Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2055-2059, March 1993 Genetics Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes (gene regulatlon/subgenomic mRNA) JAMES N. CULVER*, KIRSI LEHTOt, SHEILA M. CLOSE, MARK E. HILFt, AND WILLIAM 0. DAWSON§1 Department 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 the mRNAs are transcribed from negative-sense genomic RNA tobacco mosaic virus (TMV) genes encoding the 30-kDa cell- and share a common 3' terminus. to-cell movement protein or the coat protein greatly affected Of particular interest is that genes of TMV expressed via their expression. Higher production of 30-kDa protein was subgenomic mRNAs are independently regulated, both quan- correlated with increased proximity of the gene to the viral 3' titatively and temporally. The 30-kDa protein is produced terminus. A mutant placing the 30-kDa open reading frame 207 transiently during the early phase of the infection, 2-10 hr nucleotides nearer the 3' terminus produced at least 4 times the after infection in protoplasts (7) and within the first 24 hr in wild-type TMV 30-kDa protein level, while a mutant placing planta (8). Additionally, even though the 30-kDa protein is the 30-kDa open reading frame 470 nucleotides doser to the 3' stably associated with the cell wall (9, 10), it accumulates to terminus produced at least 8 times the wild-type TMV 30-kDa relatively low levels, ==1% oftotal plant protein. In contrast, protein level. Increases in 30-kDa protein production were not coat protein gene expression reaches a maximum 24-72 hr correlated with the subgenomic mRNA promoter (SGP) con- after infection and constitutes up to 70% oftotal plant protein trolling the 30-kDa gene, since mutants with either the native synthesis (11). The 30-kDa and coat protein genes have 30-kDa SGP or the coat protein SGP in front of the 30-kDa several notable differences. Each is controlled by different gene produced similar levels of 30-kDa protein. Lack of coat subgenomic mRNA promoter (SGP) sequences and each protein did not affect 30-kDa protein expression, since a occupies a different position within the genome. The coat mutant with the coat protein start codon removed did not protein mRNA has a short A+U-rich leader that is termi- produce increased amounts of 30-kDa protein. Effects of gene nated with a 5' 7-methylguanosine cap structure whereas the 30-kDa mRNA has a long leader (75 nt) that is not capped positioning on coat protein expression were examined by using (12). a mutant containing two different tandemly positioned tobam- Recent studies have provided some information concern- ovirus (TMV and Odontoglossum ringspot virus) coat protein ing the regulation of TMV genes expressed via subgenomic genes. Only coat protein expressed from the gene positioned mRNAs. Changing the "suboptimal" context of the start nearest the 3' viral terminus was detected. Analysis of 30-kDa codon ofthe 30-kDa mRNA to a more optimal context did not and coat protein subgenomic mRNAs revealed no proportional increase the expression ofthe 30-kDa protein, suggesting that increase in the levels of mRNA relative to the observed levels this is not a major factor in the regulation of this gene (13). of 30-kDa and coat proteins. This suggests that a translational The function of the different SGPs was examined by placing mechanism is primarily responsible for the observed effect of the 30-kDa gene under the control of the coat protein SGP genomic position on expression of 30-kDa movement and coat (12). This rearrangement did not greatly increase the expres- protein genes. sion ofthe 30-kDa gene but changed the timing ofexpression. The 30-kDa protein of this mutant, KK6, was produced late Positive-sense RNA viruses have evolved numerous strate- instead of early, suggesting that different SGPs control the gies for gene regulation. One common strategy is the use of timing rather than the level of gene expression. A mutant subgenomic mRNAs, transcribed from genomic RNA, for the identical to KK6 except with the entire coat protein ORF expression of internal open reading