© 1990 Oxford University Press Nucleic Acids Research, Vol. 18, No. 7 1825

The primary structure and expression of the second openreading frame of the polymerase gene of the coronavirusMHV-A59; a highly conserved polymerase is expressed byan efficient ribosomal frameshifting mechanism

Peter J.Bredenbeek, Catherine J.Pachuk1, Ans F.H.Noten, Jeroen Charite, Willem Luytjes,Susan R.Weiss1 and Willy J.M.Spaan*Institute of Virology, Department of Infectious Diseases and Immunology, State University of Utrecht,PO Box 80.165, Utrecht, The Netherlands and department of Microbiology, University ofPennsylvania School of Medicine, Philadelphia, PA 19104-6067, USA

Received November 7, 1989; Revised and Accepted March 2, 1990 EMBL accession no. X51939

ABSTRACT

Sequence analysis of a substantial part of thepolymerase gene of the murine coronavirus MHV-A59revealed the 3' end of an open reading frame (ORF1a)overlapping with a large ORF (ORF1b; 2733 aminoacids) which covers the 3' half of the polymerase gene.The expression of ORF1b occurs by a ribosomalframeshifting mechanism since the ORF1a/ORF1boverlapping nucleotide sequence is capable of inducingribosomal frameshifting In vitro as well as in vivo. Astem-loop structure and a pseudoknot are predicted inthe nucleotide sequence involved in ribosomalframeshifting. Comparison of the predicted amino acidsequence of MHV ORF1b with the amino acid sequencededuced from the corresponding gene of the aviancoronavirus IBV demonstrated that in contrast to theother viral genes this ORF is extremely conserved.Detailed analysis of the predicted amino acid sequencerevealed sequence elements which are conserved inmany DNA and RNA polymerases.

INTRODUCTION

The genome of mouse hepatitis virus (MHV), a coronavirus,consists of an infectious single stranded RNA molecule ofapproximately 30 kb in length (1). After entry the viral genomeis released, translated into the RNA dependent RNA polymeraseand subsequently used as the template for the transcription ofnegative stranded RNA of genome length (2, 3). This RNA thenserves as a template for the synthesis of six subgenomic mRNAsand genomic RNA. The subgenomic mRNAs form a3'-coterminal nested set. An unusual feature of the mRNAs ofcoronaviruses is the presence of an identical leader sequence.This common leader is proposed to result from a unique leader-primed transcription mechanism. The transcription and translationstrategy of coronaviruses has recently been reviewed in detail (4).

The RNA dependent RNA polymerase of coronaviruses is amultifunctional protein: it contains the activities necessary forthe transcription of negative stranded RNA, leader RNA,subgenomic mRNAs and progeny virion RNA. In addition it islikely to possess capping activity. These activities and theprotein(s) on which they reside are poorly characterized.Complementation studies using temperature sensitive (ts) mutantswhich are defective in RNA synthesis revealed six differentcomplementation groups, indicating that a large number of genesor at least activities are involved in the synthesis of the viral RNAs(5, Van der Zeijst, personal communication). Several authorshave shown the presence of membrane associated RNA dependentRNA polymerase activity in lysolecithin permeabilized MHV-A59 infected cells (6, 7) or cytoplasmic lysates of MHV infectedcells (8, 9). Brayton et al. (10) have described one early andtwo late polymerase activities in lysates of MHV-A59 infectedcells. The early polymerase activity was shown to be involvedin the synthesis of negative stranded RNA, while the late RNApolymerase activities were responsible for the synthesis ofgenomic RNA and subgenomic mRNAs.

In vitro translation of genomic RNA of MHV-A59 resultedin the synthesis of a protein with a molecular weight exceeding200 kd (11). In vitro this protein is cleaved into a 220 kd anda 28 kd protein. The p28 protein is the N-terminal cleavageproduct of the precursor protein (11, 12) and has also beenidentified in MHV-A59 infected cells (13).

