Proc. Natl. Acad. Sci. USAVol. 90, pp. 2055-2059, March 1993Genetics

Genomic position affects the expression of tobacco mosaic virusmovement and coat protein genes

(gene regulatlon/subgenomic mRNA)

JAMES N. CULVER*, KIRSI LEHTOt, SHEILA M. CLOSE, MARK E. HILFt, AND WILLIAM 0. DAWSON§1Department of Plant Pathology and Graduate Genetics Group, University of California, Riverside, CA 92521

Communicated by Robert J. Shepherd, November 23, 1992

ABSTRACT Alterations in the genomic position of thetobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affectedtheir expression. Higher production of 30-kDa protein wascorrelated with increased proximity of the gene to the viral 3'terminus. A mutant placing the 30-kDa open reading frame 207nucleotides nearer the 3' terminus produced at least 4 times thewild-type TMV 30-kDa protein level, while a mutant placingthe 30-kDa open reading frame 470 nucleotides doser to the 3'terminus produced at least 8 times the wild-type TMV 30-kDaprotein level. Increases in 30-kDa protein production were notcorrelated with the subgenomic mRNA promoter (SGP) con-trolling the 30-kDa gene, since mutants with either the native30-kDa SGP or the coat protein SGP in front of the 30-kDagene produced similar levels of 30-kDa protein. Lack of coatprotein did not affect 30-kDa protein expression, since amutant with the coat protein start codon removed did notproduce increased amounts of 30-kDa protein. Effects of genepositioning on coat protein expression were examined by usinga mutant containing two different tandemly positioned tobam-ovirus (TMV and Odontoglossum ringspot virus) coat proteingenes. Only coat protein expressed from the gene positionednearest the 3' viral terminus was detected. Analysis of 30-kDaand coat protein subgenomic mRNAs revealed no proportionalincrease in the levels of mRNA relative to the observed levelsof 30-kDa and coat proteins. This suggests that a translationalmechanism is primarily responsible for the observed effect ofgenomic position on expression of 30-kDa movement and coatprotein genes.

Positive-sense RNA viruses have evolved numerous strate-gies for gene regulation. One common strategy is the use ofsubgenomic mRNAs, transcribed from genomic RNA, for theexpression of internal open reading frames (ORFs). In thismanner the expression of multiple genes residing on a com-mon genomic RNA can be independently regulated. One suchvirus that utilizes this strategy is the type member of thetobamovirus group, tobacco mosaic virus (TMV).The genome ofTMV resides on a single strand of positive-

sense RNA, 6395 nucleotides (nt) in length, and encodes atleast four proteins (1). The TMV genome is organized suchthat two 5'-coterminal ORFs encoding 126-kDa and 18&-kDareplicase proteins are translated from the genomic RNA,while an internal ORF encoding the 30-kDa cell-to-cell-movement protein and a 3'-proximal ORF encoding the coatprotein (structural) are translated from respective subge-nomic mRNAs (2-5). A third subgenomic mRNA has beendetected for an additional internal ORF, within the 183-kDaORF, encoding a putative 54-kDa protein that has not yetbeen detected in infected plants (6). The TMV subgenomic

mRNAs are transcribed from negative-sense genomic RNAand share a common 3' terminus.Of particular interest is that genes ofTMV expressed via

subgenomic mRNAs are independently regulated, both quan-titatively and temporally. The 30-kDa protein is producedtransiently during the early phase of the infection, 2-10 hrafter infection in protoplasts (7) and within the first 24 hr inplanta (8). Additionally, even though the 30-kDa protein isstably associated with the cell wall (9, 10), it accumulates torelatively low levels, ==1% of total plant protein. In contrast,coat protein gene expression reaches a maximum 24-72 hrafter infection and constitutes up to 70% of total plant proteinsynthesis (11). The 30-kDa and coat protein genes haveseveral notable differences. Each is controlled by differentsubgenomic mRNA promoter (SGP) sequences and eachoccupies a different position within the genome. The coatprotein mRNA has a short A+U-rich leader that is termi-nated with a 5' 7-methylguanosine cap structure whereas the30-kDa mRNA has a long leader (75 nt) that is not capped(12).Recent studies have provided some information concern-

