Proc. Natl. Acad. Sci. USAVol. 81, pp. 4358-4362, July 1984Biochemistry

Striking similarities in amino acid sequence among nonstructuralproteins encoded by RNA viruses that have dissimilargenomic organization

(alfalfa mosaic virus/brome mosaic virus/tobacco mosaic virus/Sindbis virus/protein sequence homology)

JAMES HASELOFF*, PHILIP GOELET*t, DAVID ZIMMERN*, PAUL AHLQUISTt§, RANJIT DASGUPTAt¶,AND PAUL KAESBERGT¶*Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom; and tBiophysics Laboratory andDepartments of ¶Biochemistry and §Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706

Communicated by T. 0. Diener, April 5, 1984

ABSTRACT The plant viruses alfalfa mosaic virus (AMV)and brome mosaic virus (BMV) each divide their genetic infor-mation among three RNAs while tobacco mosaic virus (TMV)contains a single genomic RNA. Amino acid sequence compar-isons suggest that the single proteins encoded by AMV RNA 1and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 arerelated to the NH2-terminal two-thirds and the COOH-termi-nal one-third, respectively, of the largest protein encoded byTMV. Separating these two domains in the TMV RNA se-quence is an amber termination codon, whose partial suppres-sion allows translation of the downstream domain. Many ofthe residues that the TMV read-through domain and the seg-mented plant viruses have in common are also conserved in aread-through domain found in the nonstructural polyproteinof the animal alphaviruses Sindbis and Middelburg. We sug-gest that, despite substantial differences in gene organizationand expression, all of these viruses use related proteins forcommon functions in RNA replication. Reassortment of func-tional modules of coding and regulatory sequence from preex-isting viral or cellular sources, perhaps via RNA recombina-tion, may be an important mechanism in RNA virus evolution.

Viruses with single-stranded RNA genomes that infect high-er eukaryotic hosts form a diverse group displaying widevariation in genomic organization (reviewed in ref. 1). Thegenome of tobacco mosaic virus (TMV), for example, is asingle RNA molecule of 6.4 kilobases (kb) (ref. 2; reviewedin ref. 3). It encodes at least four proteins in three open read-ing frames. That nearest the 5' end contains an in-phase am-ber termination codon that is partly suppressed during trans-lation in vitro or in vivo to give two products, the larger(known from its molecular weight as p183) being a read-through extension of the smaller (p126). The template fortranslation of both of these proteins is the genomic RNA, thetwo remaining genes being expressed via subgenomic RNAs.The genomes of alfalfa mosaic virus (AMV) and brome

mosaic virus (BMV), in contrast, each consist of three RNAsegments, termed RNAs 1, 2, and 3 in order of decreasingsize (ref. 4-8; reviewed in ref. 9). The two larger RNAs ofeach virus are monocistronic. The smallest is dicistronic,with the 3' proximal gene in both cases encoding the coatprotein that is translated from a subgenomic mRNA. Al-though both viruses require all three RNAs for infection,AMV, unlike BMV, also requires either coat protein or thesubgenomic mRNA for coat. Conversely, all three BMVRNAs, unlike the AMV RNAs, are aminoacylatable with ty-rosine. In this respect, the BMV RNAs resemble TMV RNA(which accepts either histidine or valine according to the

strain). Each virus has a different morphology, TMV beingrod-shaped, AMV bacilliform, and BMV icosahedral.

All three viruses are thus clearly distinguished by conven-tional criteria. Nevertheless, we show in this paper that theamino acid sequences of the proteins encoded by AMV RNA1 and BMV RNA 1 are strikingly similar both to each otherand to that ofTMV p126. Furthermore, the proteins encodedby AMV RNA 2 and BMV RNA 2 are also similar to eachother and to the COOH-terminal read-through domain inTMV protein p183. We suggest that despite different strate-gies of viral gene expression, these proteins are related infunction, and perhaps origin. We also show that one of thesetwo groups of related proteins has a counterpart in a proteinexpressed by translational read-through and proteolyticprocessing that is encoded by the animal alphaviruses Sind-bis and Middelburg (ref. 10; reviewed in ref. 11). We discussthese relationships and their possible implications.

MATERIALS AND METHODSInitial homology searches were made with the matrix com-parison program DIAGON (12) run on a VAX 11/780 com-puter. Detailed alignments were made by using the interac-tive facility of DIAGON and with the objective alignmentprograms BESTFIT and GAPOUT (13).

