Sequence and analysis of a portion of the genomes of shope fibroma virus and malignant rabbit fibroma virus that is important for viral replication in lymphocytes

VIROLOGY

185, 585-595

(1991)

Sequence and Analysis of a Portion of the Genomes of Shope Fibroma Virus and Malignant Rabbit Fibroma Virus That Is Important for Viral Replication in Lymphocytes’ DAVID S. STRAYER,* Department

of Pathology

and Laboratory

Medicine, Received

HENRY

H. JERNG,

University October

of Texas

4, 1990;

KATHLEEN

AND Health

accepted

Science

August

Center

O’CONNOR3 at Houston,

Houston,

Texas

77030

9, 199 1

The 10.7-kb BamHl “C” restriction fragment of malignant rabbit fibroma virus (MV) contains genes that are important for its immunosuppressive activity. When this fragment is transferred to a related avirulent leporipoxvirus, Shope fibroma virus (SFV), recombinant viruses show clinical features characteristic of MV: they replicate in lymphocytes and alter immune function in vitro, induce disseminated tumors in recipient rabbits, and are immunosuppressivein vivo. The 10.7-kb BarnHI “C” restriction fragment of MV was sequenced in its entirety. Its DNA sequence and the 14 ORF’s derived from analyzing this sequence are discussed. Analysis of known open reading frames to which the ORF’s from MV’s Barn “C” fragment show homology permits us to identify some MV ORF’s showing high degrees of similarity to known and postulated proteins produced by vaccinia virus. Functions for some of these vaccinia proteins are known, while functions for others are hypothetical or unknown. Further analysis of genetic determinants of MV’s virulence has indicated that two overlapping restriction subfragments of the BarnHI “C” fragment can transfer MV’s virulent behavior to SFV. The 0.7-kb region in which these two subfragments overlap includes the C-terminus of MV or-f C-7 and the N terminus of MV or-f C-8. These correspond to the C- and N-termini, respectively, of SFV or-f’s D-9 and D-10 and to vaccinia orf’s D-6 (early transcription factor) and D-7 (subunit of RNA polymerase). We sequenced the region of SFV’s BarnHI “D” fragment in this area and illustrate here the comparative sequences of this portion of SFV’s genome and orfs. On the basis of comparisons between MV, SFV, and vaccinia in this area we discuss the potential significance of these observations. 0 1991 Academic Press. Inc.

INTRODUCTION

phocytes but does not kill them (Strayer et a/., 1985, 1987). It alters immune function by inhibiting lymphocyte activation and inducing T cells to elaborate a nonspecific immunosuppressive lymphokine (Strayer et al., 1988a). Shope fibroma virus (SFV) is a related leporipoxvirus. SFV-induced tumors do not disseminate and are not associated with immunologic dysfunction (Strayer et al. 1984). SFV does not replicate in lymphocytes and cannot be recovered systemically from infected animals (Strayer eta/., 1985). Rather, infected rabbits eliminate tumor within 3 weeks and are thereafter immune to subsequent MV or SFV infection. MV is a recombinant between SFV and myxoma virus (MYX) (Blocker a/., 1985) the recombination sites being located in the unique sequences near the terminal inverted repeats (TIR) (Upton and McFadden, 1986; Upton et a/., 1988). MV thus derives most of its TIR and some lateral unique sequences from SFV. Most of its central unique sequences come from MYX. When analyzed by Southern hybridization the majority of MV and SFV genomes cross-hybridize under conditions of low stringency. We have utilized the genetic relatedness of SFV and MV to study genetic factors determining the virulence of MV. A 10.7-kb BarnHI restriction fragment (the “C” fragment) that lies approximately 72 kb from the left hand end of the MV genome transfers from MV to SFV

Malignant rabbit fibroma virus (MV)4 is a leporipoxvirus that causes a malignant tumor syndrome in rabbits. MV-induced disease is characterized by widespread tumors, severe Gram negative infection, and inevitable death (Strayer eT al., 1983a-c). After intradermal inoculation in the thigh, tumors appear on the ears, face, and in the subcutaneous soft tissues. Mucous membranes overlying these tumor proliferate and undergo squamous metaplasia. Virus is recovered from internal organs of infected rabbits. Malignant fibromatosis involves severe immunologic dysfunction. MV infects and replicates in B and T lymSequence data from this article have been deposited with the EMBUGenBank Data Libraries under Accession Nos. M32743 (MV Sequence) and M74532 (SFV Sequence). ’ Supported by USPHS Grant CA44800. ’ To whom correspondence and reprint requests should be addressed at Department of Pathology, University of Texas, 643 1 Fannin Street, Houston, TX 77030. ’ Present address: Dept. of Biology, Case Western Reserve University, Cleveland, OH. 4Abbreviations used are: bp, base pair; “C” fragment, BamHl “C” fragment of MV; CA, carbonic anhydrase; “D” fragment, Hindlll “D” fragment of vaccinia; ETF, early transcription factor; kb, kilobases; kDa, kilodaltons; MV, malignant rabbit fibroma virus; MYX, rabbit myxoma virus; ORF, open reading frame; SFV. Shope fibroma virus; TIR. terminal inverted repeat; VETF, vaccinia early transcription factor. 585

0042-6822/91 CopyrIght All rights

$3.00

0 1991 by Academic Press, Inc. of reproduction in any form resewed.

STRAYER,

586

JERNG,

the abilities to replicate in lymphocytes, induce immune dysfunction in vitro and in viva, and produce disseminated infection (Strayer et al., 1988b; 1990). Further, we have found that this function may be mediated by one or both of two over-lapping restriction subfragments from the right5-hand side of the “C” fragment: a 3.6-kb Ndel fragment and a 1.9-kb Hincll fragment. Recombinant viruses generated using these smaller restriction fragments replicate in lymphocytes and alter their function in vitro and in vivo (Heard et a/., 1990). The Barn “C” fragment of MV is therefore important in the pathogenesis of MV induced malignant disease: one or more of its genes determines the ability of virus to replicate in lymphocytes and suppress their function and to cause disseminated tumors and secondary infection in vivo. We report here the sequence and open reading frame of this fragment and also of the relevant portion of SFV. We analyze the relatedness of these or-f’s to those of vaccinia. Homologies between MV and other genomes already reported, in terms of both DNA and protein structure, shed light on the functions of this region of the MV genome and suggest mechanisms by which this region may function to mediate MV’s virulence. METHODS Sequencing

strategy

Using the BamHl “C” restriction fragment cloned MV DNA, we prepared a restriction map using EcoRI, Hincll, Xhol, Ndel, andXbal. (This map is shown in Fig. 1.) Gel-purified restriction fragments (except Hincll) were cloned into pGEM vectors (pGEMblue, 3, 4, 5, and 7; Promega, Inc.), and both strands were sequenced. Hincll fragments were shotgun cloned into the Smal site of pGEM4. Using pGEM SP6 and T7 promoter sequences as templates for oligonucleotide primers, we generally sequenced 300 to 400 bp into the inserts from either end. Complementary 18-mer oligonucleotide primers 20 to 40 bases from the 3’ end of the known sequence were made for both strands (Milligen/Biosearch Cyclone) and used to prime sequencing reactions. Overlapping restriction fragments provided sequences and alignments at the ends of abutting restriction fragments. All sequencing was performed at least twice, usually on both strands. In the event of discrepancy, further resolution of sequences in question was performed. Using cloned BamHl restriction fragments from SFV, ’ We had previously referred to this portion as the left-hand side. However, for uniformity in convention with the orientation reported for vaccinia virus strain WR, we have reversed polarity compared to our previous publications describing MV genome organization.

