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J. gen. Virol. (1985), 66, 2571-2579. Printed in Great Britain 2571 Key words: tymovirus/eoat proteins/structure Comparisons between the Primary Structure of the Coat Proteins of Turnip Yellow Mosaic Virus and Eggplant Mosaic Virus By A N D R I ~ D U P I N , * DANIEL COLLOT, RI~MY PETER AND J E A N WITZ 1 Laboratoire de Physiologic VOg(tale, Institut de Botanique, 28 rue Goethe, 67083 Strasbourg Cedex and 1DOpartement de Virologic, Institut de Biologic MolOculaire et Cellulaire du C.N.R.S., 15 rue Descartes, 67084 Strasbourg Cedex, France (Accepted 28 August 1985) SUMMARY Comparison of the primary structures of eggplant mosaic virus (EMV) and turnip yellow mosaic virus (TYMV) coat proteins shows that 3 2 ~ of their amino acids are conserved. Alignment of the two sequences requires only one deletion near the N terminus and two insertions at the C terminus of T Y M V coat protein. Although the coat protein of EMV is on average less hydrophobic than that of T Y M V , structural predictions yield fairly similar conformations for the two proteins, a p a r t from the N terminus. Neither coat protein possesses an accumulation of basic residues able to form the strong ionic R N A - p r o t e i n interactions observed in several other isometric viruses. The nature of the amino acid exchanges seems to be different from that seen in families of homologous proteins. The highly conserved regions encompass a (probably weak) potential R N A - p r o t e i n interaction site. Implications for the structure and stability of small isometric viruses are discussed. INTRODUCTION Tymoviruses are small isometric plant viruses stabilized by strong p r o t e i n - p r o t e i n interactions. Their properties have been reviewed recently by Koenig & Lesemann (1979, 1981). The type m e m b e r of the group is turnip yellow mosaic virus (TYMV). The primary structure of its coat protein (Peter et al., 1972) as well as a large part of the nucleotide sequence of its R N A are known (see for instance Guilley & Briand, 1978, and references therein). N o high resolution details on its three-dimensional organization are available, but chemical cross-linking has shed some light on the R N A - p r o t e i n interaction sites (Ehresmann et al., 1980) and several antigenic determinants have been characterized (Pratt et al., 1980; Quesniaux et al., 1983a, b). The primary structure of the coat protein of a second tymovirus, eggplant mosaic virus (EMV), has been determined recently (Dupin et al., 1984). Since E M V is serologically not directly related to T Y M V (Koenig, 1976) and since the stabilities of its virions and capsids differ from those of T Y M V (Bouley et al., 1977), we compare here the primary structures of the two coat proteins and discuss the implications for various properties of the virions. METHODS The primary structures of the coat proteins ofTYMV (P-TY) and EMV (P-E) were taken from Peter et al. (1972) and Dupin et al. (1984). The amino acid sequences were established by automatic sequencing and mass spectroscopic studies of the products of cleavage of the purified proteins by cyanogen bromide, BNPS-skatole, trypsin, chymotrypsin and thermolysin. A computer program written by G. de Marcillac for a Univac 1100 computer was used to align the protein sequences. Sliding one sequence along the other, it indicated in a two-dimensional plot whether identical amino acids face each other; alignments show up as lines of dots parallel to a diagonal [this program is similar in principle to the simplest program of Staden (1982); it does not make use of a scoring matrix]. Assuming that EMV and TYMV derive directly from each other, expected amino acid substitution frequencies between the primary structures of EMV and TYMV coat proteins were calculated the following way: both aligned sequences (as in Fig. 1) encompass N amino acids, n~~,n,i, n~i and n2j are the numbers of amino acids i andj in the 0000-6621 © 1985 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 01:31:27 2572 A. D U P I N AND OTHERS first and second sequence, respectively (1 ~< i, j ~< 21 if deletions and insertions are taken into account). If substitutions are random, the number of possible substitutions involving residues i andj :~ i is nl~ n2j + n~j nzi, and the number of possible conservative exchanges is n~, n_,~.The total number of possible exchanges (conservative or not) is N 2, of which N actually occur. The expected numbers of random substitutions and conservations