Since ZIKV outