VIROLOGY

183,

773-777

Identification

(1991)

and DNA Sequence

of the Large Subunit of the Capping

Enzyme from Shope Fibroma Virus

C. UPTON, D. STUART, AND G. MCFADDEN’ Department

of Biochemistry, Received

University April

of Alberta,

19, I99 1; accepted

Edmonton, May

Alberta,

T6G 2H7

Canada

13, I99 I

A 3.6-kb region of the Shope fibroma virus (SFV) BarnHI D fragment located in the central region of the viral genome was sequenced. Three open reading frames (ORFs) were identified, D3R, D4L, and D5R. Each of these ORFs have a counterpart organized identically within the HindIll fragment D of the vaccinia virus genome (DlR, D2L, and D3R). Homology scores and assays of viral cores indicate that SFV D3R encodes the large subunit of the SFV mRNA capping 0 1991 Academic Press, Inc. enzyme.

Viruses within the family Poxviridae all share a number of common features. They are similar morphologically, replicate in the cytoplasm of infected cells, and possess a genome consisting of a single linear dsDNA molecule with inverted terminal repeats (ITR) and hairpin termini (Y-5). The different genera into which the poxviruses have been classified contain viruses that are relatively closely related in that the viral genomes can be shown to cross-hybridize. However, even in the Orthopoxviridae, the most extensively examined genus, where the composite DNA restriction maps of many of the members are almost identical, individual viruses are quite different in terms of pathogenic profile, host range, and the severity of disease produced (6). Thus poxvirus biology may be fruitfully investigated by examining both the similarities and the differences between individual viruses or virus groups. Elucidation of factors responsible for the observed differences in virulence between very closely related viruses and the examination of distantly related viruses for conserved genes and functions should both yield useful information. We previously noted that the thymidine kinase (7) and topoisomerase (8) genes of Shope fibroma virus (SFV), a Leporipoxvirus, have greater than 60% identity when compared to their counterparts from vaccinia virus (VV), the prototypical Orthopoxvirus, and that the organization of the two viral genomes in this central portion appeared to be somewhat conserved. In contrast, the region of the ITR is far less conserved and although many of the genes present within the SFV ITRs have homologues in VV they have very low homology and their relative positions within the genome have

Sequence EMBUGenBank ’ To whom

data

from this article have been deposited with Data Libraries under Accession No. M63902. reprint requests should be addressed.

not been maintained (9). In this paper we present the DNA sequence of a 3.6-kb region within the BarnHI D fragment of the SFV genome and show that it contains three open reading frames (ORF) which correspond to three contiguous ORFs from the VV HindIll D fragment, including the large subunit of the mRNA capping enzyme (10). This region was chosen in order to continue our study of evaluating the conservation of essential genes between the Lepori- and Orthopoxviruses. Comparison of these ORFs confirms that the selective pressure exerted on the various poxviral ORFs yields dramatically different levels of sequence conservation, even in a region of the poxviral genome that has completely maintained its organization between the two genera. The region of interest (SFV ORFs D3R, D4L, and D5R) is diagrammed in Fig. 1. The DNA sequence was determined by the chain termination method (11) using Sequenase (USBC, Cleveland, OH) in both orientations from sets of overlapping deletions (12) created from overlapping subclones from the BarnHI-HindIll fragment. Programs of the Genetics Computer Group (13) were used to analyze sequence data. The DNA sequence and the translation of the three ORFs are shown in Fig. 2. This DNA sequence overlaps slightly with a previously published sequence (8) in which the SFV DNA topoisomerase gene (Di R) was described. The nomenclature of the SFV and VV ORFs should not be confused: that of SFV was derived from a BarnHI map (strain Kasza) while VV uses a HindIll map (strain Copenhagen). Thus, SFV ORF D3R is equivalent to VV ORF Dl R. SFV ORF D3R was found to be 836 amino acids (aa) long, 8 aa shorter than VV ORF Dl R which is known to encode the large subunit of the VV mRNA capping enzyme (10). An alignment of these two ORFs, shown in Fig. 3A, indicates they are 60% identical overall, although the C-terminal one-third of the protein is greater

the

773

0042-6822/g

1 $3.00

Copyright 0 1991 by Academic Press, Inc All rights of reproduction 8” any form reserved

SHORT

774

COMMUNICATIONS 100

50

0 L

I

B

-----*-

Da.

