Genomic Organization

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

Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic 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 and Departments 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) strain). Each virus has a different morphology, TMV being and brome mosaic virus (BMV) each divide their genetic infor- rod-shaped, AMV bacilliform, and BMV icosahedral. mation among three RNAs while tobacco mosaic virus (TMV) All three viruses are thus clearly distinguished by conven- contains a single genomic RNA. Amino acid sequence compar- tional criteria. Nevertheless, we show in this paper that the isons suggest that the single proteins encoded by AMV RNA 1 amino acid sequences of the proteins encoded by AMV RNA and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are 1 and BMV RNA 1 are strikingly similar both to each other related to the NH2-terminal two-thirds and the COOH-termi- and to that of TMV p126. Furthermore, the proteins encoded nal one-third, respectively, of the largest protein encoded by by AMV RNA 2 and BMV RNA 2 are also similar to each TMV. Separating these two domains in the TMV RNA se- other and to the COOH-terminal read-through domain in quence is an amber termination codon, whose partial suppres- TMV protein p183. We suggest that despite different strate- sion allows translation of the downstream domain. Many of gies of viral gene expression, these proteins are related in the residues that the TMV read-through domain and the seg- function, and perhaps origin. We also show that one of these mented plant viruses have in common are also conserved in a two groups of related proteins has a counterpart in a protein read-through domain found in the nonstructural polyprotein expressed by translational read-through and proteolytic of the animal alphaviruses Sindbis and Middelburg. We sug- processing that is encoded by the animal alphaviruses Sind- gest that, despite substantial differences in gene organization bis and Middelburg (ref. 10; reviewed in ref. 11). We discuss and expression, all of these viruses use related proteins for these relationships and their possible implications. common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preex- MATERIALS AND METHODS isting viral or cellular sources, perhaps via RNA recombina- Initial homology searches were made with the matrix com- tion, may be an important mechanism in RNA virus evolution. parison program DIAGON (12) run on a VAX 11/780 computer. Detailed alignments were made by using the interac- Viruses with single-stranded RNA genomes that infect high- tive facility of DIAGON and with the objective alignment er eukaryotic hosts form a diverse group displaying wide programs BESTFIT and GAPOUT (13). variation in genomic organization (reviewed in ref. 1). The genome of tobacco mosaic virus (TMV), for example, is a RESULTS AND DISCUSSION single RNA molecule of 6.4 kilobases (kb) (ref. 2; reviewed in ref. 3). It encodes at least four proteins in three open read- Homologous Nonstructural Proteins in Three Plant Viruses. ing frames. That nearest the 5' end contains an in-phase am- The recently determined nucleotide sequences of the AMV ber termination codon that is partly suppressed during trans- (4-6), BMV (7, 8), and TMV (2) genomes, together with ear- lation in vitro or in vivo to give two products, the larger lier in vitro and in vivo viral translation studies, suggest that (known from its molecular weight as p183) being a read- each virus encodes four major proteins. For brevity, we will through extension of the smaller (p126). The template for refer to the products of AMV RNAs 1 and 2 and of BMV translation of both of these proteins is the genomic RNA, the RNAs 1 and 2 as Al and A2 and as B1 and B2, respectively, two remaining genes being expressed via subgenomic RNAs. and to the products of each dicistronic RNA 3 as A3 and B3 The genomes of alfalfa mosaic virus (AMV) and brome (products of the 5' proximal genes) and A4 and B4 (the coat mosaic virus (BMV), in contrast, each consist of three RNA proteins), respectively. Similarly, we will refer to the TMV segments, termed RNAs 1, 2, and 3 in order of decreasing open reading frames in their 5' to 3' order along the RNA as size (ref. 4-8; reviewed in ref. 9). The two larger RNAs of Ti (p126), T2 (the remainder of the first open reading frame each virus are monocistronic. The smallest is dicistronic, downstream of the amber stop codon of p126, which forms with the 3' proximal gene in both cases encoding the coat the COOH terminus of the read-through product p183), T3, protein that is translated from a subgenomic mRNA. Al- and T4 (the coat protein). though both viruses require all three RNAs for infection, The amino acid sequences of these proteins predicted AMV, unlike BMV, also requires either coat protein or the from the genomic RNA sequences were compared by using subgenomic mRNA for coat. Conversely, all three BMV the computer program DIAGON. For these comparisons the RNAs, unlike the AMV RNAs, are aminoacylatable with ty- program recorded as dots on a graph all pairs of 31 residue rosine. In this respect, the BMV RNAs resemble TMV RNA blocks whose similarity in terms of both identical and related (which accepts either histidine or valine according to the Abbreviations: AMV, alfalfa mosaic virus; BMV, brome mosaic virus; TMV, tobacco mosaic virus; kb, kilobases. The publication costs of this article were defrayed in part by page charge tPresent address: Center for Neurobiology and Behavior, College of payment. This article must therefore be hereby marked "advertisement" Physicians and Surgeons of Columbia University, New York, NY in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10032. 4358 Downloaded by guest on September 30, 2021 Biochemistry: Haseloff et aL Proc. Natl. Acad. Sci USA 81 (1984) 4359

