Analysis of nucleotides 13–96 of the human parainfluenza virus type 3 antigenomic promoter reveals positive- and negative-acting replication elements

During replication of human parainfluenza virus type 3 (HPIV3), the 96-nucleotide antigenomic promoter (AGP) of HPIV3 directs the synthesis of genomic RNA. Previous work showed that nucleotides 1–12 were critical in promoting replication of an HPIV3 minireplicon, but nucleotides 13–96 were not investigated. In this study, the role of nucleotides 13–96 in AGP function was analyzed by creating and assaying mutations in an HPIV3 minireplicon. A replication promoting element known as promoter element II (nt 79–96) was confirmed in the HPIV3 AGP. Additionally, nucleotides 13–39 were found to constitute an additional positive-acting cis-element. However, detailed analysis of the 13–39 element revealed a complicated control element with both stimulatory and repressing elements. Specifically, nucleotides 21–28 were shown to repress RNA replication, while flanking sequences had a stimulatory effect.