Parvovirus NS1 stimulates P4 expression by interaction with the terminal repeats and through DNA amplification.

Parvovirus protein NS1 is required for replication of viral DNA and plays a role in the regulation of viral gene expression. NS1 trans-activates the P38 promoter for capsid protein synthesis and has variable effects on other promoters. In this study, we examined the effects of NS1 on the regulation of its own promoter, P4. A number of plasmid constructions were made with the P4 promoter fused to reporter genes. The effects of NS1 on expression from the P4 promoter differed depending on the construction. Plasmids containing viral sequences which could not replicate showed a decrease in P4 expression on cotransfection with the NS1 gene. However, plasmids having replication-proficient viral sequences showed a three- to fivefold increase in P4 expression dependent on NS1. The effect on NS1 on P4 transcription was also evaluated at the steady-state RNA level. An infectious clone of the LuIII viral genome was modified to an NS1-NS2 null mutant (pLu272) that is competent for viral DNA replication by introducing a frameshift mutation at codon 5 of the NS1 open reading frame. The P4 transcripts of pLu272 are four nucleotides longer than the wild type and can therefore be resolved from the wild type by primer extension analysis. pLu272 allows comparison of the constitutive level of steady-state RNA produced by the pLu272 P4 promoter in the absence or presence of a template replication dependent on NS1 supplied in trans. NS1 increased P4 transcripts about six- to eightfold. Expression of P4 transcripts from clones that could not amplify depended on the presence of an intact inverted terminal repeat sequence at the left end. A clone with an intact viral left end and a defective viral right end gave an NS1-dependent threefold increase in P4 expression. Destruction of terminal hairpins at both ends resulted in no significant increase in P4 expression in the presence of NS1. Thus, the positive effect of NS1 on the steady-state levels of P4 transcripts depends on the amplification of gene copy number and the integrity of the terminal repeats.