are (nli nzj + n~j n2~)/N and nl~ n_,jN, respectively. If the pattern of substitutions follows the rules established for a large sample of protein families, these figures should be multiplied by the coefficients of the corresponding log-odds matrix (Fig. 85 in Schwartz & Dayhoff, 1978). Hydrophilicity plots were computed according to Hopp & Woods (1981). Secondary structures were predicted according to Chou & Fasman (1978), taking into account the changes of frequencies of the amino acids close to the N and C termini of helices and ~-sheets, or in their interior (Chou & Fasman, 1974). R E S U L T S AND D I S C U S S I O N Alignment o f the amino acid sequences Visual inspection of the sequences of P-E and P-TY showed that superposition of several tripeptides and one tetrapeptide could be achieved by introducing in P-TY one single deletion, facing Asp 3 (P-E), and two insertions at the C terminus to compensate for the longer polypeptide chain (Fig. 1). The computer search showed that no major homologies could be found other than those shown in Fig. 1. Assuming that P-E and P-TY derive from a common ancestor, 3 2 ~ of the residues (including one tetrapeptide and five tripeptides) are strictly conserved in P-E and P-TY. About two-thirds of the exchanges (89 of 127) require only one nucleotide change in the messenger R N A and none requires all three nucleotides of the corresponding codon to be changed. Conserved regions are indicated by boxes in Fig. 1. These are approximately evenly distributed along the sequence and the number of boxes of each size corresponds well with that expected for a 3 2 ~ homology distributed at random along 189 residues, i.e. 27.5 single amino acids, 9.6 dipeptides, 3.4 tripeptides and 0.8 tetrapeptides compared with the observed numbers of 21, 10, 5 and 1, respectively. This situation is very similar to that observed for the isometric phages Qfl and MS2, although their aligned sequences contain many deletions and insertions (Konigsberg et al., 1970; alignment 58 in Dayhoff, 1972). In contrast, for tobacco mosaic virus, a block of eight amino acids in helix R R and another of ten amino acids in helix L R are conserved in all sequenced strains. In this virus the polynucleotide chain and the protein subunits are arranged along the same basic helix, and the conserved regions correspond to the two s-helical segments that interact with three nucleotides in each subunit (Stubbs et al., 1977). In no isometric virus yet investigated by single-crystal X-ray diffraction could the R N A or the N terminus of the coat protein be seen in the electron density maps, although in each case the R N A is rigidly linked to the capsid (Harrison, 1983; Rossmann et al., 1983). The icosahedral symmetry of the surface domains of the shell does not hold for the interior of the virion, and it is possible that R N A protein interactions differ from one protein subunit to another, even if these belong to the same group of 60 related by strict icosahedral symmetry at their surface domains. Although the number of boxes in Fig. 1 follows the distribution expected for a random distribution of the conserved residues in P-E and P-TY, their location is unlikely to be defined by chance only. Indeed, we also determined the primary structure of three fragments of the coat protein of another, as yet unidentified, plant virus which we presume to be also a tymovirus. The fragments could be aligned with regions 63 to 68, 108 to 117 and 140 to 166 of Fig. 1. This virus was about as close to EMV as it was to TYMV : out of 44 residues, 23 matched with P-E and 20 with P-TY. But it is remarkable that in these regions all but one (position 160) amino acids that are common to P-TE and P-TY also occurred in the third sequence (A. Dupin & R. Peter, unpublished). The fragment corresponding to positions 140 to 166 possesses several remarkable properties: it contains the unique common tetrapeptide, together with a neighbouring tripeptide. The region 144 to 153 corresponds to an antigenic determinant that was found in dissociated TYMV protein only (see below). Peptide 133 to 153 could also be crosslinked in situ to TYMV RNA (Ehresmann et al., 1980) and contains one of the two short sites (positions 142 and 143) that were found to remain bound to viral RNA after dissociation and drastic hydrolyais Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 01:31:27 2573 Tymovirus coat protein sequences i0 1 20 40 TYMV {Ac M El. I D K E L AlP 0ID R T V T V A T VIL PIA v P O P S P L TIIiK 0IP F