The nucleotide sequence of the gene encoding the RNApolymerase (pol) of the avian coronavirus infectious bronchitisvirus (IBV) has been determined (14). The pol gene, which isabout 20 kb in length! contained two large open reading frames(ORF) ORF la and ORFlb (previously termed Fl and F2) whichpotentially encode polypeptides of 441 kd and 300 kd,respectively. The ORFs overlap by 42 nucleotides, ORFlb beingin a - 1 reading frame with respect to ORF la. Brierley et al.(15, 16) showed that a cDNA fragment spanning the

* To whom correspondence should be addressed

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ORFla/ORFlb overlap was able to direct ribosomal frameshiftingin vitro and in vivo.

Recently, we have completed the molecular cloning of thegenome of MHV-A59 and determined the nucleotide sequenceof the p28 coding region at its 5' end (1). Here we present thepredicted amino acid sequence of ORFlb and of the carboxylterminal region of ORFla of MHV-A59. In addition, wedemonstrate that a conserved nucleotide sequence of theORFla/ORFlb overlap directs ribosomal frameshifting in vitroand in vivo.

MATERIALS AND METHODSIsolation of viral genome RNAVirus obtained from roller bottle cultures of Sac(-) cells infectedat 2 p.f.u./cell was purified on sucrose gradients as describedbefore (17). Viral genomic RNA was isolated as describedpreviously (18).

cDNA synthesis and cloningFirst and second strand cDNA synthesis were carried out asdescribed by Gubler and Hoffman (19) using viral genomic RNA(50 /tg/ml) as a template and calf thymus pentanucleotides(100 /ig/ml) as random primers. Reverse transcriptase wasobtained from Promega, RNasin from Amersham, Escherichiacoli DNA ligase from New England Biolabs, RNase H and DNApolymerase I from Boehringer. After phenol/chloroformextraction and ethanol precipitation approximately 0.3 /tg cDNAwas used for homopolymeric tailing using dCTP (20). Sampleswere taken every 30 seconds during tailing and the reaction wasimmediately stopped by adding 0.1 volume of 1 % SDS containing100 mM EDTA. After phenol extraction and ethanolprecipitation, the tailed cDNA samples were annealed to Pst Idigested, oligo-dG tailed pUC9 (Pharmacia). For transformations(21) Escherichia coli strain JM109 was used.

Subcloning for M13 sequencingDNA restriction fragments were separated by agarose gelelectrophoresis and isolated by binding to NA45 membranes(Schleicher and Schuell). Purified fragments were recloned inM13 vectors. When no convenient restriction enzyme sites wereavailable plasmid DNA was digested with Rsa I and the DNAfragments were directly subcloned in Sma I cut M13mpl9. M13clones were selected by hybridization to nick translated purifiedDNA fragments of the region to be sequenced and screened bysingle track sequencing.

DNA sequencingSingle stranded DNA from M13 clones was sequenced using theKlenow fragment of DNA polymerase I (22) and [32P]a-dATPor T7 DNA polymerase (23) (Sequenase, US Biochemicals) and[35S]a-dATP. Double stranded DNA was sequenced using T7DNA polymerase according to the instructions of themanufacturer. Sequence data were analyzed using the computerprograms of Staden (24) and Wisconsin (version 5, 1987)(25).Comparison to the National Biomedical Research Foundation(NBRF) protein identification resource was made using theprogram FAST A (26).

Construction of a plasmid for the analysis of ribosomalframeshiftingTo study the potential ribosomal frameshifting in the pol geneof MHV-A59 the expression vector pBBMAC was constructed

as follows. Plasmid pMAC (a gift of Peter Rottier), whichcontained a copy of the MHV-A59 membrane (M) protein geneof which the region encoding the amino acids 121 up to 196 (27)was deleted (full details on pMAC will be published elsewhere),was digested with BamH I and filled in using the Klenowfragment of DNA polymerase I. The M protein encoding DNAfragment was purified and ligated into Sma I cut Bluescribe(+)vector (Stratagene, USA). A clone containing the MAC codingregion downstream of the T7 promoter was selected. The uniqueSma I cleavage site of the resulting expression vector pBSMACwas converted into a Bgl II site by an 8-mer linker addition,resulting in pBBMAC.

