Complete Nucleotide Sequence and Genome Organization of Bovine Parvovirus


We determined the complete nucleotide sequence of bovine parvovirus (BPV), an autonomous parvovirus. The sequence is 5,491 nucleotides long. The terminal regions contain nonidentical imperfect palindromic sequences of 150 and 121 nucleotides. In the plus strand, there are three large open reading frames (left ORF, mid ORF, and right ORF) with coding capacities of 729, 255, and 685 amino acids, respectively. As with all parvoviruses studied to date, the left ORF of BPV codes for the nonstructural protein NS-1 and the right ORF codes for the major parts of the three capsid proteins. The mid ORF probably encodes the major part of the nonstructural protein NP-1. There are promoterlike sequences at map units 4.5, 12.8, and 38.7 and polyadenylation signals at map units 61.6, 64.6, and 98.5. BPV has little DNA homology with the defective parvovirus AAV, with the human autonomous parvovirus B19, or with the other autonomous parvoviruses sequenced (canine parvovirus, feline panleukopenia virus, H-1, and minute virus of mice). Even though the overall DNA homology of BPV with other parvoviruses is low, several small regions of high homology are observed when the amino acid sequences encoded by the left and right ORFs are compared. From these comparisons, it can be shown that the evolutionary relationship among the parvoviruses is B19AAVBPVMVM. The highly conserved amino acid sequences observed among all parvoviruses may be useful in the identification and detection of parvoviruses and in the design of a general parvovirus vaccine. 

The mammalian parvoviruses are divided into two genera on the basis of the requirement for a helper virus for productive infection. The defective parvovirus, adenoassociated virus (AAV), requires coinfection with adenovi- rus or herpesvirus. The autonomous parvoviruses, which include bovine parvovirus (BPV), canine parvovirus (CPV), feline panleukopenia virus (FPV), H-1, Kilham rat virus (KRV), LuIII, and minute virus of mice (MVM), do not require a helper virus for infection (51). 
Parvoviruses are DNA viruses with a linear single-stranded genome of ca. 5,000 nucleotides (nt). The complete nt sequences of AAV2 (serotype 2 of AAV) (50), MVMp (prototype of MVM) (4), MVMi (immunosuppressive variant of MVM) (3,45), H-1 (43), and the partial nt sequences of CPV2 (type 2 of CPV) (42) and FPV (15) have been deter- mined. Also, transcription has been studied in detail for AAV2, H-1, and MVMp (16). From these studies, several common features of the parvovirus genomes are apparent. (i) There are two large open reading frames (ORFs) within the plus strand (the strand with the same polarity as the mRNAs). (ii) The mRNAs from both ORFs are polyadenyl- ated and 3' coterminated at about map unit (m.u.) 95. (iii) 
The left ORF encodes one or more noncapsid proteins which are necessary for viral DNA replication (17,23,43,52). (iv) The right ORF encodes the major capsid proteins of the virus as a nested set (4,26,43). 
BPV is an autonomous parvovirus. Four virus-specific proteins have been reported: three capsid proteins (VP1, VP2, and VP3) of Mr 80,000, 72,000 and 62,000 and one noncapsid protein (NP-1; Mr 28,000) (30, 32). However, BPV shows very little homology with other autonomous par- voviruses (FPV, H-1, KRV, LuII, and MVM) either by heteroduplex mapping or by immunological cross-reactivity of the capsid proteins, whereas the others as a group have significant homology among themselves (5,48). 
In this paper, the complete nt sequence of BPV is pre- sented. Genome organization, possible coding regions for the viral proteins, and comparison with other parvoviruses are discussed. The sequence of BPV has several small regions of conservation with other parvoviruses which ap- pear to be useful for understanding the evolutionary relation- ship of parvoviruses, in the design of vaccines, and for the clinical detection of parvoviruses. 
Recently the nucleotide sequence of B19, a human autonomous parvovirus, was published (47). The relationship of B19 with other parvoviruses is presented in the section "Comparison with B19" at the end of the Discussion. 

MATERIALS AND METHODS

Materials. Restriction enzymes were purchased from Be- thesda Research Laboratories, Inc. (Gaithersburg, Md.), and New England BioLabs, Inc. (Beverly, Mass.). Esche- richia coli DNA polymerase I (Klenow fragment) and universal sequencing primer were obtained from Bethesda Research Laboratories. Deoxynucleotides and dideoxynucleotides were purchased from Pharmacia P-L Biochemic- als, Inc. (Piscataway, N.J.). Terminal deoxynucleotide transferase, [a.-355]dATP and [a-35S]ddATP were obtained from New England Nuclear Corp. (Boston, Mass.). Various synthetic oligonucleotide primers for DNA-sequencing reac- tions were the kind gift of R. Foote, Oak Ridge National Laboratory. 
Cell culture and virus propagation. BPV was propagated in bovine fetal lung cells grown in monolayer culture and maintained in Eagle minimal essential medium supplemented with 10% fetal calf serum as described by Parris and Bates (39). 
Isolation of viral DNA. Single-stranded viral DNA was purified from virions isolated from BPV-infected cells. In- 1085 fected cells were frozen, thawed, and centrifuged at 15,000 x g for 15 min. The supernatant fraction was then centri- fuged at 25,000 rpm in a Beckman SW27 rotor for 3.5 h at 4°C to pellet the virus. The pellet was resuspended in 50 mM Tris hydrochloride, pH 8.0, and loaded on a CsCl step gradient (CsCl [2 ml at 40%, 4 ml at 35%, and 2 ml at 30%] overlaid with 1 M sucrose in the same buffer), and the gradient was centrifuged at 34,000 rpm for 18 h at 15°C in a Beckman SW41 rotor. Virus particles at a density of 1.39 to 1.41 g/cm3 (full particles) were visualized by light scattering, collected, and dialyzed against 50 mM Tris hydrochloride, pH 8.0. Viral DNA was isolated by proteinase K digestion (100 ,ug/ml, 55°C, 8 to 12 h) and phenol-chloroform extraction and ethanol precipitated. Since BPV encapsidates ca. 10% plus strands in bovine fetal lung cells (6) and the plus and minus strands readily form hybrids under the isolation conditions, the DNA thus prepared contained ca. 20% double-stranded DNA and ca. 80% single-stranded DNA of the minus polarity. Single-stranded DNA was separated from double- stranded DNA by centrifugation in an SW27.1 rotor (25,000 rpm, 15°C for 14 h) on a high-salt sucrose gradient (5 to 20% sucrose in 1 M NaCl, 10 mM Tris hydrochloride, pH 8.0, 1 mM EDTA, and 0.15% Sarkosyl). Gradient fractions were analyzed by electrophoresis on 1.0% agarose gels, and fractions containing single-stranded DNA and doublestranded DNA of monomer length were pooled separately. 
The minus single-stranded DNA was replicated in vitro to double-stranded form by E. coli DNA polymerase I (Klenow fragment) as described previously (14), by using the 3' palindrome of virion DNA as a self-primer. This is referred to as the in vitro replicative form (RF), and it was the source for cloning and sequencing of the DNA from the m.u. 5 to 100 region. 
In vivo RF DNA which contains monomer RF with both fully extended and joined termini, as well as dimeric RF (2), was obtained from BPV-infected cells by the method of Hirt (24). This is referred to as the in vivo RF, and it was the source of DNA for cloning of the m.u. 0 to 18 region. 
Cloning of BPV DNA. EcoRI fragment A (m.u. 18 to 92) and PstI fragment A (m.u. 5 to 76) were obtained from in vitro RF and first cloned into pAT153. EcoRI fragment B (m.u. 0 to 18, from in vivo RF) and EcoRI fragment C (m.u. 92 to 100, from in vitro RF) were cloned into pUC8 after the attachment of Sall linkers to the ends. Sall linkers were chosen because there is no Sall site within the BPV genome. The cloned fragments were then subcloned into M13mpl8 and M13mpl9 vectors for sequencing (55). 
S1 nuclease-resistant fragments of single-stranded virion DNA were cloned into SmaI-digested M13mpl8 vectors to permit direct sequencing of the terminal duplex regions in the virion DNA. 
All recombinant pUC plasmids and M13 phages were propagated in E. coli JM107, whereas pAT153 recombinant plasmids were propagated in E. coli HB101. Transformation was performed as described by Hanahan (21). 
Sequencing of BPV DNA. The chain termination method of Sanger et al. ( 46) was the primary method used for sequencing. A 15-mer universal primer was used for most runs. Several synthetic oligonucleotides were used to sequence overlapping clones and to obtain extended sequence data from larger fragments. Buffer gradient gels and [a-35S]dATP were used according to the method of Biggin et al. (12). 
The 5' end (m.u. 100) region was also sequenced by the method of Maxam and Gilbert (34). The pUC8 clones containing the EcoRI C fragment (m.u. 92 to 100) were linearized at the SalI site, end labeled with [ox-35S]ddATP and terminal deoxynucleotide transferase (44), and se- quenced by the chemical degradation method. 
Computer analysis. DNA sequences were analyzed by the Pustell and Kafatos (41) sequencing programs available commercially from International Biotechnologies Inc. (New Haven, Conn.). Locally homologous protein sequences were detected with the program written by Goad and Kanehisa (19) which was implemented in the ICR SEQuence program package obtained from Memorial University of New- foundland, St. John's, Newfoundland, Canada. 