150

I

I B

H

FIG, 1. Diagram of the BamHl restriction fragments of the SFV genome. The terminal inverted are indicated. ORFs DlR, DZR, D3R, D4L, and D5R are shown as arrows on an expanded topoisomerase gene (8). B, BamHl ; H, Hindlll.

than 809/o identical. This measurement of relatedness is similar to that found for the DNA topoisomerases (61%) and thymidine kinases (65%) of SFV and VV and appears to reflect the conservation of enzymatic functions active in the metabolism of poxviral DNA and RNA. The capping of the 5’ end of VV mRNA has been carefully studied, and the enzymes that per-form the reactions involved (RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7) methyltransferase) reside within a heterodimeric complex composed of the 01 R (large subunit) and D12R (small subunit) proteins (14, 15). The guanylyltransferase activity is associated with the large subunit and can be easily detected by means of a GTP-PP, exchange reaction (16). In order to demonstrate bona fide guanylyltransferase activity by SFV, viruses were grown in BGMK cells as previously described ( 17) and viral cores were isolated (18) as a source of capping enzyme. After incubation of viral cores with [cY-~~P]GTP(19), samples were electrophoresed in a gradient (5-20%) polyacryamide gel. An autoradiogram of the dried gel is shown in Fig. 3B. As in the case of VV, a single protein species was predominantly coupled with GMP for both SFV and myxoma virus (a closely related Leporipoxvirus). The molecular weight of the labeled SFV and myxoma virus proteins appear to be very close to the 95-96 kDa observed for VV (14) and in agreement with the sequencing data which showed the SFV D3R ORF to be only 8 amino acids shorter than the VV Dl R ORF. Lysine has been identified as the amino acid through which the VV guanylyltransferase is linked to GMP (20, 27) and by expression of carboxy-terminal deletions of the VV 01 R protein in Escherichia co/i this residue has been found to map in the N-terminal two-thirds of the protein (22). Therefore, the sequence of the SFV D3R ORF should be of use in the determination of the active-site lysine by identification of conserved residues between the two proteins. However, it should be noted that align-

KB

repeats BarnHI

(TIR) and the thymidine D fragment. ORF DlR

kinase is the

gene (Tk) SFV DNA

ments of protein sequences performed by amino acid homology alone will rarely reflect the correct relationship between the proteins with respect to their structural and functional entities. For instance, the alignment of SFV 03 lysine-326 with VV 01 lysine-332 (which are offset by a single amino acid and indicated by + in Fig. 3) has been penalized because this requires the insertion of two gaps in the alignment although these two residues may in fact be functionally equivalent. The VV ORFs D2L and D3R, the counterparts of the SFV ORFs D4L and D5R presented here, are known to be late genes transcribed after the onset of viral DNA replication (23, 24). Both vaccinia proteins are believed to be virion structural proteins (Oyster and Niles, personal communication). ORFs SFV D4UVV D2L and SFV D5RIVV D3R were found to be 36 and 33% identical, respectively (Figs. 4A and 4B), relatively low with respect to the score of the large capping enzyme subunits described above. However, the hydrophilicity/hydrophobicity plots of these two pairs of proteins were very similar (data not shown), suggesting a similar structural function for these SFV proteins, although it remains to be proven that the products of these SFV genes are actually present in the virion. The epidermal growth factor-like family of proteins (including TGF-(U and VGF) represent another instance where functionality has been maintained despite a relatively small number of conserved amino acids. There are 12 amino acid residues conserved throughout this family (6 of which are cysteines), but overall the SFV and myxoma growth factors are only 36 and 34% identical to the VGF, respectively (25, 26). In conclusion, the DNA sequence presented here and previously (8) indicates that this central region of the SFV and W genomes has been rigorously conserved. Each of the ORFs described in SFV has a counterpart in exactly the same genomic context

