JOURNAL OF VIROLOGY,0022-538X/98/$04.0010

Aug. 1998, p. 6742–6751 Vol. 72, No. 8

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Fowlpox Virus Encodes Nonessential Homologs of CellularAlpha-SNAP, PC-1, and an Orphan Human Homolog

of a Secreted Nematode ProteinSTEPHEN M. LAIDLAW, M. ARIF ANWAR, WARREN THOMAS, PHILIP GREEN,

KATHY SHAW, AND MICHAEL A. SKINNER*

Institute for Animal Health, Compton Laboratory, Newbury,Berkshire RG20 7NN, United Kingdom

Received 2 February 1998/Accepted 18 May 1998

The genome of fowlpox virus (FWPV), type species of the Avipoxviridae, is considerably rearranged comparedwith that of vaccinia virus (the prototypic poxvirus and type species of the Orthopoxviridae) and is 30% larger.It is likely that the genome of FWPV contains genes in addition to those found in vaccinia virus, probablyinvolved with its replication and survival in the chicken. A 7,470-bp segment of the FWPV genome has five openreading frames (ORFs), two of which encode ankyrin repeat proteins, many examples of which have been foundin poxviruses. The remaining ORFs encode homologs of cellular genes not reported in any other virus. ORF-2encodes a homolog of the yeast Sec17p and mammalian SNAP proteins, crucial to vesicular transport in theexocytic pathway. ORF-3 encodes a homolog of an orphan human protein, R31240_2, encoded on 19p13.2.ORF-3 is also homologous to three proteins (YLS2, YMV6, and C07B5.5) from the free-living nematodeCaenorhabditis elegans and to a 43-kDa antigen from the parasitic nematode Trichinella spiralis. ORF-5 encodesa homolog of the mammalian plasma cell antigen PC-1, a type II glycoprotein with exophosphodiesteraseactivity. The ORFs are present in the virulent precursor, HP1, of the sequenced attenuated virus (FP9) and areconserved in other strains of FWPV. They were shown, by deletion mutagenesis, to be nonessential to virusreplication in tissue culture. RNA encoding the viral homolog of PC-1 is expressed strongly early and late ininfection, but RNAs encoding the homologs of SNAP and R31240_2 are expressed weakly and late.

Determination of the complete genome sequences of largeDNA viruses (herpesviruses and poxviruses) has revealed thepresence of numerous homologs of cellular genes (formally,the viral genes should be described as xenologs, as they are al-most certainly related through lateral gene transfer [38], butwe will use the more common generic term). Many of the cel-lular genes are involved in the immune and inflammatory re-sponses, and subsequent study of the products of the viralhomologs has demonstrated their role in immunomodulation(reviewed by Smith [56] and by Spriggs [57]). Study of immu-nomodulation has also revealed surprising functions for someviral genes (which had no known cellular homologs), espe-cially in down-regulation of major histocompatibility com-plex (MHC) molecules (reviewed by Bonifacino [6]). Thus,various viruses express homologs of receptors for alpha andbeta interferon (2, 60), interleukins (IL) and chemokines (1,27, 28, 58), and tumor necrosis factor (54, 64). Poxviruses,however, express a b-chemokine receptor with no homology toknown cellular receptors (55). Virus homologs of the solubleeffectors themselves have also been found. Human herpesvirus8 encodes homologs of IL-6 (42) and of the chemokinesMIP-1a and MIP-1b (43). Homologs of IL-10 have been foundin herpesviruses (31, 37, 48) and in a poxvirus, orf (24). Theproducts of vaccinia virus genes E3L and K3L interfere withthe interferon response (3, 10, 16). The crmA gene of cowpoxvirus blocks activation of IL-1b by inhibiting IL-1b-convertingenzyme (45). Herpesviruses interfere with MHC-mediatedpresentation of antigens by several mechanisms. For instance,

herpes simplex virus ICP47 blocks the TAP transporter (25,30), while human cytomegalovirus proteins US2 and US11“dislocate” MHC class I molecules from the endoplasmic re-ticulum back into the cytosol, where they are degraded (70,71). The mechanisms of poxvirus down-regulation of MHCexpression or presentation have, however, received relativelylittle attention.

Fowlpox virus (FWPV) is the prototypic member of theavipoxviruses. Able to replicate fully only in avian cells, it hasbeen developed as an avian expression vector but has receivedconsiderable attention as a candidate nonreplicating virus vec-tor for use in animals and humans (39, 44). Only about aquarter of its genome, which at 260 kbp (36) is more than athird larger than that of vaccinia virus (190 kbp), has beensequenced. Thus far, no clear candidates for viral immuno-modulatory genes have been reported, though homologs ofgrowth factors and serpins have been identified (63). This mayin part be due to the dearth of available avian cytokine se-quences; avian interferons, for instance, have only recentlybeen cloned (22, 52). The molecular interactions of FWPVwith its chicken host have not been well studied, partly becauseavian molecular immunology lags behind that of mammals inimportant areas.

