Sequence, Transcriptional Analysis, and Deletion of the Bovine Adenovirus Type 1 E3 Region

VIROLOGY

244, 173±185 (1998) VY989101

ARTICLE NO.

Sequence, Transcriptional Analysis, and Deletion of the Bovine Adenovirus Type 1 E3 Region Peter S. Evans,* MaÂria Benko Í,² BalaÂzs Harrach,² and Geoffrey J. Letchworth*,1 *Department of Animal Health and Biomedical Sciences, University of Wisconsin, 1655 Linden Drive, Madison, Wisconsin 53706; and ²Veterinary Medical Research Institute, Hungarian Academy of Sciences, P.O. Box 18, Budapest, H1581, Hungary Received January 8, 1998; returned to author for revision January 28, 1998; accepted February 19, 1998 The early 3 (E3) transcriptional unit of human adenoviruses (HAV) encodes proteins that modulate host antiviral immune defenses. HAV E3 sequences are highly variable; different HAV groups encode phylogenetically unrelated proteins. The role of the E3 region of many human and animal adenoviruses is unknown because the sequences are unrelated to previously characterized viruses and the functions of proteins encoded by these regions have not been studied. We sequenced a portion of the bovine adenovirus serotype 1 (BAV-1) genome corresponding to the putative E3 region. This sequence was substantially different from other adenoviral E3 sequences, including those of two other bovine adenoviruses. However, two regions of putative sequence conservation were identified. BAV-1 E3 sequences were identified in early and late transcripts, but, unlike HAV, introns were not detected in the E3 region transcripts. Like HAV E3, a majority of the BAV-1 E3 region was not essential for growth in cell culture, as demonstrated by the construction of a recombinant BAV-1 lacking 60% of the putative E3 region. © 1998 Academic Press

geographically and temporally diverse isolates of the same serotype (Hermiston et al., 1993). Variation observed between animal adenovirus E3 regions is similar to or greater than variation between human adenovirus E3A-expressed genes of different HAV groups. Although a few ORFs with homology to human adenovirus E3 ORFs have been identified, the level of similarity is low and functional conservation has not been demonstrated (Kring and Spindler, 1996; Mittal et al., 1996). An extreme example of E3 sequence variation is seen in members of the genus Aviadenovirus and the recently proposed genus Atadenovirus (Benko Í and Harrach, 1996). These viruses lack genes in the genomic region normally occupied by E3 (Vrati et al., 1995, Chiocca et al., 1996; Hess et al., 1997). Biochemical and genetic approaches demonstrated the existence of seven proteins encoded by the HAV group C E3 region and deduced functions for five of these proteins (reviewed in Wold et al., 1995). Based upon the functional grouping of other adenoviral transcriptional units (E1 encodes viral transactivators, E2 encodes the DNA replication machinery, the major late region encodes structural proteins of the virion), the E3 region is thought to consist of a gene ``cassette,'' expressing a variety of activities that disable or impede components of host immunity (Wold and Gooding, 1991). E3encoded proteins are responsible for preventing peptideloaded MHC class I molecules from reaching the surface of infected cells (Kvist et al., 1978), down-regulation of the receptors for epidermal growth factor, insulin, insulin-like growth factor (Kuivinen et al., 1993), and the Fas ligand (Shisler et al., 1997), protection of infected cells from the effects of tumor necrosis factor (Gooding et al., 1988, 1991), inhibiting the expression of immunodominant pro-

INTRODUCTION Members of the Adenoviridae family infect a wide variety of vertebrate hosts. More than 47 human adenovirus (HAV) serotypes are divided into six groups, based primarily upon hemagglutination properties. Clinical symptoms of human infections are largely dependent upon serotype (Hierholzer, 1995). Ten bovine adenovirus (BAV) serotypes are divided into two subgroups based upon biological properties (Bartha, 1969). BAV-1, along with BAV serotypes 2, 3, and 9, belong to subgroup 1 and share a complement-fixing antigen with other mastadenoviruses. BAV infections of cattle have been linked to keratoconjunctivitis and pneumoenteritis and, recently, with outbreaks of hemorrhagic enterocolitis (Horner et al., 1989; Adair et al., 1996). Many infections must be subclinical because seropositivity in the absence of obvious disease is widespread (Mohanty, 1971). HAV early 3 (E3) genes are transcribed into two sets of mRNAs, designated E3A and E3B, that originate from a single point but differ in polyadenylation site. While E3Bexpressed genes are conserved among isolates of group A, B, C, D, and F adenoviruses, E3A-expressed genes encode cladistically unrelated polypeptides (Bailey and Mautner, 1994). Although evolutionary variation between adenovirus groups appears to be large, there is evidence that the E3A 19K glycoprotein is well conserved among

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned Accession No. AF038868. 1 To whom correspondence and reprint requests should be addressed. Fax: (608) 262-7420. E-mail: [email protected]. 173

0042-6822/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. BAV-1 E3 region maps. (A) BamHI, EcoRI restriction map of the BAV-1 genome (based upon Benko Í and Harrach, 1990). 1 map unit 5 ;350 bp. (B) The sequenced region, along with the positions of the putative BAV-1 E3 promoter (bp 348) and polyadenylation signal (bp 2279), is shown. The binding positions of BAV-1-specific oligos used in this study (Table 1) are also shown. (C) Unidentified reading frames (URFs), $ 25 codons, and not located within another URF are depicted as open boxes. All URFs begin with a methionine codon, except URF 1 7, which begins with a serine codon. F1 through F6 refer to reading frames 1±3 on the top and 1±3 on the bottom strand, respectively. Shaded boxes indicate the positions of the BAV-1 pVIII and fiber homolog genes (reading frames 1 and 4, respectively). The fiber gene continues past bp 2607.

