Tumorigenic poxviruses: Fine analysis of the recombination junctions in malignant rabbit fibroma virus, a recombinant between shope fibroma virus and myxoma virus

VIROLOGY

166,229-239

(1988)

Tumorigenic Poxviruses: Fine Analysis of the Recombination Junctions in Malignant Rabbit Fibroma Virus, a Recombinant between Shope Fibroma Virus and Myxoma Virus C. UPTON, J. L. MACEN, R. A. MARANCHUK, Department

of Biochemistry,

A. M. DELANGE,’ AND G. MCFADDEN’

University ofAlberta,

Edmonton, Alberta T6G 2H7, Canada

Received April 12, 1988; accepted May 26, 1988 Malignant rabbit fibroma virus (MRV) has been shown to be a lethal tumorigenic poxvirus of rabbits derived from a recombination event between Shope fibroma virus (SFV), which induces benign fibromas in rabbits, and myxoma virus, the agent of myxomatosis. We have cloned and sequenced all of the MRV recombination junctions, which are located near the left and right terminal inverted repeat (TIR) regions, and present a composite map of the MRV genome with respect to the relevant gene products. The two junctions closest to the MRV termini, at identical positions at the left and right ends, are at nucleotide 5272 and result in an in-frame fusion protein (ORF T-5) in which the N-terminal 232 aa are derived from an SFV sequence linked to a C-terminus derived from myxoma. At the left MRV TIR the recombination junction distal from the terminus maps to nucleotide 9946 but leaves the adjacent gene virtually unchanged from its SFV homolog. At the right terminus, the relevant junction sequences from MRV and myxoma could not be cloned in wild-type Escherichia co/i but were maintained stably in a recA recBC sbcL3 host. The SFV/myxoma junction at this location maps 5’to a growth factor gene (SFGF) which is related to those encoding epidermal growth factor and transforming growth factor-a. As a result, the myxoma growth factor gene has been deleted in MRV and replaced in fofo by the SFV gene. The recombination junction upstream from the SFGF gene creates an in-frame fusion in ORF Tl l-R in which the N-terminal amino acids are derived from myxoma and the remainder from SFV. In summary, MRV has received the following ORFs from SFV: at the left terminus T5 (fusion), T6, T7, and T8; at the right terminus, T5 (fusion), o 1988Academic press, inc. T6, T7, T8, T9-R, SFGF, and Tl 1 -R (fusion).

the histological profile of the induced fibromas are very similar to those caused by SFV, while the later metastases have an invasive profile similar to that of myxoma virus; (2) MRV has broader host range than SFV, similar to that of myxoma virus; (3) the restriction enzyme digests of MRV genomic DNA show a close but nonidentical relationship to myxoma virus DNA (Strayer eT a/., 1983a,b,c, 1985, 1987). Southern blotting experiments confirmed that MRV is indeed a natural recombinant between SFV and myxoma and showed that the genome of MRV is composed predominantly of myxoma sequences, with less than 10 kb of the MRV DNA derived from the terminal inverted repeat (TIR) region and adjacent unique sequences of SFV (Block et al., 1985). Apparently, the replacement of this small region of the myxoma genome with related sequences from SFV is sufficient to produce the observed phenotypic differences between myxoma and MRV. SFV &hang et al., 1987), myxoma virus (Upton eta/., 198713) and MRV (this paper) all possess genes that encode proteins with significant amino acid homology to the family of epidermal growth factor (EGF)-like growth factors. The importance of such EGF-like growth factors with respect to the proliferative phenotype of tumorigenic poxviruses remains to be clarified. Genes encoding EGF-like growth factors are not restricted to the tumorigenic poxviruses since vaccinia

INTRODUCTION The replication and assembly of poxviruses takes place within virus factories or virosomes that are produced in the cytoplasm of infected cells (Dales and Pogo, 1982; Moss, 1985). Despite the fact that viral replication occurs outside of the host cell nucleus, a number of poxviruses are known to be the causative agents of proliferative diseases. Of the tumorigenic poxviruses, Shope fibroma virus (SFV) has been the most intensively studied at a molecular level (reviewed by McFadden, 1988). Recently a novel Leporipoxvirus, designated malignant rabbit fibroma virus (MRV), was isolated from a SFV-inoculated rabbit that exhibited unexpected disease symptoms (Strayer and Sell, 1983). Unlike SFV, which induces benign fibromas that invariably regress in an immunocompetent rabbit, MRV causes immunosuppression of its host and produces a disseminated malignancy that is fatal. Preliminary examination of the biological properties of MRV suggested that it is related to both SFV and myxoma virus, the latter being the agent of rabbit myxomatosis: (1) early in MRV infection, ’ Present address: Department of Human Genetics, University Manitoba, Winnipeg, Manitoba, Canada. ’ To whom requests for reprints should be addressed.

of

229

0042-6822/88

$3.00

Copyright 0 ,999 by Academic Press, Inc All rights of reproduction in any form reserved.

