Cistron mapping of tobacco vein mottling virus

VIROLOGY

151.159-171 (1986)

Cistron Mapping of Tobacco Vein Mottling Virus GARY M. HELLMANN,*

SHIVANAND T. HIREMATH,* ROBERT E. RHOADS*”

JOHN G. SHAW,?

AND Departments

of *Biochemistry and TPlunt Pathology, University Lexington, Kentucky ~0536-0084 Received October 22, 1985; accepted February

of Kentucky,

2, 1986

The location and order of cistrons in the RNA of the potyvirus tobacco vein mottling virus (TVMV) were investigated. Hybrid-arrested translation, using cloned single-stranded DNA probes complementary to various regions of the viral RNA, was performed and the resulting translation products were analyzed by electrophoresis. The pattern of polypeptides produced with each probe was different from that of control reactions containing RNA alone. Immunoprecipitation of reaction products with antisera to five potyviral proteins revealed that, in some cases, portions of cistrons were masked by the DNA probe resulting in the precipitation of altered polypeptides. In other cases, the entire cistron was affected, resulting in the total loss of immunoreactive material. By correlating the location of each DNA probe with the resulting pattern of translation products, it was possible to construct a map of known cistrons and potential coding regions for additional cistrons in the genomic RNA. DNA probes representing several regions of the viral RNA were expressed in E. coli Immunoprecipitation of cell proteins revealed the presence of TVMV-related polypeptides; the results were consistent with the cistron order determined by hybrid-arrested translation experiments. Our proposed cistron order and the sizes in kilodaltons of the corresponding polypeptides are: 5-25 kDa unidentified protein-53 kDa helper component protein-50 kDa unidentified protein-70 kDa cylindrical inclusion protein-52 kDa nuclear inclusion protein-56 kDa nuclear inclusion protein-32 kDa coat protein-3’. 0 1986 Academic Press. Enc.

INTRODUCTION

Dougherty and Hiebert (198Oc) presented a general map of the potyviral genome in which the positions of the 49-kDa (49K) and 54-kDa (54K) nuclear inclusion protein, cylindrical inclusion protein (CI), and coat protein (CP) cistrons, and two additional potential coding regions, were located. The map was based on the results of in vitro translation of tobacco etch virus (TEV) and pepper mottle virus (PeMV) RNAs and analysis of the products with antisera to each of these viral proteins (Dougherty and Hiebert, 1980a, 1980b). These authors detected a number of polypeptides which reacted with more than one antiserum, presumably due to translation through contiguous cistrons, and the ideni To whom dressed.

requests

for

reprints

should

be ad-

tification of these polypeptides provided information concerning the order of cistrons in the potyviral RNAs. Subsequently, several studies have appeared in which cistrons for other potyviral proteins have been proposed. Hellmann et ah (198313)showed that, in addition to 49K, 54K, CI, and CP, tobacco vein mottling virus (TVMV) RNA encoded the aphid transmission helper component protein (HC) and that, based upon the efficiency of translation of a polypeptide immunoreactive with anti-HC antiserum, its cistron was probably located near the 5’ terminus of the RNA. More recently, Hellmann et al. (1985) used single-stranded recombinant cDNA probes for TVMV RNA in hybridarrested translation experiments and obtained evidence of a previously unidentified cistron for a 25-kDa polypeptide at the 5’ terminus of the viral RNA; this was followed by the cistron encoding HC. Based 159

0042-6822/86 $3.00 Copyright All rights

0 1986 by Academic Press. Inc. of reproduction in any form reserved.

160

HELLMANN

ET AL.

