In Vitro cleavage at or near the N-terminus of the helper component protein in the tobacco vein mottling virus polyprotein

VIROLOGY

185721-731

ln

(1991)

vitro Cleavage

at or near the N-terminus of the Helper Component in the Tobacco Vein Mottling Virus Polyprotein GOPINATH MAVANKAL

Department

of Biochemistry, Received

University April

Protein

ROBERT E. RHOADS’

AND

of Kentucky,

25, 199 1; accepted

Lexington, September

Kentucky

40536-0084

4, 199 1

Translation of tobacco vein mottling virus (TVMV) RNA in a wheat germ system resulted in two products that were not observed in a rabbit reticulocyte system. One of these was the N-terminal protein, based on its being the most abundant product and its migration on SDS-PAGE at about 34 kDa. The second product was similar or identical to helper component (HC) isolated from TVMV-infected plants, based on co-migration with HC on SDS-PAGE and immunoprecipitation with anti-HC antibodies. The N-terminus of this product was determined by radiochemical Edman degradation to be Ser-257 of the polyprotein. This assignment was supported by peptide mapping with a tryptophanspecific reagent. A similar cleavage was observed when tobacco etch virus was translated in a wheat germ system. Comparison with homologous regions in five other potyviruses indicated conservation of amino acid residues on both sides of the proposed cleavage site. Conversion of Phe-256 to Met, Pro, Arg, His, or Trp by site-directed mutagenesis of a TVMV RNA transcription template inhibited cleavage in the wheat germ system. These results suggest that in vitro cleavage occurs between Phe-256 and Ser-257 and that this cleavage is the same as the in vivo cleavage which liberates the N-terminus of HC. o 1991 Academic press, I~C

INTRODUCTION

There are two cleavages, corresponding to the Nand C-termini of the helper component (HC) that are not mediated by Nla. Using deletion analysis and clustered point mutagenesis of Cys residues, Carrington et al. (1989) located a second protease activity in the Cterminal domain of the tobacco etch virus (TEV) HC cistron and termed it HC-Pro. Sequencing of the N-terminus of the downstream product suggested that this activity cleaves at a Gly/Gly dipeptide, presumably generating the C-terminus of mature HC. The Gly/Gly cleavage site was found to be conserved among six potyviruses (Thornbury et a/., 1990). The cleavage site between HC and the N-terminal protein (NT) of the polyprotein has not yet been assigned for any potyvirus. One reason for this is that, at least in the case of TVMV, the N-terminus of HC isolated from infected plants is refractory to Edman degradation (unpublished observations). Anotherexperimental difficulty is that, unlike the other sites, cleavage at the N-terminus of HC does not occur to any significant extent in the RRtranslational system. Such lysates programmed with TVMV RNA produce a 75-kDa polypeptide that is immunologically related to HC (Hellmann et al., 1983). This polypeptide was mapped to the N-terminus of the TVMV polyprotein and proposed to be an unprocessed fusion of the NT and HC proteins (Hellmann et a/., 1986). An alternative system for the translation of potyviral RNA is that derived from wheat germ (WG). This sys-

The RNA genome of the potyvirus group is expressed as a polyprotein that is proteolytically cleaved to produce six known proteins and at least two other potential proteins (reviewed in Dougherty and Carrington, 1988; Hellen et a/., 1989; Shaw et al., 1990). The protease activities responsible for most of the cleavages are known to be virally encoded. The C-terminal domain of the small nuclear inclusion protein, Nla, is the location of the protease activity responsible for five of the cleavages and is capable of making them in a rabbit reticulocyte (RR) translational system programmed with potyviral RNA (Carrington and Dougherty, 1987a; Hellmann et al., 1988). Each of the five cleavage sites is characterized by a conserved amino acid sequence motif which is necessary and sufficient to determine cleavage specificity [Domier et al., 1986; Carrington and Dougherty, 1988; summarized in Fig. 1 for tobacco vein mottling virus (TVMV)]. Nla cleaves at Gln/(Gly or Ser) dipeptides that are downstream of this motif. Cleavage at these sites has been verified in some cases by sequencing the N-terminus of the downstream product (Allison et al., 1985, Carrington and Dougherty, 1987b, 1988; Martin et a/., 1990).

’ To whom dressed.

correspondence

and reprint

requests

should

be ad-

721

0042.6822/91 Copwght All rights

$3.00

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

722

MAVANKAL Amino residue acid numbers

2561257

Polyprotein

N

RHOADS

1112/1113

713/714

0 1)

AND

174711740

0 r

NT

HC

2224l2225

274Ol2741

. 1’

. 1.

1800/r801 .. ,r ,,

Nla

Cl

42K

Nlb

CP

C

6K Cleavage

sequences

ATHFIS

YWGIG

VRFQlS

VRFCl/S

VKFO/G

VRTCUG

VRFCUS

RR products P75h

p120

wg

P75c

Ptm

p49

P75,

WG products NT

HC*

FIG. 1. Schematic of the TVMV polyprotein and the major in vitro-generated products of TVMV RNA. The amino acid residue numbers refer to those of the proposed cleavage dipeptides. The cleavages effected by the Nla protease are shown by solid squares, that by the HC-pro by an open circle, and that identified in this report by an open diamond. The amino acid sequences flanking each cleavage site are indicated in the one-letter code. The location of the major in vitro translation products in RR and WG systems are shown relative to the TVMV polyprotein.

tern yields HC-related products which are more similar in size to HC isolated from infected plants. De Mejia et a/. (1985) found that translation in WG of the RNA of pepper mottle virus (PeMV) and the watermelon mosaic virus-l strain of papaya ringspot virus (PRSV-W) yielded products of 51 kDa which could be detected by immunoprecipitation with antisera against amorphous inclusion (Al) protein (which is presumably the same as HC). The corresponding Al-related proteins made in a RR system, on the other hand, were 78 and 100 kDa, respectively. Hellmann (1988) found that translation of TVMV RNA in WG produced a polypeptide of 44 kDa which was immunoprecipitated with anti-HC antisera. In all three cases, however, these HC- or Al-related proteins were produced in extremely low quantities and could be detected only after enrichment by immunoprecipitation. This low yield was presumably due to premature termination. Koziel et a/. (1980) found that TEV RNA yielded only a single polypeptide of 40 kDa in a WG system. Vance and Beachy (1984) found that soybean mosaic virus RNA produced more products, but the predominant one was 40 kDa as well. In the aforementioned study of De Mejia et al. (1985), translation of the RNAs of PRSV-W and PeMV yielded only a single major polypeptide in each case, having molecular weights of 60 and 30 kDa, respectively. The predominant products of TVMV RNA in WG are polypeptides of 26 and 34 kDa, in contrast to the spectrum of high molecular weight proteins synthesized in a RR system (Hellmann et a/., 1988).