RESULTS

In this paper, the convention of Armentrout et al. ( 1) is used: the 3' terminus of the minus-strand DNA is referred to as the 3' end or the map origin, and the map is drawn with the 3' end at the left. Additionally, the term virion DNA will be used to refer to the minus strand, since the method of purification yielded minus-strand DNA from total virion DNA. 
Sequence determination. We determined the complete nt sequence of BPV to be 5,491 bases long. The sequencing strategy used is shown in Fig. 1. As described in Materials and Methods, the internal fragments of BPV (i.e., PstI fragment A, m.u. 5 to 18; EcoRI fragment A, m.u. 18 to 92) were cloned from in vitro RF into pAT153. The 5' end (EcoRI fragment C, m.u. 92 to 100) was also cloned from in vitro RF, whereas the 3' end (EcoRI fragment B, m.u. 0 to 18) was cloned from in vivo RF into pUC8 after the addition of Sall linkers. Each was then subcloned into M13mpl8 and M13mpl9 vectors for shotgun sequencing. However, the 5' end clones were found to have deletions in the terminal sequences nt 5484 to 5491 were deleted). To obtain sequence data for the extreme ends, we took advantage of the fact that the terminal sequences of the single-stranded virion DNA form hairpin loop structures and that the stems of the hairpins are resistant to S1 nuclease digestion (11,22). Cloning and sequencing of the S1-resistant fragments of single-stranded virion DNA gave the sequences at nt 11 to 44, 5371 to 5422, and 5440 to 5491. The latter two sequences correspond to the stems of the 5' terminal hairpins (see Fig. 3B). The reason that the first 10 bases (nt 1 to 10) were not found in the S1-resistant fractions as would be expected (see Fig. 3A) is probably due to the high A-T content and hence low melting temperature (Tm) in this region. 
The complete sequence of the genome is given in Fig. 2. The sequence shown is that for the plus strand, which has the same polarity as BPV mRNAs. DNA sequence was obtained from both strands and across all restriction sites. The 5' end sequence was also confirmed by the Maxam and Gilbert method (34). The sequence data agree with the published restriction map of BPV (14). 
Terminal structures. The terminal hairpin structures of BPV virion DNA (minus strand) are shown in Fig. 3. In agreement with other autonomous parvoviruses (H-1, MVMp, MVMi) (3,22,45), the 3' terminus can form a T-shaped configuration; the 5' terminus can form a U-shaped configuration. However, the 3' terminus is longer (150 nt versus 115 and 116 nt) and the 5' terminus is shorter (121 nt versus 207 and 242 nt) than those of the rodent parvoviruses. It was reported (2) that the 3' terminal sequence of the rodent parvovirus DNA is unique, whereas the 5' terminal sequence is heterogeneous. The 5' end possesses two mutu- ally exclusive sequences that are inverted complements of each other (denoted as the flip and the flop orientations). We analyzed four independent 3' end clones of BPV and ob- corresponds to the 5' end of the plus strand (3' end of the minus strand). The RB series are synthetic oligonucleotide primers used in some sequencing reactions. The S1 series are a set of clones obtained by Si nuclease treatment of virion DNA (minus strand) and cloned into M13mpl8 vectors. The restriction enzymes indicated by the bars above the last line are those with single cleavage sites, and those below the line have two cleavage sites in BPV. tained identical sequences. For the 5' end, we analyzed only one clone, the sequence of which is given in Fig. 2. However, NdeI digestion (CA/TATG) of the end-labeled in vitro RF (which should cut at nt 5437 in the flip orientation, and at nt 5422 in the flop orientation) yielded two fragments of equal intensity of the expected sizes (55 and 70 bases), thus demonstrating the existence of two sequence orientations in the 5' end of BPV (data not shown). Recently, we con- structed several full-length genomic clones of BPV by cloning reannealed plus-strand and minus-strand DNAs from purified viruses into pUC8 plasmids. These recombinant clones were infectious when transfected into bovine fetal lung cells, and the 5' end sequence of one (pGC17) is in the flop orientation (B. C. Shull, unpublished results). Genomic organization. A diagram of the stop codons and ATG codon in each of the three reference frames of BPV plus-strand DNA is shown in Fig. 4. There are three large and five small ORFs. The location, the coding capacity, and the putative amino acid sequence encoded by each of the three large ORFs (i.e., left, mid, and right ORF) are sum- marized in Fig. 5. When the minus-strand DNA was ana- lyzed, like all parvoviruses studied, no ORFs of significant sizes were found. Indeed, RNA from BPV-infected cells is complementary only to the minus strand (P. R. Burd, Ph.D. thesis, Virginia Polytechnic Institute and State University, Blacksburg, 1982). 
A search for the consensus promoter sequence (TATAA) in the BPV sequence revealed three possible promoters at nt 251 (m.u. 4.5, TATAAA), nt 705 (m.u. 12.8, TATAAT), and nt 2128 (m.u. 38.7, TATTAA). In contrast, only two promot- ers (at m.u. 4 and 38) have been demonstrated for the rodent autonomous parvoviruses H-1 and MVM (8, 40), whereas three promoters were identified (at m.u. 5, 18, arnd 39) for the defective parvovirus AAV (50). 
Bensimhon et al. (9), after comparing tht w-Imoter se- quences of simian virus 40 early genes and t.of herpes simplex virus tk genes, proposed that the pro er recognition and initiation by eucaryotic RNA polyri --ses do not depend on the TATA consensus sequenc ilone. They suggested that the eucaryotic promoters sh--' have three basic components: (i) an enhancer region of li -' rately high local stability, roughly 100 base pairs (bp) eam of the cap site; (ii) an activation region (a G-C-rich igh stability domain), 50 to 70 bp upstream of the cap sitt; and (iii) an A-T-rich domain (TATA box), 30 + 5 bp ups-,!i am of the cap site, that traps and positions the RNA polymerase for initiation of transcription. 
We analyzed the promoters of AAV2, MVMp, and H-1 which have been demonstrated unambiguously to be func- tional by primer extension studies (16) for the presence of the above three components (9). All the known promoters of AAV2, MVMp, and H-1 satisfy these criteria (Table 1). When this analysis is extended to the possible promoters of BPV, all three satisfy the criteria (Table 1) and hence may all be functional. In addition to the three possible promoters mentioned above, there is a TATAAAA sequence at nt 3922 (m.u. 71.4). This one, however, does not have all three components described by Bensimhon et al. (9). Experiments to map the BPV promoters are under way. 
The most characteristic feature of the 3' ends of eucary- otic mRNAs is the presence of the sequence AAUAAA at about 10 to 30 nt before the poly(A) tract (53) TCACTCGCTC ACCAAGCTAC TGGGTTCATC CTAGATCCCA TTCCAGMGA ACCAGMGAA GGGGAGCGGG ATCTGGCTAG AGAGGACGCC GAGCCAGAAG CATCGACGAG TCACACTCCA GCTACCAAAA GAGCTCGCGT GGAGGMGGT GAGCCGTGGG ATGGMCGCA GCCGATCACC GAGGGAGACT GGATCGACTT CGAGTCGAGA CAAAAGCGAC GCAGACTGGA GCGAGAGGAG MGGGAGGAG AGGACGAGGA CATGGMGTC CAGGAGTCCG ATCCGAGCGC GTGGGGAGAG AAGCTGGGGA TCGTGGAGAA GCCGGGAGAA GAACCAATCG TCCTCTACTG CTTCGAGACC TTACCAGAAA GCGACGAGGA AGGAGACAGC GACAAAGAAA 4-end of left ORF ACAAAACACA CACCGTTTAA CGTGTTTAGC GCTCACCGAG CACTCTCTM MCAGATCTC CAGTTCTGCG GCTTCTACTG GCACTCGACT CGACTGGCCA is an essential but not sufficient signal for polyadenylation. 