teins (Zhang et al., 1994; MuÈllbacher, 1992), and encouraging apoptotic cell death in infected cells, presumably to aid in the release of virions (Tollefson et al., 1996). The proposed role for E3 is consistent with the phenotype of HAV E3 deletion mutants. In animals, these viral mutants elicit an increased inflammatory response (Ginsberg et al., 1989), but there is no effect upon viral growth in vivo or in vitro (Kelly and Lewis, 1973). The purpose of this paper is to describe the putative E3 region of bovine adenovirus type 1 (BAV-1), a virus isolated from an apparently healthy cow (Klein et al., 1959). We determined the BAV-1 E3 region sequence and compared it to other adenoviral E3 regions, as well as to sequences in protein and nucleic acid databases. We determined the structure of transcripts that cross the BAV-1 E3 region in order to locate putative BAV-1 E3 genes. Last, we used intracellular homologous recombination to delete 60% of the E3 region. Viruses carrying such deletions are viable in cell culture, indicating that the BAV-1 E3 region is not essential for growth in vitro. RESULTS The BAV-1 E3 Sequence BamHI-derived fragments of the BAV-1 strain 10 genome were cloned and oriented by restriction analysis and comparison to the published BAV-1 restriction map (Benko Í and Harrach, 1990). The putative BAV-1 E3 region

was located on the BamHI ``B'' and ``C'' fragments by comparison of the BAV-1 map (Fig. 1 and Benko Í and Harrach, 1990) and the published location of the BAV-3 E3 region (Mittal et al., 1993). The resulting sequence was deposited in GenBank under Accession No. AF038868. The sequence was 2607 bp and included the entire pVIII gene and the N-terminal 46 codons of the fiber gene. The assignment of BAV-1 reading frames to these adenovirus genes is based upon similarity with published human and animal adenovirus sequences. The BAV-1 pVIII homolog was encoded on the top strand by bp 1±666. Gapped-BLAST comparison of this reading frame to the ``nr'' database produced highly significant matches with the pVIII genes of BAV-2 (Expect value 5 1 3 10267), BAV-3 (Expect value 5 1 3 10261), HAV-2 (Expect value 5 1 3 10277), as well as other pVIII genes of human, canine, porcine, ovine, and avian adenoviruses. Gapped-BLAST was also used to identify the Nterminal-coding portion of the putative BAV-1 fiber gene (bp 2470±2607). Matches with BAV-3 (Expect value 5 1 3 1028) and HAV-2 (Expect value 5 3 3 1026) as well as other adenovirus fiber genes were observed (data not shown). The sequence bordered by the pVIII and fiber genes (base pairs 667±2469, corresponding to BAV-1 genome map units 75.2±80.4) represented the putative BAV-1 E3 region. This sequence accounted for 5% of the total genome (Benko Í et al., 1988) and consisted of 47.5% G 1

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C pairs. A putative E3 TATA box, 348-TATAAAA-354, was located on the top strand within the predicted pVIII gene. Another TATA sequence, 2271-TATAAA-2276 was located on the top strand, 200 bases upstream from the fiber gene. Additionally, a partially conserved ``lymphoid enhancer'' sequence, 266-AGCTTCCC-273, thought to be important for binding of the NF-kb transcriptional factor and E1A-independent activation of the E3 promoter (Williams et al., 1990), was identified upstream of the first TATA box. A potential polyadenylation signal, AATAAA, was identified on the top strand beginning at bp 2279. Two alternative polyadenylation sequences (TATAAA, Mason et al., 1985) were found on the top strand (starting at bp 348 and 2271). The computer program NETGENE (Brunak et al., 1991) predicted a splice donor site at bp 1100 with high confidence. However, this prediction was discounted because the NETGENE algorithm had a combined sensitivity of 33% for splice donors and acceptors and a 94% false prediction rate when tested on a HAV-5 E3 sequence containing physically mapped splice donors and acceptors (Cladaras et al., 1985, data not shown). A map of the BAV-1 genome showing the position of the E3 region, unidentified reading frames (URFs), pVIII and fiber genes, as well as the binding site of oligos used in this study, is shown in Fig. 1. The BAV-1 E3 sequence was analyzed using the GCG programs TESTCODE (Fickett, 1982), which looked for variations in sequence composition with a periodicity of 3, and CODONPREFERENCE (Gribskov et al., 1984), which compared BAV-1 E3 codon usage to a codon preference table composed from 968 bovine genes. Neither approach identified BAV E3 genes (data not shown). The BAV-1 E3 sequence (minus the pVIII and fiber encoding portions) was compared to proteins in the GenBank ``nr'' database using the gapped-BLASTx algorithm (Altshul et al., 1997). BLAST searches were performed with a series of symbol comparison tables (BLOSUM62 and the PAM 40, 120, and 250 tables). BAV-1 E3 URF 1 5 was similar to reading frames located in the E3 sequences of bovine adenovirus serotypes 2 and 10 (Esford and Haj-Ahmad, 1994; Matiz et al., manuscript submitted), as well as genes located in porcine and canine adenovirus E3 regions, but not in BAV-3. Based upon their position with respect to the pVIII gene, these sequences were expected to be similar to the HAV-5 E3A 12.5 K protein and to similar proteins encoded by HAV groups A, B, C, and D, but not F. Homologs (12.5 K) of BAV-1, BAV-10, CAV-1, PAV-3, and HAV-5, -3, and -12 encode potential proteins with similar molecular weights (average 5 12,907 kDa 6 5% standard deviation) and isoelectric points (average 5 7.13 6 7%). The GES index (Engelman et al., 1986) predicted that the BAV-1 12.5 K protein will be mostly hydrophilic, except for a portion of the C-terminus (data not shown). Potential 12.5 K homologs from human and animal adenoviruses were