In the present study we have investigated the possibility that the HC polypeptide is synthesized and processed properly in a WG translation system. The evidence indicates that the two most abundant translation products represent NT and HC. By N-terminal sequencing and peptide mapping of the HC-related protein (HC*), we have assigned the in vitro cleavage site.* This site was further confirmed by site-directed mutagenesis. Based on the similarity of HC* and HC, this is presumably the in vivo cleavage site as well.

MATERIALS

AND METHODS

Raw WG of Kenosha winter wheat was obtained from General Mills (Vallejo, CA). WG tRNA was obtained from Sigma Chemical Co. (St. Louis, MO). TVMV RNA, prepared as described in Hellmann et a/. (1980) was generously provided by Mr. Richard Spalding, University of Kentucky. Antisera to TVMV HC, coat protein, and cylindrical inclusion (Hellmann et a/., 1983) as well as RNA from a HAT isolate of TEV and a HC preparation from TVMV-infected plants (Thornbury et al., 1985) were generously provided by Drs. Thomas Pirone and David Thornbury, University of Kentucky. L-[3,4,5-3H]Leu (sp act 140 Ci/mmol), L.-[ring 2,3,4,5,6-

’ The mobility tinguish

in vitro-produced polypeptide of the same electrophoretic and immunoreactivity as HC is here designated HC* to disit from the in viva product.

IN VITRO SYNTHESIS AND N-TERMINUS OF HC

3H]Phe (sp act 1 10 Ci/mmol), L-[4,5-3H]lle (sp act 140 Ci/mmol) and [35S]methionine (sp act 1000 Ci/mmol) were purchased from NEN (Irvine, CA). Polyvinylidenedifluoride (PVDF) membranes were from Millipore (Bedford, MA). fscherichia co/i strain XLl-Blue and pBluescript SK+ were from Stratagene (La Jolla, CA). Dralll restriction enzyme was from Boehringer-Mannheim (Indianapolis, IN). T7 RNA polymerase was from Pharmacia LKB (Piscataway, NJ). Taq DNA polymerase was from Perkin-Elmer Cetus. Oligonucleotides were from the University of Kentucky Macromolecular Structure Analysis Facility. Primers for PCR were selected using the program ‘Oligo’ from National Biosciences (Plymouth, MN). The autoradiography enhancer, Fluoro-hance, was from Research Products International (Mt. Prospect, IL). IgGsorb was purchased from the Enzyme Center (Malden, MA). BNPS-skatole [2-(2’nitrophenylsulfenyl)-3-methyl-3’-bromoindolenine] was from Sigma. Dialysis tubing (Spectra/par, mwco. 1214,000 Da) was purchased from Spectrum Medical Industries (Los Angeles, CA). Thermal cycling was performed on a DNA Thermal Cycler (Perkin-Elmer Cetus). Bands on autoradiograms were quantitated on a Visage 2000 scanner (Bio Image, Ann Arbor, Ml).

Cell-free translation

systems

Translation in a RR lysate system was performed as described previously (Hellmann et al., 1980) except that the final concentration of TVMV RNA was 125 pg/ ml. WG extracts were prepared as described by Rychlik and Zagorski (1978), using a 12-hr dialysis to remove low molecular weight translation inhibitors. The extract was kept frozen at -70°C and used only once after thawing. Translation reaction mixtures contained the following: 1.5 mM magnesium acetate, 75 mM potassium acetate, 5.5 mM dithiothreitol, 0.5 mM spermidine, 50 pg/ml additional WG tRNA, 12 mM HEPESKOH, pH 7.6, 10 mh/l Tris-acetate, pH 7.6, 50 pg/ml creatine phosphokinase, 1 mM ATP, 0.2 mNI GTP, 15 ml\/l creatine phosphate, 40 PLM each of 19 amino acids (excluding the radiolabeled amino acid), 50% v/v WG extract, and 120 pg/ml TVMV RNA. When translations were performed to make products for radiomicrosequencing, the system was depleted of endogenous acetyl-CoA as described by Palmiter (1977). Products for microsequencing were synthesized in 50-to 150-~1 WG translation mixtures containing either [3H]Phe, [3H]Leu, or [3H]lle at l-2 &i/PI. The radiolabeled amino acids were dried without heating in a Speedvac (Savant) prior to addition of the other reaction components. In the case of [3H]lle, the final specific activity was adjusted to 30 Ci/mmol. [35S]Met was present at

723

13 &i/PI and a final specific activity of 500 Ci/mmol, the other 19 amino acids being present at 80 PM. Electrophoresis and fluorography of translation products were performed as described by Hellmann et a/. (1980) except that linear gradient gels of 8-l 2% actylamide were used in some cases. Immunoprecipitations were carried out as described by Hellmann et a/. (1983). For immunoblotting, proteins were resolved by SDS-PAGE, transferred electrophoretically to PVDF membranes using the protocol recommended by the manufacturer, and detected immunologically as described by Harlow and Lane (1988).