AND

O’CONNOR

we identified the 13.1 -kb SFV Barn “D” fragment as corresponding to MV’s BamHl “C” fragment. By selecting for restriction subfragments that hybridized with the 3.6-kb Ndel and 1.9-kb Hincll fragments, we identified the corresponding region of SFV’s BamHl “D” fragment and sequenced it similarly. Sequencing

All sequencing was done with Sequenase (U.S. Biochemical Corp.), following the package insert instructions. Procedures for dideoxynucleotide sequencing have been well described (Maniatis et a/., 1982). We had tried sequencing using Sequenase II but were unable to obtain reproducible sequencing reactions in this double stranded DNA system. We compiled and analyzed DNA sequences using DNA Inspector Ile (Textco, West Lebanon, New Hampshire) on a Macintosh II computer (Apple Corp.). In the analyses reported here, the term “open reading frame” (orf) refers only to open reading frames beginning with a potential initiation codon. Database

searches

Sequences from GenBank were searched to determine homology between DNA sequences from MV and known DNA sequences. In this search, the Molecular Biology Information Resource of the Department of Cell Biology, Baylor College of Medicine, was used, as adapted for a VAX785 (Digital Equipment Corp.). All searches and homologies are reported as a certain number of standard deviations from the mean. An SD value of a3 generally indicates possible homology. SD ~=6 indicates probable homology (Altschul and Erickson, 1986a,b; Lawrence et al., 1986; Doolittle, 1981). Sequences of open reading frames were compared to reported protein sequences in the Protein Identification Resource at the National Biomedical Research Foundation at Georgetown University. RESULTS Restriction

map of the Barn “C”

fragment

A restriction map of the Barn “C” fragment of MV used for cloning restriction subfragments into sequencing vectors is shown in Fig. 1. All but the smallest two of the Hincll-Hincll fragments were cloned into pGEM plasmids, as were the EcoRI-BarnHI fragments, the Xhol-BarnHI fragments, A/de1 fragments, and the Xhol-Xhol fragment. Sequence

of nucleotides

in the Barn “C”

fragment

The sequence of bases from the MV Barn “C” fragment is shown in Fig. 2. Its length is 10,706 bases.

DETERMINANTS

OF

MALIGNANT

FIBROMA

VIRUS

REPLICATION

IN LYMPHOCYTES

587

Barn

HI

Xho

I

stu Xba

5 I

Sph

I

Bgl

II

Hint

II

EC0

I 0

I 1

I 2

I 3

I 4

I

I

I

I

I

I

I

5

6

7

a

9

10

11

RI

KB

FIG. 1. Restriction map of BamHl “C” fragment of MV. The “C” fragment of the BarnHI digest of MV was cloned into pBR322 and digested with the various enzymes indicated. Individual restriction fragments were isolated from these digests and correlated with each other so as to establish the order in which these fragments appear in the larger BarnHI fragment. The order of these fragments, which was used in generating the sequence (see Fig. 2) was also confirmed after the sequence was completed.

Open reading frames Analysis of the Barn “C” fragment for open reading frames yields 14 ORF’s, labeled C-l to C-l 4 of over 75 amino acids completely contained within the fragment. ORF’s are shown, compared to a restriction map of the Hincll and Ndel fragments, in Fig. 3. There is some overlap among the smaller ORF’s in both directions and in different frames. ORF 6 is enclosed within ORF 5, for example. Overlap among larger ORF’s (>lOO amino acids) is very slight. ORF’s C-l, -3, -4, -5, -7, -8, -10, -1 1, and -13 are transcribed from left to right. The others are transcribed from right to left. Because of this it is possible that the beginning of C-l is to the left of the Barn “C” fragment and that the beginning of C-l 4 is to the right of the “C” fragment. They are illustrated in Fig. 3. As with vaccinia, MV genes tend to be arranged in tandem (Niles et a/., 1986). Adjacent open reading frames read in the same direction usually overlap by one or two bases. Thus, C-3 ends at 1798 and C-4 begins at 1797; C-5 ends at 4845 and C-7 begins at 4844. Larger overlaps between reading frames are only seen among or-f’s entirely contained within other or-f’s or when two sequential open reading frames are read in opposite directions (e.g., C-l 2 and C-l 4). This arrangement is like that observed in this portion of the WR strain of vaccinia virus (Niles et a/., 1986). Small (~50 bases) gaps are present in some areas. There are no stretches of over 50 bases of DNA that do not belong to an open reading frame. The organization of MV DNA therefore resembles that of other known poxviruses (Moss, 1990).

Homologies in nucleic acid sequences Search of GenBank for nucleic acid homology with DNA from all sources shows that MV’s “C” fragment is homologous to the HindIll “D” fragment of vaccinia strain WR. In addition “C” fragment DNA shows a high probability of homology to genes encoding mammalian carbonic anhydrases (SD = 6. l-l 3.2) heat shock proteins (SD = 7.2-8.1) fibroblast growth factor (SD = 7.2) and rabbit Ig genes (SD = 7.4). Homology

among open reading frames

Among the 14 ORF’s, all except one (C-l 2) strongly resemble open reading frames described in other systems, especially vaccinia. ORF homologies between MV’s “C” fragment and vaccinia’s HindIll “D” fragment are in order and parallel (see Table 1). Except for or-f C-9, which shows very strong similarity to various mammalian carbonic anhydrases, no significant similarities are observed between open reading frames in this pat-t of MV’s genome and nonpoxviral proteins. Little is known about some of these vaccinia proteins. Functions for others are known. These results are summarized in Table 1. Only C-l 2, an or-f of 113 amino acids contained within C-14 and read right to left, shows no discernible homology to reported vaccinia open reading frames. Comparison of MV and SFV sequences in the region of the genome responsible for transferring from MV to SFV virulent clinical behavior The SFV BamHl “D” fragment is 13.1 kb and corresponds to MV’s BamHl “C” fragment. We have com-

588

STRAYER, JERNG, AND O’CONNOR 100 200

GGATCCCGACAAAGAAGCCATTACTCGTTGCATAGAACGATACAATTCGT ACGATTCGGTCCACGACGTACGTATCTAGCGTACGAGAAGTGTTCTTCTT I --t orf C-l beglns ATCCCAAACATTACACCACGGTG&$@ATAATCTAGCCGAGTTAACGGCG GTTMCGGATAAAAAGACCTTTGTGATACATAAGAACTTACCGAGCAGCG AATCCTTCGTCTATGTCTAGACCCATGCAAGAGTACATCATCAAGAGGGC TACATTTCGACACGGTCGTGGAGCGCAGTAAAAGGTTTATMCAGGCGTC Ie AGAAGCGTTAAAGTACGAAGGTACGGATATAGGCGACCTASCGCTACT GTCACGTTTCCCGMCCTAAACGAGTTAAAAATGTTTAATAACACGGTTA GGGGCMTTTCACGTATCGACTCGTACACGGTTTTGCTGGGAAACAACAT TGGTTTTGTTATCAAATCTAGCCGCGGAAGAATACACGTGGATGTTACGT

TGAACTCAGGAATAAAGTCAGTACTACAAGTTTGACTACATACAGGAA CGGCAAGTTTGACCTAGTGGATTGGCAGTTCGCCATTCATTATTCGTTTC

CGACTATTTGTTTACGTACGTACGCATGCATCGAGATAGCTGCTACTTTT AATTATATCCACTTCGGTGACGCTATGAAAAAGATAGCCAAGGCGGTCGT TGCCGTTAGTAATCGATTACGTAATGCCATGTGCCTTGTGTAAAAAGAAA ATATAAAGCGGGCAATCCTCATAGTTGTAAGGTTAAGACGATTCTGTTGG ATCCATCTMCGGATAGAGGAGAGTACATCGTATGGTTAAAGAACGCCTG AAGATCAACTTCCGACGGAATACGTACCGGATATACTGTGTCCTAGAAAA