Here we report the presence, in an 8.5-kbp block of se-quence from the left side of the FWPV genome, of homologsof the cellular genes SNAP (involved in constitutive and reg-ulated vesicle fusion) and PC-1 (a type II membrane glyco-protein with alkaline phosphodiesterase activity, implicated innon-insulin-dependent diabetes mellitus). These homologshave never been observed in any other virus. We show thatthey are conserved in different FWPV strains but that they arenonessential for virus replication in vitro. Their potential rolesin virus-host interactions, including immunomodulation, are

* Corresponding author. Mailing address: Institute for AnimalHealth, Compton Laboratory, Nr. Newbury, Berks. RG20 7NN,United Kingdom. Phone: (1635) 577270. Fax: (1635) 577263. E-mail:michael.skinner@bbsrc.ac.uk.

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discussed. A neighboring gene is a homolog of three splicedgenes of unknown function from the free-living nematode Cae-norhabditis elegans, of a 43-kDa secreted antigen from theparasitic nematode Trichinella spiralis, and of a human gene on19p13.2 encoding the orphan protein R31240_2. The virus ho-molog, FP-CEL1, is nonessential for virus replication in vitro.

MATERIALS AND METHODS

Viruses and cells. Avipoxviruses were grown on chick embryo fibroblasts(CEFs) in the presence of 2% newborn calf serum. The pathogenic strain HP1was supplied by A. Mayr and propagated by four passages on chorioallantoicmembranes. Partially attenuated strain HP1-200 and attenuated strain HP1-438,derived from virulent HP1 by six passages on CEFs plus two passages on cho-rioallantoic membranes and 200 or 438 further passages through CEFs, respec-tively (35), were provided by A. Mayr. A twice-plaque-purified isolate of HP1-438 (FP9) was then passaged six times to constitute a stock. The vaccinal strainPoxine was provided by Duphar. The mild vaccine strain Websters FWPV M andthe Chick-N-Pox vaccine strain were obtained from Salsbury Laboratories, Inc.(now Solvay Animal Health), Charles City, Iowa. Canarypox virus (strain 229),pigeonpox virus (strains Peekham and 950), sparrowpox virus (strain 9037), andturkeypox virus were provided by the Central Veterinary Laboratory, Weybridge,United Kingdom.

Genomic cloning and sequencing determinations. Extraction and molecularcloning of FWPV FP9 DNA as well as manual sequencing of sonicated FWPVDNA in M13 with Sequenase II, 35S-dATP, and dideoxynucleotide chain termi-nators were performed as described previously (5). The project was completed bymanual sequencing of denatured plasmid DNA with specific primers. Sequencedata was assembled on a DEC Vax mainframe (VMS) with the Staden sequenc-ing package (59). Database searches were performed on the Vax with the FASTAand BLAST programs, and sequence analyses and comparisons were carried outwith the GCG package (Sequence Analysis Software Package 7.2 [1991]; Genet-ics Computer Group, Madison, Wis.).

Analysis by PCR of gene conservation in avipoxviruses. PCR was performedon virus DNA, isolated as previously described (8), with the following primerpairs: M29 (59-GCAGATCTAACCATGGAACAAGAAGCGTATAG-39) andM30 (59-CGTCTAGATTCATTGTGTTTGTATATTCTTC-39) for FP-SNAP;M123 (59-ATGATATCACCTACGGC-39) and M106 (59-CTACATGTTTATAACACAACC-39) for FP-CEL1; and M39 (59-CCCCGCATGCATCATGACAG

GAAGTAAAAC-39) and M40 (59-CCCGGATCCGTACATATACTACATGTAAG-39) for FP-PC1 (Fig. 1).

Construction of deletion plasmids for transient dominant selection. A cassettecontaining the vaccinia virus p7.5 early/late promoter upstream of the Esche-richia coli gpt gene was cloned as a 1.1-kbp SalI/AatII fragment into the SmaI siteof pNEB193 (New England Biolabs) in the same orientation as the amp gene togive pGNR. The FP-SNAP gene was amplified by PCR with M47 (59-CTATTCACGTAGCTGTCG-39) and M44 (59-CCCCGCATGCACCAGTCTACTACTTCG-39). The 2.1-kbp product was digested with PacI and SphI (within M44) andcloned into pGNR to generate pSL3. Deletions were introduced into the FP-SNAP gene by digestion with AflII and BsmI (pSL15) or with BsmI and SnaBI(pSL9) followed by blunt-ending and religation. The FP-CEL1 gene was sub-cloned from pM564 as a 4-kb BamHI/HindIII fragment into pGNR to make pSL2.Deletion of a 1-kbp SnaBI-to-ClaI fragment from pSL2, to make pSL17, resultedin deletion of most of the FP-CEL1 gene. The FP-PC1 gene was amplified witholigonucleotides M39 and M40. The 2.6-kbp product was cleaved with SphI (inM39) and BamHI (in M40) and then cloned into pGNR to give pPC1.FL.Deletions were made with XbaI-to-XbaI (pPC1.X), SpeI-to-SpeI (pPC1.S), andBstEII-to-XbaI (pPC1.B) fragments. A diagrammatic representation of the de-letions is shown in Fig. 1.