frames (ORFs). In this removed, mutant KK8, produced -10 times more 30-kDa manner the expression of multiple genes residing on a com- protein than wild-type TMV (12). The reason for this large mon genomic RNA can be independently regulated. One such increase in the production of 30-kDa protein by mutant KK8 virus that utilizes this strategy is the type member of the with the 30-kDa gene under the control of the coat protein tobamovirus group, tobacco mosaic virus (TMV). SGP and lacking the coat protein ORF was not determined. The genome of TMV resides on a single strand ofpositive- In this paper, we present evidence that levels of 30-kDa sense RNA, 6395 nucleotides (nt) in length, and encodes at protein increase proportionally with the proximity ofthe gene least four proteins (1). The TMV genome is organized such to the viral 3' terminus. This increase was not related to a that two 5'-coterminal ORFs encoding 126-kDa and 18&-kDa specific SGP or to the presence of coat protein. In addition, replicase proteins are translated from the genomic RNA, the tandem placement of two different coat protein genes while an internal ORF encoding the 30-kDa cell-to-cell- movement protein and a 3'-proximal ORF encoding the coat Abbreviations: TMV, tobacco mosaic virus; ORSV, Odontoglossum are translated from ringspot virus; ORF, open reading frame; SGP, subgenomic mRNA protein (structural) respective subge- promoter. nomic mRNAs (2-5). A third subgenomic mRNA has been *Present address: Center for Agricultural Biotechnology, University detected for an additional internal ORF, within the 183-kDa of Maryland, College Park, MD 20742. ORF, encoding a putative 54-kDa protein that has not yet tPresent address: Department of Biology, University of Turku, been detected in infected plants (6). The TMV subgenomic 20500 Turku, Finland. iPresent address: U.S. Department of Agriculture Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803. The publication costs of this article were defrayed in part by page charge §Present address: Citrus Research and Education Center, University payment. This article must therefore be hereby marked "advertisement" of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom reprint requests should be addressed. 2055 Downloaded by guest on September 24, 2021 2056 Genetics: Culver et al. Proc. Natl. Acad. Sci. USA 90 (1993) within the same genome results in high levels of coat protein RNA inoculation of the indicator host N. tabacum cv. for only the gene located nearest the viral 3' terminus. These Xanthi-nc. data suggest that tobamovirus gene expression, via subge- Protein Extraction, Cyanogen Bromide Treatment, and nomic mRNAs, is controlled by the position of the genes Western Blot Analysis. Both 30-kDa and 126-kDa proteins within the viral genome. were extracted from inoculated Xanthi leaves by published methods (8, 19). Extracted proteins were analyzed by SDS/ PAGE (20) and then electroblotted onto nitrocellulose paper MATERIALS AND METHODS (21). Blotted proteins were first probed with a 1:1000 dilution Viral Constructs. Constructs were derived from pTMV204, of antiserum specific either for the 30-kDa protein or for the a full-length infectious clone of the Ul strain of TMV (14). 126-kDa protein (8). Blots were then probed with alkaline The construction of each TMV mutant has previously been phosphatase-conjugated goat anti-rabbit antibody (Calbio- described and a diagrammatic representation is presented chem) diluted 1:1000 and proteins were visualized by the (Fig. 1). TMV nucleotide numbering is that of Goelet et al. addition ofS mg of5-bromo-4-chloro-3-indolyl phosphate and (1). All of the viral constructs used in this study differ from 10 mg of nitroblue tetrazolium dissolved in 30 ml of 10 mM wild-type TMV 204 by alterations made in the region of the Tris HCl, pH 9.5/100 mM NaCl/5 mM MgCl2. All blocking coat protein ORF. Mutant TMV [-CP] contains an alteration and incubation steps were done in Tris-buffered saline (50 in the coat protein translational start codon changing it from mM Tris HCl, pH 7.4/200 mM NaCl) with 5% dry