CGTTAACTAAGCGTTTAGACGATACCATGTCGGAAGCGTTATGGGAGCCT TAACGMATTATTAATTTTMAGATCTAGACGTAACAAATTTTACGACCG ACGCACAAACACCATCATATGATATCCATCGGAAACGGTATGTTGAAATT MGGGAACAAATTATTTACGATCTCTCAAATCATTATGGACTCTMCGTC GAAATTTAACACGGACGACCCCATTATTACAAAACTGGTATTAAACATGA CGTAAAGTTATAGAAAATAATTTAAAAGATATGTTGGTAGATGTCACTGA I e orf C-6 ends TATCAAACGGTACGTTTTACAGCGGaCGMTCGAAAGATTTTATATGT CTCGGAGGAAATGAAAGMCTCGTGTCCATTATAAACGATATACAACCGT AGTTGTTTATGCGGTACGACGRAACAGTGTGTATTACGTTCTTCTTCGGAGA GCAGTTTATTTATAGAAACGGGACAGACGATTCTTACGGAGGTGATGGAC TTGTAGCGAACTTCCAGATTTTGCGTGTAGTACCAGTAAACGTATTCGTG

3200 3300 3400 3500 3600 3700

TTTTCCAATCGTATTAATAATAAAAATCACGCTTCTATCATTATCGATAC TATCATTGGTTCGTTTTAGAACTCATTTTGCACAAACGGCGACCAATAM GTTGGATATGMMTACAAAAGMTTATTACAGGTACGCGTTTCTMACT TCTACGCCAGAAAACGTACCAGACTTCGCTTTCCAGTTAAAGGTGGAGTC CAAAAATAGGATACGTAATGAGGATAATCTCGTCGTGTTGCCGGCTCCT CGATATAGAAAGTTTCATAACGAGACATAAAAAGTTTGCGAATGTTAAGG

4300

300 TCGGGTGGGAAGGTGTTGATCACGACGATGGACGGGGACTTATTGAGTCA 400 AAAACTACATGTCCGTAGAGAAGATCCACGAGGATCAGATTTTAGTGTAT 500 GAACTTGACCAAGATATTTTCGGMTACGGGTTCGMTTAATCGACTGCG TCAAAAATGGAAGMCGTAAATCCACCAAGAACTTCTTCGAACTAAACAG 600 orf C-2 ends orf C-l ends+ 700 ACATCGTATACGTCTTCTCCAAGAGGTAAGSTAATACGGTACGGGCGC CCGGGTTAGACGGTTCCGAATCGTCTGTCGATATAGCCACCGTGTAGTCC 800 GTCTATMTCGCCTCGTTGTGTTTGTACAGATACTTGACCACCCCAAACA 900 1000 ATCTTGTAGCGCCCTTTTTCTAACACAATAAATCGTTCGTTGTATATCTT orf C-2 beglnsrl I + orf C-3 beglns 1100 TTTTATATCGTTAGGACATACTATTCTATTGTTTTGTAGCAGGTCCGTTA GTTTCTTGATATCGAACTCC&CGAGTA&AATACGACTACGATACGACA TAAGCGAACGCAACGMCATATMTCGATAAACTATGTAGCCGTCTACCA 1200 CGATGATATACGCATCGACGATTTAAUGAAAATAAAACATTTTTGTTAT 1300 CCCATGTGTTTTTATTGCGACTATATAACGTCTCCGTCTCCGGACGACGAAGGCAC GTTAGAAACGAGGATTCTATCTTCCAGTTATACGATACGCGGCAAATACG 1400 TAAACGTAGAAGAGTTTATAGCCGCCGGGTTTCCTCTATTGTGGTGTAAG MTCGCCCGGAAAAGGCACGTTCTACCGCATCAGATTCGTTMTTATACG GTCCGTCCGCCGCGTACACAAACGAAGAACTTCTMTCGATCCGTTTAAG 1500 AGATGTTTTATGTTATAAAAAAGATTGGATACGTATCGTGTTGATCCAAT CGTAAACGGGCTATTGTTTTACCCCACATCATCTCCCCTATACGCCCTGT 1600 TTGCCTCGAGATCCGCCGMACTCTTTAAAAACGTMCGTTGAGGTCCTA 1700 TGACGCACTTGGTTACGACGTTCCCCATAAMCACGTTACGTGTGTTACA AAACACGACGAGAGATTACTTACCACGTGTTACGACAAGGGTAGGTTTAA orf C-3 ends+ orf C-4 I+ begins AAAATGAAAAAGTAAAAAACTCATTTACATTAGTTAAGGCACGGATm 1800 CGCCTTTGTGTATGCGTGGTTCAACTCCCAGAT~GTAGCGACGTTGTAG GACGAGTGGTTTTGGCGCACGAACCTTACGTAATAGAGTATCATGAGGAC TGGGAACATATCATCGCTCGACTGGTCGATATGTACMCGAAGTCGCCGA 1900 2000 GTGGATTTTGAAAGACGATACGTCGCCCACGCCCGATAAATTCTTTAAAC AACTGTCCGTATCGCTTAAGGACMGAGGGTGTGCGTGTGTGGTATAGAC GAAGAAGACAATAAAATGTATAGCCGAAACTGTGTCTAATATAACAGGCG 2100 CCGTATCCCCGAGACGCTACGGGAGTTCCGTTCGAATCTCCTAATTTTAC TGGMCTACTATCTAAGCTGCAAAATAGGTGAAACAAAAAGTCACGCGTT 2200 TAGGGTACTATAAAGGATATAATCTAAACGACGTAGAAGGCGTGTTTCCT 2300 ACACTGGAAGCGTATATCAAAACTGTTATTACAACATATCACGAAGTACG TAAATGTGTTATATTGTTTGGGAAAMCCGATTTCGCAAATATACGGTCT 2400 ATTTTGGAGACCCCCGTTACAACTCTGTCATAGGATACCATCCCGCCGCCAG GGAAAAGCAATTCGAGAAAGACAAAGGATTTGAAATAGTAAATGTGTTGT I + orf C-5 beains orf C-4 ends+ &CTTTAGTGAAAAATTMCTCAGGTGTTTTAAAA~GCAAAGG~AJZT 2500 TAGAGATTAACAATAAGCCCGCGATACGTTGGGAACAAGGGTTTAGTTAT AGTAGATMCGMCACATTTTTGTTTTAAAATCCATAGGTGTTCCAMTT CCCATAGGCAAAGCGMGATCCACGATTCGTGGATATCTTTACGTGCGAT 2600 2700 GAATTAGAAAACTATATTTGTAATMTCCGACGTGTACTCTATTCGAAAC ACTACGCAATGAAGAAGACTATTCCGTCGTACGCGTATTTTTCGACGTAG 2800 ATTTAGACGTCGTATTAGACGAAATCGATTACGTAGCGGCGTTAGAGGAT TTCATATTAGAGGTTACAAAATTCGTATCTGTCTTTTCTGTAAAAGMTG 2900 CGACGCTCCGCAGAACAAGGTACTCAAATGCATGCATGAGATCAAACTTTTCTA TTACACGAACGACCGATCCGGATAAMCGAGTTTTCATATGATTTTCCCG AGAATTCTTAAGATCTTCGGAAAATCCGTTAATTAGATCGATCGATCCGG 3000 GACGTCTACACTACGATGCATACTCTCATCGCGATGAAGAAACCTTTATT GCCAATAACGATAAAATACATATCAAACAACCTCCGCATACCAATATATC 3100 CGGTGTATAGACGAAAMCCACGTTACGCGTAGTGGGTACTAGAAAGGCG