Isolation of FWPV deletion mutants by transient dominant selection. Deletionmutants were isolated by the transient dominant selection method (23), asdescribed previously (8). Cells transfected with plasmids carrying deleted FWPVgenes with the gpt gene were infected with FWPV, and then, following threerounds of plaque purification in the presence of mycophenolic acid, recombinantviruses carrying the gpt gene were isolated. The recombinants were subsequentlyplaque purified in the absence of mycophenolic acid until they lost the gpt gene,as demonstrated by the inability to replicate in the presence of mycophenolicacid.

Screening of FWPV deletion mutants. Each deletion mutant was screened byPCR with flanking primers (giving PCR products of specific sizes for wild-typeand deleted genes) or one flanking primer and one primer internal to the dele-tion (detecting only wild-type genes [Fig. 1]). The primers used are as follows:FP-SNAP flanking primers M166 (59-TCTCATAACGAGTCCAG-39) to M44(pSL9) or M29 to M30 (pSL15); FP-SNAP internal primers M166 to M30 (pSL9)or M172 (59-TTAAGAAACGTAAATAACGTTAAAG-39) to M173 (59-GGCATTCTATAGATTTTTTAGGATC-39; pSL15); FP-CEL1 flanking primers M133(59-CTAATTCTAGTTGTTAGGG-39) to M38 (59-AGTTACTATTCCAGTAATGCG-39); FP-CEL1 internal primers M133 to M44; FP-PC1 flanking primersM39 to M40; and FP-PC1 internal primers M168 (59-TCTATAATATGATGT

FIG. 1. Genomic arrangement of ORFs showing the positions of primers used to analyze conservation of the ORFs, the location of PCR probes used for Northernblotting, the FWPV genomic sequences used for deletion constructs, and the positions of flanking or internal primers used for screening of deletion mutants. ORFs(hatched or shaded boxes) are shown above the line representing the sequence, as all read from left to right in the deposited sequence. Early termination signals areindicated by asterisks. PCR fragments are indicated by thick lines, and the PCR primers generating them are indicated in boldface type above (for forward) or below(for reverse) the lines. The positions of deleted sequences are indicated by thin dashed lines.

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GTG-39) to M40. A diagrammatic representation of the primers used to screenthe deletion mutants is shown in Fig. 1.

Northern blot analysis. Probes were generated for Northern blotting by am-plification of the following fragments (Fig. 1) by PCR: ANK2, M47 to M330(59-CCCACCGTCGACTATCTTATACGGAAGAAATCTGG-39); FP-SNAP,M29 to M30; FP-CEL1, M169 (59-CGATAAACGTAAATGGAACATCG-39)to M106 (59-CTACATGTTTATAACACAACC-39); ANK3, M25 (59-CCCACCAAGCTTCCTTTTCTAATAGAGTTATTACG-39) to M27 (59-CCCGTAGTCGACCGTTTTACTTCCTGTCATG-39); and FP-PC1, M41 (59-GAAGAAAGAATAAATACCGTATTGAGGTGG-39) to M40 or M39 to M171 (59-GGTATAGTGTAAAATATATCCACC-39). All of the probe DNA fragments were puri-fied by agarose gel electrophoresis, and [32P]dCTP probes were prepared with arandom priming kit (Stratagene). CEFs were infected with wild-type or mutantvirus at a multiplicity of infection (MOI) of 10. After 4 or 24 h of incubation withor without cytosine arabinoside (present at 40 mg/ml from 1 h preinfection),RNA was extracted with an RNeasy Mini kit (Qiagen) and was stored underalcohol at 270°C until it was used. Denaturing electrophoresis of RNA wascarried out with a 1% formaldehyde–agarose gel. The gel was then blotted ontonitrocellulose and hybridized with the above probes (as described by Sambrooket al. [50]).

Growth curves. Growth curve experiments were performed with wild-type andmutant viruses at MOI of 10 for single-step experiments or 0.001 for multistepexperiments, as described previously (8).

Nucleotide sequence accession number. The FWPV genomic sequence de-scribed herein has been given GenBank accession no. AJ006408.

RESULTS

Sequence analysis. A random M13 clone of FWPV genomicDNA, MFP504, translated in six reading frames and screenedagainst the SWISSPROT database, revealed good homologywith murine PC-1 (MUSPC1B [65]). Unusually, this homologywas also observed at the nucleotide level. The homology waswith a region identified as an active site for the PC-1 nucleo-tide phosphodiesterase. A labelled prime-cut probe, generatedfrom MFP504, was used to probe a HindIII FWPV genomiclibrary in pAT153. Clone M564, containing a 6.5-kbp fragment,hybridized to the probe. Sequence determination of the clonerevealed that it contained only the 59 end of the gene encoding

the PC-1 homolog. A second clone (K03) was isolated andcloned into pUC19 containing a 3.8-kbp SmaI/BglII genomicFWPV fragment which overlapped M564 from the SmaI sitewithin the PC-1 coding sequence. This allowed completion ofthe sequence encoding the PC-1 homolog.