is clearly not functional, as it precedes any possible promot-Several additional signals have been suggested, such as ers. The one at m.u. 74.8 also may not be functional because downstream G/T clusters (13), secondary structure in the it is in the middle of the right ORF and is about 1 kilobase vicinity of AAUAAA (35, 54), and a CAYUG sequence (kb) away from the ends of the left and the mid ORFs. The upstream or downstream from the poly(A) site ( 10), but no two poly(A) signals at m.u. 61 .6 and 64.6 may be functional, consistent picture of the absolute requirements has emerged. 
as one has G/T clusters (m.u. 61.6), the other has a CAYUG There are six AATAAA sequences in the BPV genome at sequence (m.u. 64.6), and both have secondary structures m.u. 2.5, 60.0, 61.6, 64.6, 74.8, and 98.5. The one at m.u. indicate that the 1.1-kb RNA coding for NP-1 terminates in and MVM (17,43) and is necessary for viral DNA replication this region (Burd, Ph.D. thesis). The poly(A) signal at m.u. (23,52). It is detected by immunoprecipitation with homol- 98.5 must be functional because it is the only one available ogous antiserum from in vitro translation reactions of RNAs for the RNA transcribed from the right ORF, and it does from MVM-infected (17) and H-1-infected cells (37). Re- have secondary structure and a CAYUG sequence around cently, we observed two proteins of Mr 75,000 and 82,000, the AATAAA site. The poly(A) signal at m.u. 60 .0 may not one or both of which may be the BPV equivalent of NS-1 (M. be functional because it does not have G/T clusters, a Lederman and R. C. Bates, unpublished results). These CAYUG sequence, or secondary structure. proteins are detected as immunoprecipitation products of Assignment of coding domains. By analogy with the ge- BPV-infected cell lysates and of in vitro translations of RNA nomic organizations reported for other parvoviruses (AAV, from BPV-infected cells by using antiserum from a calf H-1, MVM), and also by the amino acid homologies ob- experimentally infected with BPV. These proteins are syn- served for the putative proteins (see section on Homologies), thesized early in infection, and their rate of synthesis dewe assign the left ORF of BPV to code for the major part of clines as the capsid proteins accumulate. the nonstructural protein NS-1, the right ORF to code for the In all the parvoviruses studied, the capsid proteins are major parts of the three capsid proteins, and the mid ORF to encoded by the right ORF as a nested set with the amino acid code for the major part of NP-1. 
sequence of the smaller being largely contained in the next NS-1 is coded by the left half of the genome in AAV, H-1, larger protein (27). In the autonomous parvoviruses MVM and H-1, there are two coded capsid proteins VP1 and VP2' (Mr 84,000 and 62,000). The two proteins are translated from two differently spliced mRNAs generated from the promoter at m.u. 38 (3, 29, 38, 40). VP1 initiates at m.u. 44 with the first ATG codon in its mRNA, and it contains the conserved amino acid sequence NPYL region (see section on Homol- ogies). VP2' initiates with its first ATG codon at m.u. 54 (the ATG codon at m.u. 44 for VP1 initiation is spliced out). The codon at m.u. 54 is in the most favorable sequence context for an initiation codon, according to Kozak (AnnATGG) (28), and it is located 75 nt upstream of a glycine-rich region (4, 43). The defective parvovirus AAV has three capsid proteins VP1 to 3 (Mr 92,000, 72,000, and 60,000). They are translated from two differently spliced mRNAs, both generated from the promoter at m.u. 39 (16,26). VP2 and VP3 are translated from one spliced mRNA of 2.3 kb initiated at different sites. VP2 initiates at m.u. 56 with ACG as the initiation codon; VP3 initiates at m.u. 60 with the first ATG a The number in parentheses after the promoter P indicates its map position. 
b The first number in the parentheses after the sequence indicates the position of the sequence in the genome; the second number indicates the position of the sequence relative to the cap site. Since many RNA cap sites are unknown, we assume that they are 30 bp downstream from the TATA box. IG. 4. ORFs and conserved regions in the BPV genome. The lower three lines correspond to the three possible reading frames (1, 2, and 3) of the plus-strand sequence, with the stop codons and the ATG codons indicated by short bars. Three large ORFs (LT ORF, MD ORF, and RT ORF) and five small ORFs are shown by open blocks. The second line from the top represents regions of highly conserved amino acid sequences in BPV as compared with other parvoviruses. MTTJ , Regions of high amino acid homology among all parvoviruses (AAV2, BPV, MVMp); E:1, regions of high amino acid homology between the autonomous parvoviruses (BPV, MVMp) only; f9, regions of terminal hairpin structure of BPV. codon in this mRNA (7, 26). The initiation codon for VP3 also conforms to the Kozak consensus sequence for initia- tion. The exact splicing pattern of the mRNA for VP1 is not certain, but VP1 contains the NPYL region (see section on Homologies). 
BPV has three coded capsid proteins of Mr 80,000, 72,000 and 62,000 (30). An ATG codon located similarly to the initiation codon of VP2' in MVM and H-1 (i.e., with a Kozak consensus sequence and 75 nt upstream of a glycine-rich region) is found at nt 3697 (m.u. 67.3), which presumably is used for the initiation of VP3 in BPV. Nt 3697 is the second ATG codon in the right ORF. When we searched for the initiation of VP2 in the region downstream from the NPYL region to nt 3697, no ACG codon analogous to the initiation codon for VP2 in AAV or other ATG codon was found. Therefore, we assign tentatively the initiation site for VP2 at nt 3286 (m.u. 59.8), the first ATG codon in the right ORF. The initiator for VP2 (TTGATGC) is not in an as favorable a context as the initiator for VP3 (AACATGG) for protein initiation, which may explain the relative abundance of the two proteins. The predicted molecular weights for VP2 and VP3 are 75,000 and 60,000, whereas the observed values computed from electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels are 72,000 and 62,000. If the assignment of nt 3286 for VP2 initiation is correct, then VP2 will contain the NPYL region. In other parvoviruses, the largest capsid protein is the one that contains the NPYL region. This will make BPV different from the others by having one extra larger capsid protein VP1. 
A computer search for the possible splice donor and acceptor sites (36) for VP2-VP3 mRNA revealed two possible donor sites at nt 2336 (ACA/GTGAGT) and 2441 (CCA/GTGAGT) and one possible acceptor site at nt 3241 (TTCGTTGCAG/A) just before the start of the right ORF. In either case, VP2 will initiate with the second ATG (nt 3286) and VP3 will initiate with the third ATG (nt 3697) in the spliced mRNA (the first ATG at nt 3261 is soon followed by in frame termination codon at nt 3300). 
Previous studies by partial proteolysis indicate that the amino acid sequence in VP2 is largely contained in that of VP1 (30). With the sequence data alone, we cannot assign the coding region for the N-terminal amino acid sequence specific for VP1, because there are many small ORFs and possible splice donor and acceptor sites. Wherever that coding region may be, it must overlap partially with the coding region for NP-1 (see below). 