Radiomicrosequencing Translation products were separated by SDS-PAGE and transferred to a PVDF membrane as described above, after which they were detected by fluorography in 2,5-diphenyloxazole-saturated ethyl ether. The band of interest was excised and washed with ether to remove diphenyloxazole. [3H]Leu-labeled products were sequenced at the Protein Analysis Laboratory of the Cancer Center of Wake Forest University, and [3H]Pheand [3H]lle-labeled products were sequenced at the University of Kentucky Macromolecular Structure Analysis Facility. The derivatives were collected as either ATZ- or PTH-amino acids. ATZ-amino acids were collected in n-butyl chloride, evaporated to dryness, and solubilized in a toluene-based scintillant. PTH-amino acids were collected in 100% acetonitrile to which the toluene-based scintillant was added directly. Radioactivity was determined by scintillation spectrometry.

Chemical

cleavage of HC*

The HC* polypeptide, labeled with [35S]Met in a WG translation of TVMV RNA, was obtained on a PVDF strip as described above. The strip was wetted with methanol and treated with BNPS-skatole for 2 hr as described by Crimmins et al. (1990). The membranebound and free peptides were then pooled, precipitated with 159/o trichloroacetic acid, washed with 80% acetone, and electrophoretically resolved on a 15% polyacrylamide gel.

Mutagenesis

of the P, site

E. co/i strain XLl-Blue was used for recombinant DNA manipulations. The plasmid pSK4.9 was constructed by inserting the 1.9-kb fragment flanked by the Pstl and BarnHI sites from pXBS7 (Domier et a/., 1989) into the multiple cloning site of pBluescript SK+. The resulting plasmid includes TVMV sequences corresponding to nucleotides (nt) l-l 934 downstream from two T7 promoters.

MAVANKAL

724

AND

A cDNA fragment corresponding to nucleotide residues 947-1998 of TVMV RNA was amplified by PCR from the plasmid pXBS7. The 5’ primer was 5’-TCCATCACACTGCIGTGCGACTCACNN(G/C)TCAACT-3’ (corresponding to TVMV RNA nt 947-979) and the 3’ primer was 5’-TATCCTTCCTTTGCTATGTA-3’(complementary to TVMV RNA nt 1979-1998) the italicized nucleotides representing the Dralll restriction enzymerecognition site, and the slash representing the Dralll cleavage site. The Dralll site at nt 960 and the BarnHI site at nt 1934 are unique in the PCR-amplified segment. The sequence NN(G/C) in the 5’ primer replaces the lTC at nt 97 l-973. This provided the possibility of changing the codon for Phe-256 to 32 different codons that include codons for all amino acid residues. PCR was done using a [r3*P]-labeled 5’ primer (0.3 X lo6 dpm) in a 100-~1 reaction using 0.5 ng of closed circular pXBS7 DNA, 1 @I of each primer, 0.2 ml\/l dNTPs, 10 mM Tris-Cl, pH 8.4, 50 mM KCI, and 1.5 mM MgCI,. Annealing was done at 45°C for 1 min. The PCR product was completely digested by Dralll’and BarnHI to yield a fragment corresponding to TVMV nt 960-l 934. The Dralll digestion was monitored by loss of radioactivity in the product resolved on an agarose gel. A partial digestion of BarnHI-linearized pSK4.9 with Dralll was used to remove the segment of the TVMV sequence corresponding to nt 960-l 934. A ligation of this DNA with the PCR-amplified fragment resulted in

A

II

B

RR

P75,\ P75/-

aHC

aCP

the replacement of the Dralll-BarnHI fragment in pSK4.9 with the PCR-amplified segment bearing the mutagenized cassette. Plasmids isolated from colonies resulting from this ligation were termed as the pF4.9 series of plasmids. Plasmids that were characterized by sequencing around the region of the mutation were designated pF4.9M, pF4.9R, pF4.9P, pF4.9H, and pF4.9W and contained ATG, AGG, CCG, CAC, and TGG, respectively, at nt 971-973. The nomenclature of the plasmids indicates the resulting amino acid substitution using one letter symbols. Transcripts were obtained from BarnHI-linearized pF4.9 series of plasmid DNA in transcription reactions using T7 RNA polymerase as described in Hellmann et al. (1988). The transcripts were used in translations in WG as described above, except that the concentration of the RNA was 20 pg/ml. RESULTS Translation

of TVMV RNA in WG extracts

Most studies of the in vitro translation of potyviral RNA have employed the RR system, in which products derived from the entire genome are observed (Dougherty and Hiebert, 1980a,b; Hellmann et al., 1980, 1983). The relatively few studies employing WG extracts for translation have yielded results which sug-

c

WG

lmmunoppt

Total

RHOADS

4?[

lmmunoppt Total

aCI

aHC

aCP

h cl 3

aCI kD

$ Y kD

--200 P75C P75,, \-r

92.5

HC* -

-69

FIG. 2. Translation of TVMV RNA in RR and WG systems. Translations were performed in the presence of [36S]Met and the products were resolved by SDS-PAGE on an 8-l 2% gradient gel and fluorographed. (A) RR products. Lane 1, total translation products; and Lanes 2-4, immunoprecipitation with antisera to TVMV HC, CP and Cl, respectively. (B) WG products. Lane 1, total translation products: and Lanes 2-4, immunoprecipitation with antisera to TVMV HC, CP, and Cl, respectively. Insets represent an enlargement of a portion of the lane to show resolution of the two p75 products derived from the N-terminus (~75~) and the C-terminus (~75,) of the TVMV polyprotein. (C) Proteins resolved by SDS-PAGE on a 10% acrylamide gel and transferred to a PVDF membrane. Lane 1, fluorogram of total translation products in a WG system; and Lane 2, a HC preparation from TVMV-infected plants, stained with Coomassie blue. (D) Proteins resolved by SDS-PAGE on an 8-l 5% gel. Lane 1, fluorogram of total translation products in a WG system. The relatively simpler pattern, specifically the absence of high molecular weight products compared with (B) and (D) is due to the differential transfer of proteins to PVDF membranes. Lane 2, a HC preparation from TVMV-in fected plants, stained with Coomasie blue.

