;

GEORGE A. MACKIE’ Ontario, accepted

London,

Ontario,

Canada

N6A

5Cl

July 17, 1990

The genomic RNA of clover yellow mosaic virus (CYMV) contains at least seven open reading frames (ORFs) which are organized in a more elaborate array than in other sequenced members of the potexvirus group. We have investigated the strategy by which ORFs located in the 3’ region of CYMV’s genomic RNA are differentially expressed by correlating the location of the 5’ termini of the two abundant viral subgenomic RNAs with their coding potential. We have mapped the 5’termini of the subgenomic RNAs precisely to the nucleotide level and have shown that both are capped. The larger 2.1-kb subgenomic RNA encodes as its 5’ ORF a 25-kDa polypeptide, whose function is unknown, The smaller 1 .O-kb subgenomic RNA can encode only the 23-kDa coat protein. All four ORFs in the 3’ 1095 residues of CYMV are efficiently expressed in vitro, but of these only coat protein, which can be expressed from a subgenomic RNA, is detectable in CYMV infected tissue. For this reason, we believe that expression of ORFs in the 3’ one-third of CYMV RNA are controlled at the transcriptional level. o 1990Academic PWS, IIIC.

INTRODUCTION

tato virus X (PVX; Dolja et al., 1987) daphne virus X (DVX; Guilford and Forster, 1986) narcissus mosaic virus (NMV; Short and Davies, 1983; Mackie and Bancroft, 1986), papaya mosaic virus (PMV), and foxtail mosaic virus (FoMV; Mackie eta/., 1988). We presume that sgRNAs are a means of selecting and potentially amplifying certain ORFs in the viral genome to achieve preferential expression of a subset of viral gene products. Despite their importance, the mechanism by which potexvirus sgRNAs are synthesized is unclear. By analogy to brome mosaic virus (BMV) RNA3, it seems most likely that sgRNAs result from the partial copying of a full-length negative-stranded template (Miller et al., 1985). In this regard it is significant that no negativestranded RNAs corresponding to the size of sgRNAs could be detected in tissue infected with PVX (Dolja et a/., 1987) or with CYMV, PMV, or FoMV (Mackie et a/., 1988). Rather, the full-length negative strand must contain internal sequences (promoters) which serve to direct the initiation of synthesis of sgRNAs. Conserved sequences located 5’ to the coat protein ORF have been found in several potexviruses (Skryabin et a/., 1988). Their functional significance is unproven. If these conserved sequences do represent promoters they should occur close to the 5’ end of all potexvirus sgRNAs. We have sought to clarify the apparent complex coding organization which is found in CYMV, but not in other sequenced members of the potexvirus group. We have examined strategies employed for the selective expression of certain gene products of CYMV (and possibly other potexviruses). The termini of the major

The potexviruses constitute a group of related flexible rod-shaped viruses with lengths between 470 and 580 nm which contain a single strand of positively sensed RNA. Clover yellow mosaic virus (CYMV) possesses the largest RNA yet sequenced of any member of this family (Sit et a/., 1990). The genomic RNA (gRNA) of this virus contains 7015 nucleotides, excluding a poly-A tail of 75-100 residues (AbouHaidar, 1983). An analysis of the nucleotide sequence of CYMV RNA has revealed the presence of open reading frames (ORFs) capable of encoding coat protein and at least six additional polypeptides. The largest of these, the product of ORF 1, would be a protein of at least 191 kDa. A product of approximately 182 kDa has been identified by translation of CYMV RNA in vitro (Bendena and Mackie, 1986) and after infection of pea mesophyll protoplasts in viva (Brown and Wood, 1987). The extent to which the five other ORFs are expressed in vivo, and their functional roles, if expressed, remain unclear. Systemic infection of broad bean leaves with CYMV results in the accumulation of polyribosome-associated subgenomic RNAs (sgRNAs) of approximately 2.1 and 1 .O kb (Bendena et al., 1987). The smaller is likely to be the physiologically relevant template for coat protein synthesis since CYMV RNAs enriched for the 1 .O-kb species are efficient templates for the synthesis of coat protein in vitro (Bendena et al., 1987). Comparably sized sgRNAs have been detected in plant tissue infected with other potexviruses, including po’ Author 0042-6822/90

to whom

requests

for reprints

$3.00

CopyrIght 0 1990 by Academic Press, Inc. All rights of reproductnn in any form reserved.

should

be addressed. 576

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sgRNAs encoded by CYMV were detelmined, information was correlated with the expression eral of the potential ORFs in viva and in vitro. sion of ORFs located in the 3’ region of CYMV’s appears to be controlled primarily at the level scription. MATERIALS