milk for 2 AUG to AGA (15). Mutant cp35-5 contains a deletion (nt hr at 370C. 5841-6055) in the coat protein ORF (16). Mutant S3-28 has The 30-kDa and 126-kDa proteins were quantified from (16). Western blots with a scanning laser densitometer (LKB the entire coat protein ORF (nt 5713-6191) removed Ultroscan XL). Areas (mm2) under absorbance peaks were Mutant TBN62 contains the ORF of the bacterial gene then used to determine ratios of 30-kDa to 126-kDa proteins encoding neomycin phosphotransferase (NPT II, 832 nt) for each sample. A sample represents the protein extracted inserted into an Xho I site located between the TMV Ul coat from 1 g of infected Xanthi tissue, suspended in 1 ml of protein SGP and the ORSV (another tobamovirus) coat sample buffer. Equal amounts of each sample were loaded protein SGP and ORF in pTBD2 (17). The extra ORSV SGP onto gels used for Western blotting of 30-kDa and 126-kDa consists of 203 nt; thus, the 30-kDa gene of TBN62 is proteins. positioned 1039 nt further from the 3' terminus. Mutant TMV Mutant TMV 2CP-infected tissue was ground in 10 mM 2CP contains the coat protein SGP and corresponding ORF phosphate buffer (pH 6.8). Cellular debris was pelleted by ofTMV Ul and ORSV in tandem on the same genome. TMV centrifugation (10 min, 10,000 rpm, Sorvall SS-34 rotor). The 2CP was constructed by the insertion of an ORSV fragment soluble fraction was precipitated with an equal volume of (Xba I at nt 5563 to Ear I at nt 6182), end-filled, and ligated acetone and centrifuged as before. The resulting pellet was into the Pml I site (nt 6238) of pTMV204. vacuum dried to a powder, and 2 mg was resuspended in 250 Inoculations. Infectious RNA of each construct was ob- ,ul of70o formic acid containing 4.5 mg ofcyanogen bromide. tained by in vitro transcription of viral cDNAs (14, 18). This solution was incubated overnight at room temperature Transcribed RNA was used to inoculate leaves of Nicotiana and then subjected to acetone precipitation. The pellet was tabacum (L) cv. Xanthi, a systemic host for TMV, and the suspended in Laemmli sample buffer and boiled for 5 min, viruses were propagated as described previously (16). Inoc- and the proteins were resolved by SDS/PAGE. Western blot ulated leaves of Xanthi tobacco plants used for protein analysis was performed with 1:500 dilutions ofboth TMV Ul extractions were of the same approximate developmental and ORSV coat protein antisera. stage and all inoculated plants were maintained in growth RNA Extractions and Northern Blots. Total RNA was chambers at 25°C, with a 12-hr photoperiod of -20,000 lx. extracted from infected leaf tissue as described by Logeman Local lesion assays were performed by virion or infectious et al. (22). For blot hybridization analysis, RNA samples TMV SOP SaP d UfDa MUMkD I 3OkDaTICP 5'-

1-CPJ *GAGA 3OkDa CP 1 3' cp 35- 3OkDa

S3-28 sr.p 207nt deletion F 3OkDa 470nt deletion TBN62 SOp SGP FIG. 1. Genomic organization V V of wild-type TMV 204 and mu- 3OkDa | -NPT 11 |nCP -3' tants [-CP], cp35-5, S3-28, F TBN62, and 2CP. All of the mu- I - 35nt Insert tants were constructed as DNA plasmids from pTMV204 (14). CP, 2CP SGP SGP coat protein; NPT II, neomycin phosphotransferase II; ORSV, 3OkDa I TMV CP I ORSV CP | 39 Odontoglossum ringspot virus. Downloaded by guest on September 24, 2021 Genetics: Culver et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2057 were denatured with glyoxal and dimethyl sulfoxide, elec- trophoresed in 1% agarose gels (23), and transferred to Magnagraph nylon membranes (0.45 am, Micron Separa- A~~~~~~~AM tions, Westboro, MA). Membranes were hybridized with specific TMV cDNA fragments (nt 3333-5456, specific for 30-kDa mRNA, and nt 6207-6406, specific for the viral 3' untranslated region) labeled by random priming with digox- igenin-dUTP (Boehringer Mannheim). Hybridized bands were visualized by immunochemiluminescence using an anti- digoxigenin antibody conjugated to alkaline phosphatase 30- with the addition of Lumiphos 530 (Boehringer Mannheim). mRNAs were quantified with the scanning laser densitometer (LKB Ultroscan XL).