was determined by DNA sequencing (Sanger et ak, 1977). Synthesis of doublestranded TVMV cDNA corresponding approximately to nucleotide residues 40-1060 was accomplished using a synthetic oligonucleotide primer. The cDNA was subsequently cloned into pUC8 (Shahabuddin, Shaw, and Rhoads, manuscript in preparation). The locations of these probes with respect to the TVMV genome are shown in Fig. 1A and B. Hybridization and translation. DNA: RNA hybridization reactions were performed as previously described (Hellmann et al., 1985). Reaction products were examined by agarose gel electrophoresis in order to verify that hybridization had occurred. Translation reactions were performed as previously described (Hellmann et ak, 1985) except that volumes were increased to 25 ~1, incubation times were increased to 90 min, and 5 m&f DTT was included in all reactions. The presence of DTT resulted in the appearance of a polypeptide of approximately 120 kDa which reacted strongly and exclusively with anti-C1 antiserum. Immunoprecipitation of products, electrophoresis of samples in sodium dodecyl sulfate (SDS)-containing gels, and MATERIALS AND METHODS visualization of radioactive products were Preparation of single-stranded cDNA performed as previously described (Hellprobes. Probes used in hybrid-arrested mann et aZ.,1983b). Antisera to the nuclear translation experiments were prepared in inclusion proteins were raised against puseveral ways. We had previously cloned re- rified preparations from TEV-infected tisstriction fragments of TVMV cDNA into sue. These antisera were previously shown pBR322 (Hellmann et aL, 1983a). Together, to cross-react with TVMV RNA-encoded these fragments represent a continuous proteins. length of TVMV RNA corresponding apConstruction of expression vectors. A set proximately to nucleotide residues 1100 of vectors which allow insertion of DNA through 9000 from the 5’ terminus of the fragments in all three reading frames was lo-kb genome. Inserts from these recom- used for the expression of TVMV proteins binant molecules were excised in whole or in Escherichia coli. These were derived in part and subcloned into Ml3 mp8 or from pKK223-3 (Amann et al, 1983) and mp19, yielding both sense and complemen- constructed and kindly provided by Dr. J. tary strands. The orientation of each insert Brosius, (Columbia University). The cDNA was determined by restriction analysis of inserts from pTV-Hl, pTV-H3, and pTVthe replicative forms of the Ml3 recombi- H4 (Hellmann et al, 1983a) were isolated nants and by incubation with viral RNA and subcloned into vectors with appropriunder hybridization conditions followed by ate reading frames to produce expression gel analysis. In some cases the subclones plasmids containing the inserts 6-7,3, and were shortened using the single-stranded 2, respectively (Fig. 1C). The orientation of sequential cloning protocol described by the inserts with respect to the tat promoter Dale et al. (1985), and the extent of deletion was determined by restriction enzyme upon in vitro translation and peptide mapping data, a similar arrangement near the 5’termini of papaya ringspot virus (PRSV) and PeMV RNAs was reported (de Mejia et ab, 1985). Another method, expression of cloned viral cDNAs in bacterial cells followed by immunoprecipitation, has been used to locate the coding regions for CP and 54K near the 3’ terminus of PRSV RNA (Nagel and Hiebert, 1985). Finally, the agreement of amino acid sequences in isolated TEV coat protein with nucleotide sequences in the RNA unequivocally placed the coat protein cistron at the 3’ terminus of TEV RNA (Allison et ab, 1985). We have used two procedures, hybridarrested translation with cloned cDNA probes and expression of cloned cDNA fragments in bacterial cells, to determine the locations of the coding regions of the known TVMV proteins and the positions of other potential cistrons. The results, reported herein, provide evidence that the cistron order in TVMV RNA is different from that previously proposed for the potyviral genome (Dougherty and Hiebert, 198Oc).

CISTRON

MAPPING

analysis. After ligation and transformation, two types of plasmid were isolated for each insert such that both possible insert orientations were obtained.

Expression of viral polypeptides in bacterial cells. The protocol of Amann et al. (1983) was used for analysis of proteins expressed in E. coli by recombinant plasmids. When virus-related polypeptides could not be detected by staining with Coomassie Blue, 500 j&i [35S]methionine (New England Nuclear) per 0.5 ml culture was added during induction with isopropyl-fiD-thiogalactoside. Polypeptides expressed from viral cDNA sequences cloned into Ml3 were obtained in a similar manner. Cell lysates from each type of culture were also analyzed for the presence of virus-related polypeptides by immunoprecipitation with antisera to the potyviral proteins, gel analysis, and fluorography, which were performed as previously described (Hellmann et al., 1983b). RESULTS

Hybrid Arrested Translation Subcloning of selected regions of TVMV cDNA into Ml3 vectors yielded a set of 10 single-stranded probes, complementary to about 95% of the viral genome (Figs. lA, B). TVMV RNA hybridized to various probes was translated in a rabbit reticulocyte lysate system. Total products, and those immunoprecipitated with antisera against five different potyviral proteins, were analyzed on SDS-polyacrylamide gels. In all cases examined, translation of TVMV RNA after incubation with DNA probes of the same sense as the RNA produced the same spectrum of polypeptides as did reactions containing no probe (data not shown). Incubation of DNA probes with tobacco mosaic virus RNA revealed neither hybridization with nor degradation of the RNA (data not shown). These controls allow us to conclude that alterations in translation products could be attributed to hybridization between DNA probes and TVMV RN.A and not to degradation of the RNA. Probe 1. Probe 1 DNA hybridizes with viral RNA from approximately nucleotide

A.