ing the regulation of TMV genes expressed via subgenomicmRNAs. Changing the "suboptimal" context of the startcodon ofthe 30-kDamRNA to a more optimal context did notincrease the expression ofthe 30-kDa protein, suggesting thatthis is not a major factor in the regulation of this gene (13).The function of the different SGPs was examined by placingthe 30-kDa gene under the control of the coat protein SGP(12). This rearrangement did not greatly increase the expres-sion ofthe 30-kDa gene but changed the timing of expression.The 30-kDa protein of this mutant, KK6, was produced lateinstead of early, suggesting that different SGPs control thetiming rather than the level of gene expression. A mutantidentical to KK6 except with the entire coat protein ORFremoved, mutant KK8, produced -10 times more 30-kDaprotein than wild-type TMV (12). The reason for this largeincrease in the production of 30-kDa protein by mutant KK8with the 30-kDa gene under the control of the coat proteinSGP and lacking the coat protein ORF was not determined.

In this paper, we present evidence that levels of 30-kDaprotein increase proportionally with the proximity ofthe geneto the viral 3' terminus. This increase was not related to aspecific SGP or to the presence of coat protein. In addition,the tandem placement of two different coat protein genes

Abbreviations: TMV, tobacco mosaic virus; ORSV, Odontoglossumringspot virus; ORF, open reading frame; SGP, subgenomic mRNApromoter.*Present address: Center for Agricultural Biotechnology, Universityof Maryland, College Park, MD 20742.tPresent address: Department of Biology, University of Turku,20500 Turku, Finland.iPresent address: U.S. Department of Agriculture HorticulturalResearch Laboratory, 2120 Camden Road, Orlando, FL 32803.§Present address: Citrus Research and Education Center, Universityof Florida, 700 Experiment Station Road, Lake Alfred, FL 33850.$To whom reprint requests should be addressed.

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within the same genome results in high levels of coat proteinfor only the gene located nearest the viral 3' terminus. Thesedata suggest that tobamovirus gene expression, via subge-nomic mRNAs, is controlled by the position of the geneswithin the viral genome.

MATERIALS AND METHODSViral Constructs. Constructs were derived from pTMV204,

a full-length infectious clone of the Ul strain of TMV (14).The construction of each TMV mutant has previously beendescribed and a diagrammatic representation is presented(Fig. 1). TMV nucleotide numbering is that of Goelet et al.(1). All of the viral constructs used in this study differ fromwild-type TMV 204 by alterations made in the region of thecoat protein ORF. Mutant TMV [-CP] contains an alterationin the coat protein translational start codon changing it fromAUG to AGA (15). Mutant cp35-5 contains a deletion (nt5841-6055) in the coat protein ORF (16). Mutant S3-28 hasthe entire coat protein ORF (nt 5713-6191) removed (16).Mutant TBN62 contains the ORF of the bacterial geneencoding neomycin phosphotransferase (NPT II, 832 nt)inserted into an Xho I site located between the TMV Ul coatprotein SGP and the ORSV (another tobamovirus) coatprotein SGP and ORF in pTBD2 (17). The extra ORSV SGPconsists of 203 nt; thus, the 30-kDa gene of TBN62 ispositioned 1039 nt further from the 3' terminus. Mutant TMV2CP contains the coat protein SGP and corresponding ORFofTMV Ul and ORSV in tandem on the same genome. TMV2CP was constructed by the insertion of an ORSV fragment(Xba I at nt 5563 to Ear I at nt 6182), end-filled, and ligatedinto the Pml I site (nt 6238) of pTMV204.

Inoculations. Infectious RNA of each construct was ob-tained by in vitro transcription of viral cDNAs (14, 18).Transcribed RNA was used to inoculate leaves of Nicotianatabacum (L) cv. Xanthi, a systemic host for TMV, and theviruses were propagated as described previously (16). Inoc-ulated leaves of Xanthi tobacco plants used for proteinextractions were of the same approximate developmentalstage and all inoculated plants were maintained in growthchambers at 25°C, with a 12-hr photoperiod of -20,000 lx.Local lesion assays were performed by virion or infectious

TMVd UfDa

RNA inoculation of the indicator host N. tabacum cv.Xanthi-nc.