230

UPTON ET AL.

virus, a cytolytic Orthopoxvirus, also encodes a polypeptide, designated vaccinia growth factor (VGF), which has significant amino acid homology to EGF and competes with it for binding to the EGF receptor (Blomquist et a/., 1984; Brown et al., 1985; Stroobant et a/., 1985). Deletion of the VGF gene has been shown to reduce virus virulence (Buller et a/., 1988). Data from the genomic mapping of MRV, SFV, and myxoma virus and DNA sequencing of the Shope fibroma growth factor (SFGF) and myxoma growth factor (MGF) genes indicated to us that the SFV gene lies very close to the recombination junction with myxoma in MRV. Each of these genes maps within the unique region very close to the right TIR of the viral genome (Block et a/., 1985; Chang et a/., 1987; Upton et a/., 1987a,b). Since the entire 12.4-kb TIR of SFV had been sequenced, and the position and orientation of each open reading frame (ORF) in this region of the SFV genome are known (Upton and McFadden, 1986 a,b; Upton et al., 1987 a,b), we undertook the sequencing of the four SFV/myxoma DNA junctions found in MRV to determine which SFV ORFs had been transferred in toto to MRV and to examine the precise nature of these junctions to see if fusion of similar or dissimilar genes had created novel ORFs. We present data which precisely define the DNA sequences exchanged between the SFV and myxoma genomes during the recombination event that created the novel poxvirus MRV. In a related issue, we have also examined genomic variation of this same region in a strain of SFV (Boerlage) that induces larger tumors than our standard strain Kasza (Strayer et a/., 1984). These sequences also provide an important insight into the genomic organization of these Leporipoxviruses and the relationship between them. MATERIALS

AND METHODS

search Products. The 16-nucleotide primer for sequencing the SFV/myxoma junction in A” was a gift from Cangene Corp. (Mississauga, Ontario); all other reagents for DNA sequencing were from New England Biolabs (Beverley, MA). Cloning and DNA sequencing Standard methods were used for all DNA cloning (Maniatis et a/., 1982), pUC plasmids were grown in Escherichia coliJM83 unless otherwise noted and M 13 vectors were propagated in E. co/i JM103 (Messing, 1983). Some of the restriction fragments from the right TIR/unique sequence boundary in myxoma and MRV were refractory to cloning in wild-type E. co/i (Block et al., 1985) but were successfully propagated in strain DB1256, carrying red recBC sbcB loci (described in DeLanage et a/., 1986). The Sanger dideoxynucleotide chain termination method (Sanger et al., 1980) was used for sequencing. Unidirectional nested deletions were generated by exonuclease III (Henikoff, 1984) as described previously (Upton and McFadden, 1986a). Analysis of DNA sequences was performed using the core library programs of the computer resource Bionet (Funded by Public Health Services Grant 1-441RR01 685-01 from the National Institutes of Health). Conditions

for Southern

blotting

DNA samples were run in 0.7% agarose and transferred to nitrocellulose by standard techniques. The stringency of washing conditions (1 hr in 2X SSC (SSC = 150 mll/l NaCI, 15 rnn/l sodium citrate, pH 7.0) at room temperature, followed by 1 hr in 2X SSC at 50”) were such that hybridized SFGF probe detected both the homologous SFGF gene and the heterologous MGF gene. The much weaker signal from the latter distinguished the two genes.

Cells and viruses RESULTS The rabbit cell line SIRC, the monkey cell line BGMK, and the strains of virus used in this study are described elsewhere (Wills et a/., 1983; DeLange et al., 1984). All cultures were propagated in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum. Enzymes and reagents Restriction enzymes were purchased from Boehringer-Mannheim (BM), Bethesda Research Laboratories (BRL), Pharmacia, and Amersham, and were used under normal conditions. T4 DNA ligase, T4 DNA polymerase, and exonucleases III and VII were from BM or BRL. [a-32P]dATP was supplied by NEN Re-

MRV genomic structure Elsewhere it has been shown that MRV contains two blocks of sequence information derived from SFV, one of about 4 kb at the left MRV TIR and one of at least 67 kb at the right MRVTIR (Blocketa/., 1985; Upton and McFadden 198613). As shown in Fig. 1, the left and right junctions closest to the MRV termini that demark the boundary where SFV sequences begin (at about the 5 kb position) are indistinguishable by restriction enzyme mapping, while the two junctions distal to the MRV termini are at clearly different locations. This asymmetry has been interpreted as evidence that the recombination which generated M RV consisted of two events: (1)

RECOMBINATION

L( NRV R(

I Bm I

1 HD 1 I I Em B9 I

IADI

I

HE

AC

JUNCTIONS

1 I B9 I

HA

I

AB

IN MRV

Hc I I BP ; , I

I ,

231

I I Em

BI El

AA

Terminal lnvcrtcd Rcpcat

FIG 1. Organization of the left (L) and right (R) terminal regions of the SFV and MRV genomes. For SFV: BarnHI fragments IT and 0 are shown together with subclones of the C and E BarnHI fragments. For MRV: subclones of the A and H BarnHI fragments are indicated. The shaded boxes illustrate the regions within the MRV genome (below) that have been derived by recombination from SFV sequences (above). Note that the lenqth of the MRV TIR is defined at the junction where SFV sequences stop at the left TIR (10 kb from the terminus). Bm, BarnHI; Bg, Bg/ll; BI, BgllrSm, Smal; Pv, Pvull.

an insertion/replacement of 6-7 kb of SFV sequences into the right TIR region of myxoma, and (2) a partial transposition of the modified right TIR to the left terminus, which resulted in 4 kb of SFV sequences being copied into the left TIR of MRV (Block et al., 1985). However, accurate mapping of the SFV/myxoma junction distal from the right terminus of MRV was complicated by the fact that MRV and myxoma DNA sequences near the right TIR could not be propagated in wild-type E. co/i (Block et a/., 1985). We have surveyed several cloning systems to permit the stable maintenance of these sequences and have found that E. co/i DB1256 (recA recBCsbcB) permits stable and efficient replication of these regions in pUC vectors. E. co/i DB1256 has also been used to clone palindromic poxviral sequences derived from SFV telomeres (DeLange et al., 1986) and appears to be a far more tolerant host for a variety of refractory eukaryotic DNA elements. In Fig. 2, the Smal fragments of SFV, MRV, and myxoma which encompass the TIR/unique sequence boundaries at their respective right TlRs have been aligned to the SFV growth factor gene. The SFV restriction fragment profile at the right end of the MRV Smal fragment can be clearly seen to make a defined transition into a myxoma profile at the left end. The DNA sequence of this transition point is considered later. Outer MRV junctions Both the left and right outermost junctions of MRV, namely those closest to the genome termini, were