amino acids exceeded a double matching probability (12) of a. I 10-5, compared to random pairs of 31 residue blocks drawn from pools with the same amino acid composition as comparison. Extensive sequence similar- the proteins under CD ities were detected between proteins Al, Bi, and Ti and be- 4, tween proteins A2, B2, and T2 (Fig. 1). It can be seen from 4, both the diagonal plots and from more detailed alignments (Figs. 2 and 3) that the proteins Al, Bi, and Ti share two main regions of related sequence situated within the NH2- fil 822 terminal and COOH-terminal one-thirds of the proteins, with Al. 1126 A2-* 790 substantially less homology existing in the middle thirds, where protein Bi is about 130 amino acids shorter than the b. I other two. Proteins A2, B2, and T2 share a main region of related sequence that is in the central to COOH-terminal 400 amino acids of A2 and B2. T2, which is some 300 residues N shorter than A2 and B2, apparently lacks sequences corre- 4, 4, sponding to the NH2-terminal portions of these proteins. Mutants in A2 are known to map in two complementation groups (14), suggesting the existence of two functional do- 1126I mains. It is possible that only one of these is represented in TI TI T2 1616 the TMV genome. T2 1616 The significance of the observed sequence homology is UGA supported by the conserved arrangement and clustered na- 1897 ture of homologous residues in both groups of proteins. For