Protein Extraction, Cyanogen Bromide Treatment, andWestern Blot Analysis. Both 30-kDa and 126-kDa proteinswere extracted from inoculated Xanthi leaves by publishedmethods (8, 19). Extracted proteins were analyzed by SDS/PAGE (20) and then electroblotted onto nitrocellulose paper(21). Blotted proteins were first probed with a 1:1000 dilutionof antiserum specific either for the 30-kDa protein or for the126-kDa protein (8). Blots were then probed with alkalinephosphatase-conjugated goat anti-rabbit antibody (Calbio-chem) diluted 1:1000 and proteins were visualized by theaddition of S mg of5-bromo-4-chloro-3-indolyl phosphate and10 mg of nitroblue tetrazolium dissolved in 30 ml of 10 mMTris HCl, pH 9.5/100 mM NaCl/5 mM MgCl2. All blockingand incubation steps were done in Tris-buffered saline (50mM Tris HCl, pH 7.4/200 mM NaCl) with 5% dry milk for 2hr at 370C.The 30-kDa and 126-kDa proteins were quantified from

Western blots with a scanning laser densitometer (LKBUltroscan XL). Areas (mm2) under absorbance peaks werethen used to determine ratios of 30-kDa to 126-kDa proteinsfor each sample. A sample represents the protein extractedfrom 1 g of infected Xanthi tissue, suspended in 1 ml ofsample buffer. Equal amounts of each sample were loadedonto gels used for Western blotting of 30-kDa and 126-kDaproteins.Mutant TMV 2CP-infected tissue was ground in 10 mM

phosphate buffer (pH 6.8). Cellular debris was pelleted bycentrifugation (10 min, 10,000 rpm, Sorvall SS-34 rotor). Thesoluble fraction was precipitated with an equal volume ofacetone and centrifuged as before. The resulting pellet wasvacuum dried to a powder, and 2 mg was resuspended in 250,ul of70o formic acid containing 4.5 mg ofcyanogen bromide.This solution was incubated overnight at room temperatureand then subjected to acetone precipitation. The pellet wassuspended in Laemmli sample buffer and boiled for 5 min,and the proteins were resolved by SDS/PAGE. Western blotanalysis was performed with 1:500 dilutions of both TMV Uland ORSV coat protein antisera.RNA Extractions and Northern Blots. Total RNA was

extracted from infected leaf tissue as described by Logemanet al. (22). For blot hybridization analysis, RNA samples

*GAGACP 1 3'

sr.p 207nt deletion

470nt deletionSOpV

SGPV

| -NPT 11 |nCPI

- 35nt InsertSGP SGP

3OkDa I TMV CP I ORSV CP | 39

FIG. 1. Genomic organizationof wild-type TMV 204 and mu-

-3' tants [-CP], cp35-5, S3-28,TBN62, and 2CP. All of the mu-tants were constructed as DNAplasmids from pTMV204 (14). CP,coat protein; NPT II, neomycinphosphotransferase II; ORSV,Odontoglossum ringspot virus.

SOP SaP

MUMkD I 3OkDaTICP 5'-

3OkDa

3OkDa

1-CPJ

cp 35-

S3-28F

TBN62

F2CP

3OkDa

3OkDa

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were denatured with glyoxal and dimethyl sulfoxide, elec-trophoresed in 1% agarose gels (23), and transferred toMagnagraph nylon membranes (0.45 am, Micron Separa-tions, Westboro, MA). Membranes were hybridized withspecific TMV cDNA fragments (nt 3333-5456, specific for30-kDa mRNA, and nt 6207-6406, specific for the viral 3'untranslated region) labeled by random priming with digox-igenin-dUTP (Boehringer Mannheim). Hybridized bandswere visualized by immunochemiluminescence using an anti-digoxigenin antibody conjugated to alkaline phosphatasewith the addition of Lumiphos 530 (Boehringer Mannheim).mRNAs were quantified with the scanning laser densitometer(LKB Ultroscan XL).