mapped to a HindllllSall fragment (Fig. 3A, diagonal hatching) within the inverted fragments HE and AC (Block et a/., 1985; Fig. 1). The HindlIIISall fragment containing the junction at the left TIR was subcloned from the MRV BamHl fragment H into Ml 3 mpl9, and overlapping unidirectional deletions of this subclone were used to determine the sequence of this junction region. Since mapping experiments had indicated that the myxoma/SFV junctions were at similar positions in the inverted but otherwise similar HE and AC fragments of MRV (see Fig. l), an oligonucleotide primer (16-mer) was synthesized from the known SFV sequence approximately 80 bp internal to the junction found in HB. Sequencing from this primer with the full AC clone derived from the right TIR revealed that the junction in AC is indeed at precisely the same nucleotide position as that in HE at the left end. Figure 3B shows the DNA sequence at these two junctions and Fig. 3C illustrates the resulting amino acid changes that are produced in the MRV ORF T5 which spans the junction. The DNA homology in the aligned SFV and myxoma sequences in this region is greater than 90% suggesting that the junctions were created by homologous recombination. Translation of the MRV sequence shows not only that recombination took place within related ORFs of SFV and myxoma but also that the event preserved the precise reading frame in both copies of the TIR (Fig. 3C). Translation of the MRV sequences shows that a novel polypeptide is encoded wherein the N-terminal 232 amino acids of the 484 amino acid SFV ORF T5 are

232

UPTON ET AL. SFGF Probe

TIR 4 T9-R

SFV 13.0

12.0

11.0

KB

I

I 0.5

MRV

MYX

Sm

j

A I

81 I

Sm

A

BI

81 I

H

H I

C I

DHH I II

A

A I

D I

H I

KB

Sm I

A I

cc II

SI II

D

Sm I

4 TIR

of the SFV, MRV and myxoma genomes at the right TIR/unique sequence boundary. The Smal fragments of MRV and FIG. 2. Organization myxoma sequences are unstable in wild-type E. co/i, but have been propagated in recA recBC sbcB host (see Materials and Methods). The Shope fibroma growth factor (SFGF) and myxoma growth factor (MGF) genes are indicated as large striped arrows together with SFV ORF TQ-R. The hatched box denotes the SFGF probe used in Fig. 5. A, Avail; BI, Bg/l; C, C/al; D, Ddel; H, Hincll; Sm, Smal; SI, Sstl.

fused to the remaining C-terminal polypeptide sequence from a closely related myxoma ORF. Within the translated amino acid sequence available, there is greater than 90% homology between SFV ORF T5 and its myxoma counterpart.

Left internal junction The left junction distal from the terminus is contained within subclone C of the BarnHI H fragment of MRV (Fig. 1) and was sequenced from exonuclease Ill-generated deletions of the Sstl fragment of HC (Fig. 4A, diagonally hatched box). Figures 4B and 4C show the relevant DNA sequence and the translated polypeptides for both MRV and SFV. Once again, recombination is seen to have occurred between regions of highly homologous DNA (Fig. 4B), and in this case took place very close to the N-terminus of the SFV ORF T8. The only change in the resulting MRV polypeptide is the possible addition of an extra methionine at the N-terminus. Interestingly, the myxoma/MRV DNA sequences just upstream from ORF T8 are less highly conserved.

Right internal junction As indicated in Fig. 1, the internal junction of the SFV sequences transferred to the right end of MRV lies outside of the SFV TIR within the unique internal region of the SFV genome (the last nucleotide of the TIR is at nucleotide 12,397). Previously published mapping studies of this region of the MRV genome had been restricted to Southern blottings of DNA restriction fragments purified directly from viral DNA (Block et al.,

1985). This region of the genome is of considerable interest because both SFV and myxoma virus contain growth factor genes related to EGF, TGF-cu, and vaccinia growth factor (Chang et a/., 1987; Upton et a/., 1987b). The successful propagation of these relevant myxoma and MRV sequences in recombination-deficient E. co/i(see Materials and Methods) has permitted the accurate determination of this recombinant junction as well. To assess whether the SFV growth factor gene was transferred into the MRV genome, a SFGF probe (see Fig. 2) was used for Southern blotting analysis of the MRV and myxoma genomes (Fig. 5). Blots were washed using stringencies sufficiently low to allow the detection of the MGF gene with a SFGF probe and such that the two genes could be tentatively distinguished by the intensity of the signals. In each panel, SFV (lane 6) and MRV (lane 5) DNAs show single bands of similar intensity, whereas myxoma DNA (lane 4) shows a single but more faint band. These and other blots also confirm that the MGF gene was completely deleted and replaced by DNA coding for SFGF in the MRV genome and that only one copy of the SFGF gene is present in the recombinant MRV. A 1 .O-kb fragment encompassing the SFGF-derived gene was cloned from MRV and sequenced (results not shown) but no differences were observed between the MRV growth factor gene and the published SFV sequence (Chang et a/., 1987). The MRV fragment that was sequenced to examine the recombination junction in the region of the growth factor gene is shown in Fig. 6A. The junction is present within a previously undescribed SFV ORF denoted here