NP-1 is a highly phosphorylated noncapsid protein of apparent Mr 28,000. It has been detected in the nuclei of BPV-infected cells in association with chromatin and as an in vitro translation product of BPV-specific RNAs. NP-1 shares structural homology with VP1 (but not with VP2 or VP3) as demonstrated by partial proteolysis (30,32). Polyclonal antibodies to VP1 react with NP-1, but polyclonal antibodies to VP2 or VP3 do not (R. C. Bates, unpublished results). An abundant BPV-specific RNA species of 1.1 kb, when translated in vitro, gave a protein of Mr 28,000 that could be immunoprecipitated with anti-NP-1 antibody (M. Lederman, unpublished results). When this 1.1-kb RNA (supposedly mRNA for NP-1) was analyzed by the Berk and Sharp Si nuclease mapping method, it was found to be spliced with a 0.3-kb leader and a 0.8-kb main body (Burd, Ph.D. thesis). In DNA-binding assays, NP-1 binds specifically to the 3' end region (m.u. 0 to 5) of the RF form of BPV DNA (M. Lederman, B. C. Shull, E. R. Stout, and R. C. Bates, J. Gen. Virol., in press). 
Several lines of evidence suggest that the mid ORF may be the major coding region for NP-1. (i) RNA size. By using a computer search, a possible splice acceptor site was found at the start of the mid ORF (nt 2535, TTCATCCTAG/A). The mRNA for NP-1 may start from one of the three possible promotors so that the N terminus of the protein is coded by the sequence between the first ATG and the splice and then splice to nt 2535 so that the mid ORF becomes the reading frame for the remainder of the protein. It may terminate at the poly(A) signal at nt 3385 (m.u. 61.6), giving it a main body of 850 nt after the splice site. This would be consistent with the previous observation of Burd. (ii) Protein size. The putative protein encoded by the mid ORF has Mr 30,000, which is similar in size to NP-1. ( iii) Charge. The pI of the putative protein is estimated to be 10.8; it should be a basic protein (56 Lys + Arg + His versus 33 Asp + Glu), which may explain the observed DNA-binding capability of NP-1. 
(iv) Serine content. NP-1 is a highly phosphorylated protein, and our preliminary data indicate that the serine residues are phosphorylated (M. Lederman, E. R. Stout, and R. C. Bates, unpublished results). The putative protein is also rich in serine (27 serines in a total of 255 amino acids). RNA mapping studies with labeled restriction fragments to locate the coding region for NP-1 are in progress. DNA and amino acid homologies of BPV with other parvoviruses. The overall DNA homologies of the BPV left and right ORFs with those of the other parvoviruses are not significant (ca. 28%). However, several regions of high local homology are observed when the amino acid sequences encoded by the left and right ORFs are compared. The comparisons among AAV2, BPV, and MVMp are depicted in Fig. 6 and7. Since CPV, FPV, and H-1 are highly homologous to MVM, their sequences are not shown in the figures. 
In the left ORF, there is a region of 55 amino acids within the region marked as GKRN that shows a 60% homology among AAV, BPV, and MVM (Fig. 4 and6). The GKRN region (87 amino acids), located at m.u. 29 .6 to 34.4, is in the coding region for the nonstructural protein NS-1. The bio- logical significance of this conserved region is not known. It contains a sequence analogous to the nuclear targeting signal for a yeast mating type protein (20), which may act as the signal for the nuclear transport of NS-1 (31). 
Six small blocks of strong amino acid homologies are found in the right ORF (Fig. 4 and7). Three are conserved in both the defective and autonomous parvoviruses (marked as NPYL, TPW, and PIW in Fig. 4), whereas the other three are conserved among the autonomous parvoviruses (marked as PGY, GGG, and YNN; for the PGY region, see the Discussion [The right ORF] also). 
The mid ORF of BPV is located at m.u. 46 to 60. There are several smaller ORFs (besides the major left and right ORFs) in the corresponding locations in the genomes of AAV (m.u. 
C F Y " A S N A Y S D E V L A T N N L K E K S N Q E V F S F V F K N E N VQ L N S K D I S N N S Y K K E L Q E 0 E L K S L P R C A E T T N D Q S AAV fil V I K V P S D L D 6 H L P O I S D S F V N N V A E K E N E EP P D S D N D L N L I E Q P LT E K L Q R D F L T E N R R V S K A P E WV P D K H Q P S R O L Y S A L H S F K H L L I T I R Q R T HAPD TVL S R D E L N A N N N L Q K I A Q R V N HY fiD " E N E T T V D E M T K K Q V F I F D S L V K K C L F VON T K N I' F P D V N N F V N E N K D Q N H C H V L I 0 6K F S Q A AAV A L F F V Q F E K O E S Y f N N H mL V E T T C V K S N V L 0 R F L S Q I R E K L I Q R I Y R 6 I E PL P N N F A V K T R N C A O C 6 N MNV RR N D D I V D I L K C R K A N L V A Q A I N 0 T FFT R Y H L P K N R K V A D T V L T R P E 41 S D S N C K T Y C F V C 1Y Q-N NP R Q L N V Y N S R N L A C N V Q L T P A JgK L R E I A E D N E N V T L L T Y sK K Q T KK D T K C V L F 6 N N IYY AAV K V V D E C Y I P N Y LpP Q P E L Q N N T N N E Q Y L A C L Q T E It K L V*QH L T H3S <QQ E Q N K19N Q N P N S D A P V BPV N 0 E T V S E F R K D>8 L Y N I Y T N P A E N N N P S V N 6 D L P V S N I D A D D A E A R S R P I K L S R K Q K I M A 10M F L T K K K I SS P P R D 6 6 Y F L 5 S D S 6N K T F K E 6 E R HQV S K L Y T D D Pt E T[3E T(3V T T A Q[qT K R 6R.ItIQT K K AAV R T S R Y V N L V D Kt I T S E K NI Q E D QA S Y I S F N A AS N S R S QIK A A-j3DF O K I S L T TP Y L V MN IEV R A D 6 L ,LT Y N D L V V N L S D LF M L E C M P 6 C S K T A E Q L T M I H 1 I K L ,;Y,AY E FWL M K T PT Q N M N "VM |£ K 1I T JL K E NJ K R V T S P E D N N H l P D S Y I E H PS A Q P 6 6 E N L L K N T L E I ET L R T K T A F D L I L E K A E T AAV QQV E IK S S N PI Y D LE NN Y AE A PS V F L N A F C K R I NDL F T EK T N EPEF MVN P N A R Y I a V F N Y N |H C N L PH HRE I A NE N T YC P A S T K K DUT AKC V V L Y MV S L T N F S L P D TLfPC SC A F S N V N NE S Y C C V EN R Q K R N SV H L P A ST N N A N N S P AAV C VN T NECF P F N DZV IV r N E E KKtFAI V H1 i7 L 6 S KI D Q ic c . AO D TPvI| vlT N M c WPV N SETV EF P F N D IN YKWN W E E JC I J D Y N AA L S I N RVVDN KH D|R E L SP IKL KH D V Y MY" A VER F P F N D KJ1NL D U N V A I N DFCILS P Q KA TM KLC KYN AYE QN I TT MV 5 V E N I T j P LlYQ F K F ED YAAR VF NV K D F F R N A K D H V V E H E FEYV K A K K R P BPV T V P N AF JH K T Q N F N K 4 LN F C|E | I S N Y L S H C A H I H H L S L E 6 FIAI[K Q V SV MVl R I IE R P R LNIH TL F C|N P M I C A A L V K N 6 Y Q S T V A S[3CA3B P D N AAV ACN TDDQD E T E QIDI R" N I C F T QE T MPV C NMJF L * T L D P M N S Q N T F S EN V C N H
EMF P V DEC QP NV V KI A Y Q c L C Y I H H I N C K V P D A C T a c D L VJ1 4V D L D D C I F E Q - BPV NTI L S V TA SJA JPSJD R AE I E E I P S E C L E N N R E E V D R L S A H D C S L A N Q A T N F I L D P I P E E P E E V E R D MV A AEP D D I EL9K fV E I EV D L R A C F N A E P L K K D F S E P L N L D N AMV SWV L A R E D A E P E A S T S H T P A T K R A R V E E 6 E P N D 6 T Q P I T E 6 D N I D F E S R Q K R R R L E R E E K 6 6 E D E D Iq E V Q E S D HMVI MV BPV P S A N S E K L 6 S V E K P S E E P I V L Y C F E T L P E S D E E S D S D K E N K T N T V - FIG. 6.
