Specialized cis-Acting RNA Elements Balance Genome Cyclization to Ensure Efficient Replication of Yellow Fever Virus.

Genome cyclization is essential for viral RNA (vRNA) replication of the vertebrate-infecting flaviviruses, and yet its regulatory mechanisms are not fully understood. Yellow fever virus (YFV) is a notorious pathogenic flavivirus. Here, we demonstrated that a group of cis-acting RNA elements in YFV balance genome cyclization to govern efficient vRNA replication. It was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) is conserved in the YFV clade and is important for efficient YFV propagation. By using two different replicon systems, we found that the function of the DCS-HP is determined primarily by its secondary structure and, to a lesser extent, by its base-pair composition. By combining in vitro RNA binding and chemical probing assays, we found that the DCS-HP orchestrates the balance of genome cyclization through two different mechanisms, as follows: the DCS-HP assists the correct folding of the 5' end in a linear vRNA to promote genome cyclization, and it also limits the overstabilization of the circular form through a potential crowding effect, which is influenced by the size and shape of the DCS-HP structure. We also provided evidence that an A-rich sequence downstream of the DCS-HP enhances vRNA replication and contributes to the regulation of genome cyclization. Interestingly, diversified regulatory mechanisms of genome cyclization, involving both the downstream of the 5'-cyclization sequence (CS) and the upstream of the 3'-CS elements, were identified among different subgroups of the mosquito-borne flaviviruses. In summary, our work highlighted how YFV precisely controls the balance of genome cyclization to ensure viral replication. IMPORTANCE Yellow fever virus (YFV), the prototype of the Flavivirus genus, can cause devastating yellow fever disease. Although it is preventable by vaccination, there are still tens of thousands of yellow fever cases per year, and no approved antiviral medicine is available. However, the understandings about the regulatory mechanisms of YFV replication are obscure. In this study, by a combination of bioinformatics, reverse genetics, and biochemical approaches, it was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) promotes efficient YFV replication by modulating the conformational balance of viral RNA. Interestingly, we found specialized combinations for the downstream of the 5'-cyclization sequence (CS) and upstream of the 3'-CS elements in different groups of the mosquito-borne flaviviruses. Moreover, possible evolutionary relationships among the various downstream of the 5'-CS elements were implied. This work highlighted the complexity of RNA-based regulatory mechanisms in the flaviviruses and will facilitate the design of RNA structure-targeted antiviral therapies.

Generation and Application of a Luciferase Reporter Virus Based on Yellow Fever Virus 17D.

Yellow fever virus (YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans. Despite the availability of an effective vaccine, little is known about the replication mechanism of YFV, and there are still no available specific anti-YFV medicines. Herein, by introducing the Renilla luciferase gene (Rluc) into an infectious clone of YFV vaccine strain 17D, we generated a recombinant virus 17D-Rluc.2A via reverse genetics approaches. The 17D-Rluc.2A had similar plaque morphology and comparable in vitro growth characteristics with its parental strain. Importantly, the reporter luciferase was efficiently expressed in 17D-Rluc.2A-infected mammalian and mosquito cells, and there was a good linear correlation between intracellular luciferase expression and extracellular infectious virion reproduction. Furthermore, by a combination of the 17D-Rluc.2A reporter virus and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) technology, the conserved 5'-SLA element was shown to be essential for YFV replication, highlighting the capability of 17D-Rluc.2A in the investigation of YFV replication. At last, we demonstrated that two compounds with distinct anti-viral mechanisms can effectively inhibit the viral propagation in 17D-Rluc.2A-infected cells, demonstrating its potential application in the evaluation of anti-viral medicines. Taken together, the 17D-Rluc.2A serves as a useful tool for the study of YFV replication and anti-YFV medicine development.