The sequence reported here is 7,470 bp in length, encom-passing five large open reading frames (ORFs) which encodeproteins with 406, 287, 375, 341, and 817 residues, with pre-dicted molecular masses of 46.5, 33.4, 43, 39.3, and 94 kDa,respectively (Fig. 1). The first and fourth ORFs (ORF-1 [alsocalled ANK2] and ORF-4/ANK3, respectively) encode pro-teins containing nine and five ankyrin repeats, respectively.The second ORF (ORF-2/FP-SNAP) encodes a protein withsignificant homology to yeast Sec17p and to mammalian SNAPproteins involved in vesicular transport. The third ORF (ORF-3/FP-CEL1) encodes a protein with homology to an orphanhuman gene on chromosome 19p13.2 encoding hypotheticalprotein R31240_2. ORF-3 is also homologous to three pre-dicted multiply spliced proteins (YLS2, YMV6, and C07B5.5)from the free-living nematode C. elegans and to a secreted43-kDa antigen from the parasitic nematode T. spiralis. Thefifth ORF (ORF-5/FP-PC1) encodes the PC-1 homolog. Theproperties of ORF-1/ANK2 and ORF-4/ANK3 will be report-ed elsewhere.

FWPV homolog of cellular SNAP. ORF-2/FP-SNAP has 40to 44% amino acid identity over its whole length with mam-malian and squid SNAPs and 26% identity with Sec17p fromSaccharomyces cerevisiae (Fig. 2). Cellular SNAP (14) plays acrucial role in constitutive and regulated vesicle transport be-tween several compartments within the cell (49). With NSF itappears to bind to the 7S complex formed between SNAP re-ceptors on vesicle and target membranes (v- and t-SNAREs),forming in turn the 20S fusion particle. Three isoforms ofSNAP (alpha, beta, and gamma) have been identified; alpha-

FIG. 2. Alignment of human (humalpha) (g1066084, U39412) and bovine (bovalpha) (g423230, S32367) alpha-SNAP, bovine beta-SNAP (bovbeta) (g423236,S32368), human gamma-SNAP (humgamma) (g1685288, U78107 [shown to residue 297]), and SNAPs from longfin squid (Loligo pealei) (g1078943, S52426), Drosophilamelanogaster (drosomel) (g507754, U09374), S. cerevisiae (SaccCer) (Sec17p; g542367, S39837), and FWPV (Fpvsnap). Residues found in 60% or more of the sequencesare boxed, and residues showing 85% or more homology to the upper sequence (according to Dayhoff’s PAM250 tables [19]) are shaded. The sequences were alignedwith GCG PILEUP (using default parameters) and were displayed with SeqVu (J. Gardner, Garvan Institute of Medical Research, Sydney, Australia).

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and gamma-SNAP are found in a wide range of tissues, butbeta-SNAP is specific to the brain (69).

FWPV encodes a homolog of an orphan protein on humanchromosome 19. ORF-3/FP-CEL1 has 33% amino acid iden-tity over 336 residues with the human hypothetical proteinR31240_2, 32% amino acid identity over 232 residues withC. elegans YLS2 (F09G8.2 on chromosome III), 28% identityover 298 residues with YMV6 (K04H4.6 on chromosome III),and 31% identity over 321 residues with C07B5.5 on chromo-some X (Fig. 3). It is of particular interest that the region ofFP-CEL1 homology spans three to eight coding exons of thecellular homologs (FWPV, like other poxviruses, does not un-dergo splicing, so there are no introns in the virus homolog).ORF-3/FP-CEL1 has 18% amino acid identity, over 257 resi-dues, with the 43-kDa antigen (EMBL accession no. M95499)from the parasitic nematode T. spiralis (67).

The FWPV homolog of cellular PC-1 lacks the somatomedinB domains. ORF-5/FP-PC1 is predicted to encode a type IImembrane glycoprotein which has 38 to 39% amino acid iden-tity, spanning 730 residues, with human and mouse PC-1, alsotype II membrane glycoproteins (Fig. 4). The FWPV proteinshows no homology with these mammalian genes in the N-

terminal, cytoplasmic, and transmembrane segments. Two so-matomedin B domains, found in the membrane-proximal partof the extracellular segment of mammalian PC-1, are absentfrom the FWPV homolog as well as from the rice homolog(Fig. 4).

PC-1 was first recognized on plasma cells (hence plasma cellantigen 1, or Pca-1) in mice as a disulfide-linked homodimer ofa membrane glycoprotein (molecular weight, 120,000) withrestricted tissue distribution (61). Full-length murine (65, 66)and human (9) cDNA clones were isolated and sequenced.Analysis of the sequence of PC-1 (53) showed that it containsthe active site of alkaline phosphodiesterase I (EC 3.1.4.1),obtained by peptide sequencing (15). PC-1 was shown to pos-sess both alkaline phosphodiesterase I (EC 3.1.4.1) and nucle-otide pyrophosphatase (EC 3.6.1.9) activities in mice (46, 47)and humans (26), the pyrophosphatase activity being elevatedin cultured skin fibroblasts from patients with Lowe’s syn-drome.

