Functional analysis of dengue virus cyclization sequences located at the 5′ and 3′UTRs

Flavivirus RNA replication involves cyclization of the viral genome. A model for this process includes a promoter element at the 5′ end of the genome and long-range RNA–RNA interactions. Two pairs of complementary sequences present at the ends of the viral RNA, known as 5′–3′CS and 5′–3′UAR, have been proposed to be involved in dengue virus genome cyclization. The requirement of 5′–3′CS complementarity for viral replication has been experimentally demonstrated for dengue and other mosquito borne flaviviruses. Here, we performed a functional analysis to study the role of 5′–3′UAR sequences using genomic and subgenomic dengue virus RNAs. We found that single mutations disrupting 5′–3′ complementarity greatly compromised viral RNA synthesis. Although in most of the cases incorporation of compensatory mutations re-established viral RNA replication, certain nucleotides were found to be involved in alternative secondary structures also important for viral replication. In addition, mutations within 5′ or 3′UAR in the context of an infectious dengue virus RNA resulted in spontaneous mutations that restored UAR base pairings. Together, we propose that specific UAR nucleotides as well as 5′–3′UAR complementarity constitute cis-acting signals involved in amplification of the dengue virus genome.