GACGGATGTCTATCCGTGTATTCTTCCGTTCAAAAATGGTGTGTTGGATA ACTGTGTCCACGGGTTTTAACTTAAACATGGAAAAGTTCTTAGATGATGA TAACGGAGGAAAACAAACAGAATAGAGAGTTATACGAACGAACGTTAGCC AACGGCGACGGGTAAATCGACTACGAAAAAGTTGTTACATTCGGCCATAG AAAGGCCCTAATCCGTTTATAGCAAACATGCATTTAAAACGATCCGTGTT c=lorf C-6 begins CGGATAAmTAAGAAACTCACGGMCCGTGTATCGTGGGCAGACAATGT CAATTATAAACCTGTGTTCGATAGAGTCGATAACGCTCTTATGCGGCGTA GAAATGCTTMTAAAGCGGCATACGACGACGTGAMCCCTTAGACGAAAC TACTCGTTCAATGGTATCAGAAATACCACGTTCCTCACTTGAAACTGTTT TCTCATCGTGTCCAGTTCGAGTACACATACACATCTGATATCGTCTCTCT CTGTTTCAACAAAAACTGGCTAAACATTTCMTGTACGAGTTCACGGACA orf C-5 ends-q I-torf AAGAATATTTGGAGTATATATTTATAGMGACATTACTTCTAAUACT TTGCCGCATCMTTGGCGACGTTGGATTACTTAGTAAGAAGTATCATAGA TTATAGCGTTGTTATTTGCGTTGGTAGCATCGCGTTTCAAAAAAGTGTAT CGTTGTTATTAATCTATTCAACGCGGATTACATACTGGMAACATATTCA AACTACMCGGATTATCCAGATACAACAACGCGATTTTTATTATAGACGA AAAACAAAAACAAAATCCCGTTCCTATTATTGTCCGGATCTCCCATCACA AATAAACTTTGGTGATATCATTATTCAGGGGMGMAGTATTCCAAATTT ATATCCTATTACMGATGCCCGATACGGATCTACCCGGTATACAATACCA TACAGGAAAAGGACTACATCAACGTACGAAAGATGTGCMTAACGAAATG TCTGATTAATAATTTGGATATTCTATTTCAGGAACAGGATAAGGAGTTAT ACGCTTMTATAAGTTCGAAATTTAAATATTTTATAACCAAGATAGAAAG TGATCATCAMTACATCATGCTCAGTAACGGATATTCCGAGTATAACGGG CATCGTAACCAGTMGATGAAGGCGTCGTTGGAGGACCTACTCGTGACGT TCGMTATCATGTCCGAATCGTATACGTTGAAGGMGTMGAAACATATG GGTCTATACGAAAGTTCTCGTACAAGGATATAACTCAACCCGTAMCGTC AGACGACTATAGTCTGGAGGAAATCAACACGTTGCCCTTCGATATAAAAA

ATTTTGGAGAATATATCCGATTCGTACACACMCCTCCTCATCCACATAT

3800 3900 4000 4100 4200

4400 4500 4600 4700 4800

C- 7 begins CGGCCGTAATCGAGTTATTTAGGCATCATGTCAACAACATTCCTAACATA TGAGAATAAAAGCGTACTGTTGTTTCATATTATGGGTTCTGGGAAAACGA ATATTAGTTCCGAATATAAATATTTTAAAGATATTTAACTACAGCATGGA TCTATTCGACGACTAGTTTTTATTCGATCAATTATATAACGACAACGTCATT GGCCCATAATATCTTCGGGAACAATACGGGTGAACTGATGACGGTTATAA AATACACCCATTACGCTTTCCAATATTATCAGTTTAATGTCCGACGAAGA TGTTAAACGAACACGGTGTAAACGTTCTAAAAAACATTCTAAAGGGCAGG CGGTAAAAGTTTTTTAGACACGCGCGTCGTGTACTGTAACATGTCCAAAT TTTGAAAAGAACATGAACAACGTCTCATTAGCCGTATTGGGACAATTAAA ACCCAAACCTAMAATCAACAACGGCGTCTTGTACGGAGAAGAACTCGTA TTTAAAGGGGMACATTTCATTTATTTTTCTAATTCTACGTACGGTGGAC TCTCGGGGAACGCATCCTAAACTCATCCACGGGAGACCTAAGACATTCGC ATMTTCGTTGGCGAACGACGACGGMGTCAGATTATGTTTTTGTTTTCG GTTTATGACCATTCCGGACACGTTCTCTCAATACAATCAGATATTAGGAA TATCTATTGGCCGCCGTGTATTCCGACTTCAACGACACACTATAGAGTCGTT MTTACTCTATCTCAAATTTAAAACGAAACGAATAGATCTACTCC CGTGGAGATCGTATTGGGGGAAATCGTGAGACAGTTCTTCTACCATCATT

4900 5000 5100 5200 5300 5400 5500 5600 5700 5800 5900 6000 6100 6200 6300 6400 6500

DETERMINANTS

OF

MALIGNANT

FIBROMA

CGAGGATTAAGCATAACGACGAGCGGTTGCTCGCCGCCGTAAAATCCGTA ACACTTTTTCGTGTCGAATAAAGTGTTTGATAAGTCGCTGTTGTACATGT

VIRUS

REPLICATION

IN LYMPHOCYTES

TTAACGAATACGGAGGCCGCTAAAAAATACATTAAAGAAATAGTGGACGG ACAACGACGAMTCATTACCGTTCCGTTTAAATTATCTCACGAACCGTTT

orf

C-7 ends

+I

589 6600 6700

I --a orf C-8 begins

GTATGGGGCGTGAATTTTAGAAAGGAATACAACGTCGTATCGTCTCCGTA

&AcTGATTAAAAAACACGAGACGCGTTAAAATATGTCCACGTTCGTCAA

6800

ACACGTGTATTTGCCAATTATGTTACAACCACACGAGTTAACGTTAGA; TCGGGTGGCATTATGGCAAAAAGGATAGAAATATGTTATGAC~GGAGCT TCGTAACATACAAGTACTATAAAAACGGCGACGTGGTTCAAGGAACCCTA TAAACTCAGTAGGGATGCGGGGACTGTTTCTTTCAATGATTCCAAATATT

TACGTAAAAACATACGAGACGCTGTATATAAAGAATATCTACACAGGGAA ACCCTTAGGCGAATTAGTAAACAATCACGTCGTTGTAAAAGTGCCCTGTA AACATAGAAAACGAATCGAATATAACGGTACAGTGTAATGACCTCATCTG GTTTAATACGAAACGGTGTAGTGTATTGTAACGGGGAC~TGTATCAGTG

6900 7000 7100 7200

I+

orf C-Q ends

orf

C-8 ends

-q

ACATTGAAAGAGGAGCAGCAGGGGATGAACTCAAATTTTGTGTTTC&AGC

GTCCATAGTAGACTCGTCTGGA$&GAGTGCGTACGTCGGTTGTATAGA

7300

ACAACGGATATAACGGATAAGACAATGAGTATACATACAGTCGCTATAAC ACCCTTCCGCGTTAGAAAACGACGCTTTTACGTTATACTCCTTTACAGGT CGAGGATATCATCGTACGTAACCTTCCCAGTTGTCCTGCACTTATAGGAA GTGGTAGTTCCCGCATACGTCCAGTAGTCTAAA~ATTTGGGAAGTAAAGA TACTCACTTGACTGGTAATCAGTTCTATTTTATCGTTGAGTCTTCCCGTT ATCGTAGTTGGTGTATTTTAAGTTCCAGTGTACCATTTGAACTTCTCCAA AGTTCACTCAACACATACTTACTCTTTAATGGACCACCTTGTAGTGTAGA TACGGGTGGAATTATCGCCGTAGTTTATATCCAACGGGGGGTATTTCTCG