SHORT

COMMUNICATIONS

775

1 ATCGATGATTCAAAAAAGAAACGGGGAACTGATTACATCGAGGbACTGATCTTATTATAC &I (03RPtlO 0 SK K K R G T 0 Y I E E LIL L Y 81

GMGACGTTCCTMCCCCGTACCIAACTCA7CATATCMCtAC EOVPNPVPTDDMNHEVELTF

120

121

ATTCMCCACCCGTTATTACGTT~GTACACTGTTACCTTTT~TACATCTCA~MTCG IPPPVITLSTLLPFATSPES

180

181

TATATTTTGTTTACCGTGACGAATAMGGCGTTAAAATTAGAAACAGAATAAACTTATCG 2K) YILFTVTNKGVKIRNRINLS

241

AAGATCCATGGACTCCATTTAMGAACATTCAGTTGGTGGATTCCATC~T~TATTATA 3M) KIHGLOLKNIOLVDSIONII

301

TGGGAAAAGAAAACATTGGTAAAAGAGCACAAAATAGACTCGGTAGCTCTCGTAUGTAT 360 WEKKTLVKEHKIOSVALVKY

361

TCAACTGAAGAGAAGTATATCTTCCTCGATTATAAGAMTIAATTG STEEKYIFLOYKKYLSAIKL

421

GAACTGGTAAACGTCGTTCAGGTGAAGCrCAAACACGTGACAGTGGATTTT~GTTTAAG 480 ELVNVVPVKVKHVTVOFKFK

481

TATTTCCTGGGCTCGGGAGCCCAGGCAAAAAGTTCTTTGCTACACGTATT~CCATCCA5rK) YFLGSGAOAKSSLLHVLNHP

541

MGTCCAAACCCAATCCTTCTCTCGAGTTTGAGATCATCAC~CGGAT~GA~TAGAC KSKPNPSLEFEIITTOEKID

601

TCCGCCTCTTTACGGAAGGAACTCAlTGCCTTGTTTAMtTCGTGTTTATGGCATCTCCA660 SASLRKELIALFKLVFIIASP

661

AGTAATATCATCTTAGACGTCGTGTTCAAAAACCCAGTACAAACGATTTTGCTG~GAAG 720 SNIlLDVVFKNPVOTlLLKK

721

AACGAATTACCCGGGATCGATTTAACTAACCTATACGTGAC~CG~GACGGACGGTGTA 780 NELPGIOLTNLYVTTKTOGV

781

GGGGTTCTTATAACCGTAACWUTAAGG~ATCTATTGCTTTTTCACACATCTACAGTACa'@ GVLITVTNKGIYCFFTHLOY

841

ACGATACGATACGACACGACGTTCGAGTCG~CGAGTCCGTTACGTTGTACGGGG~GCC 900 TIRYDTTFESNESVTLYGEA

901

GTTAAACAGAATAACGTATGGCAGATATATCTTATTAAGTTGATAACCCCC~GGTATCC 960 VKQNNVWOIYLIKLITPKVS

961

GATCGGTTT~GGAAAAGGAATACGTCGAGGAACGTCTCC~~CATATGTGACCGAATG t&O ORFKEKEYVEERLONICDRt4

1021

ACGTTTAAAGTAdAGAAATATGAGGGCCCTTTCGAATtACACTCCGAGATCATAGATTTA lO%l TFKVKKYEGPFESHSEIIOL

1081

CTTACGACGTACTTACCGTCTCAACCCGAGGW\GTTGTGTTGTTTTATTCGGATCA~GG1140 LTTYLPSOPEGVVLFYSDOR

'420

600

192D 1921

TACATACAGGMACAATTCGATCGGTCACGTACGTATCCAGCGTACGAGAGGTGTTCTTC lBB0 YIDETIRSVTYVSSVREVFF

lQS1

TTCGGTAAITTCGATC7GGTGCATtCGCAGTTCGCAATT FGKFDLVDWGFAIHYSFHPK

2c40