RESULTS Quantification of 30-kDa Protein. Obtaining equally in- fected samples of free-RNA mutants of TMV, mutants that LlC'OD cannot produce virions, in inoculated leaves is difficult IZN CO because often it is not evident which tissues are infected. For FIG. 2. Westem immunoblot this study we selected free-RNA mutants that replicate 126- analysis of leaf proteins from efficiently and move cell-to-cell essentially like wild-type healthy (H) tobacco leaves or TMV (15, 16). However, they move long distances, even leaves infected with wild-type within inoculated leaves, poorly. Additionally, many of the TMV 204, mutant [-CP], mutant coat protein deletion mutants are symptomless. Thus, com- cp35-5, or mutant S3-28. Proteins parison of total viral protein in different samples is affected were extracted 12 days after inoc- by the percentage of cells infected. To overcome sampling ulation, separated by SDS/ PAGE, electroblotted to nitrocel- errors in measuring 30-kDa protein, we chose to quantify lulose, and probed with antisera 30-kDa protein relative to the amount of 126-kDa protein. specific for TMV 30-kDa protein Based on the ability of these mutants to replicate and move (A) or TMV 126-kDa protein (B). within an inoculated leaf, we have found the level of 126-kDa protein to be representative of the degree of infection in start codon that prevents coat protein production but other- different samples, with 126-kDa protein levels remaining wise leaves the coat protein gene intact (15). In leaves relatively constant among the different mutants at 6 and 12 inoculated with this mutant, the level of 30-kDa protein was days after inoculation (unpublished results). Thus, the 126- not noticeably increased (Fig. 2). Ratios of30-kDa to 126-kDa kDa protein level provided an internal measure for the protein for TMV [-CP] revealed only a slight increase over relative level of infectivity in each sample. By determining wild-type TMV 204 ratios (Table 1), indicating that the the ratio of 30-kDa protein to 126-kDa protein for each presence or absence of coat protein did not greatly affect the sample, a comparison of30-kDa protein levels could be made levels of 30-kDa protein. between different TMV mutants and wild-type TMV 204. Effect of Genomic Position on 30-kDa Protein Expression. Effect of SGP on Enhanced Production of 30-kDa Protein. Another difference between wild-type TMV and mutants Mutant KK8, a mutant with the coat protein SGP controlling KK8 and S3-28 is that the 30-kDa genes of the mutants are the transcription ofthe 30-kDa gene and the coat protein ORF positioned 470 nt closer to the 3' terminus. It was possible deleted, was shown previously to produce -10 times as much that the increased production of the 30-kDa protein was due 30-kDa protein as that normally found in leaves infected with to its position relative to the viral 3' terminus. To examine wild-type TMV (12). To determine whether this increase was this the a specific to the coat protein SGP, we examined the expression possibility, level of 30-kDa protein produced by of a mutant (S3-28) with the coat protein ORF similarly coat protein deletion mutant (cp35-5) was determined (Table deleted but with the 30-kDa gene controlled by the native 1 and Fig. 2). Mutant cp35-5 has a partial deletion (207 nt) in 30-kDa SGP (Fig. 1). The level of 30-kDa protein accumu- the coat protein ORF and produces a truncated coat protein lation in leaves infected with mutant S3-28 was increased (16). This deletion is approximately half the size of the over that in leaves infected with wild-type TMV (Fig. 2). deletions in mutants KK8 and S3-28. Relative 30-kDa protein Further examination of this difference revealed that TMV accumulation of cp35-5 showed an average increase of at S3-28 produced ratios of 30-kDa to 126-kDa protein that least 4-fold over that of wild-type TMV and TMV [-CP] but averaged at least 8-fold higher than those recorded for only half the increase of mutant S3-28. Thus, a nucleotide wild-type TMV 204 (Table 1). This was approximately the deletion approximately halfthe size ofthat occurring in S3-28 same as the increase observed with mutant KK8 (12). Thus, produced half as great an increase in 30-kDa protein. coat protein deletion mutants with either the coat protein The levels of 30-kDa protein were also examined for SGP or the native 30-kDa SGP in front of the 30-kDa ORF TBN62, a mutant with the 30-kDa gene moved 1039 nt further produce equivalent levels of 30-kDa protein, demonstrating from