There is now a growing family of proteins related to PC-1;these proteins have been identified in S. cerevisiae, includingYCR026c on chromosome III (7), and in C. elegans, includ-ing C27A7.1 (g1805697/Z81041), C27A7.3 (g1805698/Z81041),

FIG. 3. Alignment of the predicted human protein R31240_2 (g1905907, AD000092) and its homologs from C. elegans (yls2_caeel [g465851, P34387]; c07b5_5[g559893, Q17778]; and ymv6_caeel [g465961, P34508 {shown from residue 163}]), T. spiralis (trichina [g345333, A44164]), and FWPV (fpvcel). The derivation andexplanations of the alignment are as described in the legend to Fig. 2. Membrane-spanning segments predicted by the TopPred II program (13) are underlined. Signalcleavage sites predicted by the AnalyzeSignalase program (N. Mantei, ETH, Zurich, Switzerland) are indicated by triangles (the suboptimal site predicted for FP-CEL1is shown by a triangle in parentheses). Well-conserved cysteines are indicated by circles.

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and C01B10.5 on chromosome III and T03G6.3 on chro-mosome X (data not shown). Other members of the familyinclude autotaxin, a human protein involved in tumor cellmotility (33, 40); PD-Ia, a rat brain phosphodiesterase/pyro-phosphatase (41); and RB13_6, a rat neural differentiation andtumor antigen (20).

Conservation of homologs of SNAP, R31240_2, and PC-1 inother FWPV strains and avipoxviruses. To investigate the pos-sibility that the genes we observed in FP9 were acquired re-cently during passage of the virus in embryo and tissue culture,

PCR analysis was conducted with different passage levels ofFP9 precursor (including the pathogenic progenitor HP1-6).The analysis was also extended to investigate the prevalence ofthe gene in other strains of FWPV (Poxine and WebstersFWPV M and Chick-N-Pox) and in other avipoxviruses (pi-geon-, canary-, turkey-, and sparrowpox viruses). FP-SNAP,FP-CEL1, and FP-PC1 were all found in HP1-6, HP1-200,Poxine, FWPV M, and Chick-N-Pox (Fig. 5). We were unableto detect these genes in other avipoxviruses, but this does notprove that the genes are absent; the sequences may have di-

FIG. 4. Alignment of the predicted FP-PC1 (fppc1) protein with human PC-1 (humpc1 [g129678]), rat B10/gp130/RB13_6 (ratrb13_6 [g1363274]), human autotaxin(humautotax [g1160616]), and rice phosphodiesterase (riceppd [g818849]). Note that human PC-1 is shown with an N-terminal cytoplasmic domain of 24 residues (9),as represented in the databases, rather than the 76 residues subsequently determined (4). The derivation and explanations of the alignment are as described in thelegend to Fig. 2. The transmembrane sequences are underlined, and the two somatomedin B repeats in human PC-1, rat B10/gp130/RB13_6, and human autotaxin areindicated by dashed lines with arrowheads. Two predicted N-linked glycosylation sites which are either totally conserved (the site at human PC-1 residue 533, whichis also conserved in C. elegans but not S. cerevisiae homologs) or partially conserved (the site at human PC-1 residue 289, which is conserved in mammalian PC-1,FP-PC1, and rice phosphodiesterase) are indicated by solid brackets. A dashed bracket indicates the location of the EF-hand motif. Cysteines conserved in human PC-1,rat B10/gp130/RB13_6, human autotaxin, and FP-PC1 are indicated by closed circles; those conserved in human PC-1, rat B10/gp130/RB13_6, and FP-PC1 are indicatedby an open circle, while those conserved in sequences excluding FP-PC1 are indicated by closed triangles. SeqVu was also used to insert pad characters manually topermit local alignment of cysteines at human PC-1 residues 478, 574, 576, and 816.

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verged to a point at which our primers were no longer func-tional.

Northern blot analysis shows that the FWPV homolog ofPC-1 is strongly expressed as RNA. Northern blot analysis withFP-SNAP sequences revealed no more than weak expression,either early or late (Fig. 6B). A weak early RNA of about 2.4kb is similar in size to the strong transcript (Fig. 6A) producedby the upstream gene, ANK2, and may therefore representreadthrough from ANK2. The weak late transcripts of 3 to 7.5

kb are also in a size range similar to that observed for ANK2,but the distribution appears to be toward transcripts largerthan observed for ANK2. Northern blot analysis of FP-CEL1showed late expression of heterogeneous transcripts, between2 and 4 kb, in the absence of cytosine arabinoside (araC).There appeared to be some weak araC-sensitive expression ofthese heterogeneous transcripts even at only 4 h postinfection(Fig. 6C). Northern blot analysis with FP-PC1 sequences (Fig.6E) revealed the presence of an abundant 3-kb early mRNA ininfected but not control cells (the ORF is 2.5 kb). The sameprobe showed strong expression, in the absence of cytosinearabinoside, of late transcripts of 2.8 to 5 kb in length. The sizeof the early FP-PC1 mRNA transcript was reduced slightly invirus carrying the 170-bp SpeI deletion and was reduced to 2 kbin virus carrying the 1,050-bp XbaI deletion and to 1 kb in viruscarrying the 2-kb BstEII-to-XbaI deletion, thus indicating thatthe transcript does indeed span the FP-PC1 coding sequence(Fig. 6F).