2D41

CATTACGCCACGGTGATGAATMTCTIAtCGAGTT~CGGCGTCCGGG~G~~TGTTG HYATVtlNNLTELTASGGKVL

2100

2101

ATTACAACAAlGGACGGGGACTTATTGACTCAATTMCGGAT~G~G~GTTCGTGATA ITTBDGOLLSOLTDKKTFVI

2160

2161

CAlAAGAACTTACCGAGCAGCGAAAACTACATGTCTGTGGAG~GAlACAl~~ATCAG mo HKNLPSSENYHSVEKIHEDO

2221

ATTTTAGTATATAATCCTTCGTCTATGTCTA~CCCATGC~~GTACATCGTCAAGAGG ILVYNPSSflSRPflOEYIVKR

2281

GTGAACTTAICCAAAATATTTTCGGAATACGGCTTCGAGTTAATCGATTGTGTTCATTTC2340 VNLTKIFSEYGFELIOCVHF

2341

GACACAATCATAGAGCGAAGTAAACGGlTCAl~ACAGtCGC DTIIERSKRFINSVSKBEER

2wl

2'+01

AAATCCACCAAGAACTTCTTCGAAtTAAATAGAAGCGTCGGAT KSTKNFFELNREALKHEGTD

24e4

2461

ATAGACGACCTGTTACGAlATTACATCGlGlACGTCTlTTCC~GG~AAGT~TAGTA IDDLLRYYIVYVFSKR +SIVDHVDKGFPLYYY

2520

2521

CGACACCGGAGCGTCTTTTTTCCCGAACCTAAACCICTTAACGGT SVPAOKKGFRFSNFINLLVT

2580

2581

TACCGGATTAGACGGTTCCGTGTCATCCGTCGATATGGATACCGlAlAGTCCGGGGC~T2640 VPNSPETDDlSlSVTYDPAl

2641

lTCCCGTATAGAGTTGlAAACAGTTlTGCTGGG~C~CAlATCTAl~TCGCClCGTT ERlSNYVlKSPFL~DIlAEN

2700

2701

GTGTTTGTACAGATATlTGATCACCCC~CATAGTTTTGTlGTCG~TCTAGCAGCGGA HKYLYKIVGFHTKNDFRAAS

2160

2761

AGAATACACATGAATGTTACGTATCATACATCTGCCTTTTTCTAACAC~TA~TCGlTC 2w SYVHINRIMCRGKELVIFRE

2821

ATCGTATATCTTTTTTATATCACATG~CATACTGTTTTATTGTTTTGTAGCAAGTCCGT 2880 DYlKKlDCPCVTKNNOLLDT

2881

TAGTTTTTTGATATCTAACTCCATCGACTATCAATACCICCGACG L K K I 0 L E MDYL) (D5R)+fi N T T I L I

2280

2940 H

0

0

0

1141

AATCMCCCGATTATAAAATAAAACTAGATAATACGACGGATCACATGAT~ACATAATA 1200 NOPDYKIKLDNTTDHMINII

2941

AlATACAAGTTAATGATTTAGA~TAAAACATTCTlATTATT~GTG~CATAATG IOVNDLKENKTFLLLSEHNE

3cw

1201

TACAGGTACATGTCTAGCGAACCCGTTATATTCGGAGAG~TTCTACGTTCTTAGAGTAC1260 YRYMSSEPVIFGENSTFLEY

3DDl

~CGTATAATCGATAAACTATGlAGClGTCTACTACCCAlTAlTTTTTATTGC~CTATA RIIDKLCSCLLPIIFYCDYI

3c60

1261

AAAAAATTCAGCGACGATAAGGTTTTCCTAAAGACTACGG~CGGG~ACTCATGTTA KKFSODKGFPKDYGTGKLML

1320

3061

TAACGTCTCCGGATGATGAAGGTACGTTAGAAACGAGGATTTTATCTTCCAGTTATATGA 3120 TSPDDEGTLETRILSSSYfll