OF TVMV

161

2

se

kb

4

6

6

13’

1 LLq B.

-

=-

6

7 TzI iF

C.

AA,

4-5

6-7

I

FIG. 1. Cloned cDNA fragments

used in hybrid-ar(A) TVMV genomic RNA (10 kb). (B) Single-stranded TVMV cDNA probes subcloned in bacteriophage Ml3 in relation to the TVMV genome; only the insert portion is represented. (C) Regions of TVMV cDNA subclonedinto pKK plasmid vectors and/or Ml3 and used in expression experiments.

rested translation and expression experiments.

residues 40-1060 (Figs. lA, B). Translation of this hybrid (Fig. 2B) produced products similar to those in control reactions (Fig. 2A) except that a polypeptide of 75 kDa was present in reduced amounts. We have previously shown that translation of TVMV RNA produces two polypeptides of ‘75 kDa, one related to CP and the other related to HC (Hellmann et aL, 1983b). Immunoprecipitation showed that the one affected by Probe 1 was the HC-related polypeptide. Probes 2 and 3. Probes 2 and 3 hybridize with nucleotide residues 1100-2100 and 2100-2860, respectively (Fig. 1B). Like Probe 1, each reduced synthesis of the HCrelated 75-kDa polypeptide but in addition produced truncated forms, one of 35 kDa (with Probe 2) and one of 68 kDa (with Probe 3) (Fig. 3). Immunoprecipitation revealed that the polypeptide of 35 kDa did not react with any of the antisera while that of 68 kDa reacted with antiserum to HC. Probes 4 and 5. These probes hybridize with nucleotide residues 2860-3380 and 3900-4430 of TVMV RNA, respectively (Fig. 1B). Hybrid-arrested translation using Probe 4 resulted in synthesis of polypeptides identical to those of control reactions except that there appeared to be reduced synthesis of discrete polypeptides larger than 98 kDa (Fig. 4A). Immunoprecipitation showed some material which reacted with each antiserum tested, but there

CONTROL

B kDa

PROBE I

FIG. 2. Hybrid-arrested translation of TVMV RNA. TVMV RNA was hybridized with various single-stranded probes and translated in a reticulocyte lysate system in the presence of [?S]methionine. Total and immunoprecipitated products were analyzed on 11% polyacrylamide gels containing SDS and detected by fluorography. (A) Translation products of RNA not hybridized with DNA. (B) Translation products of RNA after hybridization with Probe 1 DNA. Lane C, total translation products in the absence of DNA probes; lane T, total translation products; lanes HC, 49K, CI, 54K, and CP, products immunoprecipitated by antiserum to TVMV helper component, TEV 49-kDa nuclear inclusion, TVMV cylindrical inclusion, TEV 54-kDa nuclear inclusion, and TVMV coat proteins, respectively. Molecular weights were calculated from Coomassie blue-stained protein standards run in adjacent lanes.

A

B PROBE 2

PROBE

-

C

T

3

Y YhU3” &L

FIG. 3. Hybrid-arrested translation of TVMV RNA. Translation products of RNA after hybridization with Probe 2 (A) and Probe 3 (B) DNA. Eleetrophoretic analysis of total and immunoprecipitated products was the same as in Fig. 2. 162

CISTRON

MAPPING

B

A

PROBE 4

c T “% r*Glon

kDa

%a

163

OF TVMV

C

PROBE 5

T

Y Y$GIn”

?a

FIG. 4. Hybrid-arrested translation of TVMV RNA. Translation products of RNA after hybridization with Probe 4 (A) and Probe 5 (B) DNA. Electrophoretic analysis of total and immunoprecipitated products was the same as in Fig. 2.

