A conserved RNA structure at the capsid-coding sequence of Zika virus genome is required for viral replication in a host-dependent manner.

Flaviviruses are emerging and re-emerging pathogens causing widespread epidemics worldwide. Their RNA genomes play multiple roles during infection, folding into dynamic structures that regulate viral processes. To understand the mechanisms of flavivirus infection and to design genetic tools for viral countermeasures, it is important to dissect functional RNA structures present in viral genomes. Here, we investigate RNA structures within the open reading frame of the Zika virus (ZIKV) genome that regulate viral replication. We identified a functional stem-loop structure, SL1, located within the conserved C1 element in the capsid protein coding sequence of mosquito-borne flavivirus genomes. The integrity of the SL1 structure was crucial for viral RNA amplification in mosquito cells and enhanced ZIKV replication in vertebrate cells. Evolution experiments in mosquito cells with lethal SL1-disrupting mutants revealed reversions and pseudo-reversions that restored SL1 structure, confirming its role as a cis-acting RNA element. We also found that a sequence within SL1 contributes to a novel genome cyclization element unique to ZIKV. This sequence folds locally into SL1 or hybridizes with a 3' UTR sequence to extend the conserved cyclization sequence (CS1), which is known to be essential for RNA synthesis. Although the C1 element is conserved among mosquito-borne flaviviruses, the RNA structures and long-range interactions in this element required for ZIKV replication differ from those reported for dengue virus. Our studies highlight the presence of a conserved RNA element operating through distinct mechanisms in related flaviviruses. These findings offer insights into the dynamic nature of the ZIKV genome and provide information for rational flavivirus attenuation. IMPORTANCE: Flaviviruses are important human pathogens mainly vectored by arthropods. They contain RNA genomes that fold into complex structures with biological functions in viral infection. Zika virus is a flavivirus that has caused significant outbreaks and epidemics around the world. In this study, we used Zika virus to identify functional RNA structures present in the viral coding sequence. We manipulated an infectious clone from an Argentinean Zika virus isolate to dissociate protein-coding sequences from cis-acting RNA structures and discovered an RNA element in the capsid coding region that is essential for Zika virus replication in mosquito cells. Point mutations, disrupting the identified structure, impaired infection in mosquito cells and rendered viral attenuation in mammalian cells. Selection of revertant viruses in cell culture restored the RNA structure and the viral replication capacity. Our studies provide a basic understanding of the flavivirus genome organization, which is necessary for designing rational antiviral strategies.