RESULTSQuantification of 30-kDa Protein. Obtaining equally in-

fected samples of free-RNA mutants of TMV, mutants thatcannot produce virions, in inoculated leaves is difficultbecause often it is not evident which tissues are infected. Forthis study we selected free-RNA mutants that replicateefficiently and move cell-to-cell essentially like wild-typeTMV (15, 16). However, they move long distances, evenwithin inoculated leaves, poorly. Additionally, many of thecoat protein deletion mutants are symptomless. Thus, com-parison of total viral protein in different samples is affectedby the percentage of cells infected. To overcome samplingerrors in measuring 30-kDa protein, we chose to quantify30-kDa protein relative to the amount of 126-kDa protein.Based on the ability of these mutants to replicate and movewithin an inoculated leaf, we have found the level of 126-kDaprotein to be representative of the degree of infection indifferent samples, with 126-kDa protein levels remainingrelatively constant among the different mutants at 6 and 12days after inoculation (unpublished results). Thus, the 126-kDa protein level provided an internal measure for therelative level of infectivity in each sample. By determiningthe ratio of 30-kDa protein to 126-kDa protein for eachsample, a comparison of 30-kDa protein levels could be madebetween different TMV mutants and wild-type TMV 204.

Effect of SGP on Enhanced Production of 30-kDa Protein.Mutant KK8, a mutant with the coat protein SGP controllingthe transcription ofthe 30-kDa gene and the coat protein ORFdeleted, was shown previously to produce -10 times as much30-kDa protein as that normally found in leaves infected withwild-type TMV (12). To determine whether this increase wasspecific to the coat protein SGP, we examined the expressionof a mutant (S3-28) with the coat protein ORF similarlydeleted but with the 30-kDa gene controlled by the native30-kDa SGP (Fig. 1). The level of 30-kDa protein accumu-lation in leaves infected with mutant S3-28 was increasedover that in leaves infected with wild-type TMV (Fig. 2).Further examination of this difference revealed that TMVS3-28 produced ratios of 30-kDa to 126-kDa protein thataveraged at least 8-fold higher than those recorded forwild-type TMV 204 (Table 1). This was approximately thesame as the increase observed with mutant KK8 (12). Thus,coat protein deletion mutants with either the coat proteinSGP or the native 30-kDa SGP in front of the 30-kDa ORFproduce equivalent levels of 30-kDa protein, demonstratingthat the SGP controlling the 30-kDa gene does not apprecia-bly affect the levels of protein expression.

Effect of Coat Protein on 30-kDa Protein Expression. Thelarge increases in production of 30-kDa protein by bothmutants KK8 and S3-28, which each have the coat proteinORF deleted, suggests a possible role for coat protein as adown-regulator of the 30-kDa gene. To determine whetherthe lack of coat protein directly enhanced 30-kDa proteinlevels, mutant TMV [-CP] was examined (Fig. 1). TMV[-CP] has a 2-nt alteration in the coat protein translational

A~~~~~~~AM

30-

LlC'ODIZN CO

126-FIG. 2. Westem immunoblot

analysis of leaf proteins fromhealthy (H) tobacco leaves orleaves infected with wild-typeTMV 204, mutant [-CP], mutantcp35-5, or mutant S3-28. Proteinswere extracted 12 days after inoc-ulation, separated by SDS/PAGE, electroblotted to nitrocel-lulose, and probed with antiseraspecific for TMV 30-kDa protein(A) or TMV 126-kDa protein (B).

start codon that prevents coat protein production but other-wise leaves the coat protein gene intact (15). In leavesinoculated with this mutant, the level of 30-kDa protein wasnot noticeably increased (Fig. 2). Ratios of30-kDa to 126-kDaprotein for TMV [-CP] revealed only a slight increase overwild-type TMV 204 ratios (Table 1), indicating that thepresence or absence of coat protein did not greatly affect thelevels of 30-kDa protein.