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and this of sevExpresgRNA of tran-

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METHODS

Virus and RNA CYMV was isolated from leaves of infected broad bean plants by using the method of Bancroft et al. (1979) and its RNA extracted as described by Erickson and Bancroft (1978). Polyribosomes were prepared from infected and uninfected leaf tissue following the method of Palukaitis (1984). RNA was extracted from ribonucleoprotein particles or from polyribosomes as described by Bendena et al. (1987). Agarose gels were prepared and run as described by McMaster and Carmichael (1977). Conditions for blotting and hybridization are those of Mackie (1986). Molecular

cloning

of CYMV

A modification of the Gubler and Hoffman (1983) method was used to synthesize double-stranded cDNA of CYMV. First-strand synthesis was carried out at 50”, priming with an oligonucleotide (CY-1; residues 7015 to 6993) complementary to the first 23 residues 5’ to the poly-A tract in the viral RNA. Following second-strand synthesis, the double-stranded cDNAs were “blunt-ended” with T4 DNA polymerase, EcoRl methylated, ligated to EcoRl linkers, and ligated into the vector pSP65. Transformation of Escherichia co/i MM294 was performed by standard procedures (Maniatis eT a/., 1982). Construction transcripts

and expression

of truncated

A plasmid (pCY935) containing the terminal 1456 residues from CYMV’s 3’ end was opened near the 5’ end of the CYMV sequence at a unique BarnHI site (see Fig. 1a). The DNA was treated with Ba/31 nuclease (Bethesda Research Laboratories) for 40 min at 37” (enzyme to fragment ratio of 0.2 unit/pmol). Truncated fragments were fractionated on a polyacrylamide gel after digestion with Pstl (see Fig. 1a). Two sets of fragments were generated after digestion with Pstl, one containing only CYMV sequences upstream of the Pstl site and the other containing the remaining CYMV sequences and the vector. DNA fragments containing only CYMV DNA sequences were eluted from the gel and ligated into a construct of pGEM-4 containing CYMV sequences distal to the Pstl site. This regener-

FIG. 1. Schematic diagram of 5’-deleted CYMV cDNAs encoding the 3’ portion of the vtral RNA. (a) The organization of ORFs in CYMV RNA. ORFs are labeled in brackets and the A4r values of the proterns encoded by the respective ORFs are shown. The relative position of plasmid pCY935 which was used in the construction of the truncated messages IS shown in the upper portion of the diagram. (b) An expanded version of the ORFs encoded on the 3’ portion of CYMV. (c) PosItions of the 5’ termini of CYMV cDNAs In relation to the 3’ ORFs of CYMV.

ated continuous CYMV cDNAs with 5’ends of different lengths. ln vitro transcription using T7 RNA polymerase was carried out using approximately 1.2 pmol of each different deletion construct as a template to produce positive-sense RNA truncated at the 5’ end. An additional 33 residues derived from the vector were present at the 5’ end of the in vitro transcripts. No attempt was made to cap these transcripts. An aliquot of the unpurified transcription reaction was added directly to a rabbit reticulocyte lysate (Promega Biotec., Inc.) containing [3H]leucine (25 &i) and incubated at 37” for 40 min. The products of translation were separated on a standard 11% polyacrylamide gel containing 0.1% SDS (Laemmli and Favre, 1975) or on a high resolution SDS-urea gel (Fling and Gregerson, 1986) with the following modifications (suggested by G. Dallmann of this department): a 20% polyacrylamide gel containing 3 M urea and 0.1% SDS was used in a minigei apparatus while the buffer system was that of Schagger and von Jagow (1987). Isolation and analysis of total protein CYMV-infected leaves

extracts

from

Samples containing 1 g (wet weight) of CYMV-infected broad bean leaves were ground in liquid nitrogen and boiled in 3 ml of SDS sample buffer for 15 min. Samples were chilled on ice and then centrifuged to pellet leaf debris. The supernatants were collected and

578

WHITE

frozen at -50”. Aliquots (2 pg) were separated electrophoretically on a SDS-polyacrylamide minigel, transferred to nitrocellulose, blocked, and incubated with coat protein antibody (titer of l/51 2, used in a 1: 1000 dilution) as described by Dunn (1986). Immunochemical detection of peptides recognized by CYMV coat protein antibodies was accomplished using a phosphatase conjugated goat anti-rabbit serum (Dunn, 1986). The blot was rinsed in 50 mM Tris-HCI, pH 8, and 100 mM NaCl for 1 min, and subsequently incubated in 50 mM Tris-HCI, pH 8, 100 mM NaCI, 5 mM MgCI,, 1 mg/ml cY-naphthol phosphate, and 1 mg/ml Fast blue RR for 5 min at room temperature (S. D. Dunn, personal communication).

Primer

extension

analysis

Primer extension experiments to map the 5’ ends of CYMV sgRNAs were performed on total RNA extracted from CYMV particles and on total polyribosomal RNA isolated from infected leaf tissue. Both RNA preparations showed the presence of 2.1- and 1 .O-kb sgRNAs when analyzed by Northern blot analysis. No further purification of the sgRNAs was carried out since their proportion in the preparations was considered acceptable for primer extension analysis. CYMV RNAs were decapped chemically with sodium periodate and aniline under conditions identical with those used by Flavell et a/. (1980). For all experiments a 5’-32P-labeled oligonucleotide (1 pmol) was mixed with 0.5 pg of native or decapped RNA extracted from CYMV ribonucleoprotein particles or with 5 Fg of native or decapped polyribosomal RNA from infected or uninfected plants and heated at 90” for 2 min. Primers were allowed to anneal at 45” for 10 min, and then at room temperature for 10 min. The extension reaction was carried out at 50” for 40 min in a buffer containing 50 mM Tris-HCI, pH 8.3,75 mM KCI, 3 mM MgCI,, 10 mMdithiothreitol, 0.5 mM each of the four deoxyribonucleoside triphosphates, and 200 units of M-MLV reverse transcriptase (BRL Inc.). The primers were phosphorylated at their 5’ ends before synthesis of the marker sequence ladder to ensure that primer extension products and chainterminated markers of the same length would display equivalent electrophoretic mobilities.

Oligonucleotides Oligonucleotides listed in Table 1 were synthesized on an Applied Biosystems 380A DNA synthesizer according to the manufacturer’s procedures.

AND

MACKIE

RESULTS Coding properties of open reading frames residues 5920 to 7015 in CYMV RNA

spanning

Sequence analysis of CYMV RNA has revealed two ORFs immediately upstream of and in-frame with the coat protein message (Fig. 1 b; AbouHaidar and Lai, 1989; Sit eta/., 1990). These are ORF 5 (residues 6105 to 6875) and ORF 5a (residues 6129 to 6875) which coterminate with coat protein, and which are predicted to encode 28- and 27-kDa products, respectively, while coat protein is predicted to be 23 kDa (Fig. 1b). The product of ORF 5 is a candidate for a protein identified previously by Bendena et al. (1987) which coprecipitated with coat protein antibody. The electrophoretie mobility of this protein, as determined by Bendena et a/. (1987) suggested a somewhat higher molecular weight, approximately 32,000, than the size predicted by the extent of ORF 5. This difference may be due to possible shape and hydration effects. Upstream of these is ORF 4, which is predicted to generate a 6.5kDa product in a different frame. We determined whether proteins can be expressed from these ORFs which were deduced from the CYMV sequence. We prepared a set of truncated viral cDNAs in the vector pGEM4, spanning the region of the initiating codons of ORFs 4, 5, 5a, and coat protein in the 3’ region of CYMV as summarized in Fig. lc. The products of translation from the appropriate in vitro transcripts were separated on a SDS-polyacrylamide gel or on a high-resolution SDS-urea gel (Fig. 2). Longer transcripts from pCYBA5920, pCYBA6004, and pCYBA6069, which leave the AUG codon of ORF 5 intact, serve as templates for a polypeptide with an apparent molecularweight of 30 kDa (Fig. 2, lanes c to e). Our in vitro 30-kDa product translated from cDNAderived transcripts has the same electrophoretic mobility as the product described by Bendena et al. (1987) which is translated from CYMV gRNA when both products are separated in the same gel (data not shown). The readthrough product of 30 to 32 kDa described by Bendena et al. (1987) and the 30-kDa product in our experiment is therefore the predicted 28-kDa product of ORF 5. A comparison of the intensities of the products produced from the longer transcripts show that they direct the synthesis of the 30-kDa polypeptide more efficiently than that of a 29-kDa polypeptide or a coat protein polypeptide (23 kDa) encoded by the ORFs embedded within the mRNA for ORF 5 (Fig. 2, lanes c to e). Larger deletions (pCYBA6123 to pCYBA6283) which remove the initiating codon of ORF 5 do not serve as templates for the 30-kDa peptide (Fig. 2, lanes f to I). Translation of transcripts prepared

