Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. Initially, ZIKV was considered of low importance as infection was usually asymptomatic or caused mild illness. However, the emergence of the Asian/American lineage, in contrast to the African lineage, was considered a public health threat. The Asian/American lineage has been associated with neurological complications such as Guillain-Barré syndrome in adults and congenital ZIKV syndrome (e.g., microcephaly) in newborns.

Currently, it is not understood why this newly emerged Asian/American ZIKV results in a more severe disease than the African ZIKV. This project focused on understanding the molecular mechanisms underlying the differences in virulence and pathogenicity between these two ZIKV lineages in human brain cells. In order to identify the molecular determinants that influence viral replication and subsequent ZIKV virulence, the differences between an African and an American ZIKV strain were studied at different levels.

Firstly, RNA-Seq and ribosome profiling (Ribo-Seq) was performed on astrocytoma-glioblastoma cells (U251 cells) infected with an African and an American ZIKV strain to determine the transcriptional and translational profile of each virus. Two previously overlooked upstream open reading frames (uORFs) in the 5′ untranslated region (UTR) of the American ZIKV genome were identified, initiating at non-canonical CUG and UUG codons; and a single uORF in the 5′UTR of the African ZIKV, initiating at a CUG start codon. The uORFs in the American ZIKV are referred to as ‘uORF1’ and ‘uORF2’, and the one in the African ZIKV as the ‘African uORF’.

Secondly, considering their overlap with the main ORF, the viral polyprotein, it was hypothesised that these uORFs could play a role in the translational regulation of the main ORF. To investigate this, luciferase reporter assays were used, which showed that when the ZIKV 5′UTR harboured an African uORF or when uORF1 was not expressed, the main ORF was better translated compared to the wild-type, in contrast to the knock-out of uORF2.

Thirdly, the presence of these uORFs was shown to modulate virus growth and replication by using mutant viruses that modulate the expression of the different uORFs and comparing these to the wild-type American ZIKV strain by performing viral growth curves and competition assays. Interestingly, the uORF2 knock-out virus showed a similar infectivity as the wild-type American virus, whereas the uORF1 knock-out virus and the African-like virus reached higher fitness than the wild-type virus. Additionally, transient overexpression of the uORF1 peptide and its subcellular localisation within the cytoskeletal fraction suggests a contribution to the dysregulation of the cytoskeleton, which has been associated with neurodegenerative diseases.

Finally, a differential gene expression analysis was performed in which cellular genes that were differentially transcribed by using the RNA-Seq datasets, but also the genes that had a different translational efficiency by using the Ribo-Seq datasets in response to infection with different ZIKV strains were thoroughly analysed. Infection of U251 cells with an African ZIKV strain led to an early upregulation of innate immune response genes, but infection with an American ZIKV strain induced the expression of a broader range of genes related to the antiviral response.

With this first analysis of neurotropic flavivirus gene expression using ribosome profiling, the functional characterisation of novel uORFs and the comparative analysis of differentially up- or down-regulated cellular genes, novel insights were provided into important differences between the African and the Asian/American ZIKV lineages, two viruses that result in different pathologies.