RECOMBINATION

JUNCTIONS

233

IN MRV

A ORFTS

4JlJ -1

OIlP T6

5961 MO4

I-

48w

lJ.70

7101

WV ED/CD I s I

& Bn 7

h\\\\\\\\\\\\\\\\\V

MRV H’IAC

I 1 8s

r s I

1

I

rrmmrmn

MYXDNA

In

SW DNA

-

0.2kb

B llRU

TCGCCGGTCGTTGTGTRTCRCGCRRTRGTTRRTGGGTGTRRRGT :: ..,*..,..,I,::::::::::::::: :: :::::::::: .......... CCGTCGGTCGTTGTGTRTCRCGCRRTRGlTGRTRGGTGTRRRGT SFU 5170 IWX >< SFU CGRGRGRGTTTTGTTCGTTTRTGCTTRTTTGTGTRRRGRTRRRRTCCRRCRCCTTTCGRT :;: :; :: :::;::::::: ::::: ;;::;;: I. ::::::: : *..a . . . . . ::::: CGRGRGRRTCTTGTTGGTTTRCGCTGRTCTGRGTRRRGRTRRRGTCCRRTRCCTTTCTRT 5272 TRRRGRCRCCRTRRCTGRTCTTRCGTT HRU . . . . . . . . . ..I............... TRRRGRCRCCRTRRCTGRTCTTRCGTT SFU 5300

C IRU-T5 SFU-T5

SFU DNA >< HYX DNA RFLRGQGUNSSQGFNRTFNFONLERKlSYGUFNRKULOFlFTQlSlNEQN ::: : : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..~~~.~.~~~ RFLRGQGUNSSQGFNRTFflFDNLERKlSYGUFNRKULDFlFTQlSUNQQD 2+0 230 200 210 220

IlRU-TS

SLDFTPlNYCUlHNDRRTFDYLLERGRDPNUUNFLGNSCLDLRULNGNK~flUHRLLRK :: ::::::::::: ii:::::::::: ::::: : : :::::::::::::::::::::: SLDFTPlNYCUlHNDRRTFDYLLEKGRNPNUUNFLGNSCLDLRULNGNK~NTLRLLRK SFU-T5 290 300 280 260 270 250

FIG. 3. Recombinant junctions of MRV which map closest to the termini. (A) SFV (ED/CD) and MRV (HE/AC) fragments are aligned to show shared and unique restriction sites. Diagonal hatching indicates presented sequence. Small arrows represent SFV ORFs and all nucleotide numbers refer to SFV DNA, numbered from the viral hairpin (Upton et a/., 1987a). Vertical hatching and solid lines indicate myxoma and SFV sequences, respectively, in representation of the MRV DNA and ORF (large arrow) at the junction. Bg, &/II; H, ffindlll; S, Sad. (B) MRV and SFV DNA sequences at the myxoma/SFV junction. Numbers refer to SFV sequence. Dashed lines between the sequences indicates the region of SFV DNA in both viruses; paired dots show identity between SFV sequence and the MRV DNA. (C)Alignment of SFV ORF T5 and the related MRV ORF in the junction region. Numbers refer to the amino acids in the SFV ORF and symbols refer to SFV amino acid sequence. The position of the recombinant junction determined by DNA sequencing is also shown above the amino acid sequences.

as ORF Tl 1-R (indicating it is the 1 1th major ORF from the terminus at the right end of the genome). The function of the presumptive SFV T-l 1 R polypeptide is unknown and no related proteins were found in the NBRF protein database. The DNA sequence presented in Fig. 6B shows this junction region and joins this sequence to that of the previously published SFV TIR, giving a total of 13,444 contiguous nucleotides at the right terminus of SFV. Translation of the junction sequences in MRV shows that the proper reading frame of SFV ORF Tl 1-R was

retained in the exchange and thus the resulting fusion polypeptide would consist of the N-terminal 14% from the myxoma analog linked to the remaining C-terminal SFV ORF Tl 1-R polypeptide sequence (Fig. 6C). Analysis

of the SFV Boerlage deletion

SFV strain Boerlage is of interest because of its ability to induce tumors of relatively larger mass than SFV Kasza (Strayer et al., 1984). A deletion of about 0.4 kb in SFV strain Boerlage was initially observed in compar-

234

UPTON ET AL.

A

BgSI

-11 SW DNA

C

I I 9946

II P MYX DNA

alIMRV

-

ORFla

0.2kb

B SFU >< flYX CGRCGTCGCRTRGTTTRCCCTTCRRRRRGRGTTTRTRCRGRGGRTRCGRCRTCRTCTCGR NRU -...................................... :::: I........ I * I - -. . . * : CGRCGTCGCRTRGTTTRCCCTTCRRRRRGRGTTTRTRCRRRGGRTRCGRCRTCGTT----SFU 9910 9996 CCCTCCGTRTCGTRTRTTRCTTTTTCRCCCTTTGTTRTCGTTCCCRTRRRCRRCRCGGTC I,, IIIOI .II.I I I..,,, :::::::::::: :: :: I, ., .I *I .I I. I. ., CCCCGRT;R;AAtRTRTTRTTTTTTCRCCCTT---~~R~RGT~T~RRT~~TRR~R~GGTR GTTGTRGGCTTGTGRTRGRTGRGRRRCRTRRRCGGTTT-GTTCGCCRCGRTCGCCGTGRG IIIII IO III,. :::::::::::::::::::: : III,II ,,,,,, ,,, ::::::: :::: GTTGTGRTTTTGTGRTRGRTGRGRRRCRTGRCCGGTTTTGTTTRCRRCTRTCGRTGTGTG 10080

C tlRU-TB SFU-TB

IWX ONR >< SFU DNA CLUER.QRUKK.YTIRRUENnSYPLYKLFLKGKLCDUElUREGKSlRRHR IO I I I88I I'......................... TULLLRLLKGEKIISYNRGTNSYPLYKLFLKGKLCDUEIUREGKSIRRHR 1 IO 20 30

FIG 4. Recombinant junction of MRV distal from the left terminus. Alignment BarnHI; Bg,Bg/ll;C, C/al;Sm. Smal;SI, Ssti.