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aligned (Fig. 2A), and their phylogenetic relationship was calculated (Fig. 2B). The BAV-1 E3 URF 1 7 was similar to the BAV-2 E3 ORF 10 (Fig. 3). Both reading frames are predicted to encode proteins with hydrophobic, membrane-spanning domains near the C-terminus (data not shown). URF 1 7 also had limited similarity to some nonadenoviral protein sequences. Using gapped-BLASTx with the BLOSUM62 symbol comparison matrix, URF 1 7 had similarity with human CD33, a sialic acid-dependent cell adhesion protein expressed on the surface of myelomonocytic cells (SwissProt P20138, Freeman, et al., 1995). Other matches were with mouse vascular cell adhesion molecule-1 (SwissProt L22355) and rat cell adhesion molecule-105 (PIR S23969) (data not shown). The structure of BAV-1 E3-transcribing mRNAs Single-stranded DNA oligos (59 end-labeled) were used as probes to identify RNA species transcribed from the top strand of the BAV-1 sequence. Based upon the location of the pVIII and fiber genes, this strand corresponded to the rightwardly transcribed (r-) strand of human adenoviruses. Single-stranded oligo probes corresponding to the bottom strand of bp 453± 482 (oligo 21), 1343±1372 (oligo 27), and 2240±2270 (oligo 25) each recognized three bands (;2, ;3, and ;6 knt) in total RNA extracted from BAV-1-infected MDBK cells and a single band of ;2 knt in RNA extracted from BAV-1-infected, AraC-treated MDBK cells (Fig. 4A). The AraC-sensitive bands and the AraC-resistant band were initially detected by oligo 21 in RNA extracted 12 h after infection. The peak level of accumulation appeared between 24 and 48 h after infection (Fig. 4B). As a control, total RNA from BAV-1infected cells was probed with oligo 8 (59-CCCACTTTAACTGAAAGTGC), which corresponded to the bottom strand of the fiber gene. Oligo 8 recognized a single band in RNA extracted at 18 h after infection, but no bands at 12 h after infection. The mRNA species recognized by oligo 8 was sensitive to 0.05 mg/ml AraC (Fig. 4B), suggesting that it is different from the AraC-resistant band recognized by oligo 21. Oligo 21 was also used to determine the 59 end of the AraC-resistant transcript in S1 and primer extension experiments. A 266-nt S1 probe, representing the BAV-1 E3 bottom strand (bases 217±482), was synthesized by asymmetric PCR using 59 end-labeled oligo 21 as a primer and a Bgl1 fragment of the BAV-1 BamHI ``B'' fragment as a template. RNA from BAV-1-infected AraCtreated cells protected three S1 probe fragments, each of approximately 100 nt. The entire S1 probe was protected by RNA extracted from cells infected in the absence of AraC (Fig. 5A). By primer extension analysis, extension of 59 end-labeled oligo 21 produced three closely spaced bands of ;100 nt when RNA from BAV-1-infected, AraCtreated cells was used as template. When RNA from

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FIG. 2. Alignment of potential 12.5K homologs. (A) Clustal V alignment (Higgins and Sharp, 1989). The settings were: K-tuple value: 1, gap penalty: 5, window size: 10, filtering level: 2.5, open gap cost: 10, unit gap cost: 10. Fully conserved residues are in bold letters. The GenBank identification numbers are AF038868 (Accession number), bp 659±1024 (BAV-1); g913095, bp 685±873 (BAV-2), Krisztina Ursu et al., unpublished (BAV-10); g210036, bp 203±553 (CAV-1), g1732265, bp 24920±25273 (CAV-2); g1146145, bp 665±1012 (PAV-1); g1146148, bp 665±1021 (PAV-2); g606653, bp 665±1015 (PAV-3); g313361, bp 26313±26627 (HAV-12); g209901, bp 410±724 (HAV-3), g762955, bp 627±941 (HAV-7), g984529, bp 687±1001 (HAV-35); g209811, bp 27899±28216 (HAV-2); g58503, bp 528±849 (HAV-5); g1279432, bp 627±941 (HAV-19a). (B) The phylogenic relationship of BAV, CAV, PAV, and HAV group A (serotype 12)-, B (serotypes 3, 7, 35)-, and D (serotype 19)-encoded homologs of the HAV group C (serotypes 2, 5) 12.5 K protein calculated by distance matrix analysis. The PROTDIST (using Dayhoff's PAM 001 scoring matrix) and FITCH programs (using Global rearrangements option) were applied from the PHYLIP 3.5c program package (Felsenstein, 1989). The tree is unrooted. The bar represents a genetic distance of 10% between sequences in the alignment. Bootstrap values determined after studying 100 data sets are indicated at each node.

cells infected without AraC was used, longer extension products were seen (Fig. 5B). The six bands identified in these experiments defined a region on the top strand between bp 374 and 384 as the cap site for the putative BAV-1 E3 transcript (Figs. 5C and 5D). This region corresponded to 27 and 37 bases downstream from the TATA box at bp 348 (Fig. 5D). The 39 end of the AraC-resistant transcript was mapped by reverse transcriptase PCR and confirmed by sequencing. BAV-1 E3 cDNAs primed with the anchorpoly(T) oligonucleotide were amplified with the anchor primer in conjunction with oligos 5 and 15. The oligo 5 1 anchor primer pair amplified a 1308-bp band, and the oligo 15 1 anchor primer pair amplified a 732-bp band (Fig. 6A). These RT±PCR products were not observed when the total RNA preparation was treated with RNase A prior to RT±PCR. The resulting PCR product sizes are consistent with a single polyadenylation site downstream from the canonical polyadenylation signal at bp