RESULTS AND DISCUSSIONHomologous Nonstructural Proteins in Three Plant Viruses.

The recently determined nucleotide sequences of the AMV(4-6), BMV (7, 8), and TMV (2) genomes, together with ear-lier in vitro and in vivo viral translation studies, suggest thateach virus encodes four major proteins. For brevity, we willrefer to the products of AMV RNAs 1 and 2 and of BMVRNAs 1 and 2 as Al and A2 and as B1 and B2, respectively,and to the products of each dicistronic RNA 3 as A3 and B3(products of the 5' proximal genes) and A4 and B4 (the coatproteins), respectively. Similarly, we will refer to the TMVopen reading frames in their 5' to 3' order along the RNA asTi (p126), T2 (the remainder of the first open reading framedownstream of the amber stop codon of p126, which formsthe COOH terminus of the read-through product p183), T3,and T4 (the coat protein).The amino acid sequences of these proteins predicted

from the genomic RNA sequences were compared by usingthe computer program DIAGON. For these comparisons theprogram recorded as dots on a graph all pairs of 31 residueblocks whose similarity in terms of both identical and related

Abbreviations: AMV, alfalfa mosaic virus; BMV, brome mosaic vi-rus; TMV, tobacco mosaic virus; kb, kilobases.tPresent address: Center for Neurobiology and Behavior, College ofPhysicians and Surgeons of Columbia University, New York, NY10032.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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amino acids exceeded a double matching probability (12) of10-5, compared to random pairs of 31 residue blocksdrawn from pools with the same amino acid composition asthe proteins under comparison. Extensive sequence similar-ities were detected between proteins Al, Bi, and Ti and be-tween proteins A2, B2, and T2 (Fig. 1). It can be seen fromboth the diagonal plots and from more detailed alignments(Figs. 2 and 3) that the proteins Al, Bi, and Ti share twomain regions of related sequence situated within the NH2-terminal and COOH-terminal one-thirds of the proteins, withsubstantially less homology existing in the middle thirds,where protein Bi is about 130 amino acids shorter than theother two. Proteins A2, B2, and T2 share a main region ofrelated sequence that is in the central to COOH-terminal 400amino acids of A2 and B2. T2, which is some 300 residuesshorter than A2 and B2, apparently lacks sequences corre-sponding to the NH2-terminal portions of these proteins.Mutants in A2 are known to map in two complementationgroups (14), suggesting the existence of two functional do-mains. It is possible that only one of these is represented inthe TMV genome.The significance of the observed sequence homology is

supported by the conserved arrangement and clustered na-ture of homologous residues in both groups of proteins. Forexample, Al residues 836-846, Bi residues 683-693, and 9of 11 Ti residues 831-841 are identical (positions 932-942 inthe alignment shown in Fig. 2). Similarly, 13 of 15 A2 resi-dues 528-542, 13 of 15 B2 residues 463-477, and 12 of 15 T2residues 1404-1418 are identical (positions 284-298 in Fig.3). A2 and B2 have identical amino acids in about 30% ofpositions, rising to about 40% when conservative substitu-tions are also counted. These figures are clearly above thebackground of random resemblances in protein sequences(15). Al and Bi and both TMV proteins are less closely relat-ed overall (20%), but random resemblances would not be ex-pected to cluster in the manner we observe in six indepen-dent pairwise comparisons. Accordingly, we suggest that allthree representatives of both groups of proteins are structur-ally related and potentially functionally homologous, al-though there may be some latitude for specialization due todifferences in folding outside the most highly conserved do-mains.Comparisons of the sequences of the remaining AMV,

BMV, and TMV encoded proteins revealed a limited numberof matches significant at the 10-4 level between A3 and B3and between A4 and B4 that fell on the diagonal, but none ofany significance between the others. It is not clear if any ofthe proteins are related in three-dimensional structure, buthave insufficient conservation of primary sequence for theirsimilarity to be apparent, or whether they are completely un-related. The sequence relationships between the proteins ofAMV, BMV, and TMV are shown schematically in Fig. 4.Having established which parts of the Al/Bi/Ti and

A2/B2/T2 amino acid sequences were strongly conservedby analyzing the plant viral sequences, we searched for relat-ed sequences in other viruses. In particular, we examinedthe sequence of the nonstructural proteins of two alphavi-ruses, Sindbis virus and Middelburg virus, where an opal ter-mination codon interrupts a long open reading frame (10).We found that the 616-residue read-through portion of thisopen reading frame, encoding a protein known as ns72, con-tained many of the same clusters of conserved residues iden-tified in A2, B2, and T2 and that these were arranged in thesame order, giving rise to diagonal lines on a matrix compari-son (Fig. 1) and enabling us to align the sequences as shownin Fig. 3. This is consistent with conservation of functionallyimportant residues within homologous, but distantly related,proteins.