isons of restriction digests of genomic DNA from strains Kasza and Boerlage (Upton and McFadden, 198613). The deletion, characterized by the absence of a Pstl site and the concomitant loss of the Pstl terminal fragment seen in strain Kasza, appeared to be 300500 bp from several restriction digests (data not shown). This result indicated that at least part of the deletion would produce changes in ORF T6 present within the SFV TIR. Since this is one of the SFV ORFs that was transferred to MRV it was of considerable interest to examine the DNA sequence of the deleted Boerlage form. A 5-kb BarnHI + EcoRl fragment spanning the deletion was isolated from Boerlage genomic DNA and cloned into pBR322. Multiple clones were assessed for any size heterogeneity within this region. No detectable forms other than the deleted variant were observed. Subsequently, a smaller SalI fragment was subcloned into M 13 mpl9 for DNA sequencing (Fig.

of SFV EE and MRV HC fragments.

For notation, see Fig. 3. Bm,

7A, diagonally hatched box). A comparison of the aligned Boerlage and Kasza DNA sequences shows 332 nucleotides, including the Pstl site at nucleotide 6580 in the Kasza genome, deleted from the ORF T6 of strain Boerlage. The exact position of the deletion is shown in Fig. 7A (black box); the numbers refer to the last nucleotides before the deletion. The effect of this deletion upon the putative polypeptide translated from ORF T6 is shown in Fig. 7B. The N-terminal 307 aa of Boerlage ORF T6 (508 aa in SFV) are unchanged with respect to Kasza, but the remaining 201 aa at the C-terminus are replaced by an unrelated 37 aa sequence produced by a change in reading frame. This difference in the ORF TG-encoded product is to date the only genomic difference observed between these two strains of SFV.

DISCUSSION The data presented here precisely define the SFV DNA sequences transferred to myxoma virus during

RECOMBINATION A 1

JUNCTIONS

3

4

5

6

235

C

a 2

IN MRV

1

23

456

1

2

3

4

5

6

?3.7

9.46

4.26

2.26 1 98

of ethidium FIG. 5. Southern blotting analysis of the SFV, MRV, and myxoma genomes with a SFGF probe. Lanes 1,2. and 3 are photographs bromide stained agarose gels of myxoma, MRV, and SFV genomic DNAs, respectively, digested with (A) BarnHI, (6) Sstl, and (C) Smal. Lanes 4, 5. and 6 are the resoective Southern blots orobed with a 594-bo Sau3A fragment encompassing the complete 240-bp SFGF coding sequence (see Fig. 2). Size markers (in kb) refer to X DNA digested with Hi~dlll.

the creation of the recombinant tumorigenic poxvirus MRV and also describe the nature of the presumptive fusion proteins expected at the SFV/myxoma junctions (Fig. 8). By combining our data with previously published data (Upton and McFadden, 1986a,b; Upton et a/., 1987a,b), the complete sequences of these SFV DNA regions are now available. This information confirms previous speculations that the genomic organization of SFV and myxoma is very similar and is quite unlike that of vaccinia virus (the prototype Orthopoxvirus) at the region within and close to the TlRs (Upton et al., 1987a,b). It also provides important clues as to the nature of the recombination events which created MRV. From examination of the DNA sequences at each SFV-myxoma junction in MRV it is probable that they were all formed by recombination between paired homologous regions of SFV and myxoma DNA. Measured within the 100 nucleotides adjacent to each MRV junction there is 87, 70, and 809/o identity between the SFV and myxoma-derived DNA sequences at the external, internal left, and right junctions, respectively. Such homologous recombination is known to occur at high frequency in the cytoplasm of poxvirusinfected cells, between viral genomes and plasmid DNAs, as evidenced by “marker rescue” of viral genes (Panicali and Paoletti, 1982; Mackett et al., 1982) by in viva intermolecular recombination between mutant genomes (reviewed in Dales and Pogo, 1982) by intramolecular recombination (Ball, 1987) and by recombination between transfected plasmids (DeLange et al., 1986; Evans et a/., 1988). The translation of MRV DNA

derived from myxoma reveals that there are related ORFs present in both SFV and myxoma virus at each of the positions examined: ORF T5 at the external junctions, ORFT8 at the left internal junction, and ORF Tl lR at the right internal junction (Fig. 8). These are present in addition to the other related ORFs previously noted, the epidermal growth factor-like genes (SFGF and MGF) and ORF T9-R (Upton et al., 1987a,b). Thus, it appears that the major part of the TlRs of SFV and myxoma virus comprise similarly organized and closely related ORFs. This organization has been strictly maintained in the recombinant virus MRV such that the reading frame of the ORF at each junction has been preserved, resulting in the fusion of myxoma and SFV ORFs T5 and Tl l-R and an unaltered ORF T8. Transcriptional mapping data also indicate that all of these ORFs are expressed as early genes, at least in SFV and MRV (Cabirac et a/., 1986; Macaulay et al., 1987). These findings suggest that these ORFs may encode essential polypeptides, although as yet no function is known for any these gene products. In addition to the changes to the genes just described, several other complete SFV ORFs are present in MRV. SFV ORFs T6 and T7 are present in each copy of the MRVTIR, whereas SFV ORFs T9-R and SFGF are only at the right terminus of MRV (Fig. 8). The latter are outside the TIR of MRV and form the right end boundary of the internal unique sequences since the end of the left internal junction also defines the end of the MRVTIRs. This arrangement of unequal lengths of SFV sequences present in the MRV TlRs has probably

236

UPTON ET AL.