Clone P638, which contains the ORFla/ORFlb overlap (Fig.1), was digested with Pst I. The 1.3 kb cDNA insert was purified,digested with Alu I and ligated into the filled-in anddephosphorylated EcoR I site of a Bluescribe plasmid. Aftertransformation bacterial colonies containing the 160 bp Alu Ifragment spanning the ORFla/ORFlb overlap were selected bycolony hybridizations (28) using a nick-translated BamH I—KpnI 850 bp cDNA fragment of clone PI 136 as probe. Clonescontaining the expected 160 bp insertion were sequenced. Theresulting plasmid pAPO was digested with EcoR I and made bluntended with Klenow fragment of DNA polymerase. After ligationto 12-mer BamH I linkers and digestion with BamH I the DNAfragment was purified and ligated into Bgl II cut,dephosphorylated expression vector pBBM C. Plasmid DNAisolated from the resulting transformants was sequenced todetermine the orientation and the borders of the polymeraseORFla/ORFlb overlapping fragment of several clones. ClonepAPl was found to be correct and used for analysis of thepotential ribosomal frameshifting.

In vitro transcription and translationPlasmid DNA of pAPl was purified on a CsCl gradient (29) andlinearized with Hind HI. In vitro transcription and translation wereperformed as described (30).

A 1 2 3 456 7

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Figure 1. Cloning and sequencing strategy of the ORFlb region of the MHV-A59 polymerase gene. A) The upper line represents the MHV genome. Verticalbars and the numbers above indicate the junction sequences involved in the initiationof the transcriplion of the corresponding mRNA. Open boxes represent the openreading frames in the polymerase gene. B) The open bar represents the sequencedregion of the polymerase gene. The Wack triangle and the open triangle indicatethe start of the large 3'ORF (ORFlb) and the junction sequence for the initiationof mRNA2 transcription, respectively. The positions of oligonucleotides A andT12 are indicated. Negative numbers mark the distance to the start of the poly(A)-tail of the genome. The numbered bars refer to the cDNA clones used forsequencing, the sequenced areas are indicated in black.

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In vivo expression of pAPlHela cells (2 x 106) were infected with recombinant vacciniavirus vTF7—3, which contains the T7 RNA polymerase geneunder the control of a vaccinia promoter (31) at a m.o.i. of 10p.f.u. At 90 min. post infection (p.i.) the cells were transfectedwith pAPl (5 fig) as described by Gorman (29). At 14 hr. p.i.the cells were labelled for 30 min. with 60 /tCi/rrut^SJ-methionine (32). Cell lysates were prepared (33) andclarified for 90 min. at 12.000xg (4°Q.

Immunoprecipitations of proteins

Immunoprecipitations of the [35S]-methionine labelled proteinswere performed as previously described (32); 4 y\ of in vitrotranslation mixtures or 150 fiX of the cell lysates were used. Amonoclonal antibody (moab) J. 1.3 (a gift of John Fleming andStephen Stohlman; ref. 34) directed against the aminoterminalregion of the M protein of MHV-A59 and an anti-peptide serumraised against the carboxyl-terminal 18 amino acids of the Mprotein (Rottier, manuscript in preparation) were used as MHVmembrane protein specific antisera.

RESULTSMolecular cloning of the polymerase geneInitially we used a synthetic oligonucleotide complementary tothe conserved junction sequence immediately upstream of thenucleocapsid gene to prime the cDNA synthesis on purified

genomic RNA. Virus specific clones were mapped by dot blotanalysis with the purified individual MHV mRNAs (data notshown). OHgonueJeotkte A was synthesized from sequence dataobtained from a cDNA clone which only hybridized to genomicRNA. Oligonucleotide T12 was synthesized on the basis of thenucleotide sequence of RNase Tl resistant oligonucleotide T12(based on the nomenclature of ref. 35; unpublished results), whichis located in the pol gene of MHV-A59 (36). OligonucleotidesA and T12 were used to screen the random primed genomiccDNA library. Several large cDNA clones (P095, P096 andP098) hybridizing to both oligonucleotides were identified (Fig.1). In addition several cDNA clones were identified which wereonly positive with oligonucleotide A e.g. P030 and PO35. ThecDNA clones P096 and P030 contain the complete codingsequence of mRNA 2 (37) hence they map to the 3' end of thepolymerase gene. The cloning of the pol gene was completedby screening the cDNA library using a nick translated probederived from the most 5' mapped cDNA clone on the viralgenome. Clones P638, P737 and PI 136 were isolated from asecond random primed cDNA library prepared against genomicRNA from an independent stock of MHV-A59 (1).