example, Al residues 836-846, Bi residues 683-693, and 9 N " of 11 Ti residues 831-841 are identical (positions 932-942 in 0 the alignment shown in Fig. 2). Similarly, 13 of 15 A2 residues 528-542, 13 of 15 B2 residues 463-477, and 12 of 15 T2 residues 1404-1418 are identical (positions 284-298 in Fig. 4 N 3). A2 and B2 have identical amino acids in about 30% of about 40% when conservative substitu- 25131 positions, rising to UAG T2-0 1616 I A2-p 790 tions are also counted. These figures are clearly above the 1117 background of random resemblances in protein sequences (15). Al and Bi and both TMV proteins are less closely relat- FIG. 1. DIAGON amino acid homology plots comparing BI vs. ed overall (20%), but random resemblances would not be ex- Al and B2 vs. A2 (a), Al vs. Ti + T2 and A2 vs. T1 + T2 (b), and pected to cluster in the manner we observe in six indepen- the read-through portion of Sindbis virus p270 (ns72) vs. T2 and A2 dent pairwise comparisons. Accordingly, we suggest that all (c). three representatives of both groups of proteins are structur- ally related and potentially functionally homologous, al- termination. Exploitation of translational read-through prob- though there may be some latitude for specialization due to ably does not depend on chance events alone, since there is differences in folding outside the most highly conserved do- evidence for specialized natural opal suppressor tRNAs in mains. both cattle and chickens (16, 17), whereas amber suppres- Comparisons of the sequences of the remaining AMV, sion of the T1 terminator may utilize a naturally occurring BMV, and TMV encoded proteins revealed a limited number undermodified form of tyrosine tRNA (18). of matches significant at the 10-4 level between A3 and B3 Functional Implications. Although RNAs 1, 2, and 3 (to- and between A4 and B4 that fell on the diagonal, but none of gether with coat protein or its subgenomic RNA for AMV) any significance between the others. It is not clear if any of are required for a productive infection by AMV or BMV, the proteins are related in three-dimensional structure, but inoculation of cowpea protoplasts with AMV particles con- have insufficient conservation of primary sequence for their taining RNAs 1 and 2 only results in symmetric synthesis of similarity to be apparent, or whether they are completely un- plus and minus viral RNA strands (19). Inoculation with related. The sequence relationships between the proteins of RNA 1 alone, RNA 2 alone, RNAs 1 + 3, or RNAs 2 + 3 AMV, BMV, and TMV are shown schematically in Fig. 4. does not result in RNA replication. Mutations in AMV Having established which parts of the Al/Bi/Ti and RNAs 1 and 2 interfere with RNA synthesis (14, 20). Similar- A2/B2/T2 amino acid sequences were strongly conserved ly, inoculation of barley protoplasts with BMV RNAs 1 and by analyzing the plant viral sequences, we searched for relat- 2 alone resulted in synthesis of B1 and B2 translation prod- ed sequences in other viruses. In particular, we examined ucts consistent with amplification of their templates (21). the sequence of the nonstructural proteins of two alphavi- Mutants in RNA 1 of the closely related virus cowpea chlo- ruses, Sindbis virus and Middelburg virus, where an opal ter- rotic mottle virus are deficient in RNA replication (22). mination codon interrupts a long open reading frame (10). These studies indicate that the products encoded by AMV We found that the 616-residue read-through portion of this and BMV RNAs 1 and 2 are required for viral RNA replica- open reading frame, encoding a protein known as ns72, con- tion. Replication-deficient TMV mutants are also known (3), tained many of the same clusters of conserved residues iden- although these have not been mapped. tified in A2, B2, and T2 and that these were arranged in the Alphaviruses encode four early proteins that are translat- same order, giving rise to diagonal lines on a matrix compari- ed from a 42S (ca. 12 kb) mRNA apparently identical to that son (Fig. 1) and enabling us to align the sequences as shown packaged into virus particles and distinct from the 26S sub- in Fig. 3. This is consistent with conservation of functionally genomic mRNA for the structural proteins (11). Translation important residues within homologous, but distantly related, of 42S RNA results in a major polyprotein that is proteolyti- proteins. cally cleaved to give three mature products and a minor It is intriguing that the potentially homologous T2 and ns72 read-through polyprotein that is cleaved into four products proteins are both expressed by suppression of translational (10), the additional read-through protein being the one ho- Downloaded by guest on September 30, 2021 4360 Biochemistry: Haseloff et aL. Proc. Nati. Acad. Sci. USA 81 (1984)

1 0 30 50 70 90 MNADAQSTDASLSMREPLSHE-3JIQE RR~jVfE4AHDTAMIC~lFS -fGRAAA4QWALPSDKGEV L iS~~Sj~AT N IAANF-0GRRT YJSSS M-S ID CjLIAIEK-GA SQ QDIUDNQ QAA IEYAK-- fsKKKNJ?4jISTKIDAE1 Ifi~YGCC DLNLT Q YH MAYTQT[ATTS L TOGNIVNKRLDAVEFAM---- pTL1AtjUAYIDj' QI 110 130 150 170 190 SS H LTDFVYR fliG NTVgSl VV MVHNVHCC ~Ij j ALE IL4~LKSj yRK------HPEIVG[DAD APSAA V YDUSW OP I GSW FSRRDKRVHBC V RAIR1 MC RMRK QESDD -FDq NF ----.--- AVHL SL -LEYUMI-IQ fYGSLT IGG SF [AVHC NDVR KDIT-RRGIKNIKEAFDRYAELP[DDA 2 10 2 30 2 50 2 70 290 S0!R ---- D AR£LDV Gf~ fQKG HMKFI t A IMQI H K-~I jSR IIRQ DgDV --- A I fY1GFQj1CDf(11HSjV1RVLRVI F MG F EF L LLKC RDSGADE Ilj FDE EST Ij4G vIj TMR PQQSGR V1AItJ TPArf_AIgLROHTCYAA~i NiJ1ASYV N[JD F. L.Aj-F--~KLTF SES yJdS 3 10 3 30 3 50 3 70 390 F[] lKHY flNAVDLGHAAF1R jiKQDFC G NV11 -I'TYf3LGF'OIJIK HKSNGI I YNRVKGQ!IV .-----HTV GY[]HHSYQTAVR fIf~--- WQ G~JSFE1 SVHCIDGT TjLI~E>L ~CNQNTjK l4JNLRC--U1 CqN4V1WILDI KYVGCV ------SIFIEDWSLNRW KCVRVKV