OIsIEIv L F I x ol { I I Io o x [--} ol i } I_I l-I I--I I--I 70 {-I {xx oo oox{ i I-I 0IF{EIA i i {x } I-{ I-I AIAI x{ ioxoo i I AIOIT K D A EIA{ {_I I_{ T T FIG{A T S T I}{ 80 90 l-} i00 I-I }--{ l-- I--I {ooo{ {xx{ {xx x{ {x{ {x { {x o { { { { o } { { S L T I{A{N I D S V S TiLiT T FiY R H{A S{LiE S L W V{T IiH{P TiL O{AiP T F{PiTiT V{G V CiW UIP A{N {_I i01 I ii0 I {_I I 120 I { I I_I I_I I 130 { I { 140 I 150 --I }-I I-I I--I I--I I-I I--I I-I {-I {--{ I-I I-I S{T A K{P{T EII{L N V F{G O{S S Y T F{G O AIL{N{A T K{P LiT I P L{P{M N S V{N{e M L{K D S[V L{Y{T{D{ I xxl loo{ l o ol Ix oxol lol {o x{ l o l {oo o{ l ol {o [ { l { TYMV SIP V TIP{A O{IIT K T Y{G GI0 I F C IIG G A[IINIT L SIP LI[ V K CIPIL E M MIN[P R VIK D SiI OIYILIDI { i_l 151 i_l I } I 160 I 1_I } 170 I-{ I--I {-I I I_I }_} 180 I-I l-IN } { }_I I_} 190 i--I C{P K L L{A Y S A{A{P S S P S K T P{T{A T{IIQ I H{GIK{L{R L S{S P L{L O A N . . x{ TYMV i 60 I_I EMV I--I O I S LIAIS A N A I T KILIA S L{Y R HIV RILIT O C A AIT IITIP TIA AIA{I A NIPIL{T VIN I VIW V{S D{N xooo{ EMV oxo { {-{ TYMV I-{ T A I I R SiP OIP S I N A P G F HIL P{P T D S 0 O S S A I I I E LiP F 51 EMV {--I 50 I--I IA= M EID l } I l--I 30 EMV S{PK i Ixx o{ { oo L L{I S I T { A { O P T A P i }_[ o } { P AS}T{C }_} { {xox{ Ix{ {xox{ {ox x I{I{TV S{G{T{L{S M H{S P L{I T D T S T }_I } { } } i { Fig. 1. Alignment of the sequences of the coat proteins of EMV and TYMV. Common residues are indicated by boxes and conservative differences by circles. Differences that conserve only the hydrophobic or hydrophilic nature of the amino acids, without change of the sign of the charge, are indicated by crosses. The identified antigenic determinants of TYMV protein are underlined. Regions of P-TY that could be crosslinked in situ to TYMV RNA are marked with an interrupted line. of the crosslinked virions. Carbon-13 nuclear magnetic resonance studies have shown that in another tymovirus, belladonna mottle virus, glutamic and aspartic residues are in contact with the RNA (Virudachalam et al., 1983): two aspartic acid residues are conserved in all three sequences, at positions 144 and 150. This makes this region a good candidate for a protein-RNA interaction site in the tymoviruses. Fig. 1 also shows the regions of approximate homology in P-E and P-TY. Circles indicate amino acid substitutions frequently observed in closely related proteins. They often correspond to exchanges between hydrophobic side-chains of similar size or to exchanges between certain charged and uncharged hydrophilic residues (Fig. 85 in Schwartz & Dayhoff, 1978; Rice et al., 1984). We also indicate by crosses substitutions that conserve the non-polar or polar (without change of the sign of the charge) character of the residues. It is striking that only one large region of low homology exists in the sequence. It corresponds to the N-terminal part of the chain (45 approximate homology for residues 1 to 25, and 50~ for residues 1 to 50). Apart from a short stretch (residues 160 to 170) close to the C terminus, the score was above 60~o everywhere else. It reached 100~ for the region 51 to 74. Coat proteins of both EMV and TYMV contain an exceptionally large number of prolines, reflecting the high cytosine content of the viral RNAs. Neither of the two sequences contains a region rich in basic residues able to undergo strong ionic interactions with the RNA, and the amino acid composition of other tymovirus coat proteins (Paul et al., 1980) makes it likely to be a general property of the family. Such regions, deeply embedded in the RNA, have been found in several small viruses stabilized by strong RNA-protein interactions (for reviews, see Rossmann et al., 1983; Harrison, 1983). Their absence in the coat proteins of tymoviruses and poliovirus (Racaniello & Baltimore, 1981) is probably a prerequisite condition for the self-assembly in the cell of capsids that, at least for TYMV, are isomorphous with the virions. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 01:31:27 2574 A, DUPIN AND OTHERS I A 13.ol I--I--I~-~[ I n Io.51o.31 I--I--I--I 3---[ I N It.41o.21o.31 I--I~--I--Ii---I 2-~[ i D It.310.210.810.4t I--Ii'--I--I--I--I I C 10.510.tlO.110.tlt.O[ 1 I--Ii--I--I I I 12 [ I Q 11.51o.410.71o.71o.11o.81 I--I¥--I--Ii-Ii---I--Ii--I 2 I E 11.31o.210.611.olo.tlo.81o.4l 1--17l--li---I I I I 6 I a 12,210.210.610,slo.llo.slo.510.91 --1 I I I I I It I H 10.510.210.310.2t0.t l o . s l o . 2 1 0 . t 10.2I --12--1 I I I I1 I I~ I I I I 11 I I1 ] 12.810.310,vI0,slo.310,810.slo.610.a13.t I I~IY--I I 2 12 I I 14 18 I 12.slo.alo.vIo.410.21~.OlO.Slo.610.414.916,71 I I1 12 K It.110.610,710.410.