Cis-acting Signals for Replication of Nodamura Virus RNA1

The family Nodaviridae is comprised of two genera: the alphanodaviruses that infect insects and the betanodaviruses, which have been isolated only from fish. Nodamura virus (NoV), the type species of the alphanodavirus genus, can lethally infect both insects (mosquitoes, honey bees, and wax moth larvae) and mammals (suckling mice and suckling hamsters). In addition, nodavirus RNAs can replicate in a wide variety of host cells, including those of mammalian, insect, plant, and yeast origin, when introduced by transfection. The nodavirus positive strand RNA genome is naturally segregated into two segments. RNA1 encodes the viral RNA-dependent RNA polymerase (RdRp) that replicates both genome segments via negative strand intermediates, while RNA2 encodes the precursor to the viral capsid proteins. During RNA replication a subgenomic RNA3 is synthesized from RNA1 and is identical to the last 470nt of RNA1. RNA3 encodes proteins B1 and B2 from overlapping reading frames. Protein B1 has no known function and B2 has been shown to suppress RNA interference. In addition, RNA3 has been shown to be required for RNA2 replication. As a part of ongoing studies to investigate the mechanism of nodavirus RNA replication, we wished to identify cis-acting RNA elements (structures or sequences within the RNA) that are required for replication of RNA1. We examined the 3\u27 untranslated region (UTR) of NoV RNA1 for these elements based on the facts that viral UTR\u27s have been shown to be important for viral RNA replication, previous work with Flock House virus (FHV) has shown that the replication signals for both FHV RNA1 and RNA1 lie in their 3\u27UTRs, and that a stem-loop in the NoV RNA2 3\u27UTR was shown to be essential for its replication We used three different computer programs running on the RNAVLab platform to predict the RNA secondary structure of the 3\u27UTR of NoV RNA1. All three programs consistently predicted the presence of a stem-loop structure (N1-3\u27SL) near the 3\u27 end of RNA1 (nt 3162-3177). To test the relevance of the structure in RNA1 replication, we used a well-characterized reverse genetic system for the launch of NoV RNA replication in transformed yeast cells to test the effect of deleting the predicted N1-3\u27SL structure. Our results showed that the deletion of the stem-loop reduced the accumulation of both positive and negative sense RNA1 and RNA3 replication products in yeast. To determine if the loop portion, instead of the entire structure, of the N1-3\u27SL comprised the replication signal for RNA1, we mutated nucleotides in the loop region of N1-3\u27SL. When we tested this mutant in the yeast cell system, it displayed a severe reduction in RNA1 replication and RNA3 synthesis. The observation that both positive and negative strands were affected suggested that the primary defect was in negative strand synthesis and that the reduction in positive strands was secondary to this defect. We wondered whether there were also internal cis-acting replication signals in NoV RNA1. To facilitate the study of RNA1 template properties without affecting the viral RdRp ORF, we separated the mRNA and template functions of RNA1 onto two different molecules: an mRNA that expresses RdRp but cannot itself be replicated and an RNA1 template that doesn\u27t produce RdRp but can be replicated in trans by the expressed RdRp. Four RdRp mRNA constructs have displayed endogenous RNA replication template activity, making it impossible to assess replication of our RNA1 templates. An alternative strategy will be required to produce the RdRp mRNA