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abcdefghijkl 119-

45-

30-

CP-

Zl14-

m

noPq

7.53.5-

FIG. 2. Translatron in vitro of truncated CYMV transcripts. Transcriptron in vitro of truncated cDNAs was carried out with approximately 1.2 pmol of each deleted template using T7 RNA polymerase (70 U) In a volume of 30 ~1. Followrng transcription an aliquot (4 ~1) of each of the unpurified in vitro generated transcripts was added to a rabbit reticulocyte lysate containing [3H]leucine. The reaction was termrnated by the addition of an equal volume of twice-concentrated SDS sample buffer followed by boiling. In lanes a-l a portion of each translatron reactron mixture was applied to an 1 1% polyacrylamide gel contarnrng 0.1% SDS without further processing. In lanes m-q selected translation Incubations were applied to a high-resolution 20% polyacrylamide gel containing 3 n/l urea and 0.1% SDS without further processrng. The templates were (a) no added RNA; (b) 0.5 pg of BMV RNA; (c) pCYBA5920 transcript; (d) pCYBA6004 transcript; (e) pCYBA6069 transcript; (f) pCYBA6123 transcrrpt; (g) pCYBA6 153 transcript; (h) pCYBA6 174 transcript; (i) pCYBA6 187 transcript; ij) pCYBA6220 transcript; (k) pCYBA6269 transcript; (I) pCYBA6283 transcript; (m) sample as In (a); (n) sample as in (c); (0) sample as In (d); (p) sample as In(e); (q) sample as in(f). The numbers in the left margrn grve the molecular weights (in thousands) of marker proteins separated in the same gels. CP denotes the positron of authentic CYMV coat protern tn the same gel. Both fluorograms were exposed for 2 weeks.

from pCYBA6 123, which lacks the start codon for ORF 5 but possesses ORF 5a’s AUG, results in the synthesis of an approximately 29-kDa polypeptide (Fig. 2, lane f). The relative intensity of this 29-kDa product increases when ORF 5a is the first coding sequence in the message (cf. Fig. 2, lane f, with Fig. 2, lane e). This 29-kDa polypeptide therefore corresponds to the predicted anhydrous product of 27-kDa from ORF 5a. Both the 30- and 29-kDa products (Fig. 2, lanes e and f) are also immunoprecipitated efficiently with coat protein antibody (data not shown).

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Coat protein is efficiently translated as a polypeptide of approximately 23 kDa once the start sites of upstream open reading frames are deleted (transcripts prepared from pCYBA6153 to pCYBA6220; Fig. 2, lanes g to j). This electrophoretic molecular weight is in good agreement with the predicted 23-kDa product of the coat protein ORF. The intensity of the band representing coat protein increases in lanes g to j of Fig. 2 compared with the intensity of the same product translated from longer messages (Fig. 2, lanes c to f). Coat protein is no longer translated when its start codon is removed by larger deletions in pCYBA6269 and pCYBA6283 (Fig. 2, lanes k and I). Longer transcripts obtained from pCYBA5920 and pCYBA6004 were able to encode a small polypeptide of the size predicted by ORF 4 (6.5 kDa) in the CYMV sequence (Fig. 2, lanes n and 0). Any one of the four complete ORFs in the 3’ terminal 1096 residues of CYMV RNA (ORFs 4, 5, 5a, and coat protein), therefore, can be expressed efficiently in vitro, provided that it is the first coding sequence in the template RNA. Expression of the products and coat protein in vivo

of ORFs 5, 5a,

In view of the efficient synthesis of the products of ORFs 5 and 5a in vitro, we have examined leaf tissue from CYMV-infected broad bean plants for the presence of comparable proteins which are reactive with coat protein antibody. Total protein extracts from infected tissue were prepared over a period of 12 days from the time of inoculation. Samples from various time points were separated electrophoretically, transferred to nitrocellulose, and probed with coat protein antibody. This immunodetection technique was sufficiently sensitive to detect 5 ng of coat protein. Coat protein was detectable as early as 2 days after infection and continued to accumulate up to 12 days after infection (Fig. 3, lanes c to g). A portion of the infected tissue harvested at the various points after inoculation with CYMV was used to extract polyribosomal RNA. The polyribosomal RNA was analyzed by RNA blotting and the 1 .O-kb sgRNA was found to appear with the same kinetics as the coat protein (data not shown). On the Western blot a second product, smaller than coat protein, also appeared with similar kinetics as coat protein and persisted up to the 12 day time point (Fig. 3, lanes e to g). We assume that this lower band is a degradation product of coat protein. We found no proteins with apparent molecular weights of 30 or 29 kDa which would correspond to the anticipated products of ORF 5 and ORF 5a at any time up to 12 days after infection. If such proteins do accumulate in infected