IN WTRO

SYNTHESIS

AND TABLE

SIZES OF THE N-TERMINAL

N-TERMINUS

Est. Mol. Mass (A)

TVMV TEV

34.0 35.0

a.a. res. # l-256 l-304

HC

725

1

PROTEIN AND HELPER COMPONENT

NT Viral RNA

OF

PROTEIN OF TEV AND TVMV HC*

Calc. Mol. Mass

Est. Mol. Mass (B)

29.2 34.7

46.0 53.0

a.a. res. #

P75, Calc. Mol. Mass

Est. Mol. Mass

51.2 51.8

78.0 84.0

257-713 305-763

A+B 80.0 88.0

Note. The estimated molecular masses of proteins (Est. Mol. Mass) are expressed in kDa and obtained from Figs. 2 (TVMV) and 5 (TEV). Similar results are obtained if Fig. 5 is used to estimate molecular masses for TVMV proteins. The calculated molecular masses (Calc. Mol. Mass) are based on the amino acid residue numbers given, in which the initiator Met of the polyprotein is 1.

gest premature termination was occurring (see Introduction). In an attempt to obtain longer translation products and ones which were representative of the entire viral genome, we added spermidine and dithiothreitol to the WG system. Varying the amounts of these two reagents produced a system in which large polypeptides were observed (Fig. 2B). For comparison, TVMV RNA was also translated in the RR system (Fig. 2A). A number of the same polypeptides were formed in both systems, notably in the size range of 75 kDa. TVMV RNA actually produces two polypeptides of 75 kDa in the RR system (Hellmann eta/., 1983). These can be distinguished immunologically and correspond to the unprocessed precursor of NT and HC, designated p75,, and that of Nlb and CP, designated ~75, (see Fig. 1 for summary). In the present case, use of anti-HC and anti-CP antibodies demonstrated that both ~75, and ~75, were formed in both translation systems (Figs. 2A and 2B, Lanes 2 and 3). We also found that the use of 8-120~ gradient gels permitted partial resolution of p75,, and p75,, as indicated in the insets to Figs. 2A and B and by the slight differences in their mobilities in Lanes 3 vs Lanes 2. Since ~75, and ~75, are derived from the N- and C-termini of the polyprotein, respectively, their synthesis indicates that the entire TVMV genome is translated in this particular WG system. Comparison of translation products from the two systems reveals the presence of several polypeptides formed in WG but not RR. Most prominent among these are polypeptides of 34 and 46 kDa, designated NT and HC* in Fig. 2B. HC* was immunoprecipitated by anti-HC antisera (Fig. 2B, Lane 2) but not (or only weakly) by anti-CP or anti-Cl sera (Lanes 3 and 4). NT was only weakly immunoprecipitated by anti-HC and anti-CP and not at all by anti-Cl. However, considering that NT is the major polypeptide formed in the WG system, the amount of its immunoprecipitation relative to that of the specific antigens (HC*, p75,, ~75,) sug-

gests that this represents nonspecific adsorption of NT to the antibodies or to IgGsorb. Table 1 indicates that the sum of the estimated molecular masses of NT and HC* (80 kDa) is near that of p75,, (78 kDa). This, together with the observation that HC* was immunoprecipitated by anti-HC antisera, suggests that ~75, is cleaved in the WG system to NT and HC*. Assuming that correct C-terminal processing of HC occurs in the WG system, HC* may represent a properly processed HC protein. In order to compare the sizes of HC* and HC, we separated WG translation products and a partially purified HC preparation by SDS-PAGE using a 10% gel. The proteins were then transferred to a single PVDF membrane to prevent artifacts due to gel shrinkage. As shown in Fig. 2C, HC* (Lane 1) co-migrated with the major protein in the HC preparation (Lane 2). The HC preparation at this stage of the purification protocol contains HC and several plant proteins, but the one designated HC in Fig. 2C, Lane 2, has been previously identified as such with the use of specific antisera (Thornbury et al., 1985). This assignment was confirmed in the present case by immunoblotting of a third PVDF strip derived from the same electrophoretic separation (data not shown). The difference in the molecular mass estimate for HC* in this experiment (Fig. 2C, 53 kDa) compared with the previous experiment (Fig. 2B, 46 kDa) is due to the use of a 10% gel instead of an 8-12% gradient gel. As the relative change in mobility with polyacrylamide concentration can vary for different proteins, we compared the mobilities of HC* and HC under additional gel conditions. The two proteins were also found to co-migrate using gels of 8-l 5% (linear gradient; Fig. 20) 12 and 15% (data not shown). The N-terminus

of HC*

Assignment of the probable cleavage sites for the majority of the TVMV proteins has been facilitated by

726

MAVANKAL

157 STGDlFWKGFNASFQECMAlGAKIILDm

28

L

12 24 Edman cycle number FIG. 3. Radiomicrosequence analysis of HC*. PVDF strips bearing HC* labeled in three different experiments with different tritium-labeled amino acids were subjected to automated Edman degradatlon. The top, middle, and bottom panels correspond, respectively, to: 30,000 cpm of [3H]Leu-labeled HC* (600 cpmkes); 60,000 cpm of [3H]Phe-labeled HC* (3000 cpmkes); and 30,000 cpm of [3H]llelabeled HC* (1 100 cpm/res), applied to the sequenator. [3H]Leu and [3H]Phe were collected as ATZ-amino acids and [3H]lle as PTHamino acids. The radioactivity released in each Edman cycle is shown as the ordinate. The amino acid bearing the label is shown as the single letter amino acid symbol. The only sequence in the TVMV polyprotein that can account for the pattern of radioactivity is that shown at the top of the figure.

the occurrence of a remarkably conserved amino acid sequence motif, VBUQ/Z3 (Domieretal., 1986; summarized in Fig. 1). This motif does not occur, however, within the first three polypeptides of the TVMV polyprotein and consequently does not provide a basis for assigning a probable cleavage site at the N-terminus of HC. Domier et al. (1986) suggested that the TVMV HC began at residue 248 of the polyprotein, based on the

3 In this consensus sequence, B is a basic amino acid (R or K), U is an aromatic amino acid (F or Y), and Z IS a small amino acid (G or S).