was a large reduction in all polypeptides reacting with anti-C1 antiserum. Probe 5 produced a total polypeptide pattern similar to that of Probe 4, i.e., a reduction in polypeptides larger than 98 kDa (Fig. 4B). However, a prominent polypeptide of 63 kDa was observed which was not present in control reactions. This new polypeptide was immunoprecipitated exclusively with anti-C1 antiserum and appeared to be essentially the only polypeptide immunoreactive with this antiserum. (The polypeptide of 53 kDa is believed to be an artifact created by IgG heavy chain present in the electrophoresis samples.) Probes 6, 7, 8. Probes 6, ‘7, and 8 are, respectively, 2000, 2200, and 1700 nucleotide residues in length, corresponding to residues 4600-6600, 5380-7580, and 5850-7580 of TVMV RNA (Fig. 1B). Analysis of translation products of reactions containing these probes showed in each case the absence of the major polypeptide P52, a substantial increase in P57 in the case of Probes 7 and 8, and the appearance of two new polypeptides: one of 110 kDa with

Probe 7 and one of 130 kDa with Probe 8 (Fig. 5). Immunoprecipitation of each of these three reaction mixtures showed that polypeptide patterns produced with all antisera except anti-HC were altered in various ways (Fig. 5). In each case, no material reacted with anti-49K antiserum, in contrast to the prominent polypeptide of 52 kDa precipitated from control reactions (Fig. 2A). Using anti-C1 antiserum, polypeptides of 53 and 75 kDa were precipitated from Probe 6-containing reactions, those of 53, 73,90, and 110 kDa from Probe 7-containing reactions, and those of 53, 90, and 130 kDa from Probe g-containing reactions. Control reactions showed none of these same polypeptides with the exception of the 53-kDa band, previously noted. Anti-54K antiserum precipitated relatively small amounts of products from Probe 6-containing reactions. In particular there was substantial reduction in the major polypeptide of 75 kDa and elimination of the 85-kDa polypeptide. Probes 7- and g-containing reactions showed the same reduc-

HELLMANN

164

A

PROBE

6

B kDa

ET AL.

PROBE

7

C

PROBE

8

FIG. 5. Hybrid-arrested translation of TVMV RNA. Translation products of RNA after hybridization with Probe 6 (A), Probe ‘7 (B), and Probe 8 (C) DNA. Electropboretic analysis of total and immunoprecipitated products was the same as in Fig. 2.

tion and an increase in the 57-kDa polypeptide. Anti-CP antiserum precipitated the normal set of polypeptides from Probe 6-containing reactions, but, as in the case of anti-54K antiserum, showed significant reduction in polypeptides of 75 and 85 kDa. Probe 7- and S-containing reactions showed no anti-CP immunoprecipitable material larger than the 57-kDa species, notably lacking the major 75-kDa species. Some differences were observed in minor polypeptides of lower molecular weight as well, but major CP-related species of 26 and 21 kDa were observed in both hybrid-containing and control reactions, Probes 9 and 10. These probes hybridize with nucleotide residues 7250-8930 and 8350-8930, respectively (Fig. 1B). Hybridarrested translation with each of these probes produced the same pattern of high molecular weight products as seen in controls except that polypeptides of 57,26, and 21 kDa were absent (Fig. 6). These missing polypeptides were previously shown to be associated with CP and 54K proteins (Hellmann et aL, 1983b). Immunoprecipitation with either anti-CP or anti-54K antisera confirmed this observation.

Expression of Viral Polypeptides Directed by Recombinant Ml3 Phages Two regions of TVMV cDNA, designated 4-5 and 6-7, which contained the entire inserts of TVMV cDNA plasmids pTV-H2 and pTV-Hl (Hellmann et al, 1983a), respectively, were utilized in experiments involving expression by Ml3 vectors (Fig. 1C). Transformation of JMlOl cells with Ml3 containing cDNA fragment 4-5 resulted in the appearance of many light blue plaques suggesting the production of a fusion between /3-galactosidase and TVMV polypeptides. When these recombinant phages were analyzed for inserted DNA by direct gel electrophoresis (Messing, 1983), all contained the complete insert 4-5 and all were of the same orientation. Transformation of JMlOl with Ml3 containing cDNA fragment 6-7 resulted in clear plaques only. Direct gel analysis and hybridization experiments with TVMV RNA revealed that recombinant phages with full-sized cDNA inserts in each orientation were produced. However, when isolated single-stranded phage DNA possessing an insert of the same sense as viral

CISTRON

A

PROBE

9

MAPPING

B kDa

165

OF TVMV

PROBE

IO

FIG. 6. Hybrid-arrested translation of TVMV RNA. Translation products of RNA after hybridization with Probe 9 (A) and Probe 10 (B) DNA. Electrophoretic analysis of total and immunoprecipitated products was the same as in Fig. 2.