WHITE

580

a

bcdefg

AND

MACKIE

Kingsbury,

1984; Alquist

and Janda, 1984; Allison et products occurred in the control lanes (Fig. 4, lanes 1 and 2). The presence of two start codons upstream of, and in frame with, the coat protein message defining ORF 5 and ORF 5a (Abouhaidar and Lai, 1989) raised the question of whether there are sgRNAs capable of encoding either of these ORFs at their 5’end. End-labeled oligonucleotide CY-10 (complementary to residues 6192 to 6173) was annealed to the region 3’ to both AUG codons initiating these ORFs and extended on template RNA extracted from CYMV ribonucleoprotein particles or from polyribosomes from CYMV-infected tissue. No primer extension product with intensity greater than background, which could correspond to

al., 1988; Sit et a/., 1990). No specific

FIG. 3. Analysis of total proteins extracted from CYMV-infected broad bean leaves. Aliquots (2 pg) of protein extracted from CYMVinfected leaves at various times after inoculation were separated on an 1 1% polyacrylamide gel containing 0.1% SDS. The gel was blotted electrophoretically for 2 hr to nitrocellulose. lmmunochemical detection of proteins which react with CYMV coat protein antibody was carried out as described. The samples were: (a) 50 ng CYMV coat protein purified from viral particles (Bancroft et al., 1979); (b) total protein extracted at time of inoculation or total protein extracted 2 (c), 4 (d), 6 (e), 8 (f). 12 (g) days postinoculation.

1

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34

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ACGT

tissue, we estimate they occur at less than 109’0 of the level of coat protein, or are unstable.

Identification

of the 5’ termini

of CYMV sgRNAs

Previous work by Bendena ef a/. (1987) identified two prominent subgenomic RNAs of 2.1 and 1 .O kb in the polyribosomal RNA fraction from CYMV-infected broad bean leaves using a probe for the coat protein gene. We have determined the precise 5’ termini of these sgRNAs using primer extension. A purified 5’-32P-labeled oligonucleotide (CY-7) complementary to residues 6322 to 6303 of the region downstream from the initiating codon of the predicted open reading frame of coat protein was annealed to native or decapped CYMV RNAs. Two sources of RNA were used for primer extension analysis: total polyribosomes or crude viral particles including encapsidated sgRNAs. Strand extension was carried out at 50” in an effort to minimize inhibitory effects of secondary structure in the template. Electrophoretic analysis of the products showed a single major primer extension product for both the decapped ribonucleoprotein RNA and decapped polyribosomal RNA (Fig. 4, lanes 4 and 6). This product corresponds to extension to residue 6230 (a guanosine) of CYMV’s RNA sequence, 10 bases 5’ to the start codon (residues 6240-6242) for coat protein (Fig. 4). For native RNAs there is an additional extension to one residue past that of the decapped messages (Fig. 4, lanes 3 and 5). We presume this extra band represents incorporation of a residue opposite the cap structure as previously reported (Gupta and

-6230

I---

-CP

-6250

FIG. 4. Determination of the 5’ terminus of the 1 .O kb sgRNA of CYMV. RNA was extracted from CYMV ribonucleoprotein particles or isolated from polyribosomes from CYMV-infected leaves. A portion of each RNA preparation was decapped and both the native and decapped RNAs were analyzed by primer extension with oligonucleotide CY-7. The following RNAs were examined: (lane 1) no RNA; (lane 2) polyribosomal RNA from uninfected leaves; (lane 3) RNA from CYMV ribonucleoprotein particles; (lane 4) decapped RNA from CYMV ribonucleoprotein particles: (lane 5) polyribosomal RNA from CYMV-infected leaves; (lane 6) decapped polyribosomal RNA from CYMV-infected leaves. A, C, G, and T contained the products of a dideoxynucleotide sequencing reaction performed with phosphorylated oligonucleotide CY-7 as primer. The arrow in the right margin indicates the predicted initiating codon for coat protein and the numbers correspond to residues of CYMV’s gRNA according to Sit era/. (1990). The autoradiogram was exposed for 48 hr.