Effect of Genomic Position on 30-kDa Protein Expression.Another difference between wild-type TMV and mutantsKK8 and S3-28 is that the 30-kDa genes of the mutants arepositioned 470 nt closer to the 3' terminus. It was possiblethat the increased production of the 30-kDa protein was dueto its position relative to the viral 3' terminus. To examinethis possibility, the level of 30-kDa protein produced by acoat protein deletion mutant (cp35-5) was determined (Table1 and Fig. 2). Mutant cp35-5 has a partial deletion (207 nt) inthe coat protein ORF and produces a truncated coat protein(16). This deletion is approximately half the size of thedeletions in mutants KK8 and S3-28. Relative 30-kDa proteinaccumulation of cp35-5 showed an average increase of atleast 4-fold over that of wild-type TMV and TMV [-CP] butonly half the increase of mutant S3-28. Thus, a nucleotidedeletion approximately halfthe size of that occurring in S3-28produced half as great an increase in 30-kDa protein.The levels of 30-kDa protein were also examined for

TBN62, a mutant with the 30-kDa gene moved 1039 nt furtherfrom the viral 3' terminus by the insertion of the bacterialneomycin phosphotransferase II gene (Fig. 1) (17). Westernblot analysis of TBN62-infected tissue failed to detect any30-kDa protein (Table 1). In addition, TBN62 induced ne-crotic lesions in N. tabacum cv. Xanthi-nc that were smallerin size (1 mm in diameter) and 1 day slower in forming thanthe 3-mm diameter lesions produced by the wild-type virus.None of the other mutants examined induced lesions inXanthi-nc that were significantly different from lesions in-duced by the wild-type TMV. The smaller and slower-forming lesions induced by TBN62 suggest a reduced ability

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Table 1. Relative levels of 30-kDa protein, 30-kDa subgenomic mRNA, and coat protein subgenomic mRNA in tobacco leaves infectedwith TMV wild type (WT) or mutants

30-kDa protein*

Day 6 Day 12 30-kDa mRNA/ CP mRNAs,jTMV 30-kDa/126-kDa Mutant/WT 30-kDa/126-kDa Mutant/WT genomic RNAt ORSV/TMV

204 (WT) 0.36 ± 0.11 1.0 0.27 ± 0.04 1.0 -[-CP] 0.48 ± 0.10 1.3 0.38 ± 0.11 1.4 0.35 ± 0.13cp35-5 2.58 ± 0.54 7.1 1.14 ± 0.06 4.2 0.23 ± 0.05S3-28 5.33 ± 0.93 14.8 2.21 ± 0.47 8.2 0.25 ± 0.05 -TBN62 ND ND2CP - - - 1.29 0.28*Proteins were extracted 6 or 12 days after inoculation. Protein levels were determined by densitometer scans of Western immunoblots. Resultsare expressed as the ratio of 30-kDa protein to 126-kDa protein (mean ± SE of three independent samples) as the ratio of the mutant30-kDa/126-kDa value to the WT 30-kDa/126-kDa value. The 30-kDa protein was not detected (ND) for mutant TBN62.tRNA was extracted 12 days after inoculation. RNA levels were determined by densitometer scans of Northern blots. Results are expressedas the ratio of 30-kDa mRNA to genomic RNA (mean ± SE of two independent samples).tORSV and TMV coat protein (CP) mRNAs were extracted from TMV 2CP infected tissue 6 and 12 days after inoculation. Subgenomic mRNAlevels were determined by densitometer scans. Results are expressed as the ratio of ORSV mRNA to TMV mRNA (mean ± SE of fourindependent samples).

to move cell-to-cell, possibly due to reduced levels of 30-kDaprotein. The induction of smaller lesions in Xanthi-nc hasalso been observed for other mutants that position the 30-kDagene further from the viral 3' terminus (data not shown).Thus, placement of the 30-kDa gene further from the viral 3'terminus resulted in a reduction in the level of 30-kDaprotein.