RNA was shortened by random amounts using the single-stranded sequential cloning protocol (Dale et uL, 1985), the resulting transformation produced a number of blue plaques, again suggesting the synthesis of fusion polypeptides. The resulting singlestranded phage DNA from these plaques possessed the 6-7 DNA insert shortened to varying degrees. The apparent synthesis of fusion polypeptides by Ml3 carrying TVMV cDNA fragments 4-5 or 6-7 suggested that proteins encoded by given segments of viral cDNA might be identified by immunoprecipitation analysis of radioactively labeled bacterial cell lysates. Expression of cDNA segment 4-5 was induced, and the products, as well as t,hose of cells infected with wild type M13, were immunoprecipitated (Fig. 7A). Most of the proteins nonspecifically precipitated from control reactions were the same as those precipitated from recombinant MlS-infected cell lysates. However, several additional polypeptides, the most prominent of which were 68 and 55 kDa, cross-reacted specifically with anti-

CI antiserum. A similar experiment using cDNA segment 6-7 revealed specific polypeptides of 120 and 70 kDa which precipitated with anti-49K antiserum, and species of 120, 39, and 32 kDa which precipitated with anti-C1 antiserum. The other antisera produced patterns identical to those of control reactions. Expression of Viral Polypeptides Directed by Recombinant Plasm&Is Three segments of TVMV cDNA, designated 2,3, and 6-7 (Fig. lC), were subcloned into plasmid vectors and used in expression .experiments. Expression of virus-related polypeptides encoded by segment 2 DNA was first determined by analyzing cell lysates on SDS-polyacrylamide gels stained with Coomassie blue (Fig. 7B). No new proteins were detected from lysates of induced cultures containing plasmids with no insert or with plasmids containing cDNA segment 2 in the incorrect orientation. However, in the case of cultures containing plasmids with correctly inserted segment

A CONTROL

6-7

kDa

Ml3 PHAGE

-32-

B

2 (%-met)

2 (Coomassie)

kDa

PKK PLASMID

-36

UlUlUl -E-2x2

k?“feY

9

FIG. 7. Expression of cloned TVMV cDNA sequences in E. co& cells. (A) Expression by Ml3 containing no insert (control) or TVMV cDNA sequences (4-5, 6-7). Polypeptides from transformed cells induced in the presence of [%S]methionine were immunoprecipitated and analyzed on gels as described in Fig. 2. (B) Expression by pKK plasmids containing TVMV cDNA segment 2 in E. coli. B(Coomassie), stained polyacrylamide gel after electrophoresis of polypeptides from induced (I) or uninduced (U) E. coli transformed with pKK plasmids containing no insert (NI) or TVMV cDNA segment 2 in correct (2) or reverse (2x) orientation. 2(%-met), electrophoresis of [35S]methioninelabeled polypeptides from E. coli transformed with pKK plasmids containing TVMV cDNA segment 2 showing total proteins (T), products precipitated in the absence of antiserum (-AS), or with antiTVMV CP antiserum (CP), anti-TVMV HC antiserum (HC), or anti-TVMV HC antiserum in the presence of TVMV HC (HC + P). (C) Expression by pKK plasmids containing TVMV cDNA segments in E. co& (3), cDNA segment 3 in correct orientation; (6-7x), cDNA segment 6-7 in reverse orientation; (6-7), cDNA segment 6-7 in correct orientation. Analysis of polypeptides was performed as in (A). 166

CISTRON

MAPPING

6-7x

OF TVMV

167

6-7

kDa

66-

46---

PKK PLASMID

26--

FIG. ‘I-Continued

2, a new polypeptide of 36 kDa comprising approximately 15% of the total protein was visible only after induction. Proteins were also labeled with [35S]methionine and immunoprecipitated with various antisera (Fig. 7B). Immunoprecipitations in the absence of antibody or with anti-CP antiserum failed to precipitate any radioactive material. Anti-HC antiserum on the other hand, precipitated the novel 36-kDa polypeptide mentioned above. When partially purified HC was added to the immunoprecipitation reaction, the 36-kDa band was eliminated. The same types of experiments were performed using the segment 6-7 DNA (Fig. 7C). No new polypeptides were detected after gel electrophoresis and staining. However, after [35S]methionine labeling and immunoprecipitation, three new polypeptides were detected. The anti-49K antiserum precipitated polypeptides of 68 and 48 kDa; the anti-C1 antiserum precipitated one of 26 kDa. No virus-specific polypeptides were detected when cultures containing segment 6-7 in reverse orientation or segment 3 DNA sequences were analyzed in the same manner.

