Different Degrees of 5'-to-3' DAR Interactions Modulate Zika Virus Genome Cyclization and Host-Specific Replication

Mosquito-borne flaviviruses, which include many important human pathogens, such as West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV), have caused numerous emerging epidemics in recent years. Details of the viral genome functions necessary for effective viral replication in mosquito and vertebrate hosts remain obscure. Here, using ZIKV as a model, we found that the conserved "downstream of AUG region" (DAR), which is known to be an essential element for genome cyclization, is involved in viral replication in a host-specific manner. Mutational analysis of the DAR element showed that a single-nucleotide mismatch between the 5' DAR and the 3' DAR had little effect on ZIKV replication in mammalian cells but dramatically impaired viral propagation in mosquito cells. The revertant viruses passaged in mosquito cells generated compensatory mutations restoring the base pairing of the DAR, further confirming the importance of the complementarity of the DAR in mosquito cells. We demonstrate that a single-nucleotide mutation in the DAR is sufficient to destroy long-range RNA interaction of the ZIKV genome and affects de novo RNA synthesis at 28°C instead of 37°C, resulting in the different replication efficiencies of the mutant viruses in mosquito and mammalian cells. Our results reveal a novel function of the circular form of the flavivirus genome in host-specific viral replication, providing new ideas to further explore the functions of the viral genome during host adaptation.IMPORTANCE Flaviviruses naturally cycle between the mosquito vector and vertebrate hosts. The disparate hosts provide selective pressures that drive virus genome evolution to maintain efficient replication during host alteration. Host adaptation may occur at different stages of the viral life cycle, since host-specific viral protein processing and virion conformations have been reported in the individual hosts. However, the viral determinants and the underlying mechanisms associated with host-specific functions remain obscure. In this study, using Zika virus, we found that the DAR-mediated genome cyclization regulates viral replication differently and is under different selection pressures in mammalian and mosquito cells. A more constrained complementarity of the DAR is required in mosquito cells than in mammalian cells. Since the DAR element is stably maintained among mosquito-borne flaviviruses, our findings could provide new information for understanding the role of flavivirus genome cyclization in viral adaptation and RNA evolution in the two hosts.