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ACGT

.5080

ORF2*

t-5090

i -5100

1

-0RF

2

FIG. 5. Determinatron of the 5’ termrnus of the 2.1-kb sgRNA of CYMV. RNAs analyzed were as described for Fig. 4 using oligonucleotrde CY-8 as primer (Table 1). (Lane 1) No RNA; (lane 2) polyribosomal RNA from uninfected leaves; (lane 3) RNA from CYMV ribonucleoprotetn partrcles; (lane 4) decapped RNA from CYMV ribonucleoprotein particles; (lane 5) polyribosomal RNA from CYMV-infected leaves; (lane 6) decapped polyribosomal RNA from CYMV-Infected leaves. A, C, G, and T contained the products of a drdeoxynucleotide sequencing reaction performed with phosphorylated oligonucleotide CY-8 as primer. The arrows in the right margin indicate the positrons of the predicted initiating codons for ORFs 2* and 2 and the numbers correspond to residues of CYMV’s gRNA according to Sit er al (1990). The autoradiogram was exposed for 48 hr.

an RNA terminus 5’ to either initiating codon, was observed (data not shown). We conclude, therefore, that there are no abundant sgRNAs initiated adjacent to the 5’ extremity of either ORF 5 or ORF 5a. The 5’ end of the 2.1 -kb sgRNA is predicted to map close to the 5’ region of ORF 2* and ORF 2 (see Fig. 1a). A 5’-labeled oligonucleotide (CY-8, residues 5 15951 39) complementary to a region 3’ to the initiating codons of both ORFs 2* and 2 was annealed to native or decapped CYMV RNAs and extended. In the native polyribosomal RNA fraction extracted from infected tissue the largest primer extension product corresponds to residue 5095 (Fig. 5, lane 5). Below this product are two additional smaller products (corresponding to residues 5096 and 5098) which most likely represent premature stalling of the reverse transcriptase (Fig. 5, lane 5). When decapped polyribosomal RNA from infected tissue is analyzed, there is a single prominent band

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corresponding with residue 5096 (Fig. 5, lane 6). This product is one residue smaller than the largest product in the capped polyribosomal fraction (cf. Fig. 5, lane 5 with lane 6). This difference of one residue in length between the two primer extension products from native and decapped polyribosomal RNA likely results from the presence of a cap structure on native 2.1 -kb sgRNA messages. The position of the bands in lanes 5 and 6 in Fig. 5 locates the 5’ terminus of the 2.1-kb sgRNA between the potential initiating codons of ORFs 2* and 2, specifically at residue 5096, 13 residues 5’to the second AUG. Primer extension products from the analysis of native or decapped RNAs extracted from crude viral particles are similar to those obtained using polyribosomal RNA from infected tissue as templates (cf. Fig. 5, lanes 3 and 4 with lanes 5 and 6). The presence of a primer extension product corresponding to the 5’ terminus of the 2.1 -kb sgRNA in crude viral partcle RNA preparations suggests that a fraction of the 2.1 -kb sgRNA produced during CYMV infections is encapsidated (see below). The absence of a band with lower mobility which would represent the capped form of the 2. I-kb sgRNA isolated from ribonucleoprotein particles (Fig. 5, lane 3) may be due to inefficient capping of the 2.1 -kb sgRNA. The capped fraction of the message may be sequestered on polyribosomes leaving uncapped messages to be encapsidated. Faint bands near coordinates 5081 to 5084 are also visible in Fig. 5, lanes 4, 5, and 6. These bands would correspond to termini located 5’ to the first AUG codon for ORF 2*. The RNA in the 5’ region of ORF 2* was folded using the program of Zuker and Stiegler (1981). Residues 5081 to 5084 lie at the bottom of the stem of a very strong hairpin structure. Therefore, these faint bands may not represent the authentic 5’end of a subgenomic message and more likely result from stalling of the reverse transcriptase on the gRNA. In order to confirm our suspicion, we probed a Northern blot containing the same RNAs used in the primer extension experiment with an end-labeled oligonucleotide (CY-9),

TABLE

1

OLIGONUCLEOTIDESUSEDINTHISWORK Ollgonucleotide CY-1 CY-7

0-a CY-9 CY-10

Sequence 5'.ATCACCCCAAAAGTCTACGGGTC 5'.AGGTTGGACTCTATGGTTAG VGTGGTGCGGGAGTATCCTTC 5'.GCGAGAATCTCATTACC VAGCTGGGAGATGACTTTGGC

a Coordinates given are for positions complementary nucleotide on CYMV RNA as defined for the full-length sequence according to Sit ef al. (1990).