DISCUSSION

This study presents an analysis of the order and location of coding regions in TVMV RNA using two complementary methods, hybrid-arrested translation and expression of cloned viral cDNA fragments. The resulting data support the cistron order presented in Fig. 8A. The principal in vitro-synthesized polypeptides which were used to derive this cistron order are summarized in schematic form in Figs. 8B, c. The presence of a cistron at the 5’ terminus of TVMV RNA encoding an unidentified protein is indicated by results obtained from translation of the Probe 2 DNA/RNA hybrid (this study; Hellmann et al, 1985). The resulting 35-kDa truncated polypeptide, which does not react with anti-HC antiserum (Fig. 3), probably contains N-terminal amino acids of HC, but apparently possesses none of the epitopes recognized by the anti-HC antiserum. Comparative translation in wheat germ and reticulocyte systems of potyviral RNA also points to the presence of such a cistron (de Mejia et ak, 1985).

A

,25 ,

53

,

50

,

70

,

52

,

56

, 32 ,

‘?’

HC

’

?

n

Cl

’

NI

’

NI

’ CP

ANTI HC D

,

ANTI Cl

:

ANTI 49K

%-J

’

ANTI CP I

FIG. 8. Cistron map of TVMV and in vitro translation products of TVMV RNA used to derive the map. Abbreviations for polypeptides are given in the text. The molecular mass of polypeptides in kDa is given above each line. TVMV proteins precipitated by anti-TEV 49- and 54-kDa nuclear inclusion protein antisera were 52 and 56 kDa, respectively (Xu, Rhoads, and Shaw, manuscript in preparation). (A) Cistron order in TVMV. (B) Major in vitro translation products of TVMV RNA from reactions containing no hybridization probe. (C) Alterations in cell-free translation products in the presence of various DNA probes. DNA probes (Boxes l-10) were prepared and hybridized with TVMV RNA as described under Materials and Methods. Dashed lines, polypeptides abolished in the presence of each probe; solid lines, novel polypeptides synthesized in the presence of each probe. (D) Antibodies to potyviral proteins with which cell-free translation products react. Polypeptides displayed in (B) and (C), aligned above each segment in (D), were shown to react with the designated antibody.

168

CISTRON

MAPPING

The cistron encoding HC appears to be located immediately downstream of the 5’terminal cistron. This assignment is based on the precipitation by anti-HC antiserum of polypeptides produced from expression plasmids containing segment 2 DNA (Fig. 7B) and the lack of HC-related translation products when RNA regions represented by Probes 1 and 2 are masked. Polypeptides immunologically related to HC do appear when probes further downstream (Probes 3-10) are used. Based on either hybrid-arrested translation or expression data, the precise arrangement of cistrons in the RNA immediately downstream of that encoding HC was not possible. Hybrid-arrested translation using Probes 5,6,7, and 8 produced a nested set of polypeptides (63, 75, 110, and 130 kDa, respectively), the origin of each being near the putative 3’ terminus of the HC cistron. All were immunologically related to CI, indicating that all must represent translation through at least a portion of that cistron (Figs. 4 and 5). Two types of evidence suggest that the CI cistron is not adjacent to the HC cistron. First, the presence of DNA probes typically resulted in either the complete loss of translation products immunoreactive with a given antiserum (Probes 1, 2, 6, 7, 8, 9, and lo), or the elimination of characteristic virus-related polypeptides with coincident appearance of truncated forms (Probes 2, 3, 5, 6, 7, and 8). Probe 4, however, caused reduction in the amount of CI-related material relative to polypeptides precipitated by the other antisera, but not the appearance of truncated products. All other products were normal in the presence of this probe. Thus, Probe 4 is probably not within the CI cistron but immediately upstream from it. Second, expression data from both Ml3 and plasmid systems indicated that while segment 4-5 encodes material detectable only with anti-C1 antiserum, segment 6-7 encodes both CI and 49K sequences. If the CI cistron were located adjacent to the HC cistron, it could not extend into the region of the RNA represented by segment 6-‘7. Consequently, we postulate a coding region for an unidentified protein immediately downstream from the cistron