Effect of Genomic Position on Coat Protein Expression. Toexamine whether genomic position would effect the regula-tion of the coat protein gene, we determined the levels ofcoatprotein produced byTMV 2CP, a mutant having two differenttobamovirus coat protein genes in tandem on the same viralgenome. The genomic organization of TMV 2CP places theORSV coat protein ORF and SGP within the genome ofTMV(Fig. 1). The ORSV coat protein gene is positioned directly3' of the native TMV coat protein gene, thus allowing thedirect measurement of both proteins from the same sample.The use ofheterologous coat protein genes prevents the rapidrecombination that can occur between homologous coatprotein gene sequences (24). In addition, progeny RNA fromeach experiment was monitored to ensure that infectionsconsisted only of TMV 2CP.The coat proteins produced by TMV 2CP were quantified

by cyanogen bromide treatment and Western blot analysis.Pretreatment of proteins with cyanogen bromide was neces-sary to avoid the problem of crossreactivity between antiseramade against the two coat proteins of the same size. TheORSV coat protein contains three methionine residues, al-lowing the chemical cleavage of the peptide chain upontreatment with cyanogen bromide. The four resulting pep-tides were too small for visualization with the describedWestern blot technique. In contrast, the coat protein ofTMVUl contains no methionine residues and is not affected bycyanogen bromide treatment.

Analysis ofTMV 2CP-infected tissue revealed that only the3' ORSV gene produced detectable levels of coat protein(Fig. 3). The 5' native coat protein gene was not expressedsufficiently to be detected, again demonstrating a preferencefor the higher expression of genes positioned nearer the viral3' terminus.Accumulation of Subgenomic mRNAs Encoding 30-kDa

Protein and Coat Protein. The preferential expression of30-kDa and coat protein genes placed nearer the viral 3'terminus might have been the result of changes in transcrip-tional or translational control mechanisms. To investigate themechanism responsible for this phenomenon, we determinedthe levels of 30-kDa subgenomic mRNA produced by thedifferent free-RNA mutants (S3-28, cp35-5, [-CP]) as well as

the levels of the different coat protein subgenomic mRNAsproduced by TMV 2CP.The 30-kDa subgenomic mRNAs for each of the free-RNA

mutants were quantified by a comparison of genomic RNAlevels to subgenomic mRNA levels found in total RNAextracts. The level of genomic RNA is representative of thedegree of infection in each of the free-RNA mutant samples.Thus, the ratio of 30-kDa subgenomic mRNA to genomicRNA can be used to compare the levels of subgenomicmRNA found in different free-RNA viruses. However, noRNA quantification can be made for viruses that producevirions (TMV 204, TBN62, and 2CP), since a large portion ofthe genomic RNA would be sequestered in the form ofvirions, greatly shifting the genomic/subgenomic RNA ratio.Quantification of the two coat protein subgenomic mRNAsproduced by mutant TMV 2CP was done by direct compar-ison of mRNA levels within the same sample.The ratios of 30-kDa subgenomic mRNA to genomic RNA

for mutants S3-28 and cp35-5 were not greatly different fromthat forTMV [-CP], even though S3-28 and cp35-5 producedsignificantly higher levels of 30-kDa protein (Table 1 and Fig.4A). Northern blot analysis of TMV 2CP-infected tissuerevealed only a small increase in the level of ORSV coatprotein mRNA (3' gene) over that of the Ul coat proteinmRNA (5' gene) (Table 1 and Fig. 4B). However, both werepresent in levels representative of a wild-type infection, yetonly the ORSV coat protein was detected.These data demonstrate that TMV mutants that express

excess levels of 30-kDa and coat protein are not transcribingproportionally higher levels of mRNA. Thus, observed in-

TMV ORSV 2CP1 2 1 2 1 2

FIG. 3. Western immunoblot analysis of leaf tissue infected withwild-type TMV, ORSV, and TMV mutant 2CP. Proteins wereextracted 12 days after inoculation, separated by SDS/PAGE, elec-troblotted to nitrocellulose, and probed with antiserum against bothTMV and ORSV. Lanes 1, protein samples not treated with cyan-ogen bromide; lanes 2, protein samples treated with cyanogenbromide. CP, coat protein.

