Zika viruses encode multiple upstream open reading frames in the 5' viral region with a role in neurotropism

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus recently associated with congenital diseases and neurological complications. As for all flaviviruses, the ZIKV RNA genome is expected to encode a single polyprotein with all the enzymatic activities required for viral replication. Here, we report the discovery of multiple non-canonical open reading frames (ORFs) identified by ribosome profiling. In both mammalian and insect cells infected with Asian/American and African ZIKV strains, we observed translation of previously unrecognised upstream ORFs (uORFs) in the 5' region. In the Asian/American ZIKV lineage, ribosomes translated uORF1 and uORF2 that initiated from non-AUG start codons, whereas in the African ZIKV lineage, these two uORFs were fused into a single uORF (African uORF). Using a reverse genetics system, we examined the impact on ZIKV fitness of the expression of single or dual uORFs by analysing a panel of mutant viruses. We found that expression of the African uORF, and more significantly, the Asian/American uORF1, modulated virus growth and tropism in human cortical neurons and 3D organoid tissue, indicating that these novel uORFs contribute to ZIKV neurotropism. Although ZIKV uORFs are expressed in mosquito cells, they did not have a detectable effect on transmission by the mosquito vector in vivo. Our discovery of ZIKV uORFs sheds new light on ZIKV-induced neuropathogenesis and raises the question of their existence in other neurotropic flaviviruses.