(A-34) From systems to pathogenesis: zooming into molecular mechanisms of Zika virus replication and neuropathogenesis.

Introduction:: All viruses hijack host processes, often through virus-host protein interactions. These protein interactions can disrupt the physiological molecular function of the host protein. For Zika virus (ZIKV), these protein interactions may serve to facilitate virus replication while simultaneously disrupting development to cause congenital Zika syndrome and severe microcephaly. Identifying and dissecting ZIKV-host protein interactions is essential to elucidate molecular mechanisms of ZIKV replication and pathogenesis.

Materials and Methods:: We used global proteomics and in vivo models of brain development to establish that ZIKV non-structural protein 4A (NS4A) physically interacts with and disrupts ANKLE2. This interaction is significant as ANKLE2 variants are associated with congenital microcephaly in humans. In fruit flies, NS4A induces microcephaly phenotypes in an ANKLE2-dependent manner (1, 2). However, whether this protein interactions plays a role in virus replication, and whether it can be disrupted remains unknown. Using CRISPR interference and CRISPR/Cas9 knockout, we established that ANKLE2 promotes ZIKV replication across several virus strains and cell types (3). Thus, ZIKV hijacks ANKLE2 to promote virus replication, and inhibits brain development in the process. We also dissect this important virus-host protein interaction with amino acid resolution. We identify domains of NS4A and ANKLE2 responsible for the interaction by systematically performing co-immunoprecipitation assays on domain deletion mutants. We then use Alphafold predictions, molecular dynamics simulations, and computational docking to predict specific amino acid contacts responsible for the protein interaction.

Results, Conclusions, and Discussions:: Our work establishes a unique model system in which Zika virus can hijack and disrupt a major developmental pathway in the process of promoting its own replication using a single protein interaction. We will use these studies to identify NS4A and ANKLE2 variants for which the protein interaction can be disrupted, to the detriment of the virus, and the benefit of the host. Ultimately, they can be used to establish novel models of genetic susceptibility to viral microcephaly, and identify promising candidates for live attenuated ZIKV vaccines.

Acknowledgements (Optional): : Funding was provided by the NIH (R01 AI170857 to PSS) and T32GM7377 and T32AI060555 to ATF.

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