AND

RHOADS

size of HC and the distribution of Gln-Ser dipeptides in the polyprotein. Hellmann (1988) suggested a start site of residue 301 based on sequence conservation between TEV and TVMV, the occurrence of Gln-Ser and Gln-Ala dipeptides, and the sizes of major translation products from each viral RNA in WG extracts. Because of the uncertainty associated with these approaches, we undertook radiochemical sequencing of HC*. In separate experiments, HC* was labeled with [3H]Leu, [3H]Phe, and [3H]lle. The products were tra.nsferred to a PVDF membrane, and the band bearing HC* was located by fluorography, excised, and subjected to automated Edman degradation (Fig. 3). With [3H]Leu, a significant increase over background occurred at cycle 22, which, by comparison with the TVMV polyprotein sequence, would correspond to the N-terminus of HC* being residue 257. The signal-tonoise ratio was low, however, because this Leu occurs after many cycles, and each cleavage cycle is attended by a decrease in overall yield. Also, because HC* is a much larger polypeptide than those normally subjected to radiochemical sequencing, the background rises significantly by cycle 22, due to nonspecific internal cleavage. With [3H]Phe, the first increase in radioactivity occurred at residue 6, producing a higher signal-to-noise ratio. Although there was some carryover of radioactivity to the next cycle, indicating incomplete cleavage or washout of ATZ-Phe, the most straightforward interpretation is that Phe occurs at cycles 6, 10, and 14. The pattern of FX,FX,F occurs only once in the 3005amino acid residue polyprotein of TVMV and corresponds to the N-terminus of HC* being residue 257, the same site predicted from the [3H]Leu experiment. With [3H]lle, the signal-to-noise ratio was further improved, the major releases of radioactivity occurring at cycles 5 and 20. The pattern of radioactivity corresponding to a sequence of IX,,1 occurs only once in the TVMV polyprotein, corresponding to the N-terminus of HC* being residue 257. Alternative interpretations of the data in Fig. 3 were also tested (viz., Leu at cycle 15; Phe at cycles 7, 1 1, and 15; and Ile at cycles 10 and 21). In each of these cases, the pattern produced by the alternate assignment was compared to the TVMV polyprotein sequence (e.g., X,,LX,LX,, F,X,FX,F, etc.). However, none of these additional patterns occurred in the polyprotein. This indicates that only those assignments shown in Fig. 3 are correct. Thus, the patterns with all three 3H-labeled amino acids yield the same location for the N-terminus of HC*, Ser-257 of the polyprotein, and suggest that the Phe/Ser at 256/257 is the cleavage dipeptide.

IN !//TRO

SYNTHESIS

AND

N-TERMINUS

OF HC

727

C +

-

BNPS-Skatole 1 -69

-a-b -x-c

kD 92.5

(1) lP

46

-

30

(2) r’

-d -e

(b) (d)

50.4 kD

Molecular

23.5 kD

/

42.7 kD (d’)

I (b’)

41.9 kD

22.6 kD

O (c)

27.6

(d)

23.5 kD

/

&I’)

22.6 kD

2

(e)

19.3 kD

weight

1

(kD)

Predicted

50.7

51.2

, 50.4

42.5

42.7

, 41.9

32.5

27.0

24.7

23.5

20.7

19.3

kD

14.3 (3)

1

P (a’)

Observed

-

-

7 51.2 kD I

(a)

, 22.6

O

not detected

6.5

FIG. 4. Chemical cleavage analysis of HC*. [%]Met-labeled HC* was transferred to a PVDF strip, treated with a lOO-fold excess of BNPS-skatole, resolved by SDS-PAGE on a 15% gel, and fluorographed. (A) Lane 1, products obtained after treatment. Lane 2, control HC* treated identically except for the omission of the BNPS-skatole. (B) Schematic of the peptides predicted to be generated by tryptophan-specific cleavage of the TVMV polyprotein segment from amino acid residues 257 to 713. The open circle represents the positions of the Trp residues, which occur at positions 7, 208, and 378. (1) The uncleaved product or that cleaved at Trp-7. This particular cleavage is not explicitly shown as are the other two cleavages because it would not be expected to product resolvable products. The net effect of this cleavage is to produce a heterogeneity in the N-terminal peptides which is depicted in the figure as a,a’ etc. (2) The effect of cleavage at one of two sites. (3) The effect of cleavages at both sites, (C) Molecular weights of peptides a-f: observed (from (A)) and predicted (from (B)).

Chemical

cleavage

of HC*

Further confirmation that in vitro cleavage occurs between Phe-256 and Ser-257 was obtained by chemical fragmentation of HC*. For this analysis, HC* was generated in the WG system, electrophoretically purified, transferred to PVDF membranes, and subjected to par-

TEV Total

TVMV a HC

Total

aHC

kDa

P75 ft HC* NT

1

2

3

4

FIG. 5. Translation of TEV RNA in a WG system. The total translation products of TEV and TVMV RNA (Total) and the products immunoprecipitated with TVMV HC-antisera ((u HC) were resolved by SDS-PAGE on a 12% gel and fluorographed. Lanes 1 and 2. TEV products; Lanes 3 and 4, TVMV products.

tial cleavage with BNPS-skatole, a Trp-specific reagent (Fig. 4A). A series of peptides was generated, the major ones designated a-e (Lane 1). A minor peptide, x, was present as a contaminant in the uncleaved HC* preparation (Lane 2). Peptide x migrates at 38 kDa and could possibly be residual NT that initially co-purified with HC* but separated from it at the subsequent step when peptides were resolved. If so, peptide x would not be cleaved by the reagent because NT does not contain Trp residues. Because it is present in the uncleaved starting material, peptide x is not considered in the following analysis. Estimation of the molecular weights of the remaining peptides from electrophoretic mobility yielded the results shown in Fig. 4C (observed). In order to determine whether these peptide sizes were consistent with the proposed N- and C-termini of HC”, we constructed a model for HC* in which the N-terminus was Ser-257, based on the results of Fig. 3, and the C-terminus was Gly-713, based on the assignment of Carrington et al. (1989) for TEV (Fig. 4B). Trp residues occur at three locations in the hypothetical HC* sequence, residues 7, 208, and 378 (counting the N-terminal Ser as 1 in this instance). Cleavage at Trp-7 would not be expected to alter mobility detectably, but cleavage at Trp-378 would produce peptides b and f. Peptide f is too small to be detected on the gel, but peptide b is clearly present (Fig. 4A) and its mobility is close to that predicted by the model (Fig. 4C). Similarly,