A ORF TSR 11101 -112648

SN

C””

1

,292o

I:409

ORFTII-R

CC

C”

I

I I

-11 SN

DNA

I

Em MYX

DNA

hfRVORFT1l.R -

0.2 kb

B 13169 GCCRGTGTGGCCRRTTTRRCGGRCGGTGRTGCGTCCRGTRRCGCRRRTCC SFU TARCGCCTTTTTRRTGTTCTCTRTGTTGTRCGRTRRRRTGTCCCGTTCCR SFU TGRTRTCRCRCRRCRCGTCGTRGTTTTTCGCRTRCGTRTTGTTGRTRRRR SFU SFU >< HYX TTGCRRCRRTTRRCCRRCTGRCRTRRTRGRTCTRTTTCTGTRCRCTCCGT SFU I. a.. ******..... . a.. I I * ** I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .**.. TTGCRRCRRTTRRCCRRCTGRCRTRRTRGRTCCRTTTCCGTRCRCTCCGT IlRU TRTRTCCCCRCCGTTTRGRTRRGTGTRGRCRRCCTCCTTTRGRCGRGRCR SFU ::: :: :: ::::: :::::::: .I..... : :::: :::::: II...., TRTRTCCRCGTCGTTCRGRTRRTCGTRTRCGGCCGTCTTTRRRCGRGRCR HRU

TRGTRGRCGRTRRRRRCTRRTRGCCGCTTGRRCCGR SFU : ::::::::::: :::!: II :::::::::: ~cRTcGRCGRTRRRRRTTRRTRR~~R~TTGRRNGRMRU

C 1 10 20 30 10 50 NSRLKEUUYT YLNGGDITEC TEIDLLCQLU NCCNFINNTY RKNYDULCOI SFU Tll-R :: i ;; ;; . . . . . . . . . . . . . . . . . . . . . . . . . . . ::::: :: ::: IlflSRLKTRUYD YLNDUDITEC TEflDLLCQLU NCCNFINNTY RKNYDULCDI HRU Tll-R 60 70 60 90 100 NERDILSYNI ENIKKRLGFR LLDRSPSUKL RTLRLLSIIL KKLNKIRHTE SFU Tll-R 110 120 130 140 150 RCUFSDUIDG ITREENKUIG FIQEKVKVNT TYYNKRSKLP U'fLSTRllURT

SFU Tll-R

I60 LIUYGUIKUR RGT

SFU Tll-R

FIG. 6. Recombinant junction of MRV distal from the right terminus, Alignment of SFV Cc and CA fragments with MRV AA. For notation, see Fig. 3. Bg, &/II; BI, Bg/l; C, C/al. The complete amino acid sequence of SFV ORF Tl 1-R is presented together with the novel DNA sequence of this region in MRV. The right viral termini are oriented here toward the left to facilitate comparison of the different ORFs.

arisen by the recombination of a 7978-bp SFV DNA sequence into the recipient myxoma genome at the right terminus followed by the copying of 4674 bp of this region to the left end of the viral genome. From the alignment of the available myxoma and SFV DNA and polypeptide sequences it is obvious that the terminal

regions of these viruses are organized in a very similar fashion and that relatively modest changes must be responsible for the differing biological phenotypes of MRV and myxoma virus. It has not yet been determined whether the presence of SFV or SFV/myxoma fusion ORFs or the altered regulation of one of these polypep-

RECOMBINATION

SFV

SFV Kasza

I

IN MRV

237

]I02

ED

t B6 Bm

JUNCTIONS

5786

6743

s

s

I B6 s

S

E

W

I 65d *

SW Keus 6215

I

SFV

Boerlrge

ORF T6

6608

I

NSS Bm

S

s

I

s

b

S SW Boalagc

E

ORFT6

-

0.5 kb

B K-16 B-T6

1 MNRSLLSFLE , =ssz==Ls==s

NGTllSOUTlI ii==I=l==*l

RGTSKFTRHR LILSUHSDYF =11=11111. 11m*1=.11*

YRLFNGGFEU PDTUTLDTDO ..=111==1= 11==11==11

B-T6 6,

K-T6

61 UULREULTWI =i3==.=1===

YTGCSNURGS TUENIQSUII I========= 11111a==11

LROVLGIHKL 11*111*111

UKECIDYIUT 1.11=11*1.

QUEPSNCIAI 1=11*s=111

K-T6 B-T6

121 FQFRDTVHIE 12, ===.==z.L=

DLKRILHIIFF LI==LZIIII

FRKLDTHEAL ==m11.1111

IULRESHNIU .=.1.1=111

GRRFUIKSIL 11-111.=1=

K-T6 B-T6

181 DWURKEPKHI KQIKRLSERI ,B, sa=,x==zs== IPIII=I=S=

GDDUEKDTIY EUYSRYHEEI KDTPHTPPLS HNCIITIDRR =s===ss=.= =v=..s=m=. . ..=.s..== ====m==*==

K-T6 B-T6

241 RYIRRYSPDH 24, II,ZSIII=IP

RUDIGDRFTU UCflDNULYCL GGTLNGUPTS DULGYDLLTG =.==v=.=m. =.====.=s. ~..t..=.s. s====w=.==

K-T6 B-T6

301 DCTWlPDtlRQ HRRNRSRCSU NGCIYRIGGI 301 =======IlL LGRTHERTLR HESRLPIRRR

K-T6

361 ERRTUCYKNE LUURGGTIDL

K-T6

421 CLGGRTNEHS OTNHUYRYDD UCCUUERIED IlTERRRNPlC

K-T6

481 NGUKUHRUND USLVTGCTNT RYPFFIQR.

MNTRIRHUG ~========a

PEILLSSRER =111==11*.