Nucleotide sequence analysisThe cDNA clones used to determine a substantial part of thenucleotide sequence of the pol gene of MHV-A59 are shown (Fig.1). Each nucleotide of the consensus sequence (Fig. 2) wasdetermined on at least two independent cDNA clones. Analysis

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Figure 3. Comparison of the primary and secondary structure of the MH V-A59and IBV-M42 ORFla/ORFlb overlap A) Alignment of the nucleotide sequences.Numbers refer to the position of the 5' G residue in the published sequence (MHV-A59, this article; Q3V, ref. 14). B) Predicted secondary and tertiary RNA structureat the site of ribosomal frameshifting. The potential codons involved inframeshifting are underlined, and the termination codons of ORFla are boxed.Solid lines illustrate the potential pseudoknot. Differences in the nucleotidesinvolved in the predicted RNA structure of MHV in comparison to 1BV areindicated in the predicted structure for fBV to illustrate the observed covariation.

position 8443, which is just upstream of the conserved junctionsequence AAAUCUAUAC. This region separates the geneencoding the 30 kd nonstructural protein from the pol gene (37).ORFla terminates at position 318 and overlaps ORFlb for 75nucleotides.

Analysis of the ORFla/ORFlb overlapping sequenceIt has been shown that the nucleotide sequence of the IBVORFla/ORFlb overlapping region is capable of inducingribosomal frameshifting in vitro and in vivo (15, 16). A stablestem-loop structure in this overlapping region was predicted tobe involved in this translational frameshifting. Comparison ofthe nucleotide sequence of the ORFla/ORFlb overlap of MHV-A59 to the ORFla/ORFlb overlapping region of IBV-M42revealed a well conserved stretch of nucleotides (Fig. 3A). Fig.3B shows that a nearly identical stem-loop structure can bepredicted for the MHV ORFla/ORFlb overlap region. Theinsertion (MHV-A59) or deletion (IBV-M42) resulting in a gapof three nucleotides in the alignment (Fig. 3A) is located in thebulge of the stem-loop structure (Fig. 3B). Even if larger regionsof the sequence (up to 500 nucleotides) were analyzed for the

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pAP1STOP

STOP

1

IN VIVOTRANSFECTION

INFECTIONWITH vTF7

IN VITROTRANSCRIPTIONTRANSLATION

19 K

25 K w////////////////mm V///////////A

BM

30-M

30-

25 K 25 K

19 K

14.3-

19 K

14.3-

Figure 4. Analysis of ribosomal frameshifting in vitro and in vivo. A) Diagram showing the expression plasmid pAPl and the predicted sizes of protein productsthat would be expected from translation of the transcribed RNA. The black area represents the T7 promoter, regions encoded by the mutant M protein gene arehatched. Stop indicates the positions of the translation termination codons. B) In vitro translation products were analyzed directly (lane 1) or after immunoprecipitationusing moab J. 1.3 (lane 2) or the carboxyl-terminal specific anti-peptide serum (lane 4), respectively. Lane 3 and 5; immunoprecipitations using the correspondingpre-immune sera. Q Lysates from pAPl transfected and vaccinia virus vTF7-3 infected cells were immunoprecipitated using moab J.I.3 (lane 1), the anti-peptideserum (lane 3) and the pre-immune sera (lane 2 and 4). M indicates molecular weight markers.

presence of secondary structure the depicted hairpin structure stillfolds as a separate entity.

In both LBV and MHV the translation termination codon ofORFla is located in this stem-loop structure. Apart from thisconservation in secondary structure the potential tertiary structureis also well conserved. The nucleotide sequence of the loop givesrise to potential pseudoknot formation with sequences downstreamof the proposed stem-loop structure. The significance of theseproposed secondary and tertiary RNA structures is emphasizedby the presence of covariation. Mutations in one part of the stemor in the nucleotide sequence of the loop are compensated bymutations in either the stem or in the downstream sequenceinvolved in the potential pseudoknot, respectively (Fig. 3B).

Ribosomal frameshifting in vitro and in vivoTo prove that the ORFla/ORFlb overlapping region of the MHVpolymerase gene directs ribosomal frameshifting we cloned thisregion in a mutant M protein gene of MHV-A59 under the controlof a T7 promoter. Termination of translation of pAPl transcriptsat the ORFla UAA stopcodon will result in the synthesis of a

19 kd protein. However, when a —1 translational frameshiftoccurs a protein of 25 kd will be synthesized (Fig. 4A). Directanalysis of in vitro translation products revealed both proteins(Fig. 4B, lane 1). As expected moab J.I.3, directed against theN-terminus of the M protein, immunoprecipitated both the 19kd and 25 kd products, indicating that both proteins have acommon N-terminus (Fig. 4B, lane 2). Only the 25 kd proteinwas specifically immunoprecipitated by the C-terminal anti-peptide serum (Fig 4B, lane 4). None of the translation productswere immunoprecipitated by the pre-immune sera (Fig 4B, lane3 and 5). Since the methionine residues are only encoded by theregion upstream of the ORFla/ORFlb overlap the frameshiftefficiency can be easily estimated. The bands corresponding tothe 19 kd and 25 kd products were excised from lane 1 (Fig.4B) of the dried gel and from the amount of radioactivity anefficiency of approximately 40% was calculated.

To test whether the frameshift signal in the ORFla/ORFlboverlap was functional in vivo, Hela cells were infected with thevaccinia virus recombinant vTF7-3, expressing the T7 RNApolymerase and subsequently transfected with pAPl. Cells were

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r POL2 BVIBV / MHV F1/ORF1 B C

- 5OO

- 1OOO

- 15OO

- 2O0O

- 25OO

5OO 1000 1S00 2O0O 2SOO

Figure 5. Proportional dot matrix comparison of the amino acid sequences fromORFI b of MHV-A59 and of IBV-M42. Numbers of the amino acid residues areindicated at the axis. For creating the dot-matrix plot the program 'Compare'of the Genetics Computer Group (Wisconsin) (25) was used with a window sizeof 21 and a stringency of 15.

labelled with [35S]-methionine and cell lysates wereimmunoprecipitated using moab J. 1.3 and the anti-peptide serum.Moab J.I.3 specifically immunoprecipitated the expectedpolypeptides of 19 kd and 25 kd from pAPl transfected andvTF7-3 infected cells (Fig. 4C, lane 1). These polypeptides werenot present in lysates from cells that had only been infectedwith vTF7-3 (data not shown). The 25 kd protein was alsoimmunoprecipitated by the anti-peptide antiserum directed againstthe carboxyl-terminus of the M protein. (Fig. 4C, lane 3). Noneof these proteins were precipitated by the pre-immune sera (Fig.4C, lanes 2 and 4). From these data it was concluded that theMHV-A59 ORFla/ORFlb overlapping region was capable toinduce ribosomal frameshifting both in vitro and in vivo.

Computer analysis of the coronavirus polymerase genesComparison of the predicted amino acid sequence of the productsencoded by ORFlb of MHV-A59 and IBV-M42 revealed twolarge regions of high similarity (Fig. 5). The positional identityin an alignment of the amino acid sequence of ORFlb of MHVand IBV is 56%.