the viral 3' terminus by the insertion of the bacterial that the SGP controlling the 30-kDa gene does not apprecia- neomycin phosphotransferase II gene (Fig. 1) (17). Western bly affect the levels of protein expression. blot analysis of TBN62-infected tissue failed to detect any Effect of Coat Protein on 30-kDa Protein Expression. The 30-kDa protein (Table 1). In addition, TBN62 induced ne- large increases in production of 30-kDa protein by both crotic lesions in N. tabacum cv. Xanthi-nc that were smaller mutants KK8 and S3-28, which each have the coat protein in size (1 mm in diameter) and 1 day slower in forming than ORF deleted, suggests a possible role for coat protein as a the 3-mm diameter lesions produced by the wild-type virus. down-regulator of the 30-kDa gene. To determine whether None of the other mutants examined induced lesions in the lack of coat protein directly enhanced 30-kDa protein Xanthi-nc that were significantly different from lesions in- levels, mutant TMV [-CP] was examined (Fig. 1). TMV duced by the wild-type TMV. The smaller and slower- [-CP] has a 2-nt alteration in the coat protein translational forming lesions induced by TBN62 suggest a reduced ability Downloaded by guest on September 24, 2021 2058 Genetics: Culver et al. Proc. Natl. Acad. Sci. USA 90 (1993)

Table 1. Relative levels of 30-kDa protein, 30-kDa subgenomic mRNA, and coat protein subgenomic mRNA in tobacco leaves infected with TMV wild type (WT) or mutants 30-kDa protein* Day 6 Day 12 30-kDa mRNA/ CP mRNAs,j TMV 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 ± 0.13 cp35-5 2.58 ± 0.54 7.1 1.14 ± 0.06 4.2 0.23 ± 0.05 S3-28 5.33 ± 0.93 14.8 2.21 ± 0.47 8.2 0.25 ± 0.05 - TBN62 ND ND 2CP - - - 1.29 0.28 *Proteins were extracted 6 or 12 days after inoculation. Protein levels were determined by densitometer scans ofWestern immunoblots. Results are expressed as the ratio of 30-kDa protein to 126-kDa protein (mean ± SE of three independent samples) as the ratio of the mutant 30-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 expressed as 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 mRNA levels were determined by densitometer scans. Results are expressed as the ratio of ORSV mRNA to TMV mRNA (mean ± SE of four independent samples). to move cell-to-cell, possibly due to reduced levels of 30-kDa the levels of the different coat protein subgenomic mRNAs protein. The induction of smaller lesions in Xanthi-nc has produced by TMV 2CP. also been observed for other mutants that position the 30-kDa The 30-kDa subgenomic mRNAs for each ofthe free-RNA gene further from the viral 3' terminus (data not shown). mutants were quantified by a comparison of genomic RNA Thus, placement of the 30-kDa gene further from the viral 3' levels to subgenomic mRNA levels found in total RNA terminus resulted in a reduction in the level of 30-kDa extracts. The level of genomic RNA is representative of the protein. degree of infection in each of the free-RNA mutant samples. Effect of Genomic Position on Coat Protein Expression. To Thus, the ratio of 30-kDa subgenomic mRNA to genomic examine whether genomic position would effect the regula- RNA can be used to compare the levels of subgenomic tion ofthe coat protein gene, we determined the levels ofcoat mRNA found in different free-RNA viruses. However, no protein produced by TMV 2CP, a mutant having two different RNA quantification can be made for viruses that produce tobamovirus coat protein genes in tandem on the same viral virions (TMV 204, TBN62, and 2CP), since a large portion of genome. The genomic organization of TMV 2CP places the the genomic RNA would be sequestered in the form of ORSV coat protein ORF and SGP within the genome ofTMV virions, greatly shifting the genomic/subgenomic RNA ratio. (Fig. 1). The ORSV coat protein gene is positioned directly Quantification of the two coat protein subgenomic mRNAs 3' of the native TMV coat protein gene, thus allowing the produced by mutant TMV 2CP was done by direct compar- direct measurement of both proteins from the same sample. ison of mRNA levels within the same sample. The use ofheterologous coat protein genes prevents the rapid The ratios of 30-kDa subgenomic mRNA to genomic RNA recombination that can occur between homologous coat for mutants S3-28 and cp35-5 were not greatly different from protein gene sequences (24). In addition, progeny RNA from that for TMV [-CP], even though S3-28 and cp35-5 produced each experiment was monitored to ensure that infections