410 430 450 470 490 .------DKIDVLT I43l flT~~RNADAHSA1QS I1TW-- TH~TG__TI S I .-- -R ~ jI C~JK FS K K N r E N MKA ~~IlI AK SrTVIIN 2 A I .,UT SERILLEDSSSVNYWFPKNrRDN"V IVPLFDISLETSKRTROEVLVSKD 4JVLNHIRTYQAKALT YNV ~FVEI RIlJRIj,1~GVAR~~E1V~ L 5 10 5 30 5 50 5 70 590 VATIYYRVKKLYNALPFNLS DKEL-rF--C~(P'l4IGT .A.KN GFVKg[BIKD]SFOOrHERr L IFA1 N--N Y HCLfJK DC KG W C HItFfl.-- - IjFWW4GGDS

6 10 6 30 6 50 670 690 TOGCVFYP 1%l rJK VIDRETT DYSFBI'E FLKDPPLISDPV RQSLW5VYYAE HQGNA1DASNYARTLgD[2I SRAKJGWL TI5R RDjYAOSFKFLTRrLS Ljl ------___ EQ[SIP~VPJI SIRLR F TEDLFDRflIQ

710 730) 750 770 790

.1EV---- TAK fR lK VPANEl PQEEFHOAPES SS ~V~DDVKPV~fV.------DJJSV~n HJ E EOASOGAUgVvTS-R EP JG Et tAIGU ESY~~ IEL-Ul~ATJ L]RQISI UT IVQ1NHSLV ]LOAjHVSNL 8 10 830 8 50 870 890

OKRLHN d14 t_SGGEANKS [FETYH~- IDDM UVHLANGNWLIKKYDYTVGYN EIGLGPKHAtF IVOKTC N IDI---

9 10 9 30 950 9 70 990

dOFA E01IP EKAQ----CGLEAHFSOJTIVDVGCKNI CSL -4L Y~Jrf5~YL~I-3---S -ASKVSVP CD ------IHDVGG AlK AFj--- DI ADVfiMALF P~JTYNS VF~- L1 VRTj SAL --

1010 1030 1050 1070 1090 ASAVKA0JWI fC Fj0JAqjIYAj Lcfc-j~f If D5 P S fPV r LGVDK CJUKYFY------NiK.NKKE] ]iGVPSCRL A1J1 J I Fi CA 9KL C N- QfDJRRDVVH YRCPUD IAAyJ4LLKRLJCGNRDTKYQSW STRCQF R FD CLX LITJ¶11CJF NFL-IAYVV YI V CJE PYP UFAlFK!E\tJE V[TRR CPDV H YLIRRY.-----EGFE] 1110 1130 1150 1170 1190

KNQf [~IEV-- - V K KA~ HEQ 1? in PfIN[PVSVERNjA YjC ------TLCgF k~fjNY ST[TAT~JRD2INGPCNG S LTK---R ICL TID RAYLq~4QDA1 ~AKDFPVSKD WIDC KTVHEAQ IS ODNLVR C 4FKHEE - ---YC

1 2 10 1 230 LVALRH ~ LV A LRH RES CFNCEL GIIF1.CVK*V~~jIYIqACRDAGNTDDSILARSYNHNF* 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 Sindbis correspond to the four complementation groups that have virus. For example, all four groups of viruses considered been assigned to replication-defective mutants of Sindbis vi- here have capped RNAs (5'-terminal m7GpppG caps for the rus and that are required for elongation (group F), minus plant viruses, m7GpppA for alphaviruses). The alphavirus strand 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 of also 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 strand nents 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 Downloaded by guest on September 30, 2021 Biochemistry: Haseloff et aL Proc. NatL Acad Sci USA 81 (1984) 4361

10 30 50 70 90

GpI I T~v TI - --- i VF FSGGY3RHH-Gf P. Ji a 1 A 3 a gEW-ATSKSLDtPEW- T K-o-7K iI F NNQ S S ENC-NC E F RF K;L FFS -flS ~V P HC___VTIHE TEAT I PTH - - N I I R DV Y F SAMN GSA QLQL SVFKG FVAAPK QKCF-- PINST--MMNN -MD I _ KD CAKS yAAIPK QI KLIPF M T ECYK YQBLYSSSV--- PAN V C HYQ-T-MDG T P ---SYp YE- ARAEN fjAP 11 0 130 150 1 7 0 190

