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Sequence analyses of the left and right termini of LuIII virus show they are nonidentical imperfect palindromes of 122 and 211 nucleotides, respectively. The left terminus of the minus strand of LuIII DNA, uniquely in the flip conformation, can assume a T-shaped structure. The right terminus of the minus strand of LuIII DNA can assume a U-shaped structure, and it exists in either the flip or flop conformation. The termini of LuIII shared a high degree of sequence homology and showed conserved secondary structure with those of the rodent parvoviruses MVMp and H-1. LuIII, like adeno-associated virus, encapsidates equal amounts of plus- and minus-strand DNA. However, the sequence data for LuIII virus demonstrate that identical termini are not required for this encapsidation pattern.

Parvoviral genomes have been searched for sequences which may code for the nuclear transport of viral proteins. Sequences similar to those which regulate the nuclear transport of T antigen and yeast mating type protein were detected within the sequences coding for capsid and non-capsid proteins.

We have investigated the interaction between bovine parvovirus (BPV) capsid and non-capsid proteins and restriction fragments of the BPV genome by a combined DNA hybridization and immunodetection assay. 32P-labelled DNA was bound to nitrocellulose membranes bearing lysates of mock-infected and virus-infected cells whose proteins had been separated by SDS-polyacrylamide gel electrophoresis. The position of bound DNA was determined by autoradiography. The proteins on the membrane were still accessible to specific antibodies, allowing confirmation of the DNA-binding species by an immunodetection reaction. In 0.2 M-NaCl, BPV capsid proteins VP2 (72,000 daltons) and VP3 (62,000 daltons) bound the 0 to 16 map unit EcoRI fragment of BPV DNA which contained label in either the minus or plus strand. At higher salt concentration (0.5 M), only VP2 still bound DNA. Within this fragment, the capsid protein binding was restricted to those nucleotides between map units 0 and 4. No binding to capsid proteins was seen with the fragment spanning the middle of the genome and minor binding to VP3 was seen with the 5' end. Binding to the BPV non-capsid protein NP-1 was observed with the 0 to 16 map unit fragment when label was in the virion strand and to other possibly BPV-coded proteins when label was in the plus strand. The NP-1 binding was localized to map units 4 to 16. We did not detect binding to the BPV homologue(s) of the autonomous parvovirus non-capsid protein NS1, due in part to its low concentration in the cell lysates used. Points of the parvovirus replication cycle at which DNA-binding proteins may serve controlling functions are discussed.

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.

Transcription of the genome of the nondefective parvovirus BPV was examined in nuclei isolated from synchronized bovine fetal spleen cells. The relative levels of total RNA polymerase and RNA polymerase I, II, and III activities in nuclei isolated from BPV-infected and mock-infected cells were found to be similar throughout the course of infection. Hybridization of RNA synthesized in isolated nuceli indicated that BPV-specific RNA synthesis began during the period of 8 to 12 h postinfection and proceeded linearly until at least 20 h postinfection. By 20 h postinfection, 5% of the total RNA synthesized in nuclei from infected cells was virus specific. BPV-specific RNA synthesis was inhibited by 95% in the presence of 0.1 microgram of alpha-amanitin per ml, suggesting that the viral genome is transcribed by cellular RNA polymerase II.