2279 (Fig. 6B). Sequencing of the RT±PCR products with oligo 24 located this polyadenylation site at base 2298, or 14 nucleotides downstream from the AATAAA polyadenylation signal (data not shown). To probe for internal introns, the AraC-resistant E3 transcript was reverse-transcribed and amplified with a series of positive- and negative-strand oligo primers (Fig. 7). Differences in the sizes of PCR products amplified from BAV-1 genomic DNA or cDNAs would suggest the presence of an intron located between the primer binding sites. Most products amplified from cDNA and viral genomic DNA templates were of identical size, suggesting the absence of introns. Small differences in the migration of RT±PCR products using the primer pairs of 23 1 6 and 23 1 25 were not confirmed using other primer pairs (5 1 25 and 15 1 25) that amplified the same portion of the viral transcript (data not shown). Control PCRs using primer pairs designed specifically to amplify viral DNA (primer pair 23 1 9) or cDNA (primer

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FIG. 2ÐContinued

pair 23 1 anchor) amplified products of the anticipated size from the expected template (Fig. 7). Partial deletion of the BAV-1 E3 region To determine whether the E3 region was required for viral replication in cell culture, the EcoRI±BamHI fragment of the BAV-1 genome, containing bp 750±1838 of the BAV-1 E3 sequence, was replaced by a transgene carrying the HSV TK downstream from the HAV-2 mlp. Viruses carrying recombinant DNA were isolated based upon their ability to confer TK activity to the TK-negative

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BU100 cell line. This method has been used to insert foreign DNA into the genomes of other large DNA viruses (Roizman and Jenkins, 1985). The presence of a functional TK gene in the E3 region was confirmed with three experiments. First, the position of the mlpTK transgene in the BAV-1 genome was determined by digestion of purified viral DNA with BamHI and ApaI (Fig. 8A). The addition of the mlpTK transgene slowed the migration of the ;6.6-kbp BamHI ``B'' fragment by the expected amount (arrows in Fig. 8A) and introduced two ApaI sites into the ;18.2-kbp ApaI ``A'' fragment of the BAV-1 genome map (Benko Í and Harrach, 1990, Fig. 8A). Second, Bg1I-BamHI-, and EcoRI-derived fragments of recombinant BAV-1 (fragments A, B, D, and F in Fig. 8C) were recognized by the TK probe. The sizes of these fragments were consistent with the replacement of the BAV-1 E3 region with the mlpTK gene cassette. Third, plaque-purified recombinant BAV-1, as well as HSV1, but not wild-type BAV-1-1, showed a 100- to 1000fold drop in titer when plaqued on BU100 cells under an agarose overlay containing BrdU (Fig. 8D). BrdU is converted into an inhibitory form by TK-mediated phosphorylation. Recombinant BAV-1 was able to replicate in MDBK and BU100 cells to produce titers comparable to those of wild-type BAV-1 (see Fig. 8D, 0 mM BrdU), implying that the BAV-1 E3 region was dispensable. DISCUSSION The sequence presented here includes the entire BAV-1 pVIII gene and the N-terminal portion of the fiber gene. The assignment of these ORFs to adenovirus

FIG. 3. Alignment of the BAV-1 URF 1 7 with BAV-2 E3 ORF 10. Portions of BAV-1 URF 1 7 (GenBank AF038868, bp 1640±2272) and BAV-2 ORF 10 (GenBank g913095, bp 1891±2565) were aligned with the Clustal V algorithm (Higgins and Sharp, 1989), using the PAM250 symbol comparison matrix. For each alignment, the positions of residues that can be grouped by structural moiety are shaded, and the group is indicated above the alignment. The residue grouping (adapted from Von Heijne, 1987) is Ambivalent 5 (ACGPSTWY), External 5 (RNDQEHK), Internal 5 (ILMFV).

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FIG. 4. Northern blot analysis of BAV-1 E3 transcription. RNA extracted from mock (lane M)-, BAV-1-infected (lane B) and BAV-1-infected, AraC-treated (lane A) MDBK cells. (A) RNA was extracted at 24 h and separated on a 1.8% agarose/MOPS/formaldehyde gel. The membrane was cut into three pieces and probed with 32P end-labeled oligos 21, 27, and 25. The lanes marked with M and B were exposed to film for 2 h; the lanes marked with A were exposed for 23 h. The binding sites for these oligos are shown in Fig. 1. The migration of RNA sizing ladder bands is indicated on the right, and the estimated sizes of major RNA bands (;2, ;3, and ;6 knt) are indicated on the left side of the gel. (B) RNA was extracted at 0, 6, 12, 18, 24, and 48 h and separated on a 1.0% agarose/MOPS/formaldehyde gel. Two Northern blots were probed with 32P end-labeled oligos 21 and 8.

genes was based upon similarity with published human and animal adenovirus sequences. Protein VIII and fiber are expressed late during the infection cycle by spliced transcripts initiating downstream from the major late promoter. The HAV-2 pVIII gene encodes a 13-kDa polypeptide associated with the hexon protein in the virion capsid (Horwitz, 1990). The HAV-2 fiber gene encodes a 62-kDa protein that assembles as a trimer and extends outward from the viral capsid. The N-terminus of fiber contacts the penton base (Horwitz, 1990). Genes encoded by the top (rightwardly transcribed) strand, between the end of the pVIII reading frame and the beginning of the fiber reading frame, comprise the HAV E3 region. By analogy, we propose that the same portion of the BAV-1 genome is the E3 region. The size of this region of the genome varies significantly among known adenoviral isolates, from 3627 bp in group ``B'' human adenovirus serotype 35 to 196 bp of AT-rich, noncoding sequence in the ovine adenovirus isolate OAV-287 (Vrati et al., 1995). The BAV-1 E3 sequence, at 1809 bp, was intermediate in size and similar to sizes reported for the BAV-2 and BAV-3 E3 sequences (2206 and 1584 bp, respectively) as well as sequences for canine and porcine adenovirus E3s. Although human adenovirus E3B sequences are well conserved, there is extreme variation among HAV E3A sequences (reviewed in Bailey and Mautner, 1994). Animal adenovirus E3 homologs also show signs of sequence conservation and extreme variation. For example, three serologically distinct PAV isolates have remarkably similar E3 sequences (Reddy et al., 1996). On the other hand, BAV serotypes 1, 2, and 3 have remarkably different E3 sequences. In much of the E3 region, the level of nucleotide identity among the BAV-1, -2, and -3 sequences is similar to the level of identity expected for a completely unrelated sequence retaining the identical

base composition (data not shown). The extreme genetic variability of BAV E3 sequences could be due to the acquisition of entirely new genes by recombination. Inadvertent recombination between the adenoviral E3 region and the SV40 genome resulted in the first observations that E3 was nonessential in cell culture (Grunhaus and Horowitz, 1992). Alternatively, an ancestral BAV E3 region may have diverged by mutation in the absence of selection, implying that BAV E3 genes are not beneficial to the virus or detrimental to the host. In HAV group C viruses, most E3 region genes are implicated in protection of virally infected cells from host immune surveillance. Variation in the coding capacity of HAV E3 regions may reflect group-specific differences in tissue specificity and differences in local immunity at these sites (Wold et al., 1995). It is possible that E3 variation in animal adenoviruses could also reflect these differences. In the E3 region of the group C human adenoviruses, differential splicing of primary transcripts, readthrough of upstream reading frames, and translational re-initiation following termination of an upstream reading frame allows for the expression of seven E3 proteins (Wold et al., 1995). This suggested that the BAV-1 E3 region may also have a complex expression program. Thus, the structures of mRNAs transcribing the BAV-1 E3 region were determined in order to identify genes that are likely to be expressed in infected cells. Northern blot analysis revealed three top-strand transcripts that cross the E3 region. Two of these transcripts were sensitive to AraC, implying that they were late transcripts. Group C HAV late transcripts terminate after the L4 polyadenylation site located downstream from the end of the pVIII reading frame and upstream from all E3 genes except 12.5 K. However, some transcripts, containing tripartite leader exons, are spliced into acceptors downstream from the L4 polyadenylation site (Chow et al., 1979). In PAV sero-

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FIG. 5. Primer extension (PE) and S1 analysis of the BAV-1 E3 cap site. MDBK cells were infected with BAV-1, and RNA was extracted after 24 h. (A) For S1 analysis, RNA was combined with 1 3 105 cpm S1 probe (bottom strand, bases 217±482). S1 and PE products were separated on a sequencing-type gel. A sizing ladder (32P end-labeled HinfI fragments of fX174 phage) was run on each side of the gel. (B) For PE, RNA was combined with 32P end-labeled oligo 21 and extended with AMV Reverse Transcriptase. A sizing ladder was run on the left side of the gel. (C) S1- and PE-derived products were prepared with RNA from BAV-1-infected, AraC-treated MDBK cells as described in A and B. Plasmid 2-4 (see Materials and Methods) was sequenced with oligo 21 to produce the relevant, bottom-strand sequence. Sequencing, PE, and S1 products were run on the same gel. (D) The top strand of a portion of the BAV-1 E3 sequence (bases 365±390) is shown along with the position of major S1- and PE-derived products and their distance from the putative BAV-1 E3 TATA box (base 348).

types 1, 2, and 3, three E3 transcripts appear 6±8 h after infection, but these RNAs were not tested for AraC sensitivity (Reddy et al., 1996). The PAV RNA species could be homologous to the BAV-1 E3 transcripts identified in this study. PAV-1, -2, and -3, as well as BAV-1, -2, and -3 lack a putative L4 polyadenylation signal downstream from the end of the pVIII reading frame. Unlike the HAV-2 late transcripts, the AraC-sensitive BAV E3 transcripts appear to contain 2000 or 4000 nucleotides of upstream sequence. The initiation (cap) site of the AraC-resistant transcript was mapped to a 10-nt region of the top strand. Heterogeneity in the assignment of an AraC-resistant mRNA cap site could be due to the analytical methods employed or the existence of multiple cap sites. Both primer extension and S1 nuclease digestion of RNA/ DNA hybrids pointed to transcription initiation at bp 374 and 377. S1 analysis, but not primer extension, pointed to transcription initiation at bp 384. S1 mapping of the HAV-5 cap site (Cladaras et al., 1985) produced three or more nuclease-resistant fragments, the largest of which was judged to represent the cap site. By this criterion, the BAV-1 cap site should be

located at guanosine 374 (G374), which is indicated by both S1 and primer extension-derived products. Using RT±PCR and DNA sequencing, a single polyadenylation site of the AraC-resistant transcript was mapped to bp 2298, 14 bases downstream from an optimal polyadenylation signal. Thus, the length of the AraCresistant E3 transcript is 1924 bases, consistent with the results in Figs. 4A and 7A. Differential splicing of the HAV C E3 primary transcript is important for expression of multiple E3 proteins. RNA species recognized by three oligo probes on Northern blots were identical in size, which argued against splicing of the BAV-1 AraC-resistant transcript. However, as some of the HAV group C introns are small (i.e., , 400 nt in one case for group C HAV), they may not be detected by Northern analysis. RT±PCR was employed to find evidence of E3 splicing and of multiple polyadenylation sites. This technique was used to determine splicing patterns of a group B HAV (Basler and Horowitz, 1995). Using this method, internal introns were not found in the AraC-resistant transcript. The lack of introns was not due to RT±PCR amplification of unspliced precursors because Northern analysis did not indicate the presence of

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FIG. 6. Amplification of the BAV-1 E3 mRNA 39 end using RT±PCR. (A) RNA was extracted from BAV-1-infected, AraC-treated MDBK cells at 24 hpi, and cDNA was primed by the anchor-poly T oligo. PCR was performed with the anchor primer in combination with E3 top-strand primers 5 and 15. A DNA size ladder was run on the sides of the gel. (B) A map based upon the E3 sequence (top strand, bases 970±2650), including the position of putative polyadenylation signals, and the binding sites of oligos used in the RT±PCR experiment is shown along with the size of PCR products observed in the gel in A was based upon the migration of DNA size ladder bands.