~ I0.V10.410.410,210.710.710.81 I I It I I I I I I I 2 I It M 10.610.110.210.t 1 0 . 0 1 0 . 2 i o . l l o . t 10.110.91 1.6]o.31o.31 I II t l I1 I I1 I 1 I I i 11 v 10.510.110.210.zlo.110.210.110.110.t I i . t l 1.61o.11o.211.3l 12 I I lO I [2 I I ]2 1 12 I I I It P 15.210.71 z.310.alo.011.610.911.310.311.81 2.011.110.410.317.0] 5 13---]1--12--13 It I 1 14 I I1 12 12 1 I1 I1 I1 i s 14.Blo.611.511.210.711.411.zlz.910.512.01 1.81Z.310.510.514.412.51 [2 1 14 14 I Ito [ I1 I 1 12 11 I I1 14 5 14 I i T i6.6i0.711.911.310.5il.611.2]1.8[0.513.611.0[1.710.710.714.816.0i5.11 --I~1--1--1-- --I I I I L I I I I I I I I I I I 12 I]---I I I I I1--Ig--12 I It I I I I I I I--I--I--I--I--I--I--I I I I I--I--I--I--I--I--I--I--I~l-- I--I--I--I--I--I--I--I I I--I--I--I I--I 1----i w 10.z10.110.~10.010.010.010.010.010.010.~10.210.~10.010.1i0.110.210.I10.51 --I--I--I I I 12 Y 10.310.1t0.z I0.110.110.110.1 I0.110.110.410.510.110.tl z.010.210.310.510.110.71 --I~--I--Ii--I--I1 ~ I i--1~l--1--12---r v 12.s10.210.610.310.210.610.410.610.213.613.210.5 0.610.51z.511.612.710.410.211.41 i--i i I i i i i I i i I--I--I--I--I--I--I-_IA i n_l._ i ~ 1 c Q I E I G I n I x I L I K M I ~ I P I S_I T_T_Iw._~_IJ!_IVY_ I I I I I I I I I I In(i)! 351 6 I 111 9 t 7 l tnl 9 t 9 I 5 I 251 2BI 121 6 I 101 27l 331 42 IN(i)l 301 5 I B 1 7 I 7 I 121 7 I 3 I 4 I 2tl 201 tOI 5 I 9 IA'(i~)l 4___£07___1 1__~411 ~ 1 7 1 I 171 301 3e I--I--I--I--I--I-- I I I--I 2 I 5 I i gl t I 3 I 171 1.._~611_!11~1 6_&_l291 3__661 t_A417___1 t.~ll 3__zl 3__661 4___~61_~SI L l 2__!1 Fig. 2. Number of observed amino acid exchanges between the coat proteins of EMV and TYMV, and comparison with the numbers expected for random sequences of the same length and amino acid composition, but following the rules established for homologous proteins (see text). Figures along the diagonal refer to the conserved residues of each kind observed in Fig. 1. Observed numbers of exchanges are at the top and expected ones at the bottom of each square, n(i), N(i) and N(i) represent the number of positions at which amino acid i appears in Fig. 1, the total number of exchanges involving amino acid i, and the total number of residues i, respectively (insertions and deletions are not taken into account). The exchanges observed in Fig. 1 are tabulated in Fig. 2. It is striking that glycine residues are highly conserved whereas all seven cysteine positions are exchanged. The 'mutability' of several other amino acids, normalized to that of alanine, also departs from the values observed in families of homologous proteins (Dayhoff et al., 1978). To analyse such differences in more detail, we calculated the number of exchanges for random sequences possessing the amino acid composition and structural similarity of P-TY and P-E (see Methods). These figures were weighted using the log-odds matrix corresponding to 250 point accepted mutations ( P A M s ; Fig. 85 in Schwartz & Dayhoff, 1978). This evolutionary distance corresponds to an 80% difference in amino acid positions instead of the 68 % observed in Fig. 1 (corresponding to 150 PAMs), but proved well adapted to the analysis of distantly related proteins, although it overestimates Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 01:31:27 Tymovirus coat protein sequences 2575 somewhat the exchange rates in the present case. Fig. 2 shows that the exchangeability of all charged and amino-amide amino acids is much lower than expected ifP-E and P-TY behaved as homologous proteins (12 residues are conserved, instead of an expected number of three or four): this high conservation rate may at least in part compensate for the small number of these amino acids in tymovirus coat proteins. As already mentioned, glycine residues are highly conserved, especially when they occur in closely spaced doublets (positions 113 and 114, and 120 and 121, Fig. 1). Several exchanges occur at a low rate (A P, A-S, T-P), but several others are unusually frequent (S-R, S-Q, T - L and T - K , for instance). If this observation is confirmed by analysis of the primary structures of more coat proteins of distantly related viruses, it would imply that standard amino acid exchange tables such as those of Dayhoff (1972) cannot be used reliably to establish phylogenetic trees of virus families and/or to predict possible primary structures of putative common ancestors, as has been done for instance for the MS2/Q/~ family (alignment 78 in Dayhoff, 1972). It is indeed striking that the foldings of the polypeptide chains of the surface domains of tomato bushy stunt virus, southern bean mosaic virus and satellite tobacco necrosis virus are so similar that it has been suggested that these viruses derive from a common ancestor, although there is no obvious similarity in the primary structures of the three coat proteins (Rossmann et al., 1983). Even if EMV and TYMV derived from a common ancestor, the variability of their stability (Bouley et aL, 1977) would allow amino acid exchanges in their coat proteins that are different from those occurring in homologous proteins, mainly enzymes, that must preserve the stereochemistry of an active site, or immunoglobulins. Antigenic determinants No data are available on the nature of the antigenic determinants of EMV but a detailed immunological investigation of TYMV is in progress (Pratt et al., 1980; Quesniaux et al., 1983a, b). Three antigenic regions have been identified with antibodies raised against virions. They correspond to residues 1 to 13, 58 to 65 and 184 to 190 respectively, using the numbering convention of Fig. 1. Two other regions (34 to 46 and 144 to 153) have been found in dissociated protein only. Fig. 1 clearly shows why EMV and TYMV do not possess common antigenic determinants (Koenig, 1976). At best, four residues in 12 are conserved in peptide 1 to 13, five in 13 in peptide 34 to 46 and six in 10 in peptide 144 to 153. Only one residue in seven or eight is conserved in the two other sites. Only one determinant (positions 1 to 13 in Fig. 1) corresponds to a highly hydrophilic region of TYMV coat protein (Fig. 3). The second one (positions 34 to 46) cannot be assigned with accuracy. The fourth determinant (positions 144 to 153) corresponds to a moderate oscillation of hydrophilicity and the third site (positions 58 to 65) clearly corresponds to the descending part, if not the trough, of one of the deepest minima of the hydrophilicity plot. Similar observations have also been made for tobacco mosaic virus, myoglobin and lysozyme where a better correlation has been found between antigenicity and segmental mobility than between antigenicity and hydrophilicity (Westhoff et al., 1984). Structure predictions Fig. 3 and 4 show the hydrophilicity plots (Hopp & Woods, 1981) and the predicted secondary structures (Chou & Fasman, 1974, 1978) of P-E and P-TY. Secondary structure predictions proved very ambiguous at several points. The number of regions possessing a high probability of being in a helical conformation is very low. We included in Fig. 4 segments possessing at least five residues, although Chou & Fasman (1974) argued that a helix containing less than six residues may not be stable on its own. The existence of the predicted helix from P81 to A87 in P-E is questionable. Although it has a high calculated probability of being c~-helical (P~), it contains four alanines and one isoleucine in seven amino acids, rendering it very hydrophobic. In P-TY two overlapping turns are predicted in the region from position 139 to 145. We chose the turn that was also predicted in P-E. Peaks and troughs occur at similar positions in the two average hydrophilicity plots of Fig. 3, although their amplitudes may be very different, being often larger in P-TY; several regions that Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 01:31:27 2576 h. I I I l DUPIN I I AND I N I OTHERS I I I I I I I I i I I I maD 1.5 i'l 1 ti IA II I i,d,,,, 0.5 t'I i - i 0 ,> 17 1 -0.5 ,I,!,I ,, . 1 ! ~,.7 i ! ';u ~ I '. I~ .~ 7 i: l t t ,, ', , i [ Li ~' --1 . I~ f ' I , , ,7 i I ' i < ", i( :1 "~ ; , , 7" , I [1"- (; , li i i : ,' ~LI" ~' : btl I~;, 'A' 1.5 U a 1.5 1 i 0.5- i iil't N i ', i't 0 ( l' L, h I , it., --0.5 II P ,,'-' ~ ~ ,, 0 I ,r'l/I i I I I I 50 [ I I I I I i ', II I i ~ '~ ,1J t,!''I, I If fJ I ~'!q t,' h~ t ..... . ~,I,E !