GAGGGCATATAACTCAACAAATGGATTCTTGTACACGTGTCTGTCATAAC TGGGTCTCTCTAAAATTATTGTAAATGTAAGTAACGGGTTTCCCTGGGiC TGGGTTCCTTGAACACGATCCACGTTACGTTTTTATCAATAGGAGATGCC GATGGGGTTAMCCCGGTAAACACATOTGACGCGTTCTTTTGTA~TATGT TTTAMAACACGGCGAGTATAACCACACCGTCCTTATATTTACGAGCGTT TGTAATACGCTCCGTTTACGGAGTGTTCTGAACGTCCCCAACGAAAGGAG GGGAACAGAAACGTTAAAAAGTACTTTTAACGTACTTCCATTATTGTGGA ACTGTCGTGTTGTATATCTTTAAGTCTATOGGTGATTCCTGACGAGAACT

7400 7500 7600 7700 7800 7900 8000 8100

CATMTACCAAA~AAAATTMGGGTACTTCGGOTGTGGTAT

GACGACGTTTGAAACGCCTCGAGAGACCGTATTTATAGMTCGGTTGACT

8200

CTATCCCTCAGTCGAAAAAGA~T~A~GTATTTG~GATTGTGTAA~GGT~ AATCACTATGAACATGAATCCGCCTATTGTCGTTACGATTTCTAAACACT CAGAAAGGTTCCGCGCCTATTTATAAGACATCGTTTGAAGAATTAATTTT GGGAGATCAAGGAGGAAACGGATTCTAAACTTACGATAAAAAGTATAGGG TGTAAATTATTGCAAATTATGTTATATACATGAATTGATGGAAGAGGCCC

GATAATAAGCCTATTGTAGCGGCTAGGCGTAGTTCGTTCGTATTTC~GA TAACTAACTACATGTATAACAACGAAATTAAGGAGATAAAAAGAAAACTA ATTGGGCGGAAAACTAAATAAATCAGAAACGATAGACGACTGTATACGGA ACCACCTGCGTCAAGATAACGATTACCGATAAACTGTTTAATCGAAAGTA TATCGTTCGTTATTTACAATGTGGAAATTAGAAAACTGAAATCGTTACTA

8300 8400 8500 8600 8700

elorf

C-Q begins I+ orf C-10

begins

orf GATTGTGTCAATAATGACATTTACTTATTTACGTTTTATTTATAACAC

IC= orf

C-12

C-10

ends

+I I+

orf C-11

begins

GTTGTTGTATAGTAAmACGAACGCCTACAATATTCGCATTTGATGAA

8800

ends

TTATATCACGCAACACAACAGAAAGTTGTCCAAAACC&CACGTGGACGG

ACGACGCGCAAAGAGTCTCCGCCACGGCATTTAACAACCAGCGTCTACGA

8900

TGGAGTAACAAAACCTCCATCTGTTTAATATTATCCACGTTGGATAATAA GATGTCGTAGCGTATTTCGTAAGAAGAGATTGTTTTTAACGTATACGAAA

GTTCATCGCGTGTTCTAGAAAACATAGTTTTTTATACTCCGAGATCGTGC TTCCTAAAGAAAATCGAACGTCCGTTTTTATCGTCTAAGTTAAACGTTCC

9000 9100

CCTGGACGATCCAGGTACGGM~ATGATATTATATTTAT

TGCCTAAGAACGAGGAAGATCCCATU>GTCTATCGAGAGAGATMAA

9200

GAAGAAATTAATATAGACAGTAAAGATATATATATCGATTCTAGGTTCTT TTTTGTTTTTGGGAAAAACGACGTTAACGAGTCACGAAATATTAAATAAT

TGTACACTTATTTATAGAAGATTTGTTGTCTAATCGMTTTACGAAACTA TTTCTAGCGAATAGAGAAATTAAGTCATTAGTTTTTCTAGACACGTTGGA

9300 9400

TAAAATGTTTCGGAAGTGTCGGAGATAAAACCGAATTATTATTGTACGACAAA

9500

orf

AAAAGGAGTGATGTGCGACGTACTTAGGTACGTGCTCGCCGTATCGCGAC

orf

C-11

ends

+I

I+

orf

C-14

C-12

begins*1

ends

GTTACGGAGTCCGTGAAGAAAATGACCAGGTACGGATTTGATTAATCAAT

CGAGTACGTACACCAGTTTATCGTTATGTATTTTGATCACCGGATTATCT

9600

TGTATGGTTTGTATGAAGTTGCCGTCAGAATCGAATAGGCGTCCGTCGTC ATATGTTTTGACCTTTCACCACGTGCGGTACGCGCTTCGTCGTCGGGTTT GGGAGAGAAGTCCGTCTGGTGTTCGTATATCCATTCGATTGAGGTGTGTT TCCAACAGGTCTTCATCTACCGTGGCGTCTCCGTTAGATAGTCGAGCGAC

CGTTTTAAATCCTTTATAGACGGCGATCAGTCTGTTGGAATGGGAATACC TCGTCGATCGATCTAGATATTAACGCGCTCCATCCTGATTCGTTGTCTAC TAAACACTTTAAACAACTGCGTAAATTCCTTAGACTTGGTACGGATAATA GATAAAGTGTACGTTTACATACCGCCGATGTTCCGGGGTAAGTACGTGAC

9700 9800 9900 10000

TGTTTAATCGABGCTCGGCCTATAATCTGTCTCAGGGAGGCCTCGTTC

CAGGTCATGTCTAAAATAAAAATATCGTTAATGGAGAAGAAACTAATCCC

10100

CTCTCCCCCGCTAAGAGAGAATACACACGTTTTAATCAGATCCCCGTCGG GTCCTAGAGGAAAACTCTATATACGATATGTTAAACGCCGAGAAATATAA ATTTACCGGGAGATGCCAAAATACGTAAACATACGTCCGTAAATTTACAA

TGTTCTCGCGTTTGTTAAATTCGGCTACCGATTGGACTCGGGTGTTTTTC AAGTAATATATTAATACCCGACTGGTTGACGAACGGTTCG~GACCAAAC CTGCGTTCTCTAAGTTCGTTTAAAAAGGATATGTCGTCCGCGGAAGACGT

10200 10300 10400

TCCGCCTAGCGATTGACCCCTTTTGAACTGAGATAGGGCGTGTTCAGAGA

ACTTCTTTGTTTTTATAAACGTTTCAAAATCCTTGTACAGGGTTATCTGC

10500

TCGTTCGCGACCTCTTCGGGGGTTTTATCCGTCTTATCCAGGAACGCATC

GAACGTAAACGTTGCGGCCATTCGTCTGTATATTCTAAACGAGGAGATGC

10600

I + orf

-+I orf C-13

C-13

begins

ends

eiorf CGGCCTTTAACTCGGCTAACTTCGCCTTTTGATATACCTGTTCCTGTTTC

C-14

begins

TGTGTC>TTACGTACTGGAAGTATACCGTCTTTCTCGCGAACGCGGA

10700

GGATCC

FIG. 2. Sequence of the BarnHI “C” fragment of MV. Restriction fragments from the Hincll digest of MV were shotgun cloned into the Smal site of pGEM4. Other fragments, including the Barn-Xho fragments, the Xho-Xho fragment, the Eco-Barn fragments, the Ndel fragments, and the Xho-Xba fragment, were cloned into other plasmids of the pGEM series, with appropriate linkers if necessary. Sequencing was performed using Sequenase, from the two primers in the pGEM vector, and using complimentary 1%mer oligonucleotide primers to determine sequences beyond the resolution of the plasmid primers. Sequences around restriction sites were all determined using primer-generated sequences derived from overlapping restriction fragments, Sequences were assembled into the larger “C” fragment using DNA Inspector Ile DNA analysis program. ORF’s read left to right are indicated with solid arrows (+) while those read from right to left are designated with open arrows (-). Bases where orf’s begin or end are doubly underlined.

