Zika virus NS4A hijacks host ANKLE2 to promote viral replication

Abstract

Zika virus (ZIKV) is infamous among flaviviruses for its unique association with congenital birth defects, notably microcephaly. We previously mapped ZIKV-host protein interactions and identified the interaction between ZIKV NS4A and host ANKLE2, which itself has established ties to congenital microcephaly. In fruit flies, NS4A induces microcephaly phenotypes in an ANKLE2-dependent manner. This suggests that NS4A interacts with ANKLE2 to dysregulate cell behavior and contributes to abnormal host neurodevelopment. Here, we explore the role of ANKLE2 in ZIKV replication to understand the biological significance of the interaction from the viral perspective. We show that knockdown of ANKLE2 reduces replication of two ZIKV strains, across multiple MOIs. We observe that localization of ANKLE2 is drastically shifted to sites of NS4A and viral replication. We investigate which domains of ANKLE2 mediate this behavior and the interaction with NS4A. Using co-immunoprecipitation, we show that deletion of either the transmembrane or LEM domain has little impact on the interaction, but deletion of both significantly reduces interaction with NS4A. Finally, we show that the C-terminal transmembrane domains of NS4A stabilize the interaction with ANKLE2. Together, these results suggest NS4A interacts with ANKLE2 through a combination of its transmembrane and LEM domains, bringing it to sites of ZIKV replication to promote replication through an unknown mechanism. Taken together with our previous results, our findings indicate that, in the process of hijacking ANKLE2 for replication, ZIKV disrupts its physiological function to cause disease.

Molecular functions of ANKLE2 and its implications in human disease

“…This observation, along with the ubiquitous expression of ANKLE2, suggests the potential for a broader post-mitotic function in the ER. Our group has explored this unknown ER function and, recently, showed that it may be coopted for ZIKV replication ( Fishburn et al, 2022a preprint). It is enticing to speculate that the scaffolding function of ANKLE2 could be hijacked by viral proteins, including ZIKV NS4A, to mediate aspects of virus replication in the ER that are otherwise inefficient.…”

“…In humans and many other vertebrates ANKLE2 begins with an N-terminal transmembrane (TM) domain that acts as an anchor to the INM and endoplasmic reticulum (ER) membrane. In human cells loss of this TM domain causes ANKLE2 to mislocalize to the cytoplasm ( Elkhatib et al, 2017 ; Fishburn et al, 2022a preprint). Interestingly, invertebrates do not have this TM domain ( Fig.…”

“…Identifying the physical determinants of this protein–protein interaction is vital in understanding the mechanism how ZIKV NS4A inhibits ANKLE2. While these determinants are not fully established, they are of continuing interest to our group ( Fishburn et al, 2022a preprint).…”