Several short sequence motifs have been identified in particularpolymerase proteins of RNA and DNA viruses. One motif whichcontains the core sequence 'GDD' (38) has also been identifiedin the amino acid sequence of ORFlb of IBV (39). This domainis conserved at an almost identical position in the product encodedby ORFlb of MHV-A59 (Fig. 6). However, in contrast to the'GDD' amino acid sequence, both MHV and IBV contain theamino acid sequence 'SDD' at this position. Althoughoccasionally a M, C, V or L residue has been reported in theposition of the G residue (38), no serine residue has been reported

JLX F2/ORF2

622 MOWDYPKCDRA -(52)- QGTSSQDATTAFANSV -(59>- 8MILLSODOWC

-<«)- FCSQKT

1218 VQQPPQTQKSHLA -(78>- IWDEVSMLT -<1S)- YVYKJDPAOLPA -(37)-

TCYRCPKEI -<81)- TQTVD8AQQ8EYDFVIY6Q -(18)- RFNVATrRA

Figure 6. A) Localization of the 'polymerase' (B) and 'helicase' (Q domain inthe second ORFs of the IBV and MHV-A59 pol genes. B and C) Amino acidsequence of the conserved domains identified in ORFlb of MHV-A59. Conservedresidues are indicated by triangles. Numbers on the left refer to the localizationof the conserved domains in the ORFlb sequence. Fig 6B; The 'GDD' or'polymerase' motif (38, 39). The position of the serine residue which replacesthe glycine is indicated with an open triangle. Fig. 6C. The 'GKS/T' or 'helicase'motif (40, 41).

immediately upstream of the two conserved aspartic acid residues.Analyzing the ORFlb amino acid sequence of MHV-A59revealed the presence of another 'GDD' motif at position2268-2270. Although it cannot be excluded that the ORFlbencodes more than one polymerase activity, it is unlikely thatthis 'GDD' motif is part of the active site of a coronaviruspolymerase since the surrounding sequences do not meet thecriteria proposed by Argos (38). Furthermore this 'GDD'sequence is not conserved between MHV and IBV.

The amino acid residues encoded by ORFlb of IBV whichexhibit similarity to a sequence motif found in a group of proteinsfrom different organisms and which are probably involved incrucial nucleoside triphosphate dependent steps in nucleic acidreplication (40, 41) are also present at a nearly identical positionin the amino acid sequence encoded by ORFlb of MHV-A59(Fig. 6).

No other significant similarities were identified whenoverlapping regions of approximately 300 residues of the MHV-A59 ORFlb amino acid sequence were tested using the programFASTA (24) and the NBRF/PIR and NBRF/NEW proteinidentification resources (releases 19.0 and 37.0, respectively).

DISCUSSION

In this paper the primary structure of the second ORF of theputative MHV-A59 pol gene is described. Assuming that theorganization of the polymerase gene of MHV is identical to theequivalent gene of IBV, in which two large ORFs have beenidentified (14), the nucleotide sequence of the small 5' ORFpresented in this article represents the 3' end of a large ORF.This ORF starts at position 210 at the 5' end of the viral genome(Fig. 1; ref. 1).

No similarity has been detected in the predicted amino acidsequence of the 5' end of ORF la of the IBV and MHV-JHMor MHV-A59 polymerase gene (1, 12). However, the putativecarboxyl terminal region of the translation product of ORF la andalmost the complete translation product of ORFlb are wellconserved among MHV-A59 and IBV. Recently, we havedetermined a small part (0.3 kb) of the nucleotide sequence ofthe 3' end of the polymerase gene of the feline coronavirus FIPV.(De Groot, unpublished results). The deduced amino acidsequence of this region was very similar to the carboxyl terminalpart of ORFlb of MHV and IBV.In contrast to the high similarity

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in the second ORF of the pol gene, no significant similarity hasbeen observed in the amino acid sequence of the other viralnonstructural proteins. The structural proteins of coronavirusesonly show significant similarity in relatively small regions. Theoverall identity in the nucleocapsid, membrane and spike proteinis 29%, 30% and 35% respectively (reviewed by 4). This stronglysuggests selective pressure against mutations in the pol gene toconserve functional domains. Identical observations have beenmade for the picornaviruses (42), alphaviruses (43), flaviviruses(44) and negative stranded RNA viruses like rhabdoviruses (45).