significantly higher levels of 30-kDa protein (Table 1 and Fig. consisted only of TMV 2CP. 4A). Northern blot analysis of TMV 2CP-infected tissue The coat proteins produced by TMV 2CP were quantified revealed only a small increase in the level of ORSV coat by cyanogen bromide treatment and Western blot analysis. protein mRNA (3' gene) over that of the Ul coat protein Pretreatment of proteins with cyanogen bromide was neces- mRNA (5' gene) (Table 1 and Fig. 4B). However, both were sary to avoid the problem ofcrossreactivity between antisera present in levels representative of a wild-type infection, yet made against the two coat proteins of the same size. The only the ORSV coat protein was detected. ORSV coat protein contains three methionine residues, al- These data demonstrate that TMV mutants that express lowing the chemical cleavage of the peptide chain upon excess levels of 30-kDa and coat protein are not transcribing treatment with cyanogen bromide. The four resulting pep- proportionally higher levels of mRNA. Thus, observed in- tides were too small for visualization with the described Western blot technique. In contrast, the coat protein ofTMV TMV ORSV 2CP Ul contains no methionine residues and is not affected by 1 2 1 2 1 2 cyanogen bromide treatment. Analysis ofTMV 2CP-infected tissue revealed that only the 3' ORSV gene produced detectable levels of coat protein (Fig. 3). The 5' native coat protein gene was not expressed sufficiently to be detected, again demonstrating a preference for the higher expression of genes positioned nearer the viral 3' terminus. Accumulation of Subgenomic mRNAs Encoding 30-kDa Protein and Coat Protein. The preferential expression of 30-kDa and coat nearer the viral 3' FIG. 3. Western immunoblot analysis of leaf tissue infected with protein genes placed wild-type TMV, ORSV, and TMV mutant 2CP. Proteins were terminus might have been the result of changes in transcrip- extracted 12 days after inoculation, separated by SDS/PAGE, elec- tional or translational control mechanisms. To investigate the troblotted to nitrocellulose, and probed with antiserum against both mechanism responsible for this phenomenon, we determined TMV and ORSV. Lanes 1, protein samples not treated with cyan- the levels of 30-kDa subgenomic mRNA produced by the ogen bromide; lanes 2, protein samples treated with cyanogen different free-RNA mutants (S3-28, cp35-5, [-CP]) as well as bromide. CP, coat protein. Downloaded by guest on September 24, 2021 Genetics: Culver et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2059 tioned nearer to the viral 3' terminus appears to be transla- A C, B tional in nature. 0. Enhanced expression of internal ORFs positioned nearer I to the viral 3' terminus provides a means for the regulation of XIen I Cy genes expressed via subgenomic mRNAs. In tobamoviruses, the coat protein is produced at higher levels than the 30-kDa protein. The 54-kDa protein, positioned furthest from the 3' terminus, would be expected to be produced at proportion- ally lower levels than the 30-kDaprotein, which could explain why 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 generally produce structural proteins in greater amounts than nonstruc- tural proteins. In many of these virus groups the structural protein 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 variation FIG. 4. Analyses of 30-kDa and coat protein subgenomic observed between the levels ofeach protein produced. How- mRNAs. Total RNAs were extracted and visualized. (A) RNA ever, mutants that positioned the 30-kDa gene in approxi- extracted from leaf tissue infected with free-RNA mutants 35-5, mately the same location as the wild-type coat protein gene S3-28, and [-CP]. The 30-kDa subgenomic mRNAs fall within the did not produce levels of the 30-kDa protein that corre- bracketed region. (B) RNA extracted from leaf tissue infected with sponded to wild-type levels of coat protein. The levels of mutants 2CP and [-CP]. Coat protein subgenomic mRNAs are 30-kDa protein were always at least 10 times lower than the marked. Mutant [-CP] was run as a reference marker. level ofwild-type coat protein. Thus, otherfactors in addition are creases in protein levels appear to be the result of changes in to genomic position involved in the regulation of genes translational regulation. expressed from subgenomic mRNAs. This research was supported in part by Grant DMB 9005225 from DISCUSSION the National Science Foundation. The expression of the TMV 30-kDa gene was markedly 