1321

ACTGATAACGTCAGATACTTAAACAACATTTACTGCATCGCGTTTACA~CGTATACGAA 1380 TDNVRYLNNIYCIAFTNVYE

3121

TACGTGATAAATACGTAAACGTGGAGGAGTTTATCACTGCCGGGCTCCCCCTGTCGTGGT 3180 ROKYVNVEEFITAGLPLSWC

1381

GACGTAGGTAlAAAGAACGTCGlAGTTCCTATAAAGTlCATAlCGGAGTTTTCGGCCACT1440 DVGIKNVVVPIKFISEFSAT

3181

GTGTGAATCTTCCGGAAAAGGCGCATlCTACCGCATCtCATG VNLPEKAHSTASDSLIIROV

32'10

1441

GGAGAGTTAATAAAACCACGAATCGATAAAACGTTTAAGTATCTGTAC~AGAGTATTAC 15M) GELIKPRIDKTFKYLYKEYY

3241

TTllATACTATAAAAAAGATlGGATACGTATCTTAlTMTCCMTGTCCAlCAGCCATAl LYYKKDWIRlLLlOCPSAlY

3300

1501

GGCAACCAGTATCAAATCGTCGTAGCACATATAAGAGATC~~CATC~ATffiGAGAC GNOYOIVVAHIRDQNIKIGD

1560

3301

ACACGGACGAAGAAClCCTGAlCGATCCGTTTAAACTGCCTC~CAlCCTCCG~ACTGT 3360 TDEELLIDPFKLPRHPPELF

1561

GTGTTAGACGAAGACMACTATCCGACGTGGACAAtATTACGC~CGAC~ATATAGA VLDEDKLSOVGPHYANDKYR

1620

3361

TTAA~CGTTACGTTGAGATCCTACGTAAACGGGCTATTGTTTTATCC~CGTCATCAC 3420 KNVTLRSYVNGLLFYPTSSP

1621

TTGAACCCGGACGTGTCATATT7TACGAACAAGAG~CTCGAGGTcCTUAGGTATCCTA1880 LNPDVSYFTNKRTRGPLGIL

3421

CCCTATACGCTTTATTGAGTCATGTGGTTACTACGTTTATTATA~C~ATTACATGTG LYALLSHVVTTFlIKHlTCV

3480

1681

TCGMTTACGTGAAGACGTTATTAATTTCTCTGTATTCC SNYVKTLLISLYCSKTFLDN

3481

TTACAAMCACGACGAG~TTAATCACAACGTGTTACGAC~GGGTAGGTTT~CGCCT TKHDEKLITTCYDKGRFNAF

3540

1741

TCTAATAAACGAAAAGTGTTGGCGATTGATTlCGGAAACGGGGcAGATTTGGAAMATAC t@Xl SNKRKVLAIDFGNGADLEKY

3541

TCGTGTACGCCTGGTACMTTCCCAGAT~GCGACGACGTTGTAG~TG~GT~ VYAWYNSQISDDVVENEKVK

3800

I.301

TTCTACGGAGAGATATCATCCCTCGTGGCCACGGACCCCGAC~G~GCCATCGGTCGT1860 FYGEISSLVATDPDKEAIGR

3601

AAAACTTATTTGCATTAGTTAAGGCACGAATAIGAGAGACGGGTATTTTTATCTCACG~CC 36eo NLFALVKARI

17w

FIG. 2. DNi sequence and translatron of ORFs D3R, D4L, and D5R from the SFV BarnHI fragment D. Nucleotide first nucleotrde of the ORF D3R) is 1986 nucleotides from the left end of the BarnHI fragment and is equivalent reference (8). Bent arrows 0) show the STOP codons of the preceeding ORF.