Homology of the translated left ORFs among parvoviruses AAV2, BPV, and MVMp. Homologous regions are enclosed by boxes. 39.2 to 46.3, 46.8 to 53.8, and 49.6 to 56.5) and MVM (m.u. 37.6 to 44.7 and 46.6 to 52.2) also. These smaller ORFs, when combined, would have coding capacity similar to that of the mid ORF of BPV. The small ORF of MVM at m.u. 37.6 to 44.7 (denoted as mid ORF A) is known to contain sequences coding for the C-terminal region of NS-2, a nonstructural protein of Mr 24,000, similar in size to NP-1 (18). 
We searched the mid ORF of BPV and the three mid ORFs of AAV for regions homologous to the C-terminal region of NS-2 (i.e., mid ORF A in MVM), scoring conservative amino acid changes like D+E, K+->R, T+S, and AVIL as matches, and none were found. In fact, no such regions were found anywhere in the genome of BPV or AAV. When the mid ORF of BPV was compared with that of the other parvoviruses, there was no homology of the putative BPV mid ORF protein with any peptides that may be encoded anywhere in the genome of AAV, MVM, or H-1. The partial nt sequences of CPV and FPV were searched, and the results were the same. Likewise, when the three small mid ORFs of AAV were compared with those of other parvoviruses, no homologies were found either. 
The analysis of comparisons of the homologous regions of BPV with the other parvoviruses is summarized in Table 2. The similarity coefficient (SAB) between two proteins A and B is defined as (2 x number of identical residues in A and DISCUSSION Although the genomic organizations of all parvoviruses studied to date are similar, the extent of DNA sequence homology among them varies. Previous heteroduplex map- ping by electron microscopy (5) indicated that the defective parvovirus AAV has little DNA sequence homology with the autonomous parvoviruses (BPV, H-1, LuIII, KRV, and MVM). The autonomous parvoviruses H-1, LuIII, KRV, and MVM have greater than 70% of their sequences con- served, whereas BPV, like AAV, has little homology with these viruses. Immunological cross-reactivity leads to the same conclusion. AAV and BPV do not cross-react with other autonomous parvoviruses, whereas CPV, FPV, H-1, KRV, Lulll, and MVM have some cross-reactivity (48). 
From our sequence data and the published sequence data on AAV, CPV, FPV, H-1, and MVM (4, 15, 42, 43, 50), the overall DNA homologies of the BPV left and right ORFs with those of the other parvoviruses (AAV, CPV, FPV, H-1, and MVM) are not significant (approximately 28%), whereas the sequences in CPV, FPV, H-1, and MVM are quite similar to each other, with greater than 50% conservation. Based on the evidence given above, we suggest that there are three groups of parvoviruses: the defective parvoviruses with AAV as a representative, the autonomous parvovirus BPV, and the other autonomous parvoviruses (CPV, FPV, H-1, KRV, LulIl, and MVM). If the human parvovirus Bi9 is also considered (47; see Comparison with Bi9 at the end of the Discussion), it would represent an additional group. 
Several points merit discussion when the sequence of BPV is compared with those of other parvoviruses. 
Terminal structures. The terminal palindromes of BPV show very little homology with the other autonomous parvoviruses at the level of DNA sequences, but the sec- ondary structures of the termini are highly conserved. This suggests that the secondary structure rather than the primary sequence is important in the function of the palindromes of the autonomous parvoviruses. This is in agreement with the data of Lefebvre et al. (33), who noted that changes in the primary sequence of AAV terminal palindromes are toler- ated if the secondary structure is conserved. The 3' end palindrome of BPV shows two G-C-rich stems, a feature conserved in all parvoviruses sequenced to date. The high G-C content of these stems may be important in stabilizing the 3' terminal palindrome. The sequence TAAAAAT at or near the viral 3' terminus is conserved among all autono- mous parvoviruses sequenced to date. The conservation of this sequence element suggests a functional role in the replication cycle of the autonomous parvoviruses. It is interesting to note that the 3' terminus of the autonomous parvoviruses is A-T rich with twelve consecutive A or T residues in BPV and 9 of the first 10 bases being A or T in H-1 and MVM. 
The left ORF. In the left ORF, the region within the one marked as GKRN (55 amino acids) is highly conserved among all parvoviruses and this conserved sequence may be used as a diagnostic probe for parvovirus identification. 
The right ORF. There are three regions in the right ORF that are conserved among all parvoviruses (marked as NPYL, TPW, and PIW in Fig. 4), and three are conserved among the autonomous parvoviruses (marked as PGY, GGG, and YNN). 
The sequence NPYL (m.u. 62 .6 to 65.1; 47 amino acids) is present in the largest capsid protein VP1 in AAV, CPV, FPV, H-1, and MVM. Based on the assignment of coding domains presented in the Results section, we propose that VP2 of BPV also contains this region. The significance of this highly conserved sequence in the large capsid protein is unknown at present. 
A glycine-rich region (GGG, m.u. 68 .7 to 69.8; 20 amino acids) is highly conserved in all of the sequences reported for the autonomous parvoviruses. It is located about 75 nt downstream from the initiation codon for the smallest coded capsid protein VP3. A proteolytic cleavage site (which generates the smallest capsid protein sometimes detectable in the mature virus, i.e., VP4 in BPV and VP2 in MVM and H-1) is often found in the vicinity of the GGG region (15, 42). It has been suggested (42) that a run of glycines distorts the alpha helical structure of the protein; the resulting extended region could be the substrate for the proteolytic cleavage which generates the smallest capsid protein. A less- conserved form of this sequence is found in AAV (31); it is only 9 nt downstream from the initiation methionine for VP3, and VP3 is not cleaved in the mature virion. 
The PGY region (m.u. 60.4 to 62.2; 33 amino acids) is conserved among autonomous parvoviruses but not in the defective parvovirus AAV2, if the published sequence data of AAV2 ( 50) is used for the translation of the right ORF (nt 2261 to 4324, frame 2). However, a PGY region is found in a different frame (frame 1) as shown below: 
AAV nt 2332 GLVLPGYKYLGPFNGLDKGEPVNEADAAALEHVQS BPV nt 3319 GLTLPGYNYLGPFNSLFAGAPVNKADAAARKHDFG MVM nt 2398 GWVPPGYKYLGPGNSLDQGEPTNPSDAAAKEHDEA This suggests that frame 1 rather than frame 2 in this region (nt 2332 to 2427) is used in the translation of VP1 in AAV. The same suggestion has been made by Shade et al. (47). The data of Janik et al. (26) also support this hypothesis. They showed that a 186-bp deletion of XhoI fragment (plasmid pLHlXH3) resulted in the synthesis of a new protein 7 kd smaller (which is equivalent to the coding capacity of the excised 186-bp tract) than the normal VP1 protein. The results of Janik indicated that the entire XhoI fragment (nt 2234 to 2419) is translated into VP1, but the only ORF in that region is frame 1. Presumably, VP1 has to switch from reading in frame 1 to frame 2 in a small region (nt 2420 to 2464) to acquire the NPYL conserved sequence and the rest of the right ORF. However, there is no splice donor or acceptor in that small region that satisfies the consensus sequences (36). The other possibility is that ribosomal frame- shifting (as demonstrated for the expression of the Rous sarcoma virus pol gene) (25) occurs in this small region. With either ribosomal frameshifting or a low-frequency RNA splicing event (despite the absence of conventional splice donor and acceptor sites), the right ORF would be in frame 1, and reading of VP1 would be uninterrupted from nt 2191 onward. 