Coordmates" 7015to6993 6322to6303 5159to 5139 5097to 5081 6192to6173

to the oligoCYMV RNA

582

WHITE

a

MACKIE

This finding confirms the implication of our primer extension analysis of RNA from ribonucleoprotein particles which suggested that a fraction of the 2.1-kb sgRNA of CYMV was encapsidated.

b

-

7.2

FIG. 6. CYMV-encoded RNAs from infected broad bean plants. Total polyribosomal RNA isolated from CYMV-infected tissue (5 gg) and total RNA extracted from CYMV particles (0.5 Fg) was denatured, resolved by electrophoresis, and blotted to nylon. The blot was probed with a r”P]RNA probe complementary to the coat protein coding region of CYMV. The samples are: (lane a) total polyribosomal RNA from uninfected plants; (lane b) total polyribosomal RNA from CYMV-infected plants; (lane c) total RNA extracted from CYMV particles. The numbers at the right-hand margin give the inferred sizes of the CYMV RNAs identified by blotting.

complementary to residues 5097 to 508 1 corresponding to the region between the two AUG codons of ORFs 2* and 2. Under conditions of low stringency, this oligonucleotide did not hybridize to the 2.1 -kb message, although it did detect the 7.0-kb genomic RNA efficiently (data not shown). Taken together, our results show that the 2.1 -kb sgRNA encodes the entirety of ORF 2 including its AUG at residues 5110 to 51 12. In contrast, the AUG which initiates ORF 2* (residues 5085 to 5087) is not contained within this sgRNA. CYMV RNAs in infected

AND

plants

We have used an RNA probe complementary to the coat protein region of CYMV RNA to identify sgRNAs in preparations of RNA extracted from polyribosomes or from ribonucleoprotein particles isolated from CYMV infected plants. Polyribosomal RNA from infected tissue contains both a 2.1- and a 1 .O-kb sgRNAs in addition to the full-length gRNA (Fig. 6, lane b; Bendena et a/., 1987). It has been previously shown that the 1 .O-kb sgRNA of CYMV is encapsidated (Bendena et al., 1987). The 2.1 -kb sgRNA is also encapsidated in our preparations (Fig. 6, lane c). This represents the first clear demonstration that a potexvirus sgRNA other than the coat protein message can be encapsidated.

DISCUSSION We have employed several techniques to test predictions based on the nucleotide sequence of CYMV (Sit et a/., 1990) and to identify elements of the replication strategy of CYMV (and of potexviruses in general) which lead to quantitative differences in the expression of the potential viral RNAs and proteins. Bendena et al. (1987) previously identified 2.1- and 1 .O-kb sgRNAs in polyribosomal RNA extracted from infected tissue. We have determined the exact 5’ termini of the 2.1- and 1 .O-kb messages to occur at residues 5096 and 6230, respectively. Assuming that these sgRNAs possess a poly-A tail of 75-l 00 residues(AbouHaidar, 1983) similar to the genomic RNA, these sgRNAs would have predicted lengths of about 0.9 and 2.0 kb,,approximately 100 residues smaller than previously estimated by electrophoresis. Our results show that initiation of synthesis of the 2.1- and 1 .O-kb sgRNAs on a negatively stranded template begins with a guanosine within the conserved sequences 5’-CUCGCA and 5’-CUCGAA, respectively (positive-strand sequences). The conservation of the 5’-CUCGAA sequence in the region where the coat protein sgRNA for PVX is initiated may underscore its potential importance as a site of initiation (Fig. 7). Putative potexvirus subgenomic “promoter” sequences have been identified previously (Skryabin et al., 1988) and extended to CYMV (Sit et a/., 1990). Interestingly, the sgRNA promoter of the alphavirus, Venezuelan equine encephalitis virus (VEE), which shares homology with the proven sgRNA promoter of Sindbis virus (Levis et a/., 1990) also displays partial identity to the putative sgRNA promoter of CYMV (Fig. 7). These similarities suggest that the proposed CYMV .promoter consensus may be functionally significant. The 2.1and 1 .O-kb sgRNAs initiate 13 and 10 residues, respectively, from the start codon (Fig. 7). In PVX, the 5’ terminus of the coat protein sgRNA also begins with a G and is located 5 residues from the initiating codon (Skryabin et a/., 1988; Fig. 7). The significance of the small differences in leader lengths remains to be determined. It is clear, however, that the extreme 5’termini of the coat protein sgRNAs produced by CYMV (5’GAAG. .) and PVX (5’-GAAA. .) as well as the 5’terminal sequence of the 2.1-kb sgRNA of CYMV (5’GCAA. a) all closely resemble the 5’ termini (5’GA&A. .) of the gRNAs of these viruses. In CYMV, both the major sgRNAs are capped. The cap structure

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PRmwrER

5’

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end of

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sgRNAs

583

START

6240 5'...ACGGtJUAAGUUACCCAA

. . . (3nt)

5'...ACcGUU-AGaa-CaCuu

. . . (24nt)

5'....ACGGUUAAGUUu-CC!Au

. . . (10nt)

S'...aucucuACGGcUAAccUAaaugga..........................

I

. . . (Snt)

. . . AUG

cnlv

. . . CUCC$AaA

. . . (ant)

.,.