No. 1 in this sequence (the to nucleotide No. 2207 in

SHORT

776

COMMUNICATIONS

A SFV-DJR VAC-DIR SFV-D3R VAC-D 1R SFV-D3R VAC-DIR SFV-DJR VAC-DIR SFV-D3R VAC-DlR SFV-D3R VAC-DlR SFV-DJR VAC-DlR SFV-DJR VAC-DIR SFV- D3R VAC-DIR SFV-D3R VAC-DlR SFV-D3R VAC-DlR SFV-DJR VAC-DIR SFV-DJR VAC-DIR SFV-DSR VAC-DIR SFV-DSR VAC-D 1R SFV-DJR VAC-DlR SFV-D3R VAC-DIR

:~DSKKKRG:--D:fEE:ILLYEDVPNPVDTDDMNHEVELTFlDPPVlTL I I II II II MDANVVSSSTIATYIDALAKNASELEPRSTAYElNNELELVFlKPPLITL

III

STLLPFATSOESYILFTVTNK-GVKlRNRlNLSKlHGLDLKNlDLVDSlD lllllIIIIIIlllll lIIIllllIIllllII TNVVNISTIDESFlRFTVTNKEGVK~RTKIPLSKVHGLDVKNVGLVDAID NIIWEKKTLVKEHKIDSVA~VKYSTEEKYIFLDYKKYLSAIKLELVNVVO IIII lllllllI I I lllll II IIII II I NIVWEKKSLVTENRLHKECLLRLSTEERHIFLDYKKYGSSIRLELVNLID

I

VKVKHVTVDFKFKYFLGSGA(IAKSSLLHVLNHPKSKPNPSLEFEIlTT~I I I III IIIIIllll llllll lllll II III I AKTKNFTIDFKLKYFLGSGADSKSSLLHAANHPKSRPNTSLEIEFTPRDN ~~IDSAS~R~~~IA~FKLVFHA~SNlILDVVFKNI;VD~IL~K~NELP~I IIIII I II EKVPYDELIKELTTLSRHIFMASPENVlLSPPINAPIKTFMLPKPD1VGL t DLTNLYVTTKTDGVCVLlTVTNKGlYCFFTHLPYTlRYDTTFESNES~TL II Ill lllll III IllllllIIll DLENLYAVTKTDGlPITlRVTSNGLYCYFTHLGYllRYPVKRlIDSEVVV YGEAVKDNNVWIlIYLIKLITP--KVSDRFKE:EYVEERLIlNICDRHTFKV lllll I I llllll I II I III I IIII FGEAVKDKN-WTVYLIKLIEPVNAINDRLEESKYVESKLVDICDRIVFKS +

II

:~::~F;:ESHSEIIDLLTTYLPSOPEGVVLFYSD-DRNDP~Y~f:LD~:T II IIIIIIIIIIIIIII KKYEGPFTTTSEVVDMLSTYLPKOPEGVlLFYSKGPKSNlDFKIKKENTl

B

~HPII~IIYRYMSSEPVIFGENSTFLEYKKFSDDKGFPKDYGTGKLMLTDN lllllll IIII I I llllll llllll II II I DGTANVVFRYMSSEPIIFGESSIFVEYKKFSNDKGFPKEYGSGKIVLYNG VRYLNNlYClAFTNVYEDVGlKNVVVPIKFlSEFSATGELlKPRlDKTFK I lllllll IIII lllIIIII II II lllllll VNYLNNIYCLEYI:THNEVGIKSVVVPIKFIAEFLVNGEILKPRIDKTMK ~LYKE-YYGNPYGIVVAHIRDDNIKIGDVLDEDKLSDVGGHYAN-DKYRL I lllll I I I III IIIII IIIIIIII III II YINSEDYYGNDHNIIVEHLRDGSIKIGDIFNEDKLSDVGHOYANNDKFRL