OF TVMV

169

encoding HC. While these data tentatively exclude the placement of the CI cistron adjacent to the HC cistron, they do not allow precise placement of the cistron within this region of RNA. It is possible, for example, that the CI cistron lies between two regions encoding unidentified proteins. Among the products synthesized in the presence of Probe 8 was a 130-kDa CI-related polypeptide which was larger than its 120-kDa counterpart seen in control reactions. There is now good evidence that maturation of potyviral proteins involves proteolytic processing (Yeh and Gonsalves, 1985; Allison et aL, 1985; Vance and Beachy, 1984). If the polypeptide in control reactions is produced by proteolysis, Probe 8 may prevent synthesis of a region necessary for protease activity. The polypeptides found in TVMV-infected tissue which are immunologically related to the TEV 49- and 54-kDa nuclear inclusion proteins are, respectively, 52 and 56 kDa (Xu, Rhoads, and Shaw, manuscript in preparation). Synthesis of 49K-related products by plasmids containing segment 6-7 DNA places the TVMV 52-kDa nuclear inclusion protein cistron downstream of the CI cistron (Fig. 7C). Hybrid-arrested translation allows placement of this cistron near the region in the RNA represented by Probe 8, but upstream from Probe 9, since normal 49K-related products were detected in reactions containing Probe 9. The cistron encoding 54K-related polypeptides must lie beyond Probe 6 but is most likely masked in part by Probes 7 and 8, based on hybrid-arrested translation results (Fig. 5). Consequently, we place the 56-kDa nuclear inclusion protein cistron adjacent to the 52-kDa nuclear inclusion protein cistron. Immunoblots of proteins from potyvirus-infected cells have also demonstrated the proximity of these two cistrons (M. Abdel-Haider and V. Hari, personal communication; Xu, Rhoads, and Shaw, manuscript in preparation). The location of the coat protein cistron has been studied extensively by a number of methods, all of which place it at or near the 3’ terminus of the RNA (Dougherty and Hiebert, 198Oc; Nagel and Hiebert, 1985; et al., 1985). Hybrid-arrested Allison

170

HELLMANN

translation with Probes 9 and 10 (Fig. 6) provides results in complete agreement with these studies. Hybrid-arrested translation has been used previously for exploring coding regions in TVMV RNA (Hellmann et al., 1985). The mechanism of translation downstream from hybridized regions is not entirely understood, and could be the result of a number of factors. We have observed, for example, that limited degradation of TVMV RNA results in enhanced translation of P26 and P21, two polypeptides related to coat protein (unpublished observation). Cleavage of full-length RNA downstream from the hybridized region would produce new 5’ termini available for initiation of translation. Dorner and coworkers (1984) have proposed that a number of products from in vitro translation of poliovirus RNA arise by spurious initiation at internal sites in the viral RNA. Our results suggest that regardless of in vitro translation mechanism the conditions employed prevent translation through hybridized regions of TVMV RNA, while unhybridized regions are translated with nearly normal efficiency. The previously published order of potyviral cistrons (5-78-87 kDa unidentified protein-49 kDa nuclear inclusion protein41-50 kDa unidentified protein-68-70 kDa cylindrical inclusion protein-54-56 kDa nuclear inclusion protein-30-33 kDa coat protein-3’; Dougherty and Hiebert, 198Oc; de Mejia et ah, 1985) is incompatible with the data presented here. While the arrangement of terminal cistrons in TVMV is the same as those proposed earlier, the order of internal cistrons is considerably different. According to the original potyviral genomic map, one would have expected to arrest synthesis of 49K-related material using Probes 4 and 5. However, normal amounts of 49K-related products were observed using each of these probes (Fig. 4). Similarly, synthesis of CI-related material should have been eliminated by Probes 6, 7, and 8. Instead, 49K-related products were arrested, while CI-related material was synthesized in the presence of all three probes (Fig. 5). Data obtained from expression experiments were consistent with these observations in all cases.