MAVANKAL

728

cleavage at Trp-208 would produce peptides c and d. Peptide c, the only C-terminal peptide that could be detected on the gel, showed a difference of 4.7 kDa between the observed and predicted sizes. This could be due to the assignment of the C-terminus being incorrect. Peptide d, however, was close to the predicted size. Double cleavage at Trp-208 and Trp-378 would leave peptide d intact and would produce a new peptide e, which is clearly present in Fig. 4A. From the intensities of peptides d and e, it is apparent that most of the HC* was doubly (or triply) cleaved. Thus, the close agreement of predicted and observed peptide sizes for peptides other than peptide c supports the assignment of Ser-257 as the N-terminus of HC*. Translation

of TEV RNA

To address the question of whether cleavage occurs between NT and HC* in the WG system for a potyvirus other than TVMV, we translated TEV RNA (Fig. 5). The 87-kDa protein previously suggested to be the NT-HC* fusion polypeptide (Hiebert et al., 1984) was the predominant product, but major products of approximately 35 and 53 kDa were also formed (Fig. 5, Lane 1). For comparison, the translation products of TVMV RNA are also shown (Fig. 5, Lanes 3 and 4). Immunoprecipitation of the TEV translation mixture with antisera to TVMV HC revealed the presence of two HC-related polypeptides (Lane 2) presumably the analogues of ~75, and HC* of TVMV. The estimated molecular weights of these polypeptides as well as the analogue of NT are presented in Table 1. As in the case of TVMV, the combined molecular weights of the NT and HC* analogues is similar to that of the ~75, analogue. Thus, both molecular mass and immunoprecipitation data support the idea that the NT and HC* analogues are derived by proteolysis of the ~75, analogue, suggesting that both potyviral polyproteins undergo a similar processing step in the WG system. Mutagenesis

of the P, site

N-terminal sequencing of HC* suggested Phe-256/ Ser-257 to be the cleavage site. If so, amino acid substitutions at the P, residue might be expected to affect cleavage. A construct, pSK4.9, which yields a runoff transcript encoding a segment of the TVMV polyprotein corresponding to amino acids l-576, was used for site-directed mutagenesis. This represents a truncated form of p75,, designated Ap75,, of 65 kDa which should be processed to polypeptides of 29 kDa (NT) and 36 kDa (AHC). Translation of the wildtype pSK4.9 transcript in WG resulted in a major product migrating at 34 kDa (Fig. 6A, Lane l), which is the same as that

AND

RHOADS A. 4.9

Total

products

M

B

RPHW

kD

-r -

g2 69

-

46

-

1 / 48

234 44 24

7

56 19 31 /

zHC

4.9

Immunoprecipitates M

R

P

HW

- AP7fjh

30

7

8

9

I

AHC

-L

NT

10 11 12

% process,ng FIG. 6. Effect of amino acid substitution at the P, site. Transcripts from BarnHI-linearized plasmids of pSK4.9 and the pF4.9 series were translated in WG in the presence of [35S]Met and the products were resolved by SDS-PAGE on a 12% gel. (A) Total products and (B) immunoprecipitates of the total products with anti-HC antiserum of the wild-type pSK4.9 (Lanes 1 and 7) pF4.9M (Lanes 2 and a), pF4.9R (Lanes 3 and 9) pF4.9P (Lanes 4 and lo), pF4.9H (Lanes 5 and 1 l), and pF4.9W (Lanes 6 and 12) respectively. The percentage processing is computed as (NT/(2 X NT + Ap75,)) X 100, where the amount of each protein was determined from a scan of the lanes in (A). The differences in the amount of total products for different pF4.9 plasmids were due to variations in the amount of transcript RNA added to the translation reactions.

observed for NT in Fig. 2. The 36-kDa product (AHC), however, was not detected in the total translation products. Hence, an immunoprecipitation with anti-HC antiserum was performed. As seen in Lane 7, a product of 36 kDa was enriched, which is the size expected for AHC. The presence of a second band at 34 kDa is consistent with the weak or nonspecific immunoprecipitation of NT, the major translation product; this was also observed in Fig. 2B, Lane 2; and Fig. 5, Lane 4. This result suggests that AHC is either insoluble or degraded in WG. The extent of processing was therefore computed as the relative fraction of NT to total, where the amount of HC was calculated as equimolar to NT. A variety of pF4.9 plasmids were produced. Five were chosen for sequence analysis and were found to encode Met, Arg, Pro, His, or Trp in place of Phe-256. In all cases examined, NT was reduced and Ap75, was enhanced, indicating that cleavage was inhibited (Fig. 6A). Substitution by Met affected processing the least (Lane 2) and that by Pro, the most (Lane 4). Other substitutions had an intermediate effect (Lanes 3, 5, and 6). The immunoprecipitates of the total products with anti-HC antiserum show the presence of AHC in the products from constructs pF4.9M and pF4.9W but little or none from pF4.9R, pF4.9P and pF4.9H (Fig. 6B),

IN V/T/?0

in agreement with the percentage processing calculated from the data in Fig. 6A.

