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. / Ph. D.

The termini of the autonomous parvovirus LuIII, which encapsulates plus and minus DNA strands equally, were cloned and sequenced. The left and right termini of LuIII differ in nucleotide sequence and these termini can assume T- and U-shaped intra-strand base-paired structures, respectively. The LuIII termini are virtually identical in nucleotide sequence and secondary structure to those of the rodent parvoviruses MVM and H-1. The presence of non-identical LuIII termini demonstrated that identical ends are not required for the encapsidation of both DNA strands with equal frequency, as suggested for parvoviruses B19 and AAV. An infectious genomic clone of LuIII was constructed and sequenced. The LuIII genome is 5135 bases and it shares over 80% sequence identity with the sequence of the genomes of MVM and H-1. The genome organization of LuIII is virtually identical to that of the rodent parvoviruses of known sequence. The major ORFs, the left and right ORFs, are restricted to the plus strand. Promoter-like sequences are present at map units 4 and 38. The transactivation responsive element (TAR), characterized in H-1, upstream of P38, is also present in LuIII. Regulatory sequences and splice donor-acceptor consensus sequences, characterized in MVM and H-1, are also present in LuIII. This suggests that both LuIII promoters are functional, and that the transcription map for LuIII could be very similar to that of MVM. The LuIII sequence has only a single copy of a repeat present in tandem at the right end of the MVMp genome. Downstream of this sequence, an A-T rich region of 47 nt is present in LuIII. Since this A-T rich region is absent from the genomes of MVM and H-1, we propose that it represents a putative encapsidation signal responsible for the encapsidation pattern observed for LuIII. Northern analysis of BPV RNAs suggests that, like the human parvovirus B19, most, if not all, BPV transcripts initiate at promoter sequences localized at map unit 4. Amplification of BPV cDNA ends by the polymerase chain reaction resulted in a number of BPV-specific fragments. Four of these fragments were cloned and sequenced. Sequencing revealed two splices, one of which is very likely a major splice for several BPV transcripts. cDNA fragments were assigned to transcripts possibly coding for three BPV non-structural proteins. Amplification of BPV transcripts with primers specific to the mid-ORF suggests that the amino terminus of the capsid protein VP1 is not coded for by the mid-ORF as suggested by earlier studies, but instead results from one or both of the two small ORFs present upstream of the right ORF, in the same reading frame. / Ph. D.