Isolation of deletion mutants shows that FWPV homologs ofSNAP, R31240_2, and PC-1 are nonessential for virus repli-cation in vitro. Putative FP-SNAP, FP-CEL1, and FP-PC1 de-letion mutants obtained by the transdominant selection meth-od were screened by PCR (Fig. 7) with flanking primers (forthe presence of a deleted gene) and with one or both primersinternal to the deletions (to exclude retention of a wild-typegene). Of six gpt-negative plaques obtained following recom-bination with pSL15, three carried only the AflII–BsmI-deletedFP-SNAP gene (Fig. 7A and B). Of five gpt-negative plaquesscreened after transdominant selection with pSL9, one carriedonly the BsmI–SnaBI-deleted SNAP gene (data not shown).Two of six gpt-negative plaques screened after transdominantselection with pSL17 carried only the SnaBI–ClaI-deletedFP-CEL1 gene (Fig. 7C and D). Following transdominantselection with deleted FP-PC1 genes, two of four gpt-negativeplaques carried only the BstEII–XbaI-deleted FP-PC1 genefrom pPC1.B (Fig. 7E and F), only one of eight carried only theXbaI-deleted FP-PC1 gene from pPC1.X (Fig. 7E and F), andone of two appeared to carry only the SpeI-deleted FP-PC1gene from pPC1.S (Fig. 7E). These results, showing isolationof deletion mutants for the FWPV SNAP, CEL1, and PC-1homologs, indicate that all three viral homologs are nonessen-tial for replication in tissue culture.

FIG. 5. FWPV homologs of SNAP, R31240_2, and PC-1 were present beforethe prolonged tissue culture passage history of plaque-purified isolate FP9. Theyare also found in other strains of FWPV but are not amplified from otheravipoxviruses by PCR. Reactions were performed with the primers described inMaterials and Methods; products were then analyzed by agarose gel electro-phoresis alongside size marker DNAs and stained with ethidium bromide. Tem-plate DNAs were from FWPV virulent precursor HP1; from FWPV intermediateHP1-200; from FWPV attenuated plaque-purified FP9; from commercial FWPVvaccines Poxine (PX), Websters FWPV M (FPV-M), and Chick-N-Pox; fromcanarypox virus strains 89 (Can89) and 229 (Can229); from pigeonpox virusstrains Peekham (PPV-P) and 950 (PPV950); from sparrowpox virus 9037(SPV9037); and from turkeypox virus (TPV). Sizes of the products are shown.

FIG. 6. Detection of FP-ANK2, FP-SNAP, FP-CEL1, FP-ANK3, and FP-PC1 transcripts in infected cells. The autoradiographs show the results of Northern blothybridizations of whole-cell RNA samples from CEFs with random-primed probes for FP-ANK2 (A), FP-SNAP (B), FP-CEL1 (C), FP-ANK3 (D), and FP-PC1 (E andF). Samples were from uninfected cells (C) or from cells at 4 h (E, E1, X, S, and B) or 24 h (L and L1) postinfection with FWPV at an MOI of 10. Infected cellswere incubated in the absence (E, L, X, S, and B) or presence (E1 and L1) of araC. Cells were infected with wild-type FWPV FP9 or the FP-PC1 XbaI-to-XbaI deletionmutant (panel F, lane X), the FP-PC1 SpeI-to-SpeI deletion mutant (panel F, lane S), or the FP-PC1 BstEII-to-XbaI deletion mutant (panel F, lane B). The arrowsindicate the positions of 28S and 18S rRNAs. The sizes of RNA markers are indicated in kilobases.

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Growth curves. No differences in the replication kinetics ofFP-SNAP, FP-CEL1, or FP-PC1 mutants were observed withsingle (MOI, 10)- or multiple (MOI, 0.001)-step infections inCEFs (data not shown), suggesting that these genes do not playsignificant roles in replication of the virus in tissue culture.Given the close proximity of adjacent genes in poxviruses, carehas to be taken to avoid disrupting the regulatory sequences ofneighboring genes. Currently, with no observable phenotypefor any of the deletions, this is not a problem; it would requireconsideration, however, if any phenotype should be detected,for instance, in vivo. With N- and C-terminal deletions forFP-SNAP, it should be possible to determine whether anyphenotype is due to deletion of FP-SNAP or to effects on theexpression of ANK2 or FP-CEL1, respectively. It is unlikelythat the deletion in FP-CEL1 would significantly affect tran-scription of FP-SNAP, the coding sequence of which ends 260bp upstream. It is also unlikely to affect expression of ANK3,the ORF for which starts 280 bp downstream. The SpeI dele-tion in FP-PC1 is a small internal deletion (leading to a frame-shift and immediate truncation), which is very unlikely to affecttranscription of neighboring genes. The BstEII-XbaI deletionin FP-PC1 is unlikely to affect expression of the upstream gene(ANK3), as the XbaI site is 450 bp downstream of the end ofthe ANK3 ORF. The BstEII-XbaI and XbaI deletions in FP-PC1 are unlikely to affect expression of the downstream gene,as the XbaI site is 150 bp before the end of the FP-PC1 ORF.

