The nucleotide sequence of the parvovirus H-1 has been determined by the chain-terminating method of Sanger. The sequence is 5,176 nucleotides long. Two large open reading frames (1 and 2) and two smaller open reading frames (3 and 4) of potential importance were identified in the plus-strand sequence. Promoter sequences are located at map positions 4 and 38 when map positions are expressed as percent of genome length from the 3' end of the virion minus strand. The locations for the genes for the parvovirus capsid proteins and a 76,000-dalton noncapsid protein (NCVP1) were mapped by hybrid-arrested translation. The gene for the capsid proteins VP1 and VP2' is located in the 5' half of the virus genome. The gene for NCVP1 is located in the 3' half of the viral DNA.

The parvovirus early protein NS1 positively regulates the expression of the P38 promoter for the viral capsid protein gene. We have examined the trans-activation of P38 by NS1 by using fusions of P38 to the reporter gene, chloramphenicol acetyltransferase (cat). Maximal trans-activation requires a small 5' cis element (tar) between-137 and-116. The tar element has activity in both orientations when 5' to the P38 promoter, but no activity has been detected 3' to the promoter. The wild-type P38 has a biphasic response to NS1 depending on the dosage of the NS1-expressing plasmid. Promoters lacking the tar also have a biphasic response that is reduced about 10-fold, and they can be inhibited by larger doses of the NS1 plasmid. Heterologous promoters from other viruses and the Harvey-ras oncogene promoter are inhibited by NS1. Truncated and internally deleted versions of NS1 lose the trans-activation, but some of them retain the inhibitory properties. Thus transactivation can be uncoupled from inhibition. The tar element has shown no activity with the heterologous simian virus 40 early promoter. In contrast, the P38 promoter responds to a heterologous enhancer, but the enhanced promoter loses activity to trans-activation by NS1. In summary, the P38 tar element has some of the properties of an enhancer with a high preference for a 5' position and a stringent requirement for the P38 promoter. * Corresponding author. promoter specificity and has some activity in the absence of the proximal promoter elements. The inhibition of expression of P38 or heterologous promoters by NS1 and truncated or deleted mutants of NS1 will also be described.

The autonomously replicating parvoviruses contain a 5-kilobase linear single-stranded DNA genome that produces two noncapsid proteins, Ni and N2, and two overlapping capsid proteins, VP1 and VP2. To characterize the regulation of viral transcription, we began with a study of the promoter for the coat proteins (P38) at map unit 38. Various constructions containing the P38 promoter were fused to the bacterial gene for chloramphenicol acetyltransferase (cat), and the relative efficiency of expression was determined in the presence and absence of parvovirus gene products. Our results show that the P38 promoter is a weak promoter This work was supported by grant LB 506 85-56 from the state of Nebraska; Public Health Service grants RO1, CA26801, and CA36727, from the National Institutes of Health; and grant DBM-8444778 from the National Science Foundation. I thank L. Laimins for plasmids pAlocat and pSV2cat, Carol Wilson for technical assistance, and R. Hines, A. Rizzino, and E. Bresnick for reviewing the manuscript.

The nucleotide sequence of the canine parvovirus (CPV2) from map units 33 to 95 has been determined. This includes the entire coat protein gene and noncoding sequences at the 3' end of the gene, exclusive of the terminal inverted repeat. The predicted capsid protein structures are discussed and compared with those of the rodent parvoviruses H-1 and MVM.

Parasynchronous cultures of hamster embryo cells were used to study some of the events in the replication process of the parvovirus H-1. Synthesis of viral DNA, viral hemagglutinating antigen, and infectious virus were examined. It was found that initiation of DNA synthesis, on which subsequent viral hemagglutinin synthesis was dependent, occurred at a specific time in late S-phase. Production of H-1 viral protein was shown to be sensitive to inhibition by aamanitin. The implications of these findings are discussed.