| Dengue virus, a member of the Flavivirus genus of positive-strand RNA viruses, is an important arboviral pathogen worldwide. Tens of millions of cases of dengue fever resulting in hundreds of thousands of hospitalizations are estimated to occur annually. Other flaviviruses that cause disease in humans include yellow fever, Japanese encephalitis, West Nile and tick-borne encephalitis viruses. Conserved sequences and structures in the untranslated regions (UTRs) of flaviviruses have been shown to regulate the processes of viral protein and RNA synthesis; however, the role of RNA elements in the coding region in regulating these processes has not been widely explored. Flavivirus genomes are translated as a single polyprotein that is cleaved into 10 proteins, the first of which is the viral capsid protein (C). The viral polyprotein contains a number of in-frame start codons contained in the C coding region, and the first start codon is in a weak initiation context, predicting inefficient translation of the full-length polyprotein. Using a reverse genetic approach, we show that an RNA hairpin structure in the C-coding region (cHP) enhances translation from the first start codon in a manner consistent with a mechanism of ribosome stalling. An intact cHP is required for viral replication in mosquito and human cells. Moreover, mutation of the nucleotides immediately upstream and downstream of the start codon to improve the initiation context also resulted in a defect in viral replication, implicating the use of this atypical mechanism of start codon selection to overcome constraints on the sequence surrounding the start site. Finally, we demonstrate that the cHP performs a dual role in the viral life cycle, also functioning at the step of RNA synthesis. Thus, we have identified a unique coding-region RNA element that participates in two distinct stages of the viral life cycle. Since the cHP is predicted to be maintained among the flaviviruses analyzed, it likely plays a similar role for other members of the genus. Characterization of the viral determinants of replication in mammalian and mosquito cells may provide insight into normal cellular processes and allows for the rational design of vaccines and antiviral therapies. |