DISCUSSION

The sequence described here is located between 33 and 42kbp from the left end of the FWPV FP9 genome, flanking thejunction between BamHI fragments B and C (36). Mappingdata (not shown) is consistent with the 39 end of ORF-5/FP-PC1 being proximal to the left terminus of the genome andORF-1/ANK2 being distal. All of the genes in this segmentwould therefore be transcribed toward the left terminus. Thiswould be consistent with other poxviruses in which genes nearthe termini are transcribed toward the nearest terminus. Noneof the sequence described here has a counterpart in any of theother poxviruses for which sequence data has been reported,nor are there any reports of homologs in any other viruses ofany of the proteins encoded by the sequence reported here(although many genes encoding ankyrin repeat proteins havebeen identified in poxviruses).

An AT-rich core consensus sequence has been derived forvaccinia virus early promoters (17). Although no such consen-sus has been derived for FWPV, vaccinia virus promoters func-tion well in FWPV so it is assumed that similar constraints areapplied to FWPV promoters. Similarly, vaccinia virus late pro-moters contain the motif TAAAT(A/T/G) at the transcrip-tional initiation site (18). The sequence T5NT serves as aterminator for early transcription in vaccinia virus (72) andapparently in other poxviruses. No late transcriptional termi-nator which correlates with the heterogeneous length of latetranscripts has been identified. Both the FP-SNAP and FP-CEL1 genes have early terminators within their coding se-quences; FP-PC1 does not. Thus, it would be predicted thatFP-SNAP and FP-CEL1 are transcribed late, not early, andthat FP-PC1 is transcribed early and/or late. Candidate latepromoter sequences exist upstream of each of these genes.There are no candidate early promoter sequences in suitablepositions upstream of FP-SNAP and FP-CEL1, even allowingfor five mismatches from the 16-bp consensus vaccinia virus se-quence (AAAAAATGAAAAAAAA). Upstream of FP-PC1,however, there are three candidate early promoter sequenceswith start sites 13 to 20 bases upstream of the first predictedAUG codon. The core of each of these candidate promotershas five mismatches compared to the vaccinia virus consensus.Taken together, these data indicate that FP-PC1 would betranscribed early (and possibly late) as an mRNA of at least 2.5kb but that FP-SNAP and FP-CEL1 would be transcribed late.The results of Northern blotting of infected cell RNA, there-fore, correspond with this analysis for FP-PC1 (an abundant3-kb early mRNA and late transcripts of 2.8 to 5 kb), forFP-SNAP (no early transcripts and only weak late transcripts),and for FP-CEL1 (only late transcripts).

Conservation of the genes in different FWPV strains sug-gests that FP-SNAP, FP-CEL1, and FP-PC1 play importantroles in virus survival. That their deletion does not seem toaffect replication in vitro indicates that their roles are in virus-host interactions. It is unlikely that the loss of any one suchgene has a great effect on virus replication in vivo, so it will beimportant to know more about possible activities and targets.

Several roles could be postulated for the viral SNAP ho-molog. It may be required merely to supplement existing cel-lular supplies of SNAP to allow efficient replication of FWPV.Similarly, it may be required to replace cellular SNAP depletedas a consequence of the shutdown of host cell metabolismupon virus infection. It may, however, be required to interferewith the normal vesicle transport pathways, subverting them topermit virus replication or, alternatively, to inhibit vesicletransport, possibly as a way of preventing antigen presentationby MHC. Clearly the isolation of SNAP deletion mutants in-

FIG. 7. PCR analysis to screen putative FWPV FP9 mutants for deletions inFP-SNAP (A and B), FP-CEL1 (C and D), and FP-PC1 (E and F). Flankingprimers were used to show the presence of deleted genes (B, D, and E), anddeletion-internal primers were used to exclude the retention of wild-type genes(A, C, and F). DNA samples were from wild-type FP9 (wt), the appropriatedeletion plasmid (D), and gpt-negative putative mutants (1 and 2 for FP-SNAPand FP-CEL1; B1 and B2, X1 and X2, and S1 and S2 for BstEII-to-XbaI,XbaI-to-XbaI, and SpeI-to-SpeI deletions of FP-PC1, respectively). In all cases, 1is wild type and 2 is mutant. Controls were used with no template DNA (2ve) orwith a plasmid, pPC1.FL, containing full-length FP-PC1 (FL). The FP-SNAPmutants shown were produced by using pSL15 (AflIII to BsmI), and the FP-CEL1 mutants shown carry the SnaBI-to-ClaI mutation of pSL17. The sizes ofthe PCR products are shown in base pairs.

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dicates that any replacement or subversion function for SNAPin FWPV replication may not be absolute. Moreover, deletionof FP-SNAP did not result in any obvious changes to theprofiles of proteins secreted from the infected cell (data notshown).

