Long-Range Interaction of 3' Terminal Stem-Loop Structure in Nodamura virus (NoV) with Upstream RNA2 Sequence necessary for RNA replication

Viruses with RNA genomes, including West Nile and hepatitis C, depend on genomic RNA structures for their replication. A 3’-terminal stem-loop structure (3’SL) in Nodamura virus (NoV) RNA2 is essential for its replication. NoV was used as the model system for studying mechanisms of viral RNA replication, because it can replicate its RNA to high levels in many host cells. Its positive-strand RNA genome is divided into two segments: RNA1 encodes the viral RNA polymerase (RdRp), while RNA2 encodes the viral capsid protein precursor. RNA2 contains a conserved 3’-terminal stem-loop structure (3’SL). RNA folding software predicted a 3’SL that can form a long-range base-pairing interaction with nts 600 bases upstream. We hypothesize that this putative long-range interaction plays a role in viral RNA replication. We tested our hypothesis by making mutations in a full-length RNA2 cDNA clone and examining their ability to replicate in mammalian cells. Single mutations disrupt the putative interaction by altering the loop or its upstream complement, while a double mutation restores the interaction. Cells were transfected with wildtype or mutant versions of NoV RNA2, together with RNA1 as a source of RdRp. Total cellular RNA was isolated and analyzed by Northern blot hybridization using probes specific for NoV RNA2. Quantification of these replication intermediates will allow us to determine whether the long-range interaction is required for RNA2 replication. A greater understanding of the mechanism of viral RNA replication may lead to the development of more effective antiviral agents against RNA viruses that infect humans.