AAV D S G G S S N L A H H H Q Srj Q S G I R T T A G V L C F L G T S T S I7S T D S T R E S R S T R Q T P R P S S T Y K IYDIR Q 1 | BPV L I L K C N K R Q K L L M PP T N K A N S K KL T L|P 6 YN LGP F| SLF A AF1V|T DA A |R K ;F;Y D L L K 6KN MVM N H L V V PKP K Y|KY L G P G| SD Q ET S K E E Q Y K S G K N AAV p E Q EIR A lTV F Q l A R V L E P L G L V E EVK T 0fG 6 R P V E V E P D S S S BPV P Y L T N IDE L K D D T S F G I K K ALAP L P G T G D RAL K RL Y F A NK MVM P Y L Y F AEA D I Q A F KL A T E P G T S G V S P K R T S A Y I F I N Q AAV TP R LFNAF G G D AKDlGGV TP Q PLGQ FAP S G L G T N T M A T G S G A P M A D N N E AD G V A N S BPV GT A N R EFPR P S R N E V S N D I P D E A G N PI E LA T R S V V -L-TNT -ATG S G S V G R G SG V GY S T MVM A K KANRE L T S P S R A A D - S 6 G 6 G GS V G S I AAV H C D S T R M G D R I T S T N9L P T YN N QIS S ------QS ASN D N H -------F G Y S[T rY BPV G T G I F S E N I V K Q F I C D I N H L Y K S EV L -- NTGD A H R Q --------A7TT P N S Y MVM GjS Y D N Q H Y R F L G D G N V E I T A LE|T R L V L NK SEN Y C R I R V N N S V K G N M A K D D A H E Q P AAV F D F NR F H C FS R R I N N N G A R N F K N I V|;EE|Q NE]G T I - V Q T BPV FNF N Y S H F S P|N|D H D Y E F R P KGA M I V R V N L I K Q I M T D G A M| ---V N N H F C D MVM A N G V S|D N dY I C T M S Q L N L V SQ D Q E I NV V L K TEE Q1EIL G JA I K I|Y N NDL T A C!M 2v U AAV E Y L G S A H Q G P FA D V F M Vfl L T L N N G A V G R S S F Y Y F P S Q L R T G N N F S Y BPV D H RP YQ H P N D D E LIPN S I N E L -Y I-I P A P IISV D N T T NT VEH L ---V -L Y M LI MVM N N IL P L P A A N S M E 1 GF Y WN K P T I A S P Y Y F C V D R D L VT Y E Q E GT V E N V M G T P I Q F IE AAV GIF E D V P F H S S Y A H S Q S L D R L M N P L I DQ Y L Y Y L S R T N T P S GfT T Q S R L Q F S A G A S D I R D Q 3NN L PG P C BPV S D H -E t{NR I Y R I Y I Q L W R L R M D R K Q H H A S D D VR G Q K Q K N L L IQJR T K Q P N K R F Q ------- MVM El Q Q I T LLjTIJD E F A T G T Y Y F D T N S V K L T T T N R Q L G P P L L S T F P E A D T D A G T L T A G NH G T T Q M G AAV Y R Q Q R VS - SA D N N N S E Y S N T G AT K Y H L N G R D S L V N PM A S H K DIE[JK F F P Q S G V L I FL6 G SEKK T N V BPV ----N TRT N M P G I A R G T H L T LQQ STQ G A LV -V NRAIDV S G V R AW V G --S T D P I Y G G Q Q MVM V N NV S E IP A Q F C Q P H N DF ESR A G P FA P K I v K EA N G S Y S -- H G EN N A S AAV N I E K V M I -E] D EW3 E I G T T N P V A T E Q Y G S V S T N ---------fQ R RQ AjT AH-----8EN T Q G V G M VI BPV P E S DL L -L R Y S A A EQ Q N P I L E N A A R H T F T R E A R T K -|LII TGN - D GD ------Y K E N ML N Q ME MVM H G P A P YG W DO F G R D T K D G F I Q S A P L V V P P P L NG ILT NA P IGK NDI H F S NE0F N S Y G P L T A F S AAV Q D R L IAiIIRH T D G H F H P S P L M(G F G L IL IN N P S T T F S]A K F A S F I T Q Y S T BPV DS P I R V N T D T Q DS I P M HPPI I KA R I RV P V N G D S F L N I Y V T G Q V S C E V MVM H P PIV P DQ!j E L D L E I4PRIJ H I T A P F V C K NNV GEN L T D Q Y D P N GT L S R I V T Y G T F F AAV G 3 IQ - D R V GEI G K Q Q T L E S R N S V H F Q L Q Q V C - BPV V N E V E N N R Y M H S A T N VD A Y T I N N A Y A G A V Q N A D VF T R F N H H K V L - Mm W KO3 K L T M R L R N T T N N P V Y Q V A E D N N S Y M ST K W L P T A T G N JS V P L I T R P V A R N T Y -
The mid ORF. No significant homology was detected among the mid ORFs of AAV, BPV, and MVM (Table 2). Even the closely related viruses MVM, CPV, and FPV showed little homology when the regions corresponding to the coding domain for the C terminus of NS-2 in MVM (nt 1990 to 2236) were analyzed. A total of 20 conserved amino acids are scattered in a total of 82 residues. Whatever proteins these ORFs in the middle of the genomes may code for, they have little amino acid sequence homology, although they may have similar functions. 
Homology with RA-1. Although BPV has little homology with AAV, CPV, FPV, H-1, KRV, LullI, or MVM, it is highly homologous to a recently described human parvovirus RA-1 (49) isolated from the synovial fluid of a patient suffering from rheumatoid arthritis (D. Van Leeuwen, Abbott Laboratories, North Chicago, Ill., personal commu- nication). The similarity coefficients (SAB) between BPV and RA-1 in the left, right, and mid ORFs are considerably higher than the values shown in Table 2 (manuscript in preparation). The relationship between these two parvoviruses deserves further investigation. 
Comparison with B19. While the present manuscript was in preparation, the nt sequence of the human parvovirus B19 became available (47). B19 is probably an autonomous parvovirus, since it appears to replicate in erythropoietin- stimulated human bone marrow cultures without the aid of a helper virus. However, it is more similar to the defective parvovirus AAV in having inverted terminal repeats at both ends and in lacking the two conserved regions (GGG and YNN) in its right ORF which are characteristic of other autonomous parvoviruses. It does contain the conserved GKRN region in its left ORF and the conserved PGY, NPYL, TPW, and PIW regions in its right ORF. From the sequence analyses of B19, AAV2, and MVM, Shade et al. 
(47) concluded that B19 is only distantly related to the other parvoviruses and that B19 is as different from MVM and AAV2 as those two viruses are different from each other. When we compared the homology of the putative proteins translated from the left and right ORFs of B19 with the corresponding proteins from the other parvoviruses (Table 3), we found that B19 is closer to AAV than it is to BPV or to MVM and that the evolutionary relationship among them is B19+->AAV+->BPV+->MVM. The same relatioriship was deduced when the algorithm described by Goad and Kanehisa (19) was used to calculate the evolutionary dis- tance between two protein sequences (data not shown). 
In conclusion, from the genomic sequences reported so far, there seem to be four classes of parvoviruses: (i) B19; (ii) AAV; (iii) BPV; and (iv) MVM, H-1, FPV, and CPV. The highly conserved amino acid sequences observed among all parvoviruses may prove to be useful in the development of molecular probes for the detection and identification of viruses in the family Parvoviridae. For example, a synthetic peptide including a consensus amino acid sequence derived from the GKRN region found in the left ORF of all parvoviruses might be used to prepare antibodies for diagnostic purposes. Antibodies against the conserved GKRN regions of B19 and MVM did give positive reactions in an immunofluorescence assay with BPV-infected cells (M. Lederman and S. F. Cotmore, personal communication). The smaller conserved sequences in the right ORF, if shown to be associated with antigenic domains, could provide the basis for the development of peptide vaccines protective against all parvoviral infections. Synthetic oligonucleotides containing the consensus sequences could be used as probes for detection of parvoviral DNA in clinical specimens. Also, it is now possible to search the BPV sequence for regions identified as functionally important for replication and transcription in other viruses and in eucaryotic cells. 