Potential roles for the viral homolog of PC-1 are less evi-dent than for the SNAP homolog, mainly because the cel-lular role(s) for PC-1 is less well defined. PC-1 has been im-plicated in the inhibition of insulin receptor tyrosine kinase ininsulin-resistant patients with non-insulin-dependent diabetesmellitus (34). It has also been shown that PC-1 is coordinatelyup-regulated in activated T cells with two other ectoenzymesinvolved in nucleotide metabolism, CD38 NAD glycohydrolaseand CD73 59-nucleotidase (21). Autotaxin is able to hydrolyzeATP to ADP or AMP and AMP to adenosine (12). It is,therefore, able to perform the functions of CD39 apyrase, anATP diphosphohydrolase (E.C. 3.6.1.5 [68]), and CD73 ecto-59-nucleotidase (E.C. 3.1.3.5 [62]), converting ATP to adeno-sine, which can be taken up by cells (11). Acting alone or inconcert, these enzymes may provide cells with external supple-mentary sources of nucleosides. The presence in the FWPVgenome of thymidine and thymidylate kinases indicates thatthe intracellular side of the scavenger pathway can be benefi-cial. That it may be more important for FWPV than for vac-cinia virus is perhaps indicated by the observation that inser-tion into the thymidine kinase locus of attenuated FWPV isdifficult to obtain (51). We have, however, been unable to de-tect any difference between the replication rates of mutant andwild-type viruses in media which do or do not contain ATP(medium 199 or modified Eagle medium, respectively). Otherpotential roles for the PC-1 homolog must be speculative butmay include down-regulation of signal transduction (cf. PC-1in non-insulin-dependent diabetes mellitus [34]) or interfer-ence in interactions of the infected cell with the extracellularmatrix or with other cells (via extracellular phosphodiesteraseactivity). Of some relevance to this is the observation that dis-ruption of the exophosphodiesterase activity of autotaxin, bymutation of the catalytic threonine (residue 210) to alanine oraspartate, inhibits motility stimulation. This has been held to“open up the possibility of extra-cellular enzymatic cascades asa regulatory mechanism” (32).

Potential roles for FP-CEL1, the viral homolog of the un-known C. elegans proteins and of the orphan human proteinR31240_2, are even more mysterious. There is, as yet, no in-dication of potential roles, functions, or activities for the hu-man homolog of ORF-3, R31240_2, or for any of the C. eleganshomologs, but the protein from T. spiralis is known to be asecreted antigen. It is somewhat surprising that there is no suchclear homolog encoded in the completely sequenced genomeof S. cerevisiae, suggesting that it might have developed in me-tazoans. Given that the T. spiralis protein is secreted and giventhe indications that the homologs in C. elegans and humans arealso secreted (Fig. 3), it will be interesting to see if FP-CEL1 issecreted from infected cells and, if so, whether a ligand oractivity can be identified.

The origins of such clear homologs of cellular genes inFWPV are unknown. As the splicing pattern of the animalhomologs of FP-CEL1 is well conserved from humans down tonematodes, it seems most likely that the viral gene (which hasno introns) was acquired by insertion of cDNA that had beencopied from mRNA by reverse transcriptase from endogenousor exogenous retroviruses. It is tempting to speculate that theability of poxvirus genomes to carry integrated retroviral pro-viruses, which can then produce infectious retrovirus (as shownrecently for reticuloendotheliosis virus in FWPV [29]), couldfacilitate this process. Whatever the exact mechanism, the re-

lationship of the viral genes to the cellular genes would be oneof xenology. Until viruses more closely related to FWPV thanvaccinia virus, variola virus, and molluscum contagiosum virusare sequenced, it will not be clear whether these homologswere acquired by FWPV or by a progenitor. More extensivesequence analysis will also be required to establish whetherthese homologs were acquired within the avipoxvirus lineageafter divergence from the mammalian poxviruses or whetherpreexisting genes were lost by the mammalian viruses.

The discovery of homologs of cellular SNAP and PC-1genes, not found previously in any other virus, in such a smallpart of the genome of FWPV is intriguing. It remains to beseen whether such homologs play a role in virus-host relation-ships unique to the avian host. It also remains to be seenwhether the rest of the genome of FWPV and other avipoxvi-ruses will yield other such surprising homologies.

ACKNOWLEDGMENTS

We are indebted to Mike Boursnell and Matthew Binns for settingup the Fowlpox group at the Institute for Animal Health (IAH).Without their work and the resources they provided, none of this workcould have taken place. We are grateful to Mike for initially pointingout the homology of MFP504 with PC-1.

This work was supported by BBSRC funding to the IAH. M.A.A.was supported by a BBSRC special studentship.

ADDENDUM IN PROOF

Two independent sequences entered into the genome databases since submission of this article show .95% amino acididentity to the human homolog of FP-Cell, R31240_2. Both ofthese sequences (G2895897 and G2921839) have been identi-fied as DNase II (E.C. 3.1.22.1). There has also been an inde-pendent report of the cloning of human DNase II (T. Yasuda,H. Takeshita, R. Iida, T. Nakajima, O. Hosomi, Y. Nakashima,and K. Kishi, J. Biol. Chem. 273:2610–2616, 1998).

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