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Genome synthesis in paramyxoviruses, including Nipah virus (NiV), is controlled by sequence elements that reside in the non-coding nucleotides at the 5'-trailer (3'-antigenomic) end that make up the antigenomic promoter (AGP). Using a chloramphenicol acetyl transferase-based plasmid-driven minigenome system, the terminal 96 nt of NiV AGP were first mutagenized in blocks of three hexamers to enable broad mapping of the minigenome functional regions. This was followed by further dissection of these functional regions to define the cis-acting elements contained therein. Results based on RNA analysis and reporter gene activity identified a bipartite promoter structure similar to that seen in related viruses, but with some distinct differences: in NiV, each of the two discrete replication control elements was bimodal, characterized by a critical conserved region (nt 1-12 and 79-91) and a contiguous non-conserved region (nt 13-36 and 73-78), which appeared less important. The regulatory role of these less critical regions was underscored by the use of a two-step mutation strategy, which revealed the additive detrimental effect of substitutions in this part of the terminal element. The structure and sequence characteristics of the internal control element was also different: it involved four contiguous hexamers, and the region encompassing three of these (nt 79-96, corresponding to hexamers 14, 15 and 16), although analogous in position to the equivalent element in the Sendai virus AGP, was characterized by the distinct 5'-(GNNNUG)(14-15)(G)(16) motif.

Cis-acting elements in the antigenomic promoter of Nipah virus