The role of the 3′ terminus of the Sindbis virus genome in minus-strand initiation site selection

Genome replication of plus-strand RNA viruses begins with the generation of a minus-strand copy of the genome. Minus-strand synthesis must initiate at or close to the 3′ end of the genome and progress to processive elongation to yield the appropriate template for genomic RNA synthesis. The Sindbis virus genome possesses a 3′ polyadenylate tail preceded by a 19 nucleotide conserved sequence element (3′ CSE). Analyses of in vitro and in vivo synthesized minus-strand RNA presented in this manuscript identify the cytidylate residue immediately preceding the poly (A) tail as the predominant wild-type initiation site. Mutations in the poly (A) tail and the 3′ CSE caused the initiation site to shift to the poly (A) tail. Analysis of the products of non-wild-type initiation events demonstrated that they are not productively elongated. This study indicates that full-length minus-strand RNA synthesis is dependent upon initiation occurring at the appropriate site and suggests a mechanism for selection and maintenance of the wt 3′ CSE and poly (A) tail.