SYNTHESIS

AND

results

DISCUSSION The results of the translation of potyviral RNA in WG reported here differ from previous reports (Koziel et al., 1980; Hellmann eta/., 1980; Vance and Beachy, 1984; de Mejia eta/., 1985; Hellmann eta/., 1988; Hellmann, 1988) in that products which map to both the 5’ and 3’ ends of the RNA template are observed (Figs. 1 and 2). We have not systematically investigated the reason for this, but it is likely to be some combination of factors that include the source of WG, the long-term dialysis to remove low molecular weight inhibitors of translation (Rychlik and Zagorski, 1978) and the inclusion of optimal amounts of dithiothreitol and spermidine. The WG system is notorious for forming prematurely terminated products, thought to be due to RNA secondary structure-induced stalling of ribosomes (Chroboczek, 1985) and this is particularly exaggerated in the case of such an extraordinarily long mRNA as the 10-kb potyviral RNA. The polarity of translation causes the production of an excess of 5’-terminal products. Such a polarity could preclude the assignment of the NT polypeptide as the correctly processed N-terminal protein since its occurrence might be due to premature termination rather than proteolytic processing. The observation of comparable amounts of HC* and NT as major products in our WG system (Fig. 28, Lane 1) argues in favor of in vitro cleavage, because the NT and HC* polypeptides would be generated from a common precursor. Processing of p75,, however, is incomplete in the WG system, judging from the presence of both HC* and ~75,. The presence of still larger precursors of HC, CP and Cl in WG (Fig. 2B) that are not observed in RR

A

294

R

C

TEV

290

R A

K

OF

HC

729

(Fig. 2A) suggests that processing by other TVMV proteases is not as efficient in WG as in RR. As noted above, de Mejia et al. (1985) also observed HC-related products of a similar size as the respective HC proteins when PRSV-W and PeMV RNA were translated in WG. However, because they did not observe similar amounts of NT and HC, they proposed internal initiation to account for the appearance of HC and argued against a proteolytic mechanism. An important yet unresolved issue is whether HC* is identical to HC derived from infected tissue. Until the N- and C-termini of HC are chemically determined, their assignments as Ser-257 and Gly-713 must be considered tentative. However, there are a number of arguments which make these assignments likely. First, the fact that in vitro translation produces essentially homogeneous NT and HC* polypeptides (e.g., Fig. 2C, Lane 1) implies a specific protease hydrolyzing a single peptide bond at each junction of HC rather than nonspecific WG proteases. Second, the HC* and HC polypeptides electrophoretically co-migrate in four different polyacrylamide gel concentrations. A third argument is based on phylogenetic considerations. In Fig. 7 we compare polyprotein sequences from six potyviruses in the region homologous to our proposed cleavage site for TVMV. Also indicated are the regions surrounding the proposed sites of Domier et al. (1986) and Hellmann (1988). For the site of Domier et a/. (Fig. 7A), the Q/S is not found in three of the six sequences (two of six if the displaced Q/S of PPV is counted). For the site of Hellmann (Fig. 7C), the H/S is likewise not found in three of the six cases. The best conservation is for the site proposed in this report (Fig. 7B), for which F/S occurs in four of the six sequences, and the closely related Y/S appears in the other two. A compelling fourth

B Pl, Pl’

PPV

N-TERMINUS

C Pl

Pl, Pl’

V S

K

K 0

K V T

F

A

V

S

Pl’ L

302

‘0

K

M7

TEV

%VAAl

LTQ

FIG. 7. Comparison of three proposals for the cleavage site between NT and HC in potyviruses. The amino acid sequences of three regions from four potyvirus polyproteins and two strains of PVY are aligned as described by Thornbury et al. (1990). Proposed cleavage sites are indicated by an arrow flanked by the amino acid residues constituting the cleavage dipeptides, Pl and Pl’. The most conserved amino acids of the dipeptide are boxed. (A) The site proposed by Domier et a/. (1986). (B) The site proposed in this report. (C) The site proposed by Hellmann (1988).

MAVANKAL

730

argument is the inhibition of cleavage when Phe at the P, site is replaced by five different amino acid residues. The protease activity responsible for the cleavage at the N-terminus of HC has not at present been identified. It seems clear that viral sequences downstream of the hypothetical 42-kDa protein are not required, since expression in transgenic plants of a cDNA containing the 5’-terminus of the TVMV genome, encoding the NT, HC, and 42-kDa proteins, leads to the accumulation of correctly processed HC (Berger el al., 1989). Similarly, Carrington et a/. (1990) showed that transgenie expression of constructs encoding the NT and HC of TEV resulted in HC of the in viva size, indicating that the protease in question is not in the third polypeptide of the polyprotein (which is 50 kDa in the case of TEV). These authors also showed that a point mutation which abolished HC-Pro activity did not prevent N-terminal processing of HC, indicating that the protease is not the one which resides in the C-terminal domain of HC. Hellmann (1988) further delimited the sequence needed by translating truncated forms of TVMV RNA in a WG system, showing that N-terminal processing of HC still occurred when essentially all of HC was deleted. This suggests that the activity resides in NT. Carrington et a/. (1990) deleted a 19-kDa segment from the NT of TEV and observed only partial inhibition of the N-terminal processing of HC. Thus, the precise location of the protease activity is not presently known. In fact, it is not yet possible to distinguish between host and viral sources for the proteolytic activity. The observation that the cleavage occurs in WG but not RR systems is compatible with either the absence of a needed host factor or the presence of an inhibitor in RR.

ACKNOWLEDGMENTS We are grateful to Drs. John Shaw, David Thornbury, and Thomas Pirone for numerous helpful discussions and for providing viral RNAs. TVMV HC, and antisera to viral proteins. We thank Dr. Phillip Ryals, Mississippi State University, Starkville, Mississippi, for providing the plasmid pSK4.9. This work was supported by Grant 4E021 from the University of Kentucky Tobacco and Health Research Institute, and Grant 90-37262-5519 from the USDA. The Protein Analysis Laboratory of the Cancer Center of Wake Forest University is supported in part by NIH Grants CA1 2197 and RR04869, and by a grant from the North Carolina Biotechnology Center.