It is intriguing that the potentially homologous T2 and ns72proteins are both expressed by suppression of translational

a. I

4,

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b. I

4,

1126I

Al. 1126

TI T2 1616

UGA1897

N "

0

4 N

25131UAG1117

A2-* 790

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I A2-p 790

FIG. 1. DIAGON amino acid homology plots comparing BI vs.

Al and B2 vs. A2 (a), Al vs. Ti + T2 and A2 vs. T1 + T2 (b), andthe read-through portion of Sindbis virus p270 (ns72) vs. T2 and A2(c).

termination. Exploitation of translational read-through prob-ably does not depend on chance events alone, since there isevidence for specialized natural opal suppressor tRNAs inboth cattle and chickens (16, 17), whereas amber suppres-sion of the T1 terminator may utilize a naturally occurringundermodified form of tyrosine tRNA (18).

Functional Implications. Although RNAs 1, 2, and 3 (to-gether with coat protein or its subgenomic RNA for AMV)are required for a productive infection by AMV or BMV,inoculation of cowpea protoplasts with AMV particles con-

taining RNAs 1 and 2 only results in symmetric synthesis ofplus and minus viral RNA strands (19). Inoculation withRNA 1 alone, RNA 2 alone, RNAs 1 + 3, or RNAs 2 + 3does not result in RNA replication. Mutations in AMVRNAs 1 and 2 interfere with RNA synthesis (14, 20). Similar-ly, inoculation of barley protoplasts with BMV RNAs 1 and2 alone resulted in synthesis of B1 and B2 translation prod-ucts consistent with amplification of their templates (21).Mutants in RNA 1 of the closely related virus cowpea chlo-rotic mottle virus are deficient in RNA replication (22).These studies indicate that the products encoded by AMVand BMV RNAs 1 and 2 are required for viral RNA replica-tion. Replication-deficient TMV mutants are also known (3),although these have not been mapped.

Alphaviruses encode four early proteins that are translat-ed from a 42S (ca. 12 kb) mRNA apparently identical to thatpackaged into virus particles and distinct from the 26S sub-genomic mRNA for the structural proteins (11). Translationof 42S RNA results in a major polyprotein that is proteolyti-cally cleaved to give three mature products and a minorread-through polyprotein that is cleaved into four products(10), the additional read-through protein being the one ho-

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4360 Biochemistry: Haseloff et aL. Proc. Nati. Acad. Sci. USA 81 (1984)

1 0 30 50 7 0 90

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F[] lKHY flNAVDLGHAAF1R jiKQDFC G NV11 -I'TYf3LGF'OIJIK HKSN GI I YNRVKGQ!IV .-----HTV GY[]HHSYQTAVR fIf~---WQ G~JSFE1 SVHCIDGT TjLI~E>L ~CNQNTjK l4JNLRC--U1 CqN4V1WILDI KYVGCV - -----SIFIEDWSLNRW KCVRVKV

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KNQf [~IEV---VPfIN[PVSVERNjA YjC K KA~------TLCgF HEQ k~fjNY 1? ST[TAT~JRD2INGPCNGin S LTK---R ICL TID RAYLq~4QDA1 ~AKDFPVSKD WIDC KTVHEAQ IS ODNLVR C 4FKHEE - ---YC

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LVALS K YOVVI4~PLVSWIRDLEKLSSYLLDMYKVDACTQ*FIG. 2. Alignment of the entire sequences of proteins Al (AMV RNA 1 product la; top row), BL (BMV RNA 1 product la; middle row), and

Ti (TMV protein p126; bottom row). Percentage identities in the pairwise alignments (including gaps) are'Al vs. Bi, 20.8%; Al vs. Ti, 18.5%;and Bi vs. Ti, 17.7%.