DEEGRLIPNU EYUTPSNDDU YYSRYLYPNU ULRHGTHRGH DGGR*

YPTTFTNRUN RLTDDGUUKil RPLPIlPRSGR SIVJUYKGRLY CUYNNTLYUL GGRTNSRESY

FIG. 7. Position and sequence of a deletion in ORF T6 of SFV strain Boerlage. (A) DNA fragments sequenced are shown as diagonally hatched boxes. Region deleted from strain Boerlage is represented as a black box. All numbers refer to nucleotides in SFV strain Kasza. See Fig. 1 for the position of fragment ED within the TIR of SFV strain Kasza. Bg, Bg/ll; Bm, BarnHI; E, EcoRI; P, Pstl; S, SW. (B) Alignment of the amino acid sequences from ORF T6 from SFV strains Kasza (K-T6) and Boerlage (B-T6). The full sequence of Kasza ORF T6 is presented; dashed lines show the N-terminal 307 aa shared with Boerlage ORF T6.

tides is responsible for the production of SFV-like tumors in the early stages of MRV infection. However, one prime candidate for this effect is the replacement in MRV of the MGF gene by the intact SFGF gene. Viral constructs in which only the growth factor genes have been exchanged between these viruses are required to definitively settle this issue (in progress). It is also important to consider that since the SFV-derived sequences in MRV actually replace the original myxoma sequences, it may be that the loss of the homologous

myxoma sequences in MRV is involved in the change in phenotype. SFV strain Boerlage has been observed to induce larger tumors than strain Kasza although such tumors were found to regress in a comparable time frame (Strayer et al., 1984). The function of the SFV ORF T6 polypeptide is at present unknown and the stability of the novel truncated protein produced from the altered gene in Boerlage has not yet been examined. Such results and the fact that this gene has also been trans-

238

UPTON ET AL. I

2

1

41

6I

I

8

12

10

Terminal lnvcrtcd Rcpcrt

Di :

t Em I

R(

IT

Bm B9 II

1011

B9 I

BP

CD

I

CB

Sm Sm I 1

IcElcFl

14 1

I

16 (kb) 1

BIB9 II

cc

I

T9-R

SFGF Til-R

B9 CA

CG

c-(-CICIU Tl

u-u-

T2 T3A T3C T4

Bocrlrgr

-4-l-

T5

dclrtion

T6

T7

TB

m

FIG. 8. Organization of the right terminal region of SFV. Small arrows indicate major ORFs. The stippled boxes illustrate the regions within the MRV genome (L, left; R, right) that have been derived from SFV sequences. SFV sequences deleted in strain Boerlage are shown by a box with vertical stripes. Restriction sites are as in Fig. 1.

ferred to MRV suggest that ORF T6 of SFV is also a candidate for involvement in the induction of tumors by this virus. This hypothesis is made especially interesting but considerably more complex by the fact that ORF T6 is one of three related ORFs within or close to the TlRs of SFV strain Kasza (Upton and McFadden, 198613). ORF T6 has 59.8Ob identity with ORF T9-R which spans the TIR junction at the right terminus and 33.9% identity with ORF T8 which is contained entirely within the TIR. All these SFV ORFs are also within the SFV DNA sequences transferred to MRV. Since SFV ORFs T6, T8, and T9-R are so closely related it is possible that each one influences tumor production to a greater or lesser degree. The data presented in this paper conclude the analysis of the SFV-derived DNA sequences that are present in the recombinant virus MRV. While it is not yet understood what precisely determines the altered phenotype of MRV, such questions may now be addressed more readily. Both the SFGF gene and the family of ORFs related to T6 (which contains the deletion in strain Boerlage) are currently being examined individually to assess their role in virus-induced tumorigenesis. ACKNOWLEDGMENTS This work was supported by the Alberta Heritage Foundation for Medical Research (AHFMR) and by an operating grant from the National Cancer Institute of Canada. G.M. is an AHFMR scholar. We are grateful to A. Wills for excellent technical assistance.

REFERENCES BALL, L. A. (1987). High frequency

DNA. J. Viral. 81, 1788-l

recombination

in vaccinia

virus

795.

S., and Moss, B. (1982). Incomplete base-paired flip-flop terminal loops link the two DNA strands of the vaccinia virus genome into one uninterrupted polynucleotide chain. Cell 28, 3 15-324.

BAROUDY, B. M., VENKATESSAN,

BLOCK, W., UPTON, C., and MCFADDEN, G. (1985). Tumorigenic poxviruses: Genomic organization of malignant rabbit virus, a recombinant between Shope fibroma virus and myxoma virus. Virology 140,113-124. BLOMQUIST, M. C., HUNT, L. T., and BARKER,W. C. (1984). Vaccinia virus 19K protein: Relationship to several mammalian proteins, including two growth factors. Proc. Natl. Acad. Sci. USA 81, 73637367. BROWN, J. P.. TWARDZIK, D. R., MARQUARDT, H., and TODARO, G. J. (1985). Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor-a. Nature (London)313,491-492. BULLER,R. M. L., CHAKRABARTI,S., COOPER,J. A., TWARDZIK,D. R., and Moss, B. (1988). Deletion of the vaccinia virus growth factor gene reduces virus virulence. J. Viral. 62, 866-874. CAEIRAC, G. F., MULLOY, J. J., STRAYER,D. S., SELL, S., and LEIBOWIT~, J. L. (1986). Transcriptional mapping of early RNA from regions of the Shope fibroma and malignant rabbit fibroma virus genomes. Virology 153, 53-69. CHANG, W. C., UPTON, C., Hu, S., PURCHIO,A. F., and MCFADDEN, G. (1987). The genome of Shope fibroma virus, a tumorigenic poxvirus, contains a growth factor gene with sequence similarity to those encoding epidermal growth factor and transforming growth factor alpha. Mol. Cell. Biol. 7, 535-540. DALES, S.. and POGO, B. G. T. (1982). “The Biology of Poxviruses.” Springer-Verlag, New York. DELANGE. A. M., MACAULAY, C., BLOCK, W., MUELLER,T., and MCFADDEN, G. (1984). Tumorigenic poxviruses: construcfion of the composite physical map of the Shope fibroma virus genome. J. Virol.50,408-416. DERANGE, A. M., REDDY, M., SCFIABA,D., UPTON, C., and MCFADDEN, G. (1986). Replication and resolution of cloned poxvirus telomeres in vivo generates linear minichromosomes with intact viral hairpin termini. 1. Viral. 59,249-259. EVANS, D. H., STUART, D., and MCFADDEN, G. (1988). High levels of genetic recombination among co-transfected plasmid DNAs in poxvirus-infected mammalian cells. 1. V&o/. 62, 367-375. FENNER, F. (1983). Biological control, as exemplified by qallpox eradication and myxomatosis. Proc. R. Sot. London B 218, 259285. FENNER,F., and MYERS, K. (1978). Myxoma virus and myxomatosis in retrospect: The first quarter century of a new disease. ln “Viruses and Environment.” (E. Kurstak and K. Maramorosch, Eds.), pp. 539-570. Academic Press, New York.