such transcripts in total RNA preparations and because cDNA synthesis was primed with the anchor-poly(dT) oligo, which selectively binds mature mRNAs. The exis-

tence of exons located upstream from E3 and splicing into E3 exons was not ruled out by this experiment. However, results from the S1 nuclease mapping experiment, using a probe equivalent to nt 217±482, argued against a splice acceptor in this portion of E3 transcript. Additionally, the identical size of AraC-resistant RNA species recognized by three bottom-strand oligo probes in Northern blot analysis did not support the existence of a splice acceptor in the E3 region. These techniques may not be sensitive enough to rule out RNA species present in small quantities. The splicing of late (i.e., AraC-sensitive) E3 transcripts was not addressed by the RT±PCR experiments. However, late RNAs that transcribe the HAV-2 E3 region utilize many of the same splice acceptors, donors, and polyadenylation sites as early mRNAs (Chow et al., 1979). In the absence of differential splicing, an unusual mechanism may be responsible for expression of genes downstream from URF 1 5. Two mechanisms have been recognized in the HAV C E3 region. Most of the HAV C E3 genes are translated following readthrough of upstream reading frames 3.6 K and 11.6 K. Additionally, the product of the HAV group C E3B 14.5 K gene is believed to be translated from RNA f by re-initiation following translation of an upstream reading frame encoding the E3B 10.4 K protein (Wold et al., 1995). Due to the lack of conservation between the BAV E3 sequences, it was difficult to determine BAV-1 E3 genes with certainty. Two candidate reading frames appeared to be shared between the BAV-1 and BAV-2 E3 sequences. The BAV-1 URF 1 5 was similar to the BAV-2 E3 ORF 4. These reading frames may be distant homologs of the E3A 12.5 K protein encoded by the HAV group C viruses. It is interesting that the reading frame encoding

FIG. 7. RT±PCR analysis of splicing within the BAV-1 E3 (AraC-resistant) transcript. (A) RNA was extracted from BAV-1-infected, AraC-treated MDBK cells at 24 hpi. PCR was performed using aliquots of anchor-poly(T) oligo-primed cDNA preparation made from DNase-treated RNA (lane A), DNase- and RNaseA-treated RNA (lane B), or BAV-1 genomic DNA (lane C). DNA size ladders were run at the sides of each gel. (B) A map, based upon the E3 sequence, including the cap and polyadenylation sites of the AraC-resistant transcript (bp 374 and 2300, respectively) as well as the binding position of oligos used in the PCR.

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FIG. 8. Analysis of recombinant BAV-1. DNA from recombinant BAV-1 (lane T), as well as BAV-1-1 (lane B) and mock (lane M)-infected cells were digested with ApaI, BamHI, Bg1I, or EcoRI. Restricted DNA was run on 1.0% agarose gels, along with DNA sizing ladder. The sizes, in kbp, of DNA ladder bands are shown on the side of each gel. (A) One gel was stained with ethidium bromide and photographed. (B) One gel was transferred to a nylon membrane, probed with a 32P-labeled TK DNA fragment, and exposed to film. (C) A map of the predicted recombinant BAV-1 genome structure. The BamHI sites at map unit 59.7 and the EcoRI site at map unit 87.6 refer to the BAV-1 genomic DNA map (see Fig. 1, and Benko Í and Harrach, 1990). The position of the pVIII, fiber, polyadenylation site, and the inserted mlpTK transgene, as well as the predicted size of BamHI-, Bg1I-, BamHI 1 EcoRI-, or EcoRI-derived fragments from Fig. 8B are shown. (D) BAV-1-1, HSV1, and recombinant BAV-1 stocks were titrated on BU100 cells at 1021 through 1026 dilutions. Virus-infected monolayers were overlayed with media containing 0, 0.01, 0.1, or 1 mM BrdU. Plaques were fixed at 4 dpi.

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a 12.5 K homolog of a recently isolated adenovirus, BAV-10 (Horner et al., 1989), shares areas of similarity with both human and animal adenoviral 12.5 K sequences. The HAV group C 12.5 K protein can be immunoprecipitated from virus-infected cell culture (Hawkins and Wold, 1992). This protein is translated in small amounts from a rare, unspliced transcript. Expression of the 12.5 K protein is detected both early and late in infection. The function of the HAV group C 12.5 K is currently unknown, but it is not essential for viral growth in cell culture (Hawkins and Wold, 1992) or in a cotton rat model (Ginsberg et al., 1989). On the other hand, the C-terminal portion of the CAV-1 12.5 K homolog was partially deleted in an attenuated strain (Dragulev et al., 1991). A second candidate for a BAV-1 E3 gene was the URF 1 7, which showed significant sequence similarity to the BAV-2 ORF 10. BAV-1 URF 1 7 does not begin with a methionine codon, which may reflect a sequencing error or splice acceptor. The BAV-1 and BAV-2 sequences did not appear to be similar to other animal or human adenovirus E3 genes, but a portion of the BAV-1 sequence was similar to several proteins in the GenBank database. The low level of similarity observed between the BAV-1 reading frame with three cellular adhesion molecules is especially intriguing because adhesion may represent a novel target for inhibiting host immune surveillance. A third candidate for a BAV-1 E3 gene was URF 1 10. This reading frame was predicted to encode a protein containing as many as five transmembrane segments (data not shown), but lacks a signal peptide at the Nterminus. No potential URF 1 10 homologs were identified in the GenBank database or in adenovirus E3 sequences. One of the distinct characteristics of the human adenovirus E3 region is its nonessential role in viral growth in vitro (Kelly and Lewis, 1973) and in vivo (Ginsberg et al., 1989). This property has been exploited by genetic engineers, who have replaced the HAV group B and C E3 regions with foreign DNA. Such recombinant HAVs have been used for foreign gene expression in cell culture and as vaccine and gene therapy vectors in many vertebrates, including humans. It is unlikely that HAV E3 genes are truly nonessential since many of these genes are conserved. The role of animal adenovirus E3 regions in infection has not been determined; it may closely resemble that of human adenoviruses. To date, BAV-3based recombinant viruses have been constructed (Mittal et al., 1995), and these viruses are viable in cell culture and in a cotton rat model (Mittal et al., 1996). Ovine adenovirus recombinants have also been constructed (Vrati et al., 1996), but neither the location nor the existence of the OAV E3 has been determined. Our paper reports the construction of a BAV-1-based recombinant virus in which an EcoRI±BamHI fragment was deleted and replaced with foreign DNA. The position of