U L; V VNSV N NS IfjlVID ----- LCMFPDFISEGE VfFQDYIf NPJDPELYSIPLGB--RSIRS~K Q T EVL TK R VP RIM KATAAKF----jGEDCLSMDVK. WYj HI?LQGVNVAAET---D LgYfQ MPRQ dL P IDAE SL F K -IKRJNKNVSLfqSREE LEKQEQVTIGQLAIFDF0DLPAV JA UNL T ELPTL F CIYWEEF K I ITT VfAYVA eLgGPKAAALFAKTYNLVPLQEVP 2 1 0 230 250 270 290

RISNELHLER!PP TJI iiAAADERISTI FH FSIj FABDASF IIFSKFDKSQNELHHE H SK VTDTILHLE V AIA HSKjGV SgFSPJATS JFE RFIV ISSL LKNVRLNNRY----FL ADESKFDKSQ&LHLE H ALDSVIDLSRFLF|KL IQ L EI TRKT I EFFGDLDSHVPMD L LDIS DKSQN§RHCA MD MIR VTP GKHTE KRVENQAAEPLAJYHTLLD--ADFKAIISKQGDPKS DDAMAJ 310 330 350 370 390

EKY NE FN HRKRI SK FlNoD|FQRRTGDALT LG I C PNVKFVV GDDSLIGT EELP- RQEFLV1 AL GF N I4|SDF YjLSD| PH S CQRRTGDTYFGNT S FSGDDSLIIS P--VLDTDMfl VEYIERRLGFED jEE RTTIYJIGJIKTCIWY GSV NT VII S PMEKIIK-- YFPKGCEFP VQHSA TGLtIEqFGEISSTHLPTTR KFGAM S FUL TLNVVIAS EERLKTSR AAFIGDNI HGPSD--KEMAERC 410 430 450 470 490 FIPHMFC SKFLITMP 11jGGKFJLP KN I WYQ-- S IDIGG -DV R VR LFNMEM VMP--P ---SVP SKF VETE M LStF- PDP IR L RAHF SCDR4F .. E I L ---- NLMMEEA LFKK ---- QY FCY HHD I1IYY----y DPL G HI E E SLCDVAVSL1ANCAYY LEWEVHKTAPE]W A2W I II 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 SLFIDGSISIC ITPJ Lh RTFAQS JRA E AIRGEIK HIjUJGGPK* FIG. 3. Alignment of the homologous portions of the amino acid sequences of AMV 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 (bottom line). The figure is numbered for reference only; sequences begin at residue 265 of A2, 202 of B2, and 1 of T2 (immediately after the p126 termination codon) and, for ns72, 101 residues after the opal codon in the Sindbis nonstructural cistron. Percentage identities in the pairwise alignments (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 temporally outlined. Possibly, a TMV-like virus could be generated by and quantitatively. This is therefore another candidate for a the fusion of all three RNA segments of a tripartite virus to potential common function for a homologous protein. form a single RNA, providing that control sequences appro- Evolutionary Implications. The structural similarities be- priate to the expression of T2 and T3 were generated and tween these proteins may reflect either convergent evolution that the particle became adapted to carry a larger RNA. Con- due to common functions or common origins in preexisting versely, a segmented virus could be derived from a TMV- viral or host genes or some combination of these possibili- like progenitor by fission, provided that the fragments be- ties, which we consider in turn. came able to replicate and be encapsidated. General necessities of RNA replication, such as the ability The three plant viruses and the alphaviruses might also to bind RNA, might account for a degree of resemblance have descended from a common viral ancestor whose exis- among the enzymes responsible. In particular, one might tence predated the divergence of the plant and animal king- question whether the homology observed among the three doms. This would necessitate extraordinary selection pres- plant viruses and the alphaviruses is due to convergence. Se- sures at the protein level given the high mutation rate of quence comparisons alone cannot provide a definite answer. RNA virus genomes (26, 27). Alternatively, a more recent However, the demonstration of amino acid sequence homol- ancestral virus might have existed that could replicate in ogy between a protein encoded by cauliflower mosaic virus both plant and animal cells, like the reo- and rhabdoviruses and the reverse transcriptases of both retroviruses and hep- of plants that also multiply in their insect vectors (28), but adnaviruses (24), and between the replicases of cowpea mo- this does not readily account for the major differences in ge- saic virus and picornaviruses (25), suggests that several nomic organization contrasted with the conservation of pro- groups of plant and animal viruses may use similar proteins tein sequence. It seems more attractive to explain this in a in their replication. Since each group uses a distinctive set of different way. proteins to achieve the common end of replicating an RNA It is clearly necessary to postulate some form of recombi- template, we suggest that each group of proteins owes its nation to account for interconversion of the tripartite and common features to descent from a common ancestor. TMV genomes during evolution, even assuming a common The genomes of AMV and BMV, which are similar apart viral ancestry. Given recombination, it seems equally possi- from their 3' termini, clearly evoke a common viral ancestry, ble that similar genes may be incorporated independently but the TMV genome also has many structural motifs in into different viral genomes from a separate common source, common with the tripartite viruses. Each virus encodes four presumably cellular genes. There are at least two possibili- well-characterized translation products (Fig. 4), of which the ties for such a recombination mechanism. Although reverse two largest show clearly identifiable amino acid sequence transcription is not thought to be involved in normal replica- homology. The RNA termini show similarities as already tion by these viruses, rare reverse transcription such as may Downloaded by guest on September 30, 2021 4362 Biochemistry: Haseloff et al. Proc. NatL Acad Sci. USA 81 (1984)