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AND

RHOADS transgenic plants of a viral gene product that mediates insect transmission of potyviruses. Proc. Nat/. Acad. Sci. USA 86, 84028406. CARRINGTON, J. C., and DOUGHERTY. W. G. (1987a). Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. 1. Virol. 61, 2540-2548. CARRINGTON, J. C., and DOUGHERI-Y, W. G. (198713). Processing of the tobacco etch virus 49K protease requires autoproteolysis. Virology 160, 355-362. CARRINGTON, J. C., and DOUGHERP/, W. G. (1988). Aviral cleavage site cassette. Identification of amino acid sequences required for Tobacco Etch Virus polyprotein processing. Proc. Nat/. Acad. SC;. USA 85, 3391-3395. CARRINGTON, J. C.. CARY, S. M., PARKS, T. D., and DOUGHERI-Y, W. G. (1989). A second proteinase encoded by a plant potyvlrus genome. EMBO/. 8, 365-370. CARRINGTON. J. C., FREED, D. D., and OH, C-S. (1990). Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing. EMBO /. 9, 13471353. CHROBOCZEK, J. (1985). Incomplete polypeptides are formed in vitro by premature chain termination. EMBO /. 149, 565-569. CRIMMINS, D. L., MCCOURT, D. W., THOMA, R. S., SCOTT, M. G., MACKE, K., and SCHWARTZ, B. D. (1990). In situ chemical cleavage of proteins immobilized to glass-fiber and polyvinylidenedifluonde membranes: Cleavage at tryptophan residues with 2-(2’.nitrophenylsulfenyl)-3-methyl-3’-bromoindolenine to obtain internal amino acid sequence. Anal. Biochem. 187, 27-38. 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. DOMIER, L. L., FRANKLIN, K. M., HUNT, A. G., RHOADS, R. E., and SHAW, J. G. (1989). Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus. Proc. Nat/. Acad. Sci. 86, 3509-3513. DOMIER, L. L., FRANKLIN. K. M., SHAHABUDDIN, M., HELLMANN, G. M., OVERMEYER, J. H., HIREMATH, S. T., SIAW. M. F. E., LOMONOSSOFF, G. F., SHAW, J. G., and RHOADS, R. E. (1986). The nucleotide sequence of tobacco vein mottling virus RNA. Nucleic Acids Res. 14, 5417-5430. DOMIER, L. L., SHAW, J. G., and RHOADS, R. E. (1987). Potyviral proteins share amino acid sequence homology with picorna-, coma-, and caulimoviral proteins. Virology 158, 20-27. DOUGHERTY, W. G., and CARRINGTON, J. C. (1988). Expression and function of potyviral gene products. Annu. Rev. Phytopathol. 26, 123-143. 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. DOUGHERP/, W. G., and HIEBERT, E. (1980b). Translation of potyvirus RNA In a rabbit reticulocyte lysate: Identification of nuclear inclusion proteins as products of tobacco etch virus RNA translatton and cylindrical inclusion proteins as a product of the potyvirus genome. Virology 104, 174-l 82. HARLOW, E., and LANE, D. (1988). “Antibodies: A Laboratory Manual,” pp. 497-510. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. HELLEN, C. U. T., KRAUSSLICH, H-G., and WIMMER, E. (1989). Proteolytic processing of polyproteins in the replication of RNA viruses. Biochemistry 28, 988 l-9890. HELLMANN, G. M. (1988). “Proteolytlc processing in the genome ex-

IN V/T/70

SYNTHESIS

pression of tobacco vein mottling virus.” Ph.D. Dissertation, University of Kentucky, KY. HELLMANN, G. M., SHAW, J. G., LESNAW, J. A., CHU, L-Y., PIRONE, T. P., and RHOADS, R. E. (1980). Cell-free translation of tobacco vein mottling virus RNA. Virology 106, 207-2 16. HELLMANN, G. M., THORNBURY, D. W., HIEBERT, E., SHAW, J. G., PIRONE, T. P., and RHOADS, R. E. (1983). Cell-free translation of tobacco vein mottling virus RNA. II. lmmunoprecipitation of products by antisera to cylindrical inclusion, nuclear inclusion and helper component proteins. Virology 124, 434-444. HELLMANN, G. M., HIREMATH, S. T., SHAW, J. G., and RHOADS, R. E. (1986). Cistron mapping of tobacco vein mottling virus. L/iro/ogy 151,159-171. HELLMANN, G. M., SHAW, J. G., and RHOADS, R. E. (1988). ln vitro analysis of tobacco vein mottling virus Nla cistron: Evidence for a virus-encoded protease. Virology 163, 554-562. HIEBERT, E., THORNBURY, D. W., and PIRONE, T. P. (1984). Immunoprecipitation analysis of potyviral in vitro translation products using antisera to helper component of tobacco vein mottling virus and potato virus Y. virology 135, l-9. KOZIEL, M. G., HARI, V., and SIEGEL, A. (1980). In vitro translation of tobacco etch virus RNA. wrology 106, 177-l 79. MARTIN. M. T., OTIN, C. L., LAIN, S., and GARCIA, J. A. (1990). Determi-

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nation of polyprotein processing sites by amino terminal sequencing of nonstructural proteins encoded by plum pox potyvirus. Virus Res. 15, 97-l 06. PALMITER, R. D. (1977). Prevention of N-terminal acetylation of proteins synthesized in cell-free systems. 1. Biol. Chem. 252, 878 l8783. RYCHLIK, W., and ZAGORSKI, W. (1978). Protein synthesizing system from wheat germ. Efficient translation of synthetic and natural messages. Acta Biochim. Polonica 25, 129-l 46. SHAW, J. G., HUNT, A. G., PIRONE, T. P., and RHOADS, R. E. (1990). Organization and expression of potyvirus genes. In “Viral Genes and Plant Pathogenesis” (T. P. Pirone and J. G. Shaw, Eds.), pp. 107-l 23. Springer Verlag, NY. THORNBURY, D. W., HELLMANN, G. M., RHOADS, R. E., and PIRONE, T. P. (1985). Purification and characterization of potyvirus helper component. Virology 144, 260-267. THORNBURY, D. W., PATTERSON, C. A., DESSENS, 1. T., and PIRONE, T. P. (1990). Comparative sequence of the helper component (HC) region of potato virus Y and a HC-defective strain, potato virus C. Virology 178, 573-578. VANCE, V. B., and BEACHY, R. N. (1984). Translation of soybean mosaic virus RNA in vitro: Evidence of protein processing. Virology 132,271-281.