Functional analysis of promoter element 2 within the viral polymerase gene of an emerging paramyxovirus, Sosuga virus

Paramyxovirus genomes carry bipartite promoters at the 3 ends of both their genome and antigenome, thereby initiating RNA synthesis, which requires the viral polymerase to recognize two elements: the primary promoter element 1 (PE1) and the secondary promoter element 2 (PE2). We have previously shown that the antigenomic PE2 (agPE2) in many viruses in the Rubulavirinae subfamily is located within the coding region of the viral RNA polymerase L gene. Sosuga virus (SOSV), belonging to the Rubulavirinae subfamily, is highly pathogenic to humans, thus necessitating high-level containment facilities for infectious virus research. The use of a minigenome system permits studies of viral RNA synthesis at lower biosafety levels. Because minigenomes of negative-strand RNA viruses generally comprise only the untranslated regions, agPE2 within the L coding region--such as those found in Rubulavirinae like SOSV--are typically omitted. However, generating an SOSV minigenome that retains agPE2 led to a pronounced increase in activity, enabling a detailed examination of the role of agPE2 in SOSV replication. In many Rubulavirinae, the agPE2 not only acts as a promoter but also encodes part of the L protein, resulting in a distinct motif at the C-terminus of the L protein. We have further shown that this motif is preserved even in Rubulavirinae that no longer contain the agPE2 within the L gene. ImportanceParamyxoviruses are classified into three major subfamilies: Orthoparamyxovirinae, Avulavirinae and Rubulavirinae. All paramyxovirus genomes and antigenomes possess bipartite promoters, comprising two elements: promoter element 1 (PE1) at the 3 end and promoter element 2 (PE2) located internally. We previously revealed that, in many Rubulavirinae, the antigenomic PE2 lies within the coding region of the viral RNA polymerase L gene. In this study, we used Sosuga virus, a member of the Rubulavirinae subfamily, to elucidate the role of antigenomic PE2 in viral replication. Because the PE2 region encodes part of the L protein, its presence leads to a distinctive motif at the C-terminus of L protein. Notably, this motif is conserved in all Rubulavirinae, including those that do not harbor the antigenomic PE2 within their L gene, indicating its importance in viral propagation.