TABLE 1 BAV-1 Oligonucleotides Used in This Study Name

E3 position (nt)

Strand

5 6 9 12 15 21 22 23 24 25 27

977±994 1599±1580 2316±2300 1072±1058 1548±1562 482±452 684±655 382±396 2122±2142 2270±2241 1372±1343

Top Bottom Bottom Bottom Top Bottom Bottom Top Top Bottom Bottom

Note. Binding position refers to GenBank AF038868 and is listed from 5' to 3' end.

the mlpTK construct in the BAV-1 E3 region was confirmed by restriction enzyme and Southern blot analysis of viral DNA and by demonstrating that recombinant virus growth in TK-negative cells is inhibited by the presence of BrdU, a compound that is converted to a inhibitory metabolite by TK-catalyzed phosphorylation. Deletion of the EcoRI±BamHI fragment from the BAV-1 E3 region removed the C-terminal portion of the URF 1 5 (90 of 122 codons), all of URF 1 10, and the N-terminal portion of the URF 1 7 (81 of 226 codons). Many of the other top- or bottom-strand encoded URFs are deleted completely. This deletion did not prevent viral replication in cell culture. A BAV-1 E3-specific phenotype has not been discovered to date. Given the extreme sequence variation of E3 regions, the role of the BAV E3 region during infection of cattle may be novel. It may reveal new concepts in adenovirus±host relations. MATERIALS AND METHODS Reagents Oligonucleotide (oligo) primers corresponding to portions of the BAV-1 E3 sequence were synthesized by several suppliers and resuspended in water. The concentration was determined by spectroscopy (Maniatis et al., 1989). A list of oligos, along with their binding sites in the E3 sequence (GenBank Accession No. AF038868), is shown in Table 1. An anchor-poly(T) oligo (59-GACCACGCGTATCGATGTCGAC(T)16V, where V 5 A, C, G) and an anchor oligo (59-GACCACGCGTATCGATGTCGAC) were from Boehringer Mannheim (Indianapolis, IN). DNA and RNA molecular sizing ladders were purchased from GIBCO-BRL (Gaithersburg, MD) and Promega (Madison, WI). Cell and virus culture Viruses were grown in Madin±Darby bovine kidney (MDBK) cells (ATCC, CCL 22). BU100, an MDBK deriva-

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tive that lacks thymidine kinase activity (Bello et al., 1987), was kindly provided by Dr. W. Lawrence of the University of Pennsylvania. Cells were grown in Eagle's modified MEM (MEM) supplemented with 5% fetal bovine serum (FBS, HyClone, Logan, UT). BU100 cells were maintained in media supplemented with 0.3 mM BrdU but BrdU was removed 1 passage prior to experiments. Serum lots were tested for BAV-1-neutralizing activity by plaque reduction assay. BAV-1 strain 10 (Klein et al., 1959) was purchased from ATCC. This virus was passaged twice on MDBK cells to generate sufficient viral DNA for molecular cloning. The ATCC stock also was purified by two successive endpoint dilutions on MDBK cells to generate a cloned population (designated BAV-1-1), which was used for transcriptional studies and to produce recombinant viruses. Cells were infected with virus in a minimal volume (10 to 13 mL per cm2) for 1 h. For virus titration, infected cell monolayers were covered with MEM containing 0.5% agarose, 25 mM MgCl2 (Williams, 1971), and 5% FBS. After 4 days, plaques were fixed with 10% formaldehyde and stained with crystal violet. AraC was added to infected cells at 0.05 mg/ml to inhibit late gene expression (Mittal et al., 1993). Cloning and sequencing of the BAV-1 E3 region BAV-1 genomic DNA was extracted from infected MDBK cells by a modification of the ``Hirt'' technique (Shinagawa et al., 1983), digested with BamHI, and cloned into BamHI-linearized Bluescript (KSI) (Stratagene). Clones carrying BAV-1 BamHI fragments were identified by b-galactosidase (blue/white) screening, restriction enzyme analysis, and sequencing. ExoIII and Bal31 exonuclease-derived subclones were prepared by standard techniques (Maniatis et al., 1989). Plasmid clones were sequenced on both strands by the dideoxynucleotide termination method. The entire nucleotide sequence was determined at least twice for both strands and was assembled and analyzed with Lasergene (DNASTAR, Madison WI) and Genetics Computer Group (GCG, Madison, WI) software programs. Phylogenetic calculations were performed by the PHYLIP 3.5c program (Felsenstein, 1989). RNA extraction, Northern blotting, primer extension, S1 nuclease, and RT±PCR analysis Total RNA for S1, primer extension, and RT±PCR analysis was extracted from BAV-1 and mock-infected cells by cesium chloride gradient centrifugation of guanidinium-treated cell lysates (Chirgwin et al., 1979). Primer extension and S1 nuclease assays were performed as described (Kingston, 1989; Greene and Struhl, 1989). For primer extension, 20 mg of total RNA was used as template, and the 59 end-labeled oligo was extended at 42°C. For S1 nuclease analysis, a gel-purified, 32P end-labeled, single-stranded DNA

