UC Davis

Flaviviruses are arthropod-transmitted positive-sense single stranded RNA viruses, capable of causing significant human disease. Their RNA genomes are translated by host machinery and produce a viral polypeptide of 10 viral proteins, 3 structural and 7 non-structural. The non-structural proteins are essential for mediating virus genome replication, host defense silencing, and cell remodeling. Much of this is accomplished through physical interactions between viral protein and host proteins, which interrupts or otherwise impacts their cellular function for virus benefit. Inhibition of host protein function during these interactions can be connected to viral pathogenesis and disease. In the case of Zika virus (ZIKV) the primary disease outcome of concern is birth defects or fetal demise that occur in fetuses that are infected in utero via vertical transmission from the infected mother. Birth defects arising from ZIKV infection are broad and collectively termed congenital Zika syndrome (CZS). Most infamous of these is microcephaly, a condition where brain and head are not fully grown at birth. Microcephaly is associated with a wide range of debilitating development problems that last throughout life. The molecular mechanisms that contribute to microcephaly observed in CZS cases are not fully understood and likely multifactorial. To understand if ZIKV-host protein-protein interactions are contributing to neurodevelopmental defect, global proteomics was previously performed on ZIKV proteins to identify host interactors. This revealed the interaction between ZIKV non-structural 4A (NS4A) and the host protein ankyrin repeat and LEM domain containing 2 (ANKLE2). ANKLE2 is involved in nuclear envelope dynamics during mitosis and asymmetric cell division during neurogenesis, a key step in brain development. Mutations in ANKLE2 are associated with primary congenital microcephaly in humans. Expression of ZIKV NS4A in vivo results in abnormal brain development in fruit flies. This phenotype is rescued by overexpression of human ANKLE2. Together this data supports the hypothesis that NS4A physically interacts with ANKLE2, inhibiting its function during neurodevelopment to cause microcephaly. However, the basis for the physical interaction and the specific ways NS4A inhibits ANKLE2 function through it are still mysterious. Beyond this is the question of why NS4A interacts with ANKLE2 to begin with? Is the interaction, and the pathogenesis that follows, purely coincidental, or does NS4A interact with ANKLE2 to serve in some aspect of ZIKV replication? This work serves to explore the questions revolving the NS4A-ANKLE2 interaction. First, we establish that ANKLE2 has the opportunity to impact ZIKV replication by showing colocalization of ANKLE2 with ZIKV factors during virus replication. To explore if ANKLE2 is actively participating in some aspect of virus replication we then genetically deplete ANKLE2 using CRISPRi knockdown or CRISPR mutagenesis. Cells with diminished or depleted ANKLE2 were infected with ZIKV and had reduced replication across multiple conditions and cell lines. This work provides substantial evidence that ANKLE2 supports ZIKV replication in human cells. During a flavivirus transmission cycle the virus must efficiently replicate in both human and mosquito cells. In collaboration with another group, we show that depletion of the Ankle2 ortholog in mosquito Aag2 cells also leads to reduced ZIKV replication, supporting that Ankle2 is beneficial to virus replication across hosts. Further, we show that physical interaction between ANKLE2 and NS4A is conserved across four other mosquito-borne flaviviruses and that ANKLE2 plays a role in the replication of some of these viruses. In order to investigate the physical determinants of the ANKLE2-NS4A interaction we developed a series of truncation mutants that serially express fewer domains of each protein. To test physical interaction between the proteins, we performed co-transfection and co-immunoprecipitation experiments. This revealed the N-terminal region of ANKLE2 interacts with the C-terminal region of NS4A. Further, we show that this non-interacting ANKLE2 mutant does not colocalize with ZIKV NS4A during infection. However, the data suggest that there are multiple contact sites between ANKLE2 and NS4A, across separate domains. ANKLE2 is a scaffolding protein that modulates the cell cycle through physical protein interactions. To explore how these interactions may be perturbed during infection and how new interactions may be driven to benefit virus replication, we performed affinity-purification and mass spectrometry on ANKLE2 with and without ZIKV infection. These revealed hundreds of candidate protein interactions which enlighten both how ZIKV inhibits normal ANKLE2 function and also potential pathways through which ANKLE2 promotes virus replication. Altogether, this work vastly expands on our understanding of the ZIKV NS4A-ANKLE2 interaction and supplies avenues for future exploration.