RNA RNA 2 RNA 3 2. Goelet, P., Lomonossoff, G. P., Butler, P. J. G., Akam, BMV Tyr Tyr CO WTyr M. a 2o 3a CP E., Gait, M. J. & Karn, J. (1982) Proc. Natl. Acad. Sci. USA 79, 5818-5822. 3. Hirth, L. & Richards, K. E. (1981) Adv. Virus Res. 26, 145- TMV His or Val 199. p126 8 CP 4. Cornelissen, B., Brederode, F., Moorman, R. & Bol, J. (1983) Nucleic Acids Res. 11, 1253-1265. 5. Cornelissen, B., Brederode, F., Veeneman, G., van Boom, J. Sindbis -1 _nil- _ I._ I WM v (A) & Bol, J. (1983) Nucleic Acids Res. 11, 3019-3025. p230 p270 r/t p130 6. Barker, R., Jarvis, N., Thompson, D., Loesch-Fries, L. & 3 6 9 12kb Hall, T. (1983) Nucleic Acids Res. 11, 2881-2891. 7. Ahlquist, P., Dasgupta, R. & Kaesberg, P. (1984) J. Mol. Biol. 172, 369-383. FIG. 4. genomes Schematic diagram of the of BMV (AMV is 8. Ahlquist, P., Luckow, V. & Kaesberg, P. (1981) J. Mol. Biol. similar except that the AMV RNAs cannot be aminoacylated), 153, 23-38. TMV, and Sindbis virus. Protein homologies are marked by con- necting lines. 9. van Vloten-Doting, L. & Jaspars, E. M. J. (1977) in Compre- Genes expressed by suppression of translational ter- hensive Virology, eds. Fraenkel-Conrat, H. & Wagner, R. R. mination are showh stippled and those expressed via subgenomic (Plenum, New York), Vol. 11, pp. 1-53. RNAs are crosshatched. All caps. RNAs shown bear 5' 3' poly(A) 10. Strauss, E. G., Rice, C. M. & Strauss, J. H. (1983) Proc. Natl. or amino acid accepting structures are marked as appropriate. Acad. Sci. USA 80, 5271-5275. 11. Schlesinger, R. W., ed. (1980) The Togaviruses (Academic, occur during pseudogene formation by cellular mRNAs (29) New York). could be followed by recombination at the DNA level. An- 12. Staden, R. (1982) Nucleic Acids Res. 10, 2951-2%1. other distinct possibility is that recombination may occur by 13. Devereaux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Ac- some as yet unspecified mechanism at the RNA level. There ids Res. 12, 387-395. is genetic and biochemical evidence for RNA recombination 14. Roosien, J. & van Vloten-Doting, L. (1982) J. Gen. Virol. 63, 189-198. in picornaviruses (30-32), and it is also implicated in the gen- 15. Doolittle, R. F. (1981) Science 214, 149-159. eration of defective interfering RNAs (DI RNAs) in many 16. Diamond, A., Dudock, B. & Hatfield, D. (1981) Cell 25, 497- viruses, including alphaviruses (33, 34). Either or both of 506. these mechanisms might modify viral genomes by recombi- 17. Hatfield, D. L., Dudock, B. S. & Eden, F. C. (1983) Proc. nation with cellular genes or be responsible for assembling Natl. Acad. Sci. USA 80, 4940-4944. genes progressively to form the viruses in the first place. The 18. Bienz, M. & Kubli, E. (1981) Nature (London) 294, 188-190. recent discovery of a Sindbis virus DI RNA with a covalent- 19. Nassuth, A. & Bol, J. (1983) Virology 124, 75-85. ly attached cellular tRNA at its 5' end (34) is direct evidence 20. Nassuth, A., Alblas, F. & Bol, J. (1981) J. Gen. Virol. 