ET AL. ACKNOWLEDGMENTS We are indebted to Dr. David Thornbury and Dr. Thomas Pirone for providing antisera to TVMV coat, helper component, and cylindrical inclusion proteins; Dr. Ernest Hiebert, University of Florida, for providing antisera to TEV 49- and 54-kDa nuclear inclusion proteins, and Dr. Leslie Domier and Mr. Muhammad Shahabuddin for Ml3 DNA probes. This work was supported by Grant 4E021 from the University of Kentucky Tobacco and Health Research Institute and Grants &3-CRCR-1-1-1258 and 58-‘7B30-3-538 from the USDA.

REFERENCES ALLISON, R. F., SORENSON,J. C., KELLY, M. E., ARMSTRONG, F. B., and DOUGHERTY, W. G. (1985). Sequence determination of the eapsid protein gene and flanking regions of tobacco etch virus: Evidence for synthesis and processing of a polyprotein in potyvirus genome expression. Proc Nati Ad Sci USA 82,3969-3972. AMANN, E., BROSIUS, J., and PTASHNE, M. (1983). Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Eschtichia coli. Gene 25, 16’7-178. DALE, R. M. K., MCCLURE, B. A., and HOUCHINS, J. P. (1985). A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: Application to sequencing the corn mitochondrial185 rDNA. Plasmid 13, 31-40. DE MEJIA, M. V. G., HIEBERT, E., PURCIFULL, D. E., THORNBURY, D. W., and PIRONE, T. P. (1985). Identification of potyviral amorphous inclusion protein as a nonstructural virus-specific protein related to helper component. Virology 142,34-43. DORNER, A. J., SEMLER, B. L., JACKSON, R. J., HANECAK, R., DUPREY, E., and WIMMER, E. (1984). In vitro translation of poliovirus RNA: Utilization of internal initiation sites in reticulocyte lysate. J. Viral. 50,507-514. DOUGHERTY, W. G., and HIEBERT, E. (1980a). Translation of potyvirus RNA in a rabbit reticulocyte lysate: Reaction conditions and identification of capsid protein as one of the products of in vitro translation of tobacco etch and pepper mottle viral RNAs. virology 101,466-474. DOUGHERTY, W. G., and HIEBERT, E. (1980b). Translation of potyvirus RNA in a rabbit reticuloeyte lysate: Identification of nuclear inclusion proteins as products of tobacco etch virus RNA translation and cylindrical inclusion protein as a product of the potyvirus genome. virology 104,174-182. DOUGHERTY, W. G., and HIEBERT, E. (198Oc). Translation of potyvirus RNA in a rabbit reticulocyte lysate: Cell-free translation strategy and a genetic map of the potyviral genome. Virology 104.183-194.

CISTRON

MAPPING

H-ANN, G. M., SHAHABUDDIN, M., SHAW, J. G., and RHOADS, R. E. (1983a). Molecular cloning of DNA complementary to tobacco vein mottling virus RNA. Virology 128,210-220. HELLMANN, G. M., SHAW, J. G., and RHOADS, R. E. (1985). On the origin of the helper component of tobacco vein mottling virus: Translational initiation near the B-terminus of the viral RNA and termination by UAG codons. Virology 143,23-34. HELLMANN, G. M., THORNBURY, D. W., HIEBERT, E., SHAW, J. G., PIRONE, T. P., and RHOADS, R. E. (1983b). Cell-free translation of tobacco vein mottling virus RNA. II. Immunoprecipitation of products by antisera to cylindrical inclusion, nuclear inclusion and helper component proteins. Virology 124, 434-444. MESSING, J. (1983). New Ml3 vectors for cloning. In “Methods in Enzymology” (R. Wu, L. Grossman,

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and K. Moldave, eds.), Vol. 101, pp. 20-89. Academic Press, Orlando, Fla. NAGEL, J., and HIEBERT, E. (1985). Complementary DNA cloning and expression of the papaya ringspot potyvirus sequences encoding capsid protein and a nuclear inclusion-like protein in Escherichia coli. Virology 143,435-441. SANGER, F., NICKLEN, S., and COULSON, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc Natl. Acad Sci. USA 74,5463-5467. VANCE, V. B., and BEACHY, R. N. (1984). Translation of soybean mosaic virus RNA in vitro: Evidence of protein processing. Virology 132,271-281. YEH, S. D., and GONSALVES, D. (1985). Translation of papaya ringspot virus RNA in vitro: Detection of a possible polyprotein that is processed for capsid protein, cylindrical-inclusion protein, and amorphous-inclusion protein. Virology 143, 260-271.