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. * Corresponding author. 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 presented. 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 appear to be useful for understanding the evolutionary relationship 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 Bethesda Research Laboratories, Inc. (Gaithersburg, Md.), and New England BioLabs, Inc. (Beverly, Mass.). Escherichia 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 Biochemicals, 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 reactions were the kind gift of R. Foote, Oak Ridge National Laboratory. Cell culture and virus propagation. BPV was p...
A phosphorylated protein (NP-1) with an Mr of 28,000 has been detected in nuclei of bovine parvovirus (BPV)-infected cells in association with chromatin. No protein in this size range was detected after infection of appropriate cells with several autonomous rodent parvoviruses although the BPV-specific protein is similar in size to noncapsid proteins associated with rabbit parvovirus or adeno-associated virus infection. Structural homology between NP-1 and a BPV capsid protein could be detected by electrophoretic analysis of the products of proteolysis with chymotrypsin. This protein can be detected after in vitro translation of RNA from BPV-infected cells and BPV-specific RNA. Homology between the in vivo-and in vitrosynthesized species was shown by the similarity of the chymotryptic products.
The distribution of terminal-sequence orientations in the viral DNA of bovine parvovirus (BPV), an autonomous parvovirus, was studied by end labeling and restriction enzyme digestion and also by cloning. The left (3') end of the minus strand of BPV was found in two alternative sequence orientations (designated as flip and flop, which are reverse complements of each other), with a 10-fold excess of flip. This is in contrast to the autonomous rodent parvoviruses which encapsidate'minus-strand DNA with only the flip orientation at this end. The right (5') end of the minus strand of BPV contained both sequence orientations with equal frequencies, as in the rodent parvoviruses. Sequence inversions were also detected'at both ends of the plus strand,'which makes up about 10% of the encapsidated BPV DNA. Each terminus of BPV DNA had a characteristic ratio of flip to flop forms, and this ratio was restored in the progeny DNA resulting from transfection with genomic clones of different defined terminal conformations. Replicative-form DNA showed the same distribution of terminal-sequence orientations as the reannealed plus and minus virion DNAs, suggesting that the distribution of flip and flop forms observed in virion DNA is not due to selective encapsidation, but rather to the specific distribution of replicative forms. The current replication model for autonomous parvoviruses, which was based on the available data for the rodent parvoviruses, cannot account for the observed distribution of BPV DNA. An alternative model is suggested.
Genomic clones of the autonomous parvovirus bovine parvovirus (BPV) were constructed by blunt-end ligation of reannealed virion plus and minus DNA strands into the plasmid pUC8. These clones were stable during propagation in Escherichia coli JM107. All clones tested were found to be infectious by the criteria of plaque titer and progressive cytopathic effect after transfection into bovine fetal lung cells. Sequencing of the recombinant plasmids demonstrated that all of the BPV inserts had left-end (3')-terminal deletions of up to 34 bases. DNA isolated from progeny virions arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogenous, cloned DNA. Full-length genomic clones with 3' flip and 3' flop conformations were constructed and were found to have equal infectivity. Analysis of low-molecular-weight DNA isolated from lysates of cells transfected with these clones demonstrated that rescue and replication of BPV DNA could be detected 3 to 8 days after transfection. Expression of capsid proteins from transfected genomes was demonstrated by hemagglutination, indirect immunofluorescence, and immunoprecipitation of [35S]methionine-labeled cell lysates. Use of appropriate antiserum for immunoprecipitation showed the synthesis of BPV capsid and noncapsid proteins after transfection. Independently, a series of genomic clones with increasingly larger 3'-terminal deletions was prepared from separately subcloned 3'-terminal fragments. Transfection of these clones into bovine fetal lung cells revealed that deletions of up to 34 bases at the 3' end lowered but did not abolish infectivity, while deletions of greater than 52 bases were lethal. End-label analysis showed that the 34-base deletion was repaired to wild-type length in the progeny virus.
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