53, 207- that such genetic exchanges are possible, whatever their 214. 21. Kiberstis, P. A., Loesch-Fries, L. S. & Hall, T. C. (1981) Vi- mechanism. On a formal basis, the differences in genomic rology 112, 804-808. organization of these four viruses can be regarded as permu- 22. Dawson, W. 0. (1981) Virology 115, 130-136. tations of modules of related genetic information and of con- 23. Cross, R. K. (1983) Virology 130, 452-463. trolling elements appropriate to their distribution along one 24. Toh, H., Hayashida, H. & Miyata, T. (1983) Nature (London) or more viral RNAs. Ultimately, all of the modules of infor- 305, 827-829. mation whose reassortment we observe as viruses with dif- 25. Franssen, H., Leunissen, J., Goldbach, R., Lomonossoff, G. ferent structures may be cellular in origin. In that case, the & Zimmern, D. (1984) EMBO J. 3, 855-861. sequence conservation displayed by the proteins considered 26. Domingo, E., Sabo, D., Taniguchi, T. & Weissmann, C. (1978) here may reflect strong cellular evolutionary conservation, Cell 13, 735-744. 27. Holland, J., Spindler, K., maintained after transduction by residual functional con- Horodyski, F., Grabeau, E., Nichol, S. & VandePol, S. (1982) Science 215, 1577-1585. straints and by the need to interact with the products of other 28. Matthews, R. E. F. (1981) Plant Virology (Academic, New genes that remain in the more slowly evolving host cell. York), 2nd Ed., pp. 591-598. 29. Hollis, G. F., Hieter, P. A., McBride, 0. W., Swan, D. & Leder, P. (1982) Nature (London) 296, 321-325. Note Added in Proof. Examination of the recently published com- 30. Cooper, P. D. (1977) in Comprehensive Virology, eds. Fraen- plete Sindbis virus RNA sequence (35) shows that the Sindbis nsP1 kel-Conrat, H. & Wagner, R. R. (Plenum, New York), Vol. 9, and nsP2 proteins are related to the Al/B1/T1 group of proteins pp. 133-207. shown aligned in Fig. 2 above (unpublished results). 31. King, A. M. Q., McCahon, D., Slade, W. R. & Newman, J. W. I. (1982) Cell 29, 921-928. P.K. acknowledges the support of National Institutes of Health 32. Tolskaya, E. A., Romanova, L. A., Kolesnikova, M. S. & Public Health Service Grants AI-1466 and AI-15342 and Career Agol, V. I. (1983) Virology 124, 121-132. Award AI-21942 during this work. J.H. is a Commonwealth Scien- 33. Lehtovaara, P., Soderlund, H., Keranen, S., Pettersson, R. F. tific and Industrial Research Organisation Postdoctoral Fellow and & Kaariainen, L. (1981) Proc. Natl. Acad. Sci. USA 78, 5353- P.G. was a Thomas C. Usher Fellow. 5355. 34. Monroe, S. S. & Schlesinger, S. (1983) Proc. Natl. Acad. Sci. USA 80, 3279-3283. 1. Strauss, E. G. & Strauss, J. H. (1983) Curr. Top. Microbiol. 35. Strauss, E. G., Rice, M. & Strauss, J. H. (1984) Virology 133, Immunol. 105, 1-97. 92-110. Downloaded by guest on September 30, 2021