5'-proximal hot spot for an inducible positive-to-negative-strand template switch by coronavirus RNA-dependent RNA polymerase.

Coronaviruses have a positive-strand RNA genome and replicate through the use of a 3' nested set of subgenomic mRNAs each possessing a leader (65 to 90 nucleotides [nt] in length, depending on the viral species) identical to and derived from the genomic leader. One widely supported model for leader acquisition states that a template switch takes place during the generation of negative-strand antileader-containing templates used subsequently for subgenomic mRNA synthesis. In this process, the switch is largely driven by canonical heptameric donor sequences at intergenic sites on the genome that match an acceptor sequence at the 3' end of the genomic leader. With experimentally placed 22-nt-long donor sequences within a bovine coronavirus defective interfering (DI) RNA we have shown that matching sites occurring anywhere within a 65-nt-wide 5'-proximal genomic acceptor hot spot (nt 33 through 97) can be used for production of templates for subgenomic mRNA synthesis from the DI RNA. Here we report that with the same experimental approach, template switches can be induced in trans from an internal site in the DI RNA to the negative-strand antigenome of the helper virus. For these, a 3'-proximal 89-nt acceptor hot spot on the viral antigenome (nt 35 through 123), largely complementary to that described above, was found. Molecules resulting from these switches were not templates for subgenomic mRNA synthesis but, rather, ambisense chimeras potentially exceeding the viral genome in length. The results suggest the existence of a coronavirus 5'-proximal partially double-stranded template switch-facilitating structure of discrete width that contains both the viral genome and antigenome.