mologous to A2, B2, and T2. These four early proteins may cation such as those revealed by genetic analysis in Sindbiscorrespond to the four complementation groups that have virus. For example, all four groups of viruses consideredbeen assigned to replication-defective mutants of Sindbis vi- here have capped RNAs (5'-terminal m7GpppG caps for therus and that are required for elongation (group F), minus plant viruses, m7GpppA for alphaviruses). The alphavirusstrand synthesis (group B), and subgenomic RNA synthesis cap structure is unusual among animal cell or viral RNAs in(groups A and U) (11). Thus, protein ns72 of alphaviruses is lacking a ribose methylation on the 5'-terminal nucleotide ofalso implicated in RNA replication. the chain proper, and there is evidence that capping is per-Although the available genetic evidence is compatible with formed by an early viral function (23). An enzyme involved

the idea that either or both groups of proteins may be compo- in capping (perhaps coupled to the initiation of plus strandnents of the viral replicases, they might alternatively, or in synthesis) might thus be encoded by all four groups of virus-addition, be involved in more specialized roles in RNA repli- es. All four groups also use subgenomic RNA synthesis to

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RISNELHLER!PP ITJiiAAADERISTI FH FSIj FABDASF IIFSKFDKSQNELHHEH SK VTDTILHLE V AIA HSKjGV SgFSPJAT JFES RFIV ISSL LKNVRLNNRY----FL ADESKFDKSQ&LHLEH KL IQ L EIALDSVIDLSRFLF| TRKT I EFFGDLDSHVPMD L LDIS DKSQN§RHCAMD MIR VTP GKHTE KRVENQAAEPLAJYHTLLD--ADFKAIISKQGDPKS DDAMAJ

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EKY NE FN HRKRI SK FlNoD|FQRRTGDALT LG I C PNVKFVV GDDSLIGT EELP-RQEFLV1AL GF N I4|SDF YjLSD| PH S SCQRRTGDTYFGNT FSGDDSLIIS P--VLDTDMfl

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TGLtIEqFGEISSTHLPTTR KFGAM S FUL TLNVVIAS EERLKTSR AAFIGDNI HGPSD--KEMAERC410 430 450 470 490

FIPHMFC SKFLITMP 11jGGKFJLP KN I WYQ-- S IDIGG-DV RVRLFN MME VMP--P ---SVP SKF VETEM LStF- PDP IR L RAHF SCDR4F .. E I L ----NLMMEEA LFKK---- QY FCY HHD yI1IYY---- DPL G HI E E SLCDVAVSL1ANCAYY WLEWEVHKTAPE]A2W III VGAVIGERP YF C ILQDS AC RE- DPL F G LP DE D D R- DE JTAWFRVGIT EVDN

510 530 550

S- EAALI SLGIFAGKTLCKECLFNEKHESNVKIKPRRVKKSHSDARSRARRA*H KY KPWIFEEVRAALAAFELSENFLRFSDCYCTEGIRVYQMSDPVCKF--->109 residues to C terminus

GS sVY LSkVL SLFIDGSISICITPJ Lh RTFAQS JRA E AIRGEIK HIjUJGGPK*FIG. 3. Alignment of the homologous portions of the amino acid sequences ofAMV RNA 2 product 2a (A2; top line), BMV RNA 2 product

2a (B2; second line), the read-through domain of TMV p183 (T2; third line), and ns72, the read-through portion of Sindbis virus p270 (bottomline). The figure is numbered for reference only; sequences begin at residue 265 of A2, 202 of B2, and 1 of T2 (immediately after the p126termination codon) and, for ns72, 101 residues after the opal codon in the Sindbis nonstructural cistron. Percentage identities in the pairwisealignments (including gaps) are A2 vs. B2, 30.8%; A2 vs. T2, 21.6%; A2 vs. ns72, 18.5%; B2 vs. T2, 21.4%; B2 vs. ns72, 18.3%; T2 vs. ns72,20.0o.

control expression of their structural proteins temporallyand quantitatively. This is therefore another candidate for apotential common function for a homologous protein.

Evolutionary Implications. The structural similarities be-tween these proteins may reflect either convergent evolutiondue to common functions or common origins in preexistingviral or host genes or some combination of these possibili-ties, which we consider in turn.

