The genomic promoter of human parainfluenza virus type 3 (HPIV3) contains multiple cis-elements controlling transcription and replication. Previous work showed that regions 1 to 12 and 79 to 96 were critical in promoting replication of an HPIV3 minireplicon, while the intergenic sequence and N gene start signal (IS/Ngs, bases 49 to 61) were important for transcription. Because these data were collected primarily using point mutations, not every base from position 1 to 96 was analyzed, and some important control elements may have been missed. To clarify the role of bases 13 to 78 in transcription and replication, a series of mutations were made which collectively scanned this entire region. Mutation of bases 13 to 28 resulted in markedly decreased HPIV3 minireplicon replication, indicating these bases constitute an additional cis-element involved in the synthesis of the HPIV3 antigenomic RNA. The position dependence of the IS/Ngs was also examined. Analysis of mutants in which the IS/Ngs was shifted 5 or 3 showed that this segment could be moved without significantly disrupting transcription initiation. Additional mutants which contained two successive IS/Ngs segments were created to test whether the polymerase accessed the gene start signal by proceeding along the template 3 to 5 or by binding internally at the gene start signal. Based on analysis of the double gene start mutants, we propose a model of internal transcription initiation in which the polymerase enters the template at approximately the location of the natural N gene start but then scans the template bidirectionally to find a gene start signal and initiate transcription.Human parainfluenza virus type 3 (HPIV3) is an enveloped, nonsegmented, negative-sense RNA virus within the Respirovirus genus of the subfamily Paramyxovirinae, family Paramyxoviridae, order Mononegavirales. HPIV3 is an important cause of lower respiratory tract infections in young children and is being increasingly recognized as a cause of serious lower respiratory tract infections in the immunocompromised and elderly (4,14,15,35).During replication of the mononegaviruses, the nucleocapsid (N) protein-encapsidated genomic and antigenomic RNAs serve as templates for the synthesis of cRNA. Specific factors affecting RNA replication vary among the different families and subfamilies of the Mononegavirales. For the Paramyxovirinae, which includes the Respirovirus (HPIV3 and Sendai virus [SeV]), Morbillivirus (measles virus), and Rubulavirus (simian virus 5 [SV5]) genera, these factors include a requirement for specific sequence elements at the 3Ј ends of the genome and antigenome, proper spacing between sequence elements, and adherence to the rule of six (20,39).The genomic and antigenomic promoters (the 3Ј ends of the genome and antigenome; GP and AGP, respectively) of all members of the Paramyxovirinae studied thus far contain two critical regions: conserved region I (CRI) and conserved region II (CRII) (16,22,24,26,33,34,38). Conserved region I is at the extreme 3Ј end of the genomic ...