FIG. 1 .

 1 FIG.1. Sequenciffg strategy and restriction map of BPV. The sequences were determined in the direction of the arrows. 0 m.u. 




  CATATTGGCT GCCCGGGCCA ATAATGCGAG CGGCTGCGCC GCTGCGCGCT TCGCGCGCGC ATTGTMGGC CTAATCGGGC AGCCAATATG GTGGATGCAG GAAATGAGTA ATMAAATAT AGAGCCTGTG ATTAAGTGAA ACGACATAGG CGAGGACAGG TGGACCAATC promoter 1 ACGTTAAAGG TCAAGCTAAT GATTMCCGG GGGGTGGAGA CTAGAGGGTA TATAAAGAM GMCCAGGAA GTCAAAAATC AGACTGCTGC AGACCTCGAC AGAGAACACA CGTCTCTGCG CTCTGGTGAG TGAAGATGGC TACTGGGGGG CTAGCTAAAT GGGTGGGGGC TGTGCAGGCC TTTGGGCATC CATCTTGGTC CTACGTCATC AAGCTACGTC ATGGTCACGT GACCATGATT GAGAGAGGCT TCTATGAACT ATGTGGGGGA GAGMTTGGA ATATGCTCCA CGMCTACCT GGAGACMAT GGACTGCTGG AGATGTGGGA GAGCAGAGAG AGCATGCTAG AGAGTTGGAG TCCGGGAGAT GGACACAGTT GGGCCATGGC ATGGGCAGGG TGGAGAGCTG CTAGAGAGAC CATGCAGAGA AGACAGTATG TCMCGAGCC GACATTTAGT TGCTGGAGCC AGAGCGAGCT GGGGGAGAAC GGATGGCATG promoter 2 start of left ORF-* TGCATATAAT TATTGGGGGT CCGGGACTGA CCAGACAAAA TGCAGCCATC TCGAGGTCTA TACTCTGCAC TTCATTCGTT TAAACACCTT CTGATCACTA TTAGACAGAG MCACACGCT CCAGACACTG TGCTATCTAG AGATGAGCTG MTGCATGGA ACTGGCTGCA AAAGATAGCT CAGAGGGTGA TGAGGGGAGA CATGGATGAC ATTGTAGACA TCCTGAAGTG CAGAAAGGCC MCGGCACGC TGGTAGCTCA GGCAATCMC GGCACAGAAT TCATCACTAG ATATATGCTA CCTAAAMTA GAAAGGTGGC TGACACAGTC TTMCGAGAC ACACTACACC AGMCAGAGC TACGACTCTA CTTGGGGTAA MCATACGGC TTCGCGGTCT GTAACGGCGA GACGGTCTCC GAGTTCACAA GGAMGATCT CTGGAAAGTC CTTTACAACA TCTACACAGC GCATCCAGCT GAGMCATGC TCAACTCAAA CCCAAGCGTG TGGGGAGACC TTCCAAGGGT MGCGCTAAC CGCATCGACG CGGACGACGC GGAGGCCAGA TCAAGGCCGA TTMATTATC CAGAAAACAA AAAATCATGG CTGAGGTGAT TCAGCGAGCC ACTGATGGGT TACTGTTMC CTACMTGAT TTGGTGGTAC ATTTGTCTGA TTTGATGTTA ATGCTTGAGG GCATGCCGGG CGGGAGTAAA ACTGCTGAAC AGCTGCTTAC CATGATTCAC ATTAAATTGT GTGCCMAATA CMTGCTTAT GMTTTATGC TGATGAAAAC TCCTGCTACT CAAAACATGA ATCCGGGAGC ACCACACTAT GATTGCCAGG GAAACTTGGT CTTTAAGCTG CTCMCTTAC AAGGATACAA TCCTTGGCAA GTGGGGCACT GGTTAGTCAT GATGCTTTCT AAAAAAACGG GGAAAAGAM TTCTACTCTT TTCTATGGGC CGGCGAGCAC AGGGAAAACC MTCTCGCTA AGGCCATCTG CCACGCAGTG GGGCTATACG GGTGCGTGAA CCACAACAAC AAACAGTTTC CTTTTMCGA TGCACCCMC AAAATGATCC TGTGGTGGGA GGAATGCATC ATGACTACAG ACTACGTAGA GGCAGCAMG TGTGTGCTCG GGGGMCTCA CGTCAGGGTG GACGTGAMC ACAAAGATTC TAGGGAGCTG CCTCAGATTC CAGTGCTTCT TTCGAGCAAC CACGACGTGT ATACCGTCGT GGGGGGAMC GCCACGTTTG GAGTTCACGC GGCGCCGCTC AAAGAAAGAA TCACTCAAAT GMTTTTATG AAGCAGCTCC CTMCACTTT TGGAGAAATC ACTCCGGGCA TGATTTCAAA TTGGCTGTCT CACTGCGCGC ACATTCACCA promoter 3 AGAACATCTG TCGCTGGAAG GCTTTGCTAT TAAATGGGAC GTGCAGAGCG TGGGGAACAG CTTTCCTTTA CAGACTCTCT GTCCTGGCCA TTCACAGAAC TGGACATTCA GCGAAAACGG CGTCTGCTGG CACTGCGGAG GTTTCATCCA GCCMCACCA GAATCAGACA CTGACTCTGA CGGAGATCCT GACCCTGACG GTGCTGTTGC TGGCGATAGC GATACTTCTG CTMCAGTGA GTCTACAGTA TCGTTTAGCA GTAACGACTC AGGACTAGGA TCCGTCACTT CATCAGCTCC ATCGGTACCT GACAGAGCCG AGGAAATAGA GGAGATTCCC AGTGAGTGCT TGGAGTGGAT GCGGGAGGAG GTTGATAGAC TGAGCGCTCA CGACATCMC start of mid ORF -4 


(A) 3 'FIG. 3 .

 33 FIG. 3. Terminal structures of BPV DNA (minus strand). 




  BPV LEFT ORF (BP 731-2920; CALCULATED MOLECULARWEIGHT = 81523.88 




  C F I P T P E S D SV D P D P D P A V A 6 D S D T S E V S F S S MY" S EN NE1E PK 1T P IN LL C ASRPFrT TPK |T P LS NE^AL T PL EE ADE|[3NE"S TP NTPVV A E aN T[EE AAV KMCJ L 


FIG. 7 .

 7 FIG. 7. Homology of the translated right ORFs among parvoviruses AAV2, BPV, and MVMp. Homologous regions are enclosed by boxes. 




  Complete nt sequence of BPV (plus strand). 