STRAYER,

590

c-3 -

JERNG,

AND

O’CONNOR

C-4c-5 -C-6 -

c-7

d&~ -

c-9 c-10

C-II

-c-12

c-13c-14

2.9

1.3

FIG. 3. They are based on replicate -3, -4, -5,

2.8

0.5

1.55

Open reading frames shown here against the leftmost end of in lymphocytes and -7, -8, -10, -1 1 and

0.65

1.0

1.0

3.55

1.96

1.35

1.1

0.95

0.4

of the BamHl “C” fragment of MV. ORF’s for the “C” fragment of MV were determined using DNA Inspector Ile. the background of the Noel (above) and Hincll (below) restriction maps of the “C” fragment. ORF’s are numbered the ORF. The region of overlap between the two restriction fragments which transfer from MV to SFV the ability to suppress immune function, the 3.55-kb Ndel fragment and the 1.9-kb Hincll fragment, are highlighted. ORF’s C-l, -13 are read from left to right. The others are read from right to left.

pleted the sequence of SFV’s “D” fragment in the region corresponding to the area of overlap between MV’s 3.6-kb A/de1 and 1.9-kb Hincll subfragments. It includes the 3’ end of the genes encoding SFV orf D-9 and MV or-f C-7, a small intergenic sequence, and the 5’ ends of the genes encoding SFV or-f D-10 and MV orf C-8. This part of SFV’s genome is very similar to that of MV and is shown in Fig. 4, juxtaposed to the MV DNA and or-f sequences in this area. The nucleotide sequences of SFV and MV in this area are 92.6% identical. There are no amino acid differences between the latter two or-f’s (C-8 for MV and D-10 for SFV) in this region. D-9 and C-7 differ by 6 amino acids in this part of their structure. The 32-bp intergenic region shows four differences between the two viruses, including two bp present in MV but absent in SFV.

DISCWSSION We report here the genome structure and open reading frame organization of a pathogenetically important region of two related poxviruses. Investigators have begun to dissect the molecular determinants of virulence in a number of poxviral systems (Buller and Palumbo (199 1)). Thus, a number of investigators have reported that specific poxviral genes determine the animal hosts or host cells susceptible to infection (Drillien et al., 1980; Spehner et al., 1988; Perkus et al., 1990) host translational activity (Person-Fernandez and Beaud, 1986; Bablanian et al., 1981) and specific

aspects of host responsiveness to virus infection, e.g., hemorrhage or inflammatory reactions (Pickup et a/., 1986; Palumbo et a/,, 1989). Two nonessential rabbitpox virus genes are important in the ability of this virus to cause disease in mice (Bloom et al., 1991). Poxviruses encode a family of epidermal growth factor analogs. These EGF-like proteins appear to be very important to the behavior of several poxviruses (Buller et al., 1988a,b), including MV and MYX (A. Opgenot-th et al., in preparation). The ability to elicit most, if not all, of the clinical symptoms associated with MV infection is transferred from MV to SFV with portions of MV’s 10.7-kb BarnHI “C” fragment. Mapping of the portions of MV included in the recombinant viruses generated by this transfer process shows that the central right hand portion of the “C” fragment is necessary for this function (Strayer et a/., 1988b). The left side of the Ram “C” fragment is absent in the more virulent MV-SFV recombinant viruses. The significance of this observation is currently being investigated. Our observations on immunologic dysfunction induced by MV (Strayer et al., 1983c; 1986) suggest that MV’s virulence at least in part reflects its ability to alter certain functions of cells it infects. Lymphocytes infected with MV become largely refractory to activating stimuli. MV inhibits one or more cell activation steps that occur between the recognition of activating agents and cytokine production and DNA synthesis. Thus, production of IL-2 in response to concanavalin A

DETERMINANTS TABLE

OF

MALIGNANT

FIBROMA

1

HOMOLOGIES BETWEEN MV AND VACCINIA ORF’s AND THE FUNCTIONS OF VACCINIA PROTEINS FROM THE HNDIII “D” FRAGMENT Vaccinia Protein

MV ORF

MW

&Da)

D-l

96.7

c-2 c-3

16.7 27.8

D-2 D-3

16.9 28.0

c-4

25.3

D-4

25.0

c-5

90.5

D-5

90.3

C-6 c-7

15.9 73.2

ORFg” D-6

8.6 68.4

c-a

i a.4

D-7

17.9

C-9”

32.3

D-8

35.4

c-10

25.2

D-9

25.0

C-l 1 C-l 3 c-14

30.4 15.3 42.4

D-10 ORFb* D-l 1

28.9 7.8 68

Function

(if known)

Large subunit, mRNA capping enzyme Virion protein Essential protein, function unknown Homologous to carboxyl end of D-5 DNA binding protein, function unknown Function unknown Early transcription factor subunit Small subunit, DNAdependent RNA polymerase Virion membrane protein, CA analog Possible DNA binding protein Function unknown Function unknown Nucleoside triphosphatase

Note. Homologies between vaccinia and MV ORF’s are summarized above. Nomenclature for the vaccinia ORF’s is taken from the article by Niles et a/. (1966). Functions of vaccinia proteins to which MV ORF’s show homology are indicated, where known. These are summarized by Lee-Chen and Niles (1966). MV ORF’s C-l and C-14 are homologous to the carboxyl termini of their respective vaccinia homologs. Amino termini of these homologs from vaccinia virus extend beyond the ends of MV’s Barn “C” fragment. Thus, these offs probably extend beyond the ends of MV’s “C” fragment. These MV and vaccinia on’s should be considered to be partially homologous at the moment. a Referred to as D-72 and bB-69, respectively, in GenBank. c Carbonix anhydrases homologous to C-20: Rabbit CA II (SD = 8.8); Human CA II (SD = 8.7); Rabbit CA I (SD = 8.2); Mouse CA I (SD = 8.0); Chicken CA II (SD = 6.2); Mouse CA II (SD = 5.9); Horse CA Ill (SD = 5.6); Human CA Ill (SD = 5.5); Sheep CA II (SD = 5.5): Bovine CA II (SD = 5.5); Sheep CA VI (SD = 5.0); Rhesus CA I (SD = 4.7); Horse CA I (SD = 4.7); Human CA I (SD = 4.3).

activation is inhibited while expression of IL-2 receptor is not altered. This aspect of MV’s effects on lymphocytes appears to be inseparable from its ability to replicate in lymphocytes: they both map to the overlapping region between the 3.6-kb Ndel and the 1.9-kb Hincll subfragments of MV’s “C” fragment. Therefore, it was of interest to analyze the structure of this fragment to see if we could infer the functions conferred by this DNA from structural homologies with proteins and DNA of known activities. Our deductions of possible or