/ '<, !i v . I,,' / ., t! I' rr r (b) I !'ti'i I I I I ! ~', 't4 -I -1.5 ', i ~i #'~ ' i ,~.. i I I 100 Residue number I I I I 1 I I 150 Fig. 3. Hydrophilicity plots of (a) EMV and (b) TYMV coat proteins. The hydrophilicities were averaged over six contiguous residues. The position of the known antigenic determinants of TYMV and dissociated TYMV protein is indicated by the horizontal bars. are hydrophobic in P-TY contain one hydrophilic residue in P-E, rendering P-TY more hydrophobic than P-E, on average. P-TY contains three clusters of very hydrophilic amino acids, giving rise to the three highest peaks in Fig. 3. These include the N terminus of P-TY, much more hydrophilic than that of P-E. The region 75 to 95 is very hydrophobic in both proteins, containing no hydrophilic amino acid. It is the longest stretch of this kind found in the published primary structures of coat proteins of simple isometric viruses. A similar rather approximate similarity can also be seen in the predicted secondary structures (Fig. 4). Both proteins contain a few rather short a-helices ( 2 0 ~ of the residues of P-TY; 22 to 29% for P-E) but many/~-sheets and turns, a feature c o m m o n to several viral proteins (Argos, 1981). Both C and N termini possess different predicted structures in the two proteins and at several places the helical segments do not face each other in Fig. 4 (P105 to 1108 of P-E has a high Pc(, but is too short to form a stable helix). The N-terminal arms of P-E and P-TY are among the less conserved parts of the primary structure (Fig. 1 and 4). This difference results from many exchanges between hydrophobic and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 01:31:27 2577 Tymovirus coat protein sequences ~ A A A A A A A A A A A A - - _ - - _ - - _ - - ~-= ~.~.---------= ~------ ~---= • wv :====:========== - - - I £ ~.1 1 1 1 £ £ £ £ £ A A A A A A A A£ £ £ ~ , ~ . ~ I I / , £ £ £ AcMEDTAI I R S P Q P S I N A P G F H L P P T D S Q Q S S A I ELPFQFEATTFGATETAA 10 20 30 40 50 rrMv AcME, I D K E L A P Q D R T V T V A T V L P A V P G P S P L T I K Q P F Q S E V L F A S T K D A E A ikhhAhAhAhAhA t£££££~,£~.£££.~£~,£~,L££££££££££££££AAAAAAAAAAAAAAAAAAAAAAAAAAAA ££££££££££££~£zzz zz ~Z~AAAAAAAAAAAA~Z~ EMV Q I S L A S A N A I T K L A S L Y R H V R L T Q C A A T I T P T A A A I A ~ P L T V N IVWVSD~ 60 70 100 90 80 S L T I A N I D S V S T L T T F Y R H A S L E S L W V T I HPTLQAPTFPTTVGVCWVPAN ~,11R -- _=E =-~_==-=-=-AAAAAAAAAAAAAAAAAAAA t £ 11 I £ £ ~.£ ££~AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA=~ ~-=-~~- .AAAAAAAAAAAA--- =---------- z---~ z ~------------- z~-z . . . . --AAAAAflAAAAAA ..... ~-=-------- =. . . . . "= -=m-=~-~- ~V STAKPTE I LNVFGGSS YTFGGA L,NATKPLT I PLPMNSVN CMLKDSVLYTD T~V SPVTPAQITKTYGGQ I FC I GGAIi~TLSPLIVKCPLEMMNPRVKDS I QYLD 110 _=-- 120 130 140 ~,11 ~,~.~,~.~.i£ i_--=-~ =-_--~=_AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAA~ . 1 ~ . ~ I ~ ~-~-£ ~ 9.1£ ~ .~£ £ 9.1 ,~l £ ~ ~,£ £ ....... ~AAAAAAAAAAAAAAAA. ~ ........ AAAAAAAAAA -££111~.19.£111 ENV CPKLLAYSAAPSSPSKT PTAT I Ol HGKLRLSSP LLQAI~ TIrMV SPKLLIS ITAQPTAPPASTCI ITVSGTLS~HSPLITDTST 460 150 180 170 _--:_=_--=-=-=---AAAAAAAAAAAA_=_= ~ ~ ~._=_==-_=_=_==-=-:_-- ~ ^ A A A A A A A A A ~ . A A A A A A A A~A~ ~ ~ ~ ~ ~ ~ ^ A A A A A A A A A A A A A A ^ Fig. 4. Predicted secondary structures of EMV and TYMV coat proteins. ~,, c(-helix; A, fl-sheet; =-, flturn ; , disordered. Where two equally probable possibilities occur, both structures are indicated. E2 E2 E . . . . ~ K6 . I 4 ~ D3 ...... / A M 1 R8 I7 ~ T4 L8 ~"-"-'~ ~ D5 Fig. 5. Helical wheels (Schiffer & Edmundson, 1967) for the N terminus of the coat proteins of EMV and TYMV. The orientation and length of each vector correspond to the hydrophilicity of the amino acid, according to Hopp & Woods (1981). The numbering of the residues corresponds to that of Fig. 1; position 3 in TYMV coat protein is a deletion. hydrophilic side chains. It is also seen in the helical wheel diagrams (Schiffer & Edmundson, 1967) of the first nine (P-TY) or 10 (P-E) residues preceding the first proline : in P-E each of the three hydrophilic amino acids is surrounded by hydrophobic residues, an unfavourable distribution for a stable c(-helix (Fig. 5). In the present state of our knowledge of the architecture of tymoviruses, it is not possible to say if the difference between the N-terminal arms of P-E and P-TY contributes to the observed difference in the stabilities of T Y M V and EMV (Bouley et al., 1977). The localization of the N-terminal arm of P-TY in the capsid has been controversial. Peptides 1 to 13 (numbering of Fig. 1) could be successfully crosslinked in situ to the viral R N A either by irradiation at p H 4.8 (but not at pH 7.0) or by bisulphite treatment at neutral p H Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 01:31:27 2578 A. D U P I N AND OTHERS (Ehresmann et al., 1980). Immunochemical studies (Quesniaux et al., 1983a), on the other hand, led to the suggestion that it corresponds to an antigenic determinant of the virion, i.e. that it is located at the outer surface of the capsid. These studies were based on tests of the inhibitory capacity of peptides in reaction with