RECOMBINATION FENNER, F., and RATCLIFFE,F. N. (1965). “Myxomatosis.” Cambridge Univ. Press, London. HENIKOFF,S. (1984). Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing Gene 28, 351359. MACAULAY, C., UPTON, C., and MCFADDEN, G. (1987). Tumorigenic poxviruses: Transcriptional mapping of the terminal inverted repeats of Shope fibroma virus. Virology 158, 381-393. MACKE~T, M., SMITH, G. L., and Moss, B. (1982). Vaccinia virus: A selectable eukaryotic cloning and expression vector. froc. Nat/. Acad. Sci. USA 79,7415-7419. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MCFADDEN, G. (1988). Poxviruses of rabbits. In “Virus Diseases in Laboratoty and Captive Animals” (G. Darai, Ed.), pp 37-61. Martnus Nijhoff, Boston. MESSING, J. (1983). New Ml3 vectors for cloning. In “Methods in Enzymology” (R. Wu, L. Grossman, and K. Moldave, Eds.), Vol. 101, pp. 20-77. Academic Press, New York. Moss, B. (1985). Replication of poxviruses. In “Virology” (B. N. Fields, Ed.), pp. 685-703. Raven Press, New York. PANICALI, D., and PAOLETI, E. (1982). Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc. Nat/. Acad. Sci. USA 79,4927-4931. SANGER, F., COULSON, A. R., BARELL, B. G., SMITH, A. J. H., and ROE, B. A. (1980). Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143, 16 l-l 78. STRAYER, D. S., CABIRAC, G., SELL, S., and LEIBOWITZ,1. L. (1983a). Malignant rabbit fibroma virus: Observations on the culture and histopathologic characteristics of a new virus-induced rabbit tumor.J. Nat/. Cancer/W. 71, 91-104. STRAYER,D. S., and SELL, S. (1983). lmmunohistology of malignant rabbit fibroma virus-A comparative study with rabbit myxoma virus. 1. Nat/. Cancer inst. 71, 105-l 16. STRAYER,D. S., SELL, S., SKALETSKY,E., and LEIBOWITZ,J. L. (1983b). Immunologic dysfunction during viral oncogenesis. I, Nonspecific

JUNCTIONS

IN MRV

239

immunosuppression caused by malignant rabbit fibroma virus. J. Immunol. 131,2595-2600. STRAYER,D. S., SKALETSKY,E., CABIRAC, G. F., SHARP, P. A., CORBEIL, L. B., SELL, S., and LEIBOWI-~~,1. L. (1983~). Malignant rabbit fibroma virus causes secondary immunosuppression in rabbits. J. Immunol. 130,399-404. STRAYER, D. S., SKALETSKY,E., and LEIBOWITZ.J. L. (1985). In vitro growth of two related leporipoxviruses in lymphoid cells. Virology 145,330-334. STRAYER,D. S., SKALETSKY,E., and LEIBOWITZ,J. L. (1987). Growth of malignant rabbit fibroma virus In lymphoid cells. virology 158, 147-157. STRAYER.D. S., SKALETSKY,E.. and SELL, S. (1984). Strain differences in Shope fibroma virus: An immunologic study. Amer. 1. fatho/. 160,342-358. STROOBANT, P., RICE A. P., GULLICK, W. I.. CHENG, D. J., KERR, I, M., and WATERFIELD,M. D. (1985). Purification and characterization of vaccinia virus growth factor. Cell42, 383-393. UPTON, C., DE~NGE, A. M., and MCFADDEN, G. (1987a). Tumorigenic poxviruses: Genomic organization and DNA sequence of the telomerit region of the Shope fibroma virus genome. Virology 160, 20-30. UPTON, C., MACEN, J. L., and MCFADDEN, G. (1987b). Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha./. Viro/ 61, 1271-1275. UPTON, C., and MCFADDEN, G. (1986a). DNAsequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species. Mol. Cell. i3iol. 6, 265-276. UPTON, C., and MCFADDEN, G. (1986b). Tumorigenic poxviruses: Analysis of viral DNA sequences implicated in the tumorigenicity of Shope fibroma virus and malignant rabbit virus. Virology 152, 308-321. VIERA, J., and MESSING, I. (1982). The pUC plasmids and Ml3 mp7derived system for insertional mutagenesis and sequencing with universal primers. Gene 19, 259-268. WILLS, A., DERANGE,A. M., GREGSON,C., MACAULAY, C.. and MCFADDEN, G. (1983). Physical characterization and molecular cloning of the Shope fibroma virus DNA genome. virology 130,403-414.