The dengue virus type 3 (DENV-3) vaccine candidate, rDEN3Δ30, was previously found to be underattenuated in both SCID-HuH-7 mice and rhesus monkeys. Herein, two strategies have been employed to generate attenuated rDEN3 vaccine candidates which retain the full complement of structural and nonstructural proteins of DENV-3 and thus are able to induce humoral or cellular immunity to each of the DENV-3 proteins. First, using the predicted secondary structure of the 3' untranslated region (3'-UTR) of DENV-3 to design novel deletions, nine deletion mutant viruses were engineered and found to be viable. Four of nine deletion mutants replicated efficiently in Vero cells and were genetically stable. Second, chimeric rDENV-3 viruses were generated by replacement of the 3'-UTR of the rDENV-3 cDNA clone with that of rDENV-4 or rDEN4Δ30 yielding the rDEN3-3'D4 and rDEN3-3'D4Δ30 viruses, respectively. Immunization of rhesus monkeys with either of two deletion mutant viruses, rDEN3Δ30/31 and rDEN3Δ86, or with rDEN3-3'D4Δ30 resulted in infection without detectable viremia, with each virus inducing a strong neutralizing antibody response capable of conferring protection from DENV-3 challenge. The rDEN3Δ30/31 virus showed a strong host range restriction phenotype with complete loss of replication in C6/36 mosquito cells despite robust replication in Vero cells. In addition, rDEN3Δ30/31 had reduced replication in Toxorynchites mosquitoes following intrathoracic inoculation. The results are discussed in the context of vaccine development and the physical structure of the DENV 3'-UTR.

Several studies have generated computer-based predictions of secondary structure of the 39 untranslated region (UTR) of Dengue virus (DEN); however, experimental verification of the formation of these structures in vitro is lacking. This study assessed the congruence of Mfold predictions of secondary structure of the core region of the DEN type 4 39 UTR with nuclease maps of this region. Maps and predictions were largely consistent. Maps supported the existence of previously predicted pseudoknots and identified putative regions of dynamic folding. Additionally, this study investigated previously identified conserved elements in the flavivirus 39 UTR that differ among viruses with different modes of transmission. Specific regions of mosquito-borne DEN type 4 were either deleted or replaced with homologous sequences from tick-borne Langat virus. All of these mutations caused substantial distortion of secondary structure, yet viruses carrying these mutations were viable.

Nodamura virus (NoV; family Nodaviridae) contains a bipartite positive-strand RNA genome that replicates via negative-strand intermediates. The specific structural and sequence determinants for initiation of nodavirus RNA replication have not yet been identified. For the related nodavirus Flock House virus (FHV) undefined sequences within the 3′-terminal 50 nucleotides (nt) of FHV RNA2 are essential for its replication. We previously showed that a conserved stem-loop structure (3′SL) is predicted to form near the 3′ end of the RNA2 segments of seven nodaviruses, including NoV. We hypothesized that the 3′SL structure from NoV RNA2 is an essential cis-acting element for RNA replication. To determine whether the structure can actually form within RNA2, we analyzed the secondary structure of NoV RNA2 in vitro transcripts using nuclease mapping. The resulting nuclease maps were 86% consistent with the predicted 3′SL structure, suggesting that it can form in solution. We used a well-defined reverse genetic system for launch of NoV replication in yeast cells to test the function of the 3′SL in the viral life cycle. Deletion of the nucleotides that comprise the 3′SL from a NoV2-GFP chimeric replicon resulted in a severe defect in RNA2 replication. A minimal replicon containing the 5′-terminal 17 nt and the 3′-terminal 54 nt of RNA2 (including the predicted 3′SL) retained the ability to replicate in yeast, suggesting that this region is able to direct replication of a heterologous mRNA. These data suggest that the 3′SL plays an essential role in replication of NoV RNA2. The conservation of the predicted 3′SL suggests that this common motif may play a role in RNA replication for the other members of the Nodaviridae.

Virulence-attenuating viruses (hypoviruses) of the filamentous fungus Cryphonectria parasitica, the causative agent of chestnut blight, have become a premier model for understanding the molecular biology of mycoviruses. However, a major gap exists in current understanding of structure and function of the untranslated regions (UTRs) of the hypovirus RNA genome, despite considerable evidence that secondary and tertiary UTR structure plays a crucial role in the control of translation and genome replication in other systems. In this study we have used structure prediction software coupled with RNase digestion studies to develop validated structural models for the 5′ UTRs of the two best-characterized members of the Hypoviridae, CHV1-EP713 and CHV1-Euro7. These two hypovirus strains exhibit significant variation in virulence attenuation despite sharing >90 % sequence identity. Our models reveal highly structured regions in the 5′ UTR of both strains, with numerous stem-loops suggestive of internal ribosome entry sites. However, considerable differences in the size and complexity of structural elements exist between the two strains. These data will guide future, mutagenesis-based studies of the structural requirements for hypovirus genome replication and translation.