I

II

NPDVSYFTNKRTRGPLGILSNYVKTLLISLYCSKTFLDNSNKRKVLAIDF II IIIIIIIIIIIIIIIIIIIIIIIIII llllllll IIIIIIIIIII NPEVSYFTNKRTRGPLGILSNYVKTLLISMYCSKTFLDDSNKRKVLAIDF GNGADLEKYFYGEISSLVATDPDKEAIGRCIERYNSLNSGIKSKYYKFDY IIIIIIIIIIIIII IIIIIII II I III1 llllll IIIIIII GNGADLEKYFYGEIALLVATDPDADAIARGNERYNKLNSGIKTKYYKFDY IDETIRSVTYVSSVREVFFFGKFDLVDWOFAIHYSFHPKHYATVHNNLTE lllllll I IIIIIIll IIII IIIIIIIIIIII llIIIIIII IDETIRSDTFVSSVREVFYFGKFNIIDWDFAIHYSFHPRHYATVMNNLSE

I

LTASGGKVLITTMDGDLLSDLTDKKTFVIHKNLPSSENYHSVEKIHEDDI IIIIIIIIIIIIIIII II lllllll IIIIIIIIIIIIIIIII LTASGGKVLITTMDGDKLSKLTDKKTFIIHKNLPSSENYMSVEKIADDR~

I I

LVYNPSSflSRPMPEYIVKRVNLTKIFSEYGFELIDCVHFDTIIERSKRF~ IIIII II II III I I IIII I I I I IIIIIII VVYNPSTHSTPHTEYIlKKNDlVRVFNEYGFVLVDNVDFATllERSKKFl

II

NSVSKMEERKSTKNFFELNREALKHEGTDIDDLLRYYIVYVFSKP I IIIllllllllllllIIII 111111111111 NGASTMEDRPSTKNFFELNRGAIKCEGLDVEDLLSYYVVYVFSKR+ I-

FIG. 3. (A) An alignment of the SFV BarnHI D3R and VV (strain Copenhagen) HindIll Dl R ORFs. These protein sequences are 60% identical. Bent arrows (A) indicate the end of the ORFs. Lowercase letters beneath the alignment indicate amino acid differences present in VV strain WR (10). + demonstrate an example of unaligned but possibly functionally equivalent amino acids (see text). (B) [(Y-~‘P]GMP labeling of viral cores. Autoradiogram of a 5520% acrylamide protein gel (27). Lanes 1 and 6, W WR; lanes 2 and 5, as lane 1 (1 in 10 dilution); lane 3, SFV; lane 4, myxoma virus. Positions of marker proteins are indicated by arrowheads; 97.4, 66.2, 45.0, 31 .O, 21.5. and 14.1 kDa.

SHORT COMMUNICATIONS

A

777

REFERENCES

SFV-DYL VAC-DPL

~ELDIKKLTDLLDNNKTVCPCDIKKIYDERFIVLE-KGRCHIRNIHVYSS IIll IIll I NSINIDIKKITDLL-NSSILF~D~VD~LL~KY~:::R~SNGTPTVAlliI~KT

SFV-D4L

AARR~r;r:THFGVI~Y~YKHNEAIfDH~~SKTVYNSIREIAI;~YTVSf~T

VAC-D2L

HARFDNKSIYRIAKFLFllNRPDVIKLLF-----LEDVEPLLPDKSINISI k

SFV-D4L VAC-DPL

CONDIT, R. C., and NILES, E. G., Curr. Top. Microbial. lmmunol. 163, l-40 (1990). 2. MOSS, B., Curr. Top. Microbial. Immunol. 163, 41-70 (1990). 1.