antisera collected from rabbits immunized by injections of virions. It is known, however, that about 4 0 ~ of the virions release their R N A and partially dissociate when TYMV is incubated for 2 h at 37 °C in a neutral buffer of physiological ionic strength (Lyttleton & Matthews, 1958). It is likely that antisera raised against virions also contain antibodies against dissociated protein subunits; these antibodies may be detectable by sensitive modern immunological methods. A definite immunological proof that peptides 1 to 13 are at the surface of the virion would require raising antibodies against this purified peptide and testing their activity against the virions. The core of the region that could be crosslinked in situ to T Y M V R N A (Ehresmann et al., 1980) is the couple Lys-Asp (positions 143 to 144) located in a predicted fl-turn: it could make specific contacts with the R N A by a combination of hydrogen bonds and charge neutralization (H61~ne & Lancelot, 1982). According to the secondary structure predictions, none of the regions of P-TY that could be crosslinked in situ to T Y M V R N A is able to form the e-helix-turn-e-helix super-secondary structure, a suggested common feature of repressor and other DNA-binding proteins (Sauer et al., 1982). Conclusion The coat proteins of the distantly related tymoviruses EMV and TYMV possess similar structures although the observed amino acid differences and similarities do not correspond to those found in homologous proteins such as enzyme families. One or two more tymovirus coat proteins should be sequenced in order to ascertain which regions of the polypeptide chain are common to all viruses of the family. However, many of the similarities observed between the structures of P-E and P-TY are unlikely to have occurred by chance. Analysis of the primary structure of P-E confirms several observations already made on the structure of P-TY and helps understanding of why tymoviruses are stabilized by weak R N A protein and strong protein-protein interactions (Kaper, 1975) with little interpenetration of TYMV R N A and protein in the virion (Jacrot et al., 1978). The possibility of crystallizing virions as well as empty protein shells possessing the same surface structure, combined with the existence of short conserved potential R N A protein interaction sites, makes this group of viruses a very interesting candidate both for high resolution crystallographic studies and for studies on the relative stabilities of virions and capsids possessing R N A s and coat proteins modified by directed mutagenesis. We thank Professor R. E. F. Matthews for stimulating discussions, Mr Daney de Marcillac for computing Fig. 3 and 4, and Mrs C. Peter for her help in the preparation of this manuscript. REFERENCES ARGOS,P. ([981). Secondary structure prediction of plant virus coat proteins. Virology ll0, 55-62. BOULEY,J. P., BRIAND,J. P. &WITZ,J. (I977). The stability of eggplant mosaic virus: action of urea and alkaline pH on top and bottom components. Virology 78, 425 432. CHOU,P. V. & FASMAN,G. O. (1974). Prediction of protein conformation. Biochemistry 13, 222-245. CHOU,P. v. & FASMAN,G. D. (1978). Empirical predictions of protein conformation. Annual Review oJBiochemistry 47, 251 276. DAYHOFF,M. O. (editor) (1972). Atlas q/' Protein Sequence and Structure. Washington, D.C. : NBR Foundation, Georgetown University. DAYHOFF, M. O., SCHWARTZ,R. M. & ORCUTT, B. C. (1978). A model of evolutionary change in proteins. In Atlas of Protein Sequence antl Structure, vol. 5, supplement 3, pp. 345 352. Edited by M. O. Dayhoff. Washington, D.C.: N B R Foundation, Georgetown University. DUPIN, A., PETER, R., COLLOT, D., DAS, B. C., PETER, C., BOUILLON,P. & DURANTON,H. (1984). The primary structure of the eggplant mosaic virus (EM V) coat protein. Comptes rendus hebdomadaires des skances de l'Acadkmie des sciences, s~rie C 298, 219-221. EHRESMANN, B., BRIAND,J. P., REINBOLT, J. & WITZ, J. (1980). Identification of binding sites of turnip yellow mosaic virus protein and R N A by crosslinks induced in situ. European Journal of Biochemistry 108, 123 129. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 01:31:27 Tymovirus coat protein sequences 2579 GUILLEY, H. & BRIAND,J. P. (1978). Nucleotide sequence of turnip yellow mosaic virus coat protein m R N A . Cell 15, 113-122. HARRISON, S. C. (1983). Virus structure: high resolution perspectives. Advances in Virus Research 28, 175-240. H/~LENE, C. & LANCELOT, G. (1982). Interactions between functional groups in protein nucleic acid associations. Progress in Biophysics and Molecular Biology 39, 1 68. HOPP, T. P. & WOODS, K. R. (1981). Prediction of protein antigenic determinants from amino acid sequences. Proceedings of the National Academy of Sciences, U.S.A. 78, 3824-3828. JACROT, B., CHAUVIN, C. & WITZ, J. (1978). Comparative neutron small angle scattering study of small spherical RNA viruses. Nature, London 266, 417-421. KAPER, J. M. (1975). The Chemical Basis of Virus Structure, Dissociation and Reassembly. Frontiers of Biology series. Edited by A. Neuberger & E. L. Tatum. Amsterdam: North-Holland. KOENIG, R. (1976). A loop-structure in the serological classification system of tymoviruses. Virology 72, 1-5. KOENI6, g. & LESEMANN,D. E. (1979). Tymovirus group. Commonwealth Mycological Institute/Association of Applied Biologists Descriptions of Plant Viruses, no. 214. KOENIG, R. & LESEMANN, D. E. (1981). Tymoviruses. In Handbook of Plant Virus Injections and Comparative Diagnosis, pp. 33-60. Edited by E. Kurstak. Amsterdam: Elsevier/North-Holland. KONIGSBERG, w., MATTA,T., KATZE, J. & WEBER, K. (1970). Amino acid sequence of the Qfl coat protein. Nature, London 227, 271-273. LVTrLETON, J. W. & MATTHEWS,R. E. F. (1958). Release of nucleic acid from turnip yellow mosaic virus under mild conditions. Virology 6, 460 471. PAUL, H. L., GIBBS, A. & WITTMAN-LIEBOLD, B. (1980). The relationship of certain tymoviruses assessed from the amino acid composition of their coat protiens. Intervirology 13, 99 109. PETER, R., STEHELIN, D., REINBOLT, J., COLLOT, D. & DURANTON, H. (1972). Primary structure of turnip yellow mosaic virus coat protein. Virology 49, 615 617. PRATT, D., BRIAND, J. P. & VAN REGENMORTEL, M. H. V. (1980). Immunochemical studies of turnip yellow mosaic virus. I. Localization of four antigenic regions in the protein subunit. Molecular Immunology 17, 1167 1171. QUESNIAUX, V., BRIAND, J. P. & VAN REGENMORTEL, M. H. V. (1983a). Immunochemical studies of turnip yellow mosaic virus. II. Localization of a viral epitope in the N-terminal residues of the coat protein. Molecular Immunology 20, 179-185. QUESNIAUX, V., JAEGLE, M. & VAN REGENMORTEL, M. H. V. (1983 b). Immunochemical studies of turnip yellow mosaic virl~. III. Localization of two viral epitopes in residues 57 64 and 183 189 of the coat protein. Biochimica et ~iophysica acta 743, 226 231, RACANIELLO,V. & BALTIMORE,D. (1981). Molecular cloning of poliovirus cDN A and determination of the complete nucleotide sequence of the viral genome. Proceedings oJthe National Academy of Sciences, U.S.A. 78, 48874891. RICE, D. M., SCHULZ, G. E. & GUEST, J. R. (1984). Structural relationship between glutathione reductase and lipoamide dehydrogenase. Journal of Molecular Biology 174, 483-496. ROSSMANN, M. G., ABAD-ZAPATERO, C., MURTHY, M. R., LILJAS, L., JONES, A. & STRANDBERG, B. (1983). Structural comparisons of some small spherical viruses. Journal of Molecular Biology 165, 711 736. SAWER, R. T., YOCUM, R. R., DOOLITTLE, R. F., LEWIS, M. & FABO, C. O. (1982). Homology among DNA-binding proteins suggests use of a conserved super-secondary structure. Nature, London 298, 447-451. SCHIFFER, M. & EDMUNDSON,A. B. (1967). Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophysical Journal 7, 121 135. SCHWARTZ,R. M. & DAYHOFF,M. O. (1978). Matrices for detecting distant relationships. In Atlas oJProtein Sequence and Structure, vol. 5, supplement 3, pp. 353 359. Edited by M O. Dayhoff. Washington, D.C.: NBR Foundation, Georgetown University. STADEN, R. (1982). An interactive graphics program for comparing and aligning nucleic acid and amino acid sequences. Nucleic Acids Research 10, 2951-2961. STUBBS, G., WARREN, S- & HOLMES,K. C. (1977). Structure of R N A and R N A binding site in tobacco mosaic virus from 4 ]~ map calculated from X-ray fibre diagrams. Nature, London 267, 216 221. VIRUDACHALAM, R., SITARAMAN, K., HEUSS, K. L., ARGOS, P. & MARKLE¥, J. L. (1983). Carbon-13 and proton nuclear magnetic resonance spectroscopy of plant viruses: evidence for protein-nucleic acid interactions in belladonna mottle virus and detection of polyamines in turnip yellow mosaic virus. Virology 130, 360-371. WESTHOFF, E., ALTSCHUH, D., MORAS, D., BLOOMER, A. C., MONDRAGON, A., KLUG, A. & VAN REGENMORTEL, M. H. V. (1984). Correlation between segmental mobility and the location of antigenic determinants in proteins. Nature, London 311, 123 126. (Received 22 March 1985) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 01:31:27