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probe was hybridized to 20 mg total RNA in solution containing 80% deionized formamide at 30°C overnight. Hybrids were digested with 150 units of S1 nuclease for 1 h at 30°C. Primer extension and S1 nuclease digestion products were separated on a 6% sequencing gel. For RT±PCR analysis of E3 transcripts, total RNA was treated with DNase (100 unit/ml, 15 min). To demonstrate the absence of viral DNA, some samples were treated with RNase A (0.8 mg/ml, 15 min). The remaining nucleic acids were phenol/chloroform extracted and concentrated by ethanol precipitation. Complementary DNA synthesis was primed with an anchor-poly(T) oligo at 52°C. PCR was performed by cycling 30 times between 94°C (30 s), 55°C (1 min), and 72°C (3 min). Total RNA for Northern blot analysis was extracted with phenol and chloroform from acidic guanidiniumtreated lysates (Chomczynski and Sacchi, 1987). Equivalent amounts of RNA were separated on denaturing agarose gels as described (Thurston et al., 1988) and blotted to nylon membranes in 103 SSC overnight (203 SSC is 3 M NaCl, 0.3 M Na citrate, pH 7.0). Blots were soaked in prehybridization solution (Severson, 1997) and exposed to 32P 59 end-labeled oligonucleotide probes overnight at 42°C or a 32P internally labeled restriction fragment at 65°C. Blots were washed at hybridization temperature with 23 or 0.23 SSC containing 0.1% SDS. Construction of E3-deleted derivatives of BAV-1 Intracellular homologous recombination between a plasmid and viral DNA was used to delete the BAV-1 E3 region and replace it with a gene cassette containing the thymidine kinase gene of herpes simplex virus (HSV TK) driven by the HAV-2 major late promoter (HAV-2 mlp). To allow for homologous recombination with the BAV-1 genome, this gene cassette (called mlpTK) was assembled in a pBluescript I (KS) plasmid (Stratagene, LaJolla, CA) flanked by the EcoRI ``C'' and BamHI ``C'' genomic fragments of BAV-1. The plasmid, called 99-1, was constructed in the following steps: Plasmid ATS, a pBluescript I (KS)-based plasmid containing a 528-bp, TaqI± Sau3A fragment of HAV-2 (a generous gift of Dr. P. Farnham, University of Wisconsin) was digested with TaqI and PvuII to release a 292-bp fragment (corresponding to bp 5780±6071 of the HAV-2 genome, GenBank g209811). This fragment, which contains the HAV-2 mlp, was subcloned into AccI- and EcoRV-digested pBluescript I (KS) to produce plasmid 49-3. The single HincII site in the pBluescript I(KS) polylinker was destroyed by this cloning step. A 57-bp TaqI±TaqI fragment of pBluescript I (KS) (bp 736±792 of GenBank g209811) inadvertently ligated into the AccI and TaqI sites, thus creating another HincII site in plasmid 49-3. The HSV1 TK ORF was PCR-amplified from the cloned HSV1 EcoRI ``N'' fragment (plasmid SG87, a generous gift of Dr. R. Kintner and Dr. Curtis Brandt, University of Wisconsin) using

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primers 17 (59-CGCGAATTCACCATGGCTTCGTACCCCTGCC) and 18 (59-CGCGGATCCTCAGTTAGCCTCCCCCATCTCC). These PCR primers incorporated EcoRI and BamHI sites into the 59 and 39 ends of the ORF, respectively. Both plasmid 49-3 and the HSV TK PCR product were digested with EcoRI and BamHI and ligated to produce plasmid 52-4. A 1434-bp, HincII-, BamHI-derived fragment of plasmid 52-4, carrying the mlpTK transgene, was cloned into SmaI-, BamHI-digested plasmid 23-1, which carried the BAV-1 EcoRI ``C'' fragment (flanking E3 on the left) in pBluescript I (KS). The product of this ligation was designated plasmid 89-1. The ;4950-bp BAV-1 BamHI ``C'' fragment (flanking E3 on the right) was gel-purified from a BamHI digest of plasmid 2±4, a pBluescript I (KS)-based plasmid which carried only this fragment, and ligated with BamHI-linearized plasmid 89-1 to produce plasmid 99-1. The orientation of the BamHI ``C'' fragment was determined by EcoRI digestion. To produce recombinant virus, the TK-deficient BU100 cells were electroporated (Invitrogen Electroporator II, 450 V, 250 mF capacitor, cells washed and suspended in MEM without FBS) in the presence of 20 mg plasmid 99-1. The following day, these cells were infected with BAV-1-1. Viruses carrying the mlpTK transgene were selected by passaging the progeny virus three times on BU100 cells in the presence of HAT media (100 mM hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine in MEM1 5% FBS) followed by three rounds of plaque purification on MDBK cells. DNA from recombinant viruses was extracted from infected MDBK cells, digested with restriction enzymes, and separated by agarose gel electrophoresis. For Southern analysis, the restricted DNA was transferred to nylon membranes and hybridized to a 32P-labeled TK probe. The TK probe was PCRamplified with plasmid SG87 using oligos 17 and 18 as primers and labeled with 32P by nick translation (Maniatis et al., 1989). ACKNOWLEDGMENTS We thank Jared Fishel and Matthew Hancock for excellent technical assistance and Drs. Lee Martin, Debra MacKenzie, Rebecca Pearlman, and members of the Letchworth laboratory for helpful suggestions. Research support was provided by USDA Special Grant 89-3416-4853, USDA National Research Initiative Grants 92-37204-7900 and 9437204-0857, a USDA Animal Health grant, and a Shaw scholarship from the Milwaukee foundation to G.J.L., and the National Research Fund of Hungary OTKA T016882. Salary support to P.S.E. came from the University of Wisconsin graduate school and the Cell and Molecular Biology program training grant.

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