VIRUS

REPLICATION

IN LYMPHOCYTES

591

probable functions of the proteins from this region are based largely on analyses of sequence homologies with both DNA and proteins. The 16.1 -kb /-/indIll “D” fragment of vaccinia virus (Niles el al., 1986) is homologous to the entire Barn “C” fragment of MV. Homology between or-f’s C-l of MV and D-l of vaccinia begins 224 bases from the left end of the “C” fragment and extends to the initiation site of C-l 4, 292 bases from the right end of the “C” fragment. This distance represents 10,657 bp in MV. The corresponding distance between these areas of homologies in vaccinia or-f’s is 10,484 bp. Analysis of ORF structure shows very strong similarity between many ORF’s in MV and those in vaccinia. By implication, there should be considerable functional similarity between corresponding proteins. On the basis of these homologies, one may tentatively suggest functions for some of the ORF’s from MV. ORF C-14 is homologous to much of vaccinia’s D-l 1, including the nucleotide binding region between D-l 1 amino acids 348 and 413, and so is likely to encode a nucleoside triphosphatase (Rodriguez et a/., 1986; Broyles and Moss, 1987). C-8 is highly homologous to vaccinia D-7 and so probably represents an RNA polymerase subunit (Jones et al., 1987; Ahn et al., 1990). C-l most likely represents a subunit of the mRNA capping enzyme (Morgan eta/., 1984). Similarly, in the area involved in the transfer of virulence between MV and SFV, orf C-7 probably represents part of MV’s early transcription factor, as its homolog, D-6 does for vaccinia. Functions have not yet been determined for some proteins in vaccinia’s “D” fragment. Thus, the roles of ORF’s in the “C” fragment corresponding to such vaccinia ORF’s are unclear. Vaccinia virion proteins D-2 and D-3 are of unknown function (Lee-Chen and Niles, 1988a). Activities of DNA binding proteins D-5 (Roseman and Hruby, 1987; Evans and Traktman (1987)) and possibly D-4 and D-9 are also unknown (Lee-Chen and Niles, 1988a,b). There are strong homologies between or-f C-9 and mammalian carbonic anhydrases. Vaccinia protein D-8 is a virion membrane protein that also resembles carbonic anhydrases (Niles and Seto, 1988; Lee-Chen and Niles, 1988b). Similarities between enzymes and structural proteins have been noted by in other systems (Piatigorsky and Wistow, 1989). Variable differences are noted between MV or-f’s and their vaccinia homologs. In some cases, such as C-7 and D-6, variant amino acids account for 20% of the total protein. This or-f is relatively strongly conserved among poxviruses (Tartaglia et a/., 1990) and in vaccinia, it is a unit of the heterodimer VETF (Broyles and Fesler, 1990). However most or-f’s show substantially

WV sequence begim at 7603 lilen-mtleuser TCTAACTCTACTTACGGTGGTTTGATCATCAAATACATCATGCTCAGTAA 7600/5878 TCTAATTCTACGTACGGTGGACTGATCATCAAATACATCATGCTCAGTAA Iilemetlwaer th4vs3quenm beglneatm3

ay) gty tur SW glu tvr asn gv sar& giy thr gpro lys teu tte CGGGTATTCCGAGTATAACGGGTCTCAAGGAACCTATCCTAAACTCATTC

am

CGGATATTCCGAGTATAACGGGTCTCGGGGAACGCATCCTAAACTCATCC gly ty eer glu tyr mgty sergggty thr!&pro C

leu ile

k&l gh aepkulwml tl-s tyl aslear Iw alaaenssp~gglyser hisglyb pro lya W *ala ile vat thr serly~ mettys ala = ACGGGAAACCTAAAACGTTCGCCATCGTGACTAGTAAMTGAAAGCGTCG CTGGAAGACCTACTCGTAACATATAATTCGTTGGCGAATGACGATGGAAG 7700/5978 ACGGGAGACCTAAGACATTCGCCATCGTAACCAGTAAGATGAAGGCGTCG TTGGAGGACCTACTCGTGACGTATAATTCGTTGGCGAACGACGACGGAAG lw gkr asp leu lw val thr tyr BYI ser lw ala am asp asp gy aer hi gly gg pro lye thr pb ala ile val U-u ear lys met lye ala ear

gin ile met phe leu phe ser sBr asn ile met s8r glu ser tyr TCAGATTATGTTTCTGTTCTCGTCGAATATCATGTCCGAATCGTACACGT 7800/6078 LE[xT END @I? N@cfG38me TCAGATTATGTTTTTGTTTTCGTCGAATATCATGTCCGAATCGTATACGT gin

ile

gln

tyr

met pi18 lau

asn glu

le

phe

Ied

ser ser

gly

am

arg ser

ile

met ser

ile

arg

glu 5Br

lys pk

ser

thr

tyT asn glu

pheaenaspW

ile

ila

IEu

glu

gty arg ser

ile

arg

serlwaspasp

tyr

tyr

thr

ku

tyr

tys

asp

lys

gL

W

ib

asn

arg

glu

ikt

serleuaspasp

tyr

se4

ile

ty7

teu glu m

ile

lys

ik

asp

ile

asn

W

arg

aen

arg

ile

tyr

sar

gln phe phe

ty

his

Me

ile

sa

leu glu e

arg

ile

set asp sex

thr

ile

asn

tyr

ttx

asn

lys

gb

ile

valaspglykisphepheval

ile

lye

lys

lw

g!u

98~ hii

ile

val

giu

asp

gly hi

pro phe

val

ptw phe

by

val

gly val

s8r

tysval

asa

ORF D-104 U-rpheval

lyshkval

tyr

lp

his

val

ty~

lw

asp

thr

phe sar

m

ile

pco val

ap

asn

phe

val

met

tyr

thr

h

ile pro

Iw

ala

fLr

asp

val

thr phe sBr

ty

sar aq,

pro val

pro p4-18 asp

a.9

ile

MI

&s

tyr

iys

leu

leu

lw

leu

ala

tyr

&

lw

val

tyr

lys pha

sw asp

iys

thr

lys

lb5

AAAAAAATTACTCTATCTCAAATTTAAAACGAA AAAAAAATTACTCTATCTCAAATTTAAAACGAA

pro phe asp

i-9

he

tys

leu

leu

tyr

ku

lys pb

C

thr

pro tks

tts

k

val

gh

ile

val

!eu gty &

ile

vd

ik

val

ml

prophe

pro

pro

pro

his

pro

l-is

L

bal

gh4

ile

val

Ieu gty glu

Iw~alavdtyssarvalkuttva5nilrglualaalaty3lys

GCTCGCCGCCGTAAAATCCGTATTAACGAATACGGAGGCCGCTAMAAAT k&alavd

p4-m

asp@

lyseervallwWasnWghalaalatyatye

set

hleu

lyr

@g

ty

baspgg*

ile

ib

W

GATAAGTCGCTGTTGTACATGTACAACGACGACGAAATCATTACCGT?CCGTT

Ip

ilemetleu

pro

gln

leu

dn

Ied

phe

glu

ile met lw

asp

ty~

lys

am

ser

val

1w.d leu

val

ser

tyr m

tyr E

asp

gh

ile

ib

U-r

Ml

pm p4-e

sar pro STOP orf D-9

ACAACGTTGTATCATCTCCG&WCTGATTAAAAA.TACGATACGCGTTA

glu

an

AA-T~TCCACGTTCGTCACACGTGTATTTGCCAATTATGTTACAAC 8500/6779 BO@NU INIB @ID NdolClOo~ QVERBAP AAAT~TCCACGTTCGTCAAACACGTGTATTTGCCAATTATGTTACAAC met eer W phe val BEGIN ORF C-B-,

pro

ACTCACCGCCGTAAAATCCGTATTAACGAATACGGAGGCCGCTAAAAAAT

lys val

asn ptm arg

larpro

ik

GATAAGTCGCTGTTGTACGCGTACAAAGACGAAATAATTACTGTCCCGTT

TAAATTATCTCATGAACCGTTTGTATGGGGCGTGAATTTTAGPAAGGAAT 8400/6678 TAAATTATCTCACGAACCGTTTGTATGGGGCGTGAATTTTAGAAAGGAAT MS leu eer his clh pro phe val try gly val asn phe arg IF BEGIN metsar

thr

g!n pro

leu

ACATTAAAGAAATAGTGGACGGTCACTTTTTCGTATCGAATAAGGTATTT 8300/6578 ACATTAAAGAAATAGTGGACGGACACTTTTTCGTGTCGAATAAAGTGTTT ty

gln

leu

ti

thr

asp glu arg

serasn

thr

CACAACCTCCTCATCCACATATCGTGGAGATCGTATTGGGGGAAATCGTG tyr

CGACAGTTCTTCTACCATCATTCGAGGATTAAACAAAACGACGAACGATT 8200/6478 AGACAGTTCTTCTACCATCATTCGAGGATTAAGCATAACGACGAGCGGTT aq gln phe phe tyr his tis sef arg ik @ !& asn asp glu arg tyile