CYHV 25K

. . . CUvag

. . . (Ont)

. . . AUG I 5650

PVKCP

. . . AUG I 31 (26

mc!P

. . . . CUC+KA

(25nt)

AUG I 5109

CP

S mRNA)

FIG. 7. Comparison of nucleotide sequences surrounding the 5’ termini of CYMV and PVX and VEE sgRNAs. Portions of the genomic (+)-strand RNA of CYMV and of PVX (Sit et al., 1990) are aligned in three regions and portions of VEE gRNA are aligned in two regions. The sequences forming the context about the initiating residue of the sgRNAs for PVX and CYMV are aligned in the center of the diagram 5’to the initiating codons. The initiating residue of VEE’s sgRNA resides within the sgRNA promoter sequence. The 5’terminal residue of each sgRNA IS underlined. Sequences proposed as promoters for PVX (Skryabin et a/., 1988) CYMV (Sit et a/., 1990) and VEE (Kinney eta/., 1986) are located at the 5’ end of the sequences shown. Gaps were introduced for best alignment. Nucleotides identical with the putative CYMV 1 .O-kb sgRNA promoter and Its initiation region are in upper case and boldface.

may play an important role in regulating the translational efficiency and/or the stability of the sgRNAs. A surprising feature of CYMV is the presence of two open reading frames (ORFs 2* and 5) capable of encoding multiple nested polypeptides by virtue of several in-frame AUG codons. The 5’ end of the 2.1 -kb sgRNA is located downstream of the predicted initiation site of ORF 2*, but is upstream of the second inframe AUG of ORF 2. Thus, the larger of the two ORFs (ORF 2*) predicted by sequence analysis is not likely to be the ORF translated in viva and this would mean that ORF 1 of CYMV does not overlap the second functional ORF (ORF 2) on the viral message. This arrangement is in agreement with the organization of most other sequenced potexviruses, including PVX (Huisman et a/., 1988), white clover mosaic virus (WCIMV; Forster et al., 1988), and NMV (Zuidema et al., 1989) with the exception of PMV (Sit et al., 1989). A more extreme example of in-frame overlapping ORFs occurs in ORF 5, which contains two internally nested ORFs (ORF 5a and coat protein). We have shown that ORFs 4, 5, 5a, and coat protein are expressible in vitro. In all cases there was efficient translation of the first ORF on the message and weak expression of downstream ORFs. This is in agreement with the scanning model for initiation of eukaryotic translation (Kozak, 1986). The downstream products which were expressed may have been translated from shorter messages produced by RNA cleavage in the lysate (Bendena et al., 1985). This example illustrates the critical importance of sgRNAs for differential expression of 3’ ORFs on the polycistronic viral message

and shows that major quantitative differences in expression must be transcriptionally mediated. The only major subgenomic RNA whose 5’end maps in ORF 5 is the 1 .O-kb sgRNA which can only encode coat protein. This finding explains why coat protein is synthesized efficiently in infected leaves while the potential products of ORF 5 and ORF 5a (30 and 29 kDa in our experiment; see Fig. 2) are undetectable in vivo, even though they can be efficiently expressed in vitro. ORFs in the 3’ region of CYMV seem to be organized functionally in a similar manner to other sequenced potexviruses where there is a noncoding region between ORF 4 and the capsid coding ORF. In both cases in CYMV where multiple in-frame overlapping ORFs are present (ORFs 2* and 5) only the last ORF of the series is encoded on the sgRNA message. Bendena et a/. (1987) have shown that the 1 .O-kb sgRNA of CYMV can be encapsidated. We have clearly demonstrated that a portion of the 2.1 -kb sgRNA produced during an infection is also encapsidated. ln vitro studies on CYMV gRNA have suggested that there is a 5’ nucleation event followed by 5’ to 3’ encapsidation (AbouHaidar, 1981). The fact that both sgRNAs are derived from the 3’ portion of the genomic RNA challenges the notion of exclusive nucleation at the 5’ end of the gRNA. ln viva there may be different factors governing encapsidation.

ACKNOWLEDGMENTS We thank Dr. J. B. Bancroft for encouragement, drscusston, and gifts of material, and Reg Johnston for preparation of virus and polyribosomes. We also extend thanks to Dr. Stan Dunn and Gary Dallman

584

WHITE

for their guidance in immunological and high-resolution electrophoretie techniques. This work is supported by a grant to G.A.M. from the Medical Research Council (MRC) and a Natural Sciences and Engineering Research Council of Canada grant to 1. B. Bancroft and G.A.M. K.A.W. is a recipient of an MRC studentship.

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