The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA 
of approximately 15 kb, contains six transcription units flanked at the 3' and 
5' ends by a short (approximately 50- to 60-nucleotide) extracistronic sequence, 
dubbed the positive and negative leader regions. These leader template regions, 
present at the 3' end of the genome and the antigenome, have been shown to 
contain essential signals governing RNA replication activity. Whether they are 
sufficient to promote replication is still open to question. By using a series 
of Sendai virus defective interfering RNAs carrying a nested set of deletions in 
the promoter regions, it is shown here that for both the genomic and antigenomic 
promoters, a 3'-end RNA sequence of 96 nucleotides is required to allow 
replication. Sequence comparison of active and inactive promoters led to the 
identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 
to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5'-3' 
positive-strand]. Sequential mutation of each hexamer into its complementary 
sequence confirmed their essential role. The three hexamers are required, and 
their relative positioning is important, since displacing them by 6 nucleotides 
destroyed promoter function. RNAs carrying degenerate nucleotides in the three 
hexamers were used as replication templates. They led to the selection of 
actively replicating RNA species exclusively carrying the basic motif (GNNNNN)3 
from nucleotides 79 to 96. These results clearly show that, apart from the 
region from nucleotides 1 to 31, previously identified as governing Sendai virus 
replication activity, a second element, spanning at the most nucleotides 79 to 
96, appears essential. Thus, the paramyxovirus replication promoters are not 
confined to the leader template regions, as seems to be the case for the 
rhabdoviruses.