1. Goelet, P., Lomonossoff, G. P., Butler, P. J. G., Akam, altered by deletions or insertions near the coat protein gene. M. E., Gait, M. J. & Karn, J. (1982) Proc. Natl. Acad. Sci. Comparisons of 30-kDa protein levels produced by coat USA 79, 5818-5822. protein-deficient mutants indicated that increased 30-kDa 2. Hunter, T. R., Hunt, T., Knowland, J. & Zimmemr, D. (1976) Nature (London) 260, 759-764. protein levels were not due to the absence ofcoat protein but 3. Siegel, A., Montgomery, V. H. I. & Kolacz, K. (1976) Virology rather to positioning of the 30-kDa gene closer to the 3' 73, 363-371. terminus of the virus. The level of 30-kDa protein enhance- 4. Bruening, G., Beachy, R. N., Scalla, R. & Zaitlin, M. (1976) ment of the mutants examined appeared generally to be Virology 71, 498-517. proportional to the number of nucleotides deleted between 5. Beachy, R. N. & Zaitlin, M. (1977) Virology 81, 160-169. gene of the 6. Sulzinski, M. A., Gabaro, K. A., Palukaitis, P. & Zaitlin, M. the 30-kDa and the 3' nontranslated region (1985) Virology 145, 132-140. genome. In addition, a mutant having two different tandemly 7. Watanabe, Y., Emori, Y., Ooshika, I., Meshi, T., Ohno, T. & positioned coat protein genes produced detectable levels of Okada, Y. (1984) Virology 133, 18-24. protein only from the gene nearer the 3' terminus. Thus, the 8. Lehto, K., Bubrick, P. & Dawson, W. 0. (1990) Virology 174, genomic position of both 30-kDa and coat protein genes 290-293. relative to the 3' terminus greatly affects their expression. 9. Tomenius, K., Clapham, D. & Meshi, T. (1987) Virology 160, 363-371. This conclusion is also supported by observations of other 10. Moser, O., Gagey, M. J., Godfroy-Colburn, T., Ellwart- mutants that position the 30-kDa gene at different distances Tschurtz, M., Nitschko, H. & Mundry, K. W. (1988) J. Gen. from the 3' terminus. One mutant with an additional 30-kDa Virol. 69, 1367-1373. ORF placed between the coat protein gene and the 3' 11. Siegel, A., Hari, V. & Kolacz, K. (1978) Virology 85, 494-503. nontranslated region produced increased levels of 30-kDa 12. Lehto, K., Grantham, G. L. & Dawson, W. 0. (1990) Virology protein (25). Also, the fusion of the 30-kDa ORF in frame to 174, 145-157. 13. Lehto, K. & Dawson, W. 0. (1990) Virology 174, 169-176. approximately two-thirds ofthe coat protein ORF resulted in 14. Dawson, W. O., Beck, D. L., Knorr, D. A. & Grantham, G. L. a substantially greater level of fusion protein than that of the (1986) Proc. Natl. Acad. Sci. USA 83, 1832-1836. native 30-kDa protein (25). 15. Culver, J. N. & Dawson, W. 0. (1989) Virology 173, 755-758. In other viral systems, altered transcriptional levels have 16. Dawson, W. O., Bubrick, P. & Grantham, G. L. (1988) Phy- been associated with the genomic position of SGPs. The topathology 78, 783-789. multiple insertion of sequences containing the coat protein 17. Donson, J., Kearney, C. M., Hilf, M. E. & Dawson, W. 0. SGP of brome mosaic virus into different positions within (1991) Proc. Natl. Acad. Sci. USA 88, 7204-7208. brome mosaic virus RNA 3 resulted in of addi- 18. Ahlquist, P. & Janda, M. (1984) Mol. Cell. Biol. 4, 2876-2882. production 19. Godefroy-Colburn, T., Gagey, M., Berna, A. & Stussi-Garaud, tional 3'-coterminal RNAs, with the promoter positioned C. (1986) J. Gen. Virol. 67, 2233-2239. nearest the 3' terminus accumulating the highest level of 20. Laemmli, U. K. (1970) Nature (London) 227, 680-685. RNA (26). In contrast, Raju and Huang (27) found that 21. Dunn, S. D. (1986) Anal. Biochem. 157, 144-153. Sindbis virus SGPs positioned nearest the 5' terminus of the 22. Logemann, J., Scheli, J. & Willmitzer, L. (1987) Anal. Bio- genomic RNA expressed the highest level of RNA, suggest- chem. 163, 16-20. ing different kinds oftranscriptional regulation. However, for 23. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular TMV we have found relatively small effects on transcrip- Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). tional levels in relation to SGP position, certainly too small 24. Beck, D. L. & Dawson, W. 0. (1991) Virology 177, 462-469. to account for the dramatic differences in protein production. 25. Lehto, K. & Dawson, W. 0. (1990) Virology 175, 30-40. Thus, the primary regulatory mechanism responsible for the 26. French, R. & Ahlquist, P. (1988) J. Virol. 62, 2411-2420. higher expression of 30-kDa and coat protein genes posi- 27. Raju, R. & Huang, H. (1991) J. Gen. Virol. 65, 2501-2510. Downloaded by guest on September 24, 2021