General necessities ofRNA replication, such as the abilityto bind RNA, might account for a degree of resemblanceamong the enzymes responsible. In particular, one mightquestion whether the homology observed among the threeplant viruses and the alphaviruses is due to convergence. Se-quence comparisons alone cannot provide a definite answer.However, the demonstration of amino acid sequence homol-ogy between a protein encoded by cauliflower mosaic virusand the reverse transcriptases of both retroviruses and hep-adnaviruses (24), and between the replicases of cowpea mo-saic virus and picornaviruses (25), suggests that severalgroups of plant and animal viruses may use similar proteinsin their replication. Since each group uses a distinctive set ofproteins to achieve the common end of replicating an RNAtemplate, we suggest that each group of proteins owes itscommon features to descent from a common ancestor.The genomes of AMV and BMV, which are similar apart

from their 3' termini, clearly evoke a common viral ancestry,but the TMV genome also has many structural motifs incommon with the tripartite viruses. Each virus encodes fourwell-characterized translation products (Fig. 4), of which thetwo largest show clearly identifiable amino acid sequencehomology. The RNA termini show similarities as already

outlined. Possibly, a TMV-like virus could be generated bythe fusion of all three RNA segments of a tripartite virus toform a single RNA, providing that control sequences appro-priate to the expression of T2 and T3 were generated andthat the particle became adapted to carry a larger RNA. Con-versely, a segmented virus could be derived from a TMV-like progenitor by fission, provided that the fragments be-came able to replicate and be encapsidated.The three plant viruses and the alphaviruses might also

have descended from a common viral ancestor whose exis-tence predated the divergence of the plant and animal king-doms. This would necessitate extraordinary selection pres-sures at the protein level given the high mutation rate ofRNA virus genomes (26, 27). Alternatively, a more recentancestral virus might have existed that could replicate inboth plant and animal cells, like the reo- and rhabdovirusesof plants that also multiply in their insect vectors (28), butthis does not readily account for the major differences in ge-nomic organization contrasted with the conservation of pro-tein sequence. It seems more attractive to explain this in adifferent way.

It is clearly necessary to postulate some form of recombi-nation to account for interconversion of the tripartite andTMV genomes during evolution, even assuming a commonviral ancestry. Given recombination, it seems equally possi-ble that similar genes may be incorporated independentlyinto different viral genomes from a separate common source,presumably cellular genes. There are at least two possibili-ties for such a recombination mechanism. Although reversetranscription is not thought to be involved in normal replica-tion by these viruses, rare reverse transcription such as may

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RNA RNA 2 RNA 3BMV Tyr Tyr CO WTyr

a 2o 3a CP

TMV His or Valp126 8 CP

Sindbis - _ __nil vI. I WM (A)p230 p270 r/t p130

3 6 9 12kb

FIG. 4. Schematic diagram of the genomes of BMV (AMV issimilar except that the AMV RNAs cannot be aminoacylated),TMV, and Sindbis virus. Protein homologies are marked by con-necting lines. Genes expressed by suppression of translational ter-mination are showh stippled and those expressed via subgenomicRNAs are crosshatched. All RNAs shown bear 5' caps. 3' poly(A)or amino acid accepting structures are marked as appropriate.

occur during pseudogene formation by cellular mRNAs (29)could be followed by recombination at the DNA level. An-other distinct possibility is that recombination may occur bysome as yet unspecified mechanism at the RNA level. Thereis genetic and biochemical evidence for RNA recombinationin picornaviruses (30-32), and it is also implicated in the gen-eration of defective interfering RNAs (DI RNAs) in manyviruses, including alphaviruses (33, 34). Either or both ofthese mechanisms might modify viral genomes by recombi-nation with cellular genes or be responsible for assemblinggenes progressively to form the viruses in the first place. Therecent discovery of a Sindbis virus DI RNA with a covalent-ly attached cellular tRNA at its 5' end (34) is direct evidencethat such genetic exchanges are possible, whatever theirmechanism. On a formal basis, the differences in genomicorganization of these four viruses can be regarded as permu-tations of modules of related genetic information and of con-trolling elements appropriate to their distribution along oneor more viral RNAs. Ultimately, all of the modules of infor-mation whose reassortment we observe as viruses with dif-ferent structures may be cellular in origin. In that case, thesequence conservation displayed by the proteins consideredhere may reflect strong cellular evolutionary conservation,maintained after transduction by residual functional con-straints and by the need to interact with the products of othergenes that remain in the more slowly evolving host cell.

Note Added in Proof. Examination of the recently published com-plete Sindbis virus RNA sequence (35) shows that the Sindbis nsP1and nsP2 proteins are related to the Al/B1/T1 group of proteinsshown aligned in Fig. 2 above (unpublished results).

P.K. acknowledges the support of National Institutes of HealthPublic Health Service Grants AI-1466 and AI-15342 and CareerAward AI-21942 during this work. J.H. is a Commonwealth Scien-tific and Industrial Research Organisation Postdoctoral Fellow andP.G. was a Thomas C. Usher Fellow.

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