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A C,

XIen I

B0.

Cy I

FIG. 4. Analyses of 30-kDa and coat protein subgenomicmRNAs. Total RNAs were extracted and visualized. (A) RNAextracted from leaf tissue infected with free-RNA mutants 35-5,S3-28, and [-CP]. The 30-kDa subgenomic mRNAs fall within thebracketed region. (B) RNA extracted from leaf tissue infected withmutants 2CP and [-CP]. Coat protein subgenomic mRNAs aremarked. Mutant [-CP] was run as a reference marker.

creases in protein levels appear to be the result of changes intranslational regulation.

DISCUSSIONThe expression of the TMV 30-kDa gene was markedlyaltered by deletions or insertions near the coat protein gene.Comparisons of 30-kDa protein levels produced by coatprotein-deficient mutants indicated that increased 30-kDaprotein levels were not due to the absence of coat protein butrather to positioning of the 30-kDa gene closer to the 3'terminus of the virus. The level of 30-kDa protein enhance-ment of the mutants examined appeared generally to beproportional to the number of nucleotides deleted betweenthe 30-kDa gene and the 3' nontranslated region of thegenome. In addition, a mutant having two different tandemlypositioned coat protein genes produced detectable levels ofprotein only from the gene nearer the 3' terminus. Thus, thegenomic position of both 30-kDa and coat protein genesrelative to the 3' terminus greatly affects their expression.

This conclusion is also supported by observations of othermutants that position the 30-kDa gene at different distancesfrom the 3' terminus. One mutant with an additional 30-kDaORF placed between the coat protein gene and the 3'nontranslated region produced increased levels of 30-kDaprotein (25). Also, the fusion of the 30-kDa ORF in frame toapproximately two-thirds of the coat protein ORF resulted ina substantially greater level of fusion protein than that of thenative 30-kDa protein (25).

In other viral systems, altered transcriptional levels havebeen associated with the genomic position of SGPs. Themultiple insertion of sequences containing the coat proteinSGP of brome mosaic virus into different positions withinbrome mosaic virus RNA 3 resulted in production of addi-tional 3'-coterminal RNAs, with the promoter positionednearest the 3' terminus accumulating the highest level ofRNA (26). In contrast, Raju and Huang (27) found thatSindbis virus SGPs positioned nearest the 5' terminus of thegenomic RNA expressed the highest level of RNA, suggest-ing different kinds oftranscriptional regulation. However, forTMV we have found relatively small effects on transcrip-tional levels in relation to SGP position, certainly too smallto account for the dramatic differences in protein production.Thus, the primary regulatory mechanism responsible for thehigher expression of 30-kDa and coat protein genes posi-

tioned nearer to the viral 3' terminus appears to be transla-tional in nature.Enhanced expression of internal ORFs positioned nearer

to the viral 3' terminus provides a means for the regulation ofgenes expressed via subgenomic mRNAs. In tobamoviruses,the coat protein is produced at higher levels than the 30-kDaprotein. The 54-kDa protein, positioned furthest from the 3'terminus, would be expected to be produced at proportion-ally lower levels than the 30-kDa protein, which could explainwhy this protein has not been detected in vivo. This gener-alization perhaps can be extended to other virus groups.Viruses that express genes via subgenomic mRNAs generallyproduce structural proteins in greater amounts than nonstruc-tural proteins. In many of these virus groups the structuralprotein gene is positioned near the 3' terminus ofthe genome.The relative positions within the TMV genome of the

30-kDa and coat protein genes determine part ofthe variationobserved between the levels of each protein produced. How-ever, mutants that positioned the 30-kDa gene in approxi-mately the same location as the wild-type coat protein genedid not produce levels of the 30-kDa protein that corre-sponded to wild-type levels of coat protein. The levels of30-kDa protein were always at least 10 times lower than thelevel ofwild-type coat protein. Thus, other factors in additionto genomic position are involved in the regulation of genesexpressed from subgenomic mRNAs.

This research was supported in part by Grant DMB 9005225 fromthe National Science Foundation.

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