The role of mobility in flavivirus protease substrate selection

Abstract

Flaviviruses, including Zika virus and dengue virus, continue to threaten public health. The associated viral proteases, known as NS2B‐NS3pro, are essential to the viral life cycles and can interfere with host responses, making the proteases drug targets of considerable interest. Although the viruses are highly homologous, Zika virus is capable of severely damaging developing fetuses as well as causing neurological abnormalities in adults whereas dengue causes a sometime fatal hemorrhagic fever. While the proteases are highly identical, the catalytic efficiency and the substrates selection between Zika and dengue proteases are highly divergent, leading us to hypothesize that unique properties in the viral proteases may contribute the disparate viral pathologies. This work has made significant progress towards uncovering the role of conformation and flexibility in NS2B‐NS3pro from the function of these two flaviviruses on multiple fronts. First, NS2B‐NS3 proteases transitions between two widely different conformational states: an ‘open’ (inactive) conformation and a ‘closed’ (active) conformation. We developed versions of NS2B‐NS3pro that allow us to trap the enzyme in distinct conformations. Our data suggests that the enzymatic activity is dependent not only on the movement of NS2B, but may also rely on the flexibility of the protease core. Locking the enzyme into the ‘closed’ conformation dramatically increased activity. Second, to elucidate how Zika virus protease affects host cellular pathways and consequent pathologies, we used unbiased N‐terminomics to identify 31 human proteins cleaved by the NS2B‐NS3 protease. In particular, autophagy‐related protein 16‐1 (ATG16L1) and eukaryotic translation initiation factor 4 gamma (eIF4G1) are dramatically depleted during Zika virus infection. ATG16L1 and eIF4G1 mediate type‐II interferon production and host‐cell translation, respectively, likely aiding immune system evasion and driving the Zika life cycle. Intriguingly, the NS2B co‐factor region from Zika virus protease is essential for recognition of host cell substrates. Replacing the NS2B region in another flavivirus protease enabled recognition of novel Zika‐specific substrates by hybrid proteases, suggesting that the co‐factor is the principal determinant in ZVP substrate selection.