The 'S/GDD' as well as the nucleotide triphosphate binding'GKS/T' amino acid sequence motif, which are encoded in thepol genes of coronaviruses, are also well conserved in thepolymerases of viruses belonging to the picornavirus-like andalphavirus-like superfamily of (+) stranded RNA viruses (46,47). However, because of the quasi-helical nucleocapsidmorphology and the expression of the viral genes by multiplesubgenomic mRNAs, coronaviruses could not be assigned toeither superfamily of RNA viruses (47).

During the replication of coronaviruses multiple subgenomicRNAs are synthesized to position internal ORFs on the genomeat the 5' end of an mRNA. No subgenomic mRNA containingORFlb of the MHV polymerase gene at its 5'-proximal end hasbeen detected in infected cells. Using an expression vector whichcontained the ORFla/ORFlb overlap of MHV inserted in framewithin an MHV-A59 mutant M protein gene construct, we wereable to demonstrate that the ORFla/ORFlb spanning sequencewas capable of directing ribosomal frameshifting in vitro and invivo. Brierley et al. (15, 16) have shown that the ORFla/ORFlboverlap region of IBV is also capable of inducing frameshiftingin vitro and in vivo. Comparison of the nucleotide sequences ofthe overlapping region of IBV and MHV revealed that the signalsused for ribosomal frameshifting in coronaviruses are wellconserved. In both MHV and IBV a stable stem-loop structurecan be formed downstream of the conserved sequenceUUUAAAC. This sequence functions probably as the actual sitefor ribosomal frameshifting in MHV since mutations in thissequence have been shown to influence the ribosomalframeshifting in the IBV ORFla/ORFlb overlapping region (16).It has also been shown to function as a site for ribosomalframeshifting in Rous sarcoma virus (48). The observedcovariation between EBV and MHV in the predicted stem-loopstructure and the pseudoknot underlines the importance of thesestructures in translational frameshifting. Pseudoknots are alsopredicted to be involved in the ribosomal frameshifting for theexpression of the polymerase gene of many retroviruses and theluteoviruses (16, Ten Dam and Pleij pers. communication).Recently, it has been shown for IBV that the proposed pseudoknotin the ORFla/ORFlb overlapping region is essential forribosomal frameshifting (16).

Frameshifting is more efficient in the coronaviral system(35—40% frameshifting) than in the retroviral system where only5 — 10% frameshifting has been observed (49). Ribosomalframeshifting is an elegant mechanism for regulating the synthesisof several proteins in a well balanced manner. In manyretroviruses the polymerase is produced after translationalframeshifting which results in the expression of a gag-pol fusionprotein (48). Based on sequence comparison, it is postulated inthis article that the polymerase function of MHV-A59 is encodeddownstream of the ribosomal frameshifting sequence andexpressed as a fusion protein. It is tempting to speculate that thisfusion protein is the actual polymerase. Cleavage of this functional

polyprotein could result in an inactive pol protein. Such amechanism would explain the observed requirements forcontinuous de novo protein synthesis during the replication ofMHV-A59 (3, 6).

The determination of the nucleotide sequence and the predictedamino acid sequence of MHV-A59 ORFlb will provide a basisfor obtaining monospecific antisera against protein(s) encodedby ORFlb. These sera will be important for furthercharacterization of the proteins involved in the discontinuoustranscription of coronaviruses.

ACKNOWLEDGMENTS

The authors would like to thank G. Weeda for the determinationof the nucleotide sequence of oligonucleotide T12, P. Rottier formaking available pM C, oligonucleotides for sequencing pAPland the antisera directed against the membrane protein, L.Heijnen , H. Vennema and J. den Boon for technical assistancewith the vaccinia experiments, E. Snijder for assistance with thecomputer analysis, E.B. ten Dam and C.W.A. Pleij forcommunicating data prior to publication and B.A.M. van derZeijst for continuing interest and support. Part of this work wassupported by NIH Grant Al-17418 and NS-21954 to S.R.W. andTraining Grant T32 Al-07325 to C.P.

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