	TGAGCMAAA AAATGTTTAG ACACTTGGTA TAGAAACATG ATGTATCACT TTGCCTTGGG GGGTGATTGT GAAAATGTA ATTACTGGGA TGATGTGTAC 3200
	start of VP2 +-end of mid ORF AAAAAACACT TGGCTAATGT AGACACTTAT TCTGTTGCAG MGAGATAAC TGATTCTGAA ATGCTGGAAG CGGCAGAAGC TGTTGATGCC GCCAACCAAT 3300 start of right ORF _
	polyadenylation signal AAAGCTAATT CAAAAAAAGG CCTGACATTA CCTGGCTACA ATTATTTGGG TCCATTCMT TCTTTATTCG CGGGCGCGCC AGTGAATAAA GCAGACGCGG 3400
	CAGCGCGAAA ACACGACTTT GGCTATTCCG ACTTGTTAAA GGAGGGAAAG AATCCCTACC TATACTTTAA CACACACGAC CAAAACCTCA TAGACGAACT 3500
	polyadenylation signal CAAAGACGAC ACTTCCTTTG GCGGGAAACT CGCAAGAGGA GTGTTTCAAA TAAAAAAAGC ACTGGCGCCA GCTCTGCCAG GCACATCCAA GGGAGGAGAC 3600
	start of VP3
	AGAGCCTTAA AAAGAAAGCT ATACTTTGCG CGCTCAAACA AGGGCGCCAA AAAAGCAAAC AGAGMCCTG CACCAAGCAC ATCCAATCAA CAGAACATGG 3700
	AGGTATCAM TGATATACCT AACGACGAGG CTGGCAATCA GCCAATTGAA CTGGCGACTC GGTCCGTGGT AGGATCGGGT TCGGTGGGTG GCGGCGGCCG 3800
	AGGGGGCTCT GGAGTGGGCT ACTCAACTGG CGGATGGACG GGGGGCACCA TATTTAGCGA GMCATTGTG GTCACTAAAA ACACTCGCCA GTTTATATGT 3900
	GACATCAAAA ACGGCCATCT CTATAAAAGC GAGGTGCTTA ACACTGGTGA CACGGCTCAC AGGCAATATG CCATTACCAC TCCGTGGAGC TATTTTAATT 4000
	TCAACCAGTA CAGCTCTCAC TTTAGTCCTA ATGACTGGCA ACACCTGGTC AACGACTACG AGAGATTCAG ACCAAAAGCC ATGATAGTGA GAGTCTACM 4100
	CCTGCAAATA AAACAGATCA TGACAGACGG AGCCATGGGG ACCGTCTACA ACAATGATCT GACTGCAGGC ATGCACATCT TCTGCGACGG GGATCACAGA 4200
	TACCCGTACG TACAGCATCC ATGGGATGAC CAATGCATGC CAGAGCTGCC AAACAGCATA TGGGAGCTAC CACAATATGC TTACATACCA GCTCCAATAT 4300
	CAGTCGTAGA CAACAACACT ACAAACACAG TAGAGAACA CCTACTGAA GGAGTGCCTC TGTACATGCT GGAAAACTCT GACCACGAAG TGCTACGCM 4400
	CGGGAGAATC TACAGAATTT ACATTCAACT TTGGAGACTG CGMTGGATA GAAAACAACA TCACATTCAG CATGCCTCAG ATGATGTACA ATCCACTGGT 4500
	CAGAAGCAGA AGAATCTACT CATACAGCGG ACCAMCAAC CAAACAAGCA ACGATTCCAG AATGCAGCAC TAAGAACCAG CMCTGGATG TCAGGACCGG 4600
	GGATCGCAAG GGGAACACAC MCGCMCTC TACAAACACA ATCTGCAGGA GCACTGGTGA CCATGGTAAC TMCGGAGCA GACGTCTCCG GGGTGAGAGC 4700
	CGTGCGAGTA GGATACTCGA CGGATCCAAT CTACGGGGGA CAGCAACCAG AGTCAGACCT GCTAAGACTG AGATACTCCG CATCAGCAGC AGMGGACAA 4800
	CAAAACCCAA TATTAGAAM CGCAGCAAGA CACACTTTTA CCAGAGAGGC GAGAACGAAA CTGATCACGG GATCCAACGG AGCAGACGGA GACTACAAAG 4900
	MTGGTGGAT GCTACCMAC CAGATGTGGG ACTCGGCGCC TATCTCGAGA TACAATCCAA TATGGGTCAA GGTACCGAGA GTCMCAGM AGACACTCCT 5000
	GGACACACAA GACGGATCCA TTCCMTGTC ACATCCGCCA GGAACCATCT TCATCAAGCT AGCAAGATT CCAGTACCAG GAAACGGAGA CTCGTTCCTC 5100
	AACATCTACG TCACCGGACA AGTCTCCTGC GMGTCGTCT GGGAGGTAGA AAAGAGGGGC ACCAAMATT GGAGACCGGA ATACATGCAC TCGGCAACAA 5200
	ACATGTCCGT CGATGCATAC ACCATCMCA ACGCAGGCGT CTACGCAGGC GCGGTACAAA ACGCAGACGT CATGCAGACG AGATTCMTC ACCACAAAGT 5300
	+-end of right ORF CCTGTAGGGG GCCGCMGAA AAAAGTCACT CTATACACCG CTTTATATTT TTATACACTG ATTCACTCTT TCCACCATCT TTTAGTTGTA TTCTTAGTTA 5400 terminal hairpin
	polyadenylation signal TCMTAAAGG CGCGAAGCGC CATAMAAAT TATGTCATAT GGCGCTTCGC GCCTTTATTG ATMCTAAGA ATACAACTAA MGATGGTGG A 5491
	FIG. 2.

2.5(m.u. 61.6 and 64.6). Our preliminary RNA mapping data GCAAAGGGAC TMTGAAATA TTCAATGGAC TAAAACAATC ATTTCAGTCA AAAGCGATTG ACGGGAAACT TGATTGGGAG GGGGTGAGAG AATTATTATT 3100 
TABLE 1 .

 1 Analysis of promoter sequences in parvoviruses 


	Virus	Promoter	Enhancer	Activator	TATA box
	AAV2	P (5.5)a P (18.0)	GGGAGGTC (186, -99)b GCTCCCCA (791, -82)	GCGACACC (231, -54)b GGGCGTGG (820, -53)	TATTTAA (255, -30)b TATTTAA (843, -30)
		P (39.0)	GGTGGAGC (1757, -94)	CCCGCCCC (1803, -48)	ATATAAG (1821, -30)
	MVMp	P (3.4)	GCGGTTCA (121, -84)	GGCGCGAA (144, -61)	TATATAA (175, -30)
		P (38.4)	GGGGCAAA (1921, -85)	GGGCGGAG (1951, -55)	TATAAAT (1976, -30)
	H-1	P (3.5)	GCGGTTCA (123, -85)	GGCGGGAA (147, -61)	TATATAA (178,-30)
		P (38.2)	GGGGCAAA (1924, -85)	GGGCGGAG (1954, -55)	TATAAAT (1979, -30)
	BPV	P (4.5)	GGCGAGGA (179, -100)	CCGGGGGG (227, -52)	TATATAA (249, -30)
		P (12.8)	CGAGCCGA (645, -89)	GGGGGAGA (681, -53)	

ATATAAT (704, -30) P (38.7) CCGGGCAT (2054, -104) GCGCGCAC (2085, -73) TATTAAA (2128, -30) 
TABLE 2 .

 2 Similarity coefficientsa between putative parvoviral proteins of AAV, BPV, and MVM SAB for proteins of: 


	ORF	Virus	AAV	BPV	MVM
	Left	AAV	1	0.15	0.15
		BPV	0.15	1	0.13
		MVM	0.15	0.13	1
	Right	AAVb	1	0.27	0.18
		BPV	0.27	1	0.20
		MVM	0.18	0.20	1
	Mid	AAV	1	NSC	NS
		BPV	NS	1	NS
		MVM	NS	NS	1
	a The similarity coefficient SAB between two proteins A and B is defined as
	(2 x number of identical residues in A and B)/ [(number of residues in A) +
	(number of residues in B)].			
	b The putative amino acid sequence used for AAV2 is translated from nt
	2191 to 2436 in frame 1 and then nt 2438 to 4324 in frame 2 (see the Discussion
	[The right ORF]).			
	c NS, Not significant.			
	B)/[(number of residues in A) + (number of residues in
	B)].				

TABLE 3 .

 3 Similarity coefficients between putative parvoviral proteins of B19, AAV, BPV, and MVM SAB for proteins of: 


	ORF	Virus	B19	AAV	BPV	MVM
	Left	B19	1	0.20	0.17	0.13
		AAV	0.20	1	0.15	0.15
		BPV	0.17	0.15	1	0.13
		MVM	0.13	0.15	0.13	1
	Right	B19	1	0.19	0.16	0.13
		AAV	0.19	1	0.27	0.18
		BPV	0.16	0.27	1	0.20
		MVM	0.13	0.18	0.20	1

ACKNOWLEDGMENTS

We are grateful to R. Foote for preparing the synthetic oligonu- cleotides and to S. Boyle for making the ICR SEQuence program available to us. This work was supported by American Cancer Society grant MV-220.
Katherine C Chen, Bruce C Shull, Elizabeth A Moses, Muriel Lederman, AND Ernest R Stout, Robert C Bates
Complete Nucleotide Sequence and Genome Organization of Bovine Parvovirus