trp pi-18 met

CACAACCTCCTCATCCGCATATAGTGGAGATTGTATTGGGGGAGATCGTA

set asp ser

ly5 *

arg

ACGTTGCCCTTCGATAT

gluglu

ile

ile

ACGTTGCCCTTTGATAT

AGAAACAAATAGGATCTACTCCATTCTGGAGAGTATATCCGATTCGTACA 8100/6378 AGAAACGAATAGGATCTACTCCATTTTGGAGAATATATCCGATTCGTACA gh

aan

TATAACTCAACCCGTAAACGTCTATCTATTGGCCGCCGTGTATTCCGACT tyr

gluglu

serleu

glu val

t-3 W

TCAACGATACTATAGAGTCGTTGGATGATTATAGCCTGGAGGAAATCAAC 8000/6278 TCAACGACACTATAGAGTCGTTAGACGACTATAGTCTGGAGGAAATCAAC ph3asnaepW

arg

TATAACCCAACCCGTAAACGTCTATTTATTGGCCACTGTGTATTCTGATT

tys phe ser

sBIleu

glu vd

TGAAGGAAGTGAGAAACATATGGTTTATGACCATCCCCGATACATTCTCT QVERLAP IP@OQN+ 1 TGAAGGAAGTAAGAAACATATGGTTTATGACCATTCCGGACACGTTCTCT

CAATACAATCAGATATTAGGAAGGTCTATAAGAAAGTTCTCGTACAAGGA 7900/6178 CAATACAATCAGATATTAGGAAGGTCTATACGAAAGTTCTCGTACAAGGA gln

lya

leu

ACAACGTCGTATCGTCTCCG~P&ACTGATTAAAAAACACGAGACGCGTTA tyr esn ml val ser ser proBlOP01fC7

po

hi5 gh

leu

thr

leu

glu

val

arg

lys

asn

ila

arg as+3 ala

Ml

ty~

ml

tyr

CACACGAGTTAACGTTAGAAGTACGTAAAAACATACGAGACGCTGTATATA REQOBN=a 0 CACACGAGTTAACGTTAGAAGTACGTAAAAACATACGAGACGCTGTATATA gln

pro

his gb leu

W

leu

gh

val

arg

tfs

m

ile

arg asp

ala

FIG. 4. Comparison of nucleotide and amino acid sequences of SFV BarnHI “D” fragment (upper) and MV BarnHI “C” fragment (lower) where MV’s 1.95.kb Hincll and 3.6-kb Ndel restriction fragments overlap. Base sequences are given from 7600 for SFV and from 5878 for MV. Amino acids in the several orf’s are shown as three letter designations, as are translation initiation and termination sites. Differences in amino acids are indicated in bold type and are underlined. Bases not present in a sequence but represented in the other virus sequence are designated by(e).

592

DETERMINANTS

OF

MALIGNANT

FIBROMA

greater dissimilarity. In the pairs of C-6 and D-72 on the one hand and C-13 and B-69 on the other, the corresponding MV orf is considerably larger than itsvaccinia virus counterpart. For orf pairs such as C-9 and D-8, areas of homology and the extent of similarity are limited. Along these lines, we examined the corresponding area of SFV, the BarnHI “D” fragment” and present here a portion of this sequence. With few exceptions, SFV orf’s are remarkably similar to MV or-f’s throughout this area, notably so as in many instances both differ markedly in structure from corresponding vaccinia orf’s (D. Strayer and H. Jerng, unpublished observations). The area of the genome responsible for transferring from MV to SFV the virulence associated with the former is 0.7 kb at the carboxyl end of or-f’s that are probably early transcription factors (ETF) and the amino terminus of or-f’s probably representing a subunit of RNA polymerase. Differences between SFV and MV in this region are limited and are entirely in the former pair of orf’s, as well as in several bases in the intergenic DNA. Small differences in the ETF could mediate virulence by virtue of differential abilities of one or the other to regulate expression of important viral genes. Cellular enzymes could preferentially modify MV’s or SFV’s ETF to render it more or less active, more or less capable of binding virus DNA, etc. Alternatively, one factor could interact better than the other with a host transcription or DNA-binding factor and thus either be modified and/or in turn modify viral and host gene expression. We have found that MV infection selectively alters expression of specific host genes (Strayer, 1991). This effect of MV on cellular gene expression could reflect interactions between viral transcriptional regulatory factors and those of the host. The degree to which structural differences in these proteins determine functional differences is yet to be determined. In some cases large structural differences may not greatly alter the function of the protein in question. However there are many instances in which mutations in a solitary amino acid change protein activity or specificity dramatically. For example, a single conservative amino acid change in the ATP-binding region of v-e& protein of Friend leukemia virus (val+ile) compietely changes the tissue specificity of transformation caused by this virus (Shu et a/., 1990). This has been underscored recently by two reports from groups working with poxviruses. A change from ala to asp in the 14-kDa vaccinia envelope protein greatly reduces B The entire sequence of the SFV BamHl “D” fragment is not yet complete and is available only in preliminary form. Hence, although the orf structure of this fragment has been determined we have not finished the final sequence, except in the area reported here.

VIRUS

REPLICATION

IN LYMPHOCYTES

593

plaque size (Gong et al., 1989). Changing from ala to thr or val at position 498 in vaccinia DNA polymerase alters aphidicolin resistance of resultant mutant viruses (Taddie and Traktman, 1991). In the latter case, both mutations would be considered relatively conservative ones. The analyses of protein structure and function performed by others with this vaccinia DNA are helpful to us in understanding the functions of this portion of the MV genome. This structural analysis is a prelude to functional analysis of the “C” fragment of MV. Because of the importance of this region of MV’s genome for virus to replication in lymphocytes, suppression of lymphocyte function, and induction of disseminated tumors, further studies of the actions of this portion of the viral genome should yield valuable insights into the means by which viruses regulate host gene function to produce the type of disease patterns that they do. ACKNOWLEDGMENTS The authors thank Drs. Steve Broyles, Brian Knoll, Grant McFadden, and Ed Niles who were kind enough to discuss with us these results and their interpretation, as well as work ongoing in their laboratories relevant to this manuscript. Dr. Justin Mulloy generously gave us the cloned “C” fragment of MV and the cloned “D” fragment of SFV. The kindness and support of the people at the Molecular Biology Information Resource Center at Baylor College of Medicine, Houston, Texas was invaluable. This work was supported by NIH Grant CA44800. Note added in proof A compilation of open reading frame sequence homologies between orf’s of MV’s BarnHI “C” fragment and vaccinia strain WR Hindlll “D” fragment is available upon request from the authors. A recent publication (Tartaglia et al., 1990) describes the sequences of a fowlpox Hindlll fragment homologous to vaccinia’s Hind “D” fragment. Inspection of DNA sequences and ORF’s presented in this report indicates that they too closely resemble the MV sequences reported here. Unfortunately, fowlpox orf sequences were not yet available to us for computer analysis at the time of preparation of this manuscript. A detailed comparison of their homology to MV and SFV orf’s will be performed in due course.

REFERENCES AHN, B.-Y., JONES, E. V., and Moss, B. (1990). Identification of the vaccinia virus gene encoding an 18.kilodalton subunit of RNA polymerase and demonstration of a 5’ poly (A) leader on its early transcript. J. Viral. 64, 301 g-3024. ALTSCHUL, S. F., and ERICKSON, B. W. (1986a). Optimal sequence alignment using affine gap costs. &A/. Math. Biol. 48, 603-616. ALTSCHUL, S. F., and ERICKSON, B. W. (198613). A nonlinear measure of subalignment similarity and its significance levels. Bull. Math. Biol. 48, 617-632. BABLANIAN, R., COPPOLA, G., SCRIBANI. S., and ESTEBAN, M. (1981). Inhibition of protein synthesis byvaccinia virus. IV. The role of low molecular weight viral RNA in the inhibition of protein synthesis. Virology 112, 13-24. 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.

594

STRAYER,

JERNG,

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