3. DERANGE, A. M., and munol. 163, 7 l-92 4. TRAKTMAN, P., Curr.

DD;;PSN---~V---TV~~NI~W~F~KKDAPVSYYYLPFGKDVHDVIS' I IIIII II NNTEYPMEGPIGTKIALLELFNAFRTG1SE-PIPYYYLPLRKDINNIVTK' r

5. 6.

B ~NTT~LIH~DDIDVN~LKENKTFLLLSEHNERIIDKLCSC~LPII~YCDY I I II II I II "DIFI-VKDNKYPKVDNDDNEVFILLGNHNDFIRLKL-TKLKEHVFFSE;

7. 8. 9.

VAC-D3R

1TSI;~DE~T:ETRILSSSYHIRDKYVNVEEFITAGLPL~~VNLPEKAHS I IIIII II IVTPDTYGSLCVELNGSSFDHGGRY1EVEEFIDAGRDVRWCSTSNHISED

SFV-D5R

TASDSLIIRDVLYYKK--DUI---RILLIDCPSAIYTDEELLI~FKLPR

VAC-D3R

IPE;IHTDKF:I:DIYTF;AFKNK;LVFV&PSL-G;DSH;T&LLS!m

SFV-D5R

HPPELFK:VTLrtSY::GLL:YPTSSPL~A~~S~ACTTF--II~~~~C~TK

VAC-D3R

----YYRNSVARDtlVNDRIFNDDSFLKY-LLEHLIRSHYRVSKHITIVRY

SFV-D5R VAC-D3R SFV-DSR

k

10. 11. 12. 13.

VAC-D3R

15.

4. (A) Alignment of the SFV BarnHI D4L and W (strain Copenhagen) Hindill D2L ORFs. (6) Alignment of the SFV BarnHI D5R and W (strain Copenhagen) HindIll D3R ORFs. Bent arrows (A) indicate the end of the ORFs. Lowercase letters beneath the alignments indicate amino acid differences present in VV strain WR (IO). FIG.

16. 17. 18.

within the W genome. In addition, we have found that the degree of similarity between any two homologues is quite variable and in the case of the capping enzyme, the regions of strongest conservation probably reflect the catalytic functions of the protein. ACKNOWLEDGMENTS We thank Stewart Shuman for helpful discussions, Ed Niles for communicating results prior to publication, and Robert Maranchuk for excellent technical assistance. G.M. is supported by the Alberta Heritage Fund for Medical Research. This work was funded by operating grants from the National Cancer Institute and Medical Research Council of Canada.

G., Curr.

Top.

Microbial.

lm-

(1990).

Top. Microbial. lmmunol. 163, 93-l 24 (1990). TURNER,P. C., and MOYER, R. W., Curr. Top. Microbial. lmmunol. 163, 125-152 (1990). FENNER,F., WITTEK, R., and DUMBELL. K. R., “The Orthopoxviruses.” Academic Press, San Diego, 1989. UPTON, C.. and MCFADDEN, G., J. Viral. 60, 920-927 (1986). UPTON, C., OPGENORTH,A., TRAKTMAN,P., and MCFADDEN, G., Virology 176, 439-447 (1990). HOWARD, S. T., CHAN, Y. S., and SMITH, G. L., Virology 180, 633-647 (1991). NILE% E. G., CONDIT,R. C., CARO, P., DAVIDSON,K., MATUSICK,L., and SETO, J., Virology 153, 96-l 12 (1986). SANGER,F., COULSON, A. R., BARELL, 6. G.. SMITH, A. J. H., and ROE, B. A.,). Mol. t?iol. 143, 161-178 (1980). HENIKOFF,S., Gene 28, 351-359 (1984). DEVEREUX,J., HAEBERLI,P., and SMITHIES,O., Nucleic Acids Res. 12,387-395(1984).

74.

SFV-D5R

MCFADDEN,

19.

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