The positions of sequences necessary for transcription from the promoter located at map unit 4 of the bovine parvovirus (BPV) genome were determined. The autonomous parvoviruses, of which BPV is a member, contain two transcriptional units with promoters active in temporal order during infection. BPV proteins also appear in the same temporal order; the nonstructural (NS) proteins are produced before the capsid proteins. Northern blot analysis of BPV RNAs suggest that, like human parvovirus B19, all transcripts of BPV are initiated from a single promoter. A reporter construct was created by cloning the sequences from BPV containing the TAT A box located at nucleotides (nt) 250 - 254 upstream of the luciferase gene. A series of mutants were generated by deletion of restriction endonuclease fragments. Expression was assayed by transient expression of the constructs in transfected bovine fetal lung (BFL) cells, derived from the natural host. The data indicates that expression can be directed from the sequences containing the TAT A box. Analysis of expression of the deletion clones show that sequences from nt 120 - 170 of the BPV genome are also required for transcription. A search of these sequences reveals at least two consensus binding sites for cellular transcription factors. These are AP-I and the major late transcription factor (MLTF). MLTF has been shown to induce transcription from the early promoter of the dependovirus adena-associated virus (AA V). The presence of viral proteins provided in trans decreases expression from all constructs, with one exception. Expression, when nt 0 - 50 are deleted, is increased in the presence of mutant BPV NS-I. Feedback inhibition of P4 expression by NS-I, the P4 gene product, is seen in H-l, a rat parvovirus. The expression of BPV proteins in synchronized HeLa cells, which are not a natural tissue culture host was examined to determine the effect of a non-permissive host on BPV transcription.

Expression of viral proteins in some parvoviruses is blocked in non-permissive cells. A block in transcription is seen during infection of non-permissive cells by B 19. AA V P5 expression is negatively regulated without co-infection of a helper virus, a non-permissive state. Luciferase activity was 570/0, compared to BFL cells. and a similar decrease in expression in the presence of viral proteins was observed. Viral nonstructural and capsid proteins could be detected by immunofluorescence, but only in the cytoplasm, suggesting that expression of viral proteins necessary for replication was not the block to a productive infection, but rather their translocation to the nucleus, as seen during restrictive H-l infection of transformed cells. BPV proteins have been observed localized to nuclear foci of transfected, synchronized BFL cells. The subcellular localization of viral proteins was detected by indirect immunofluorescence labeling using antiserum that recognizes both nonstructural and capsid proteins. Punctate nuclear staining has not been observed routinely during BPV infection or transfection of actively dividing cells. AA V proteins expressed in either BFL and HeLa cells, synchronized by hydroxyurea, were also observed in distinct nuclear foci. The same pattern of localization has been observed during co-infection of cells with AA V and adeno-virus, and during H-I infection.

Gel mobility shift assays show that a cellular protein from BFL cells synchronized in S-phase recognizes and binds the right terminus of BPV. The right terminus, in hairpin and double-stranded linear forms, is an effective competitor, indicating the complex is specific, and suggesting that sequence, rather than structure may be the recognition signal for this cellular protein. The left terminus of BPV, in the hairpin conformation is also an efficient competitor for complex formation. It has been shown that a cellular protein forms a complex with the left terminus and that the right terminus is an efficient competitor for complex formation. This data taken together suggests the same cellular factor may recognize both termini, and correlates with the observation that both can act as origins of replication, and could be recognized by similar proteins. The heterologous terminus of AAV in the hairpin conformation, is not an efficient competitor for complex formation between the right terminus of BPV and a BFL cell protein. The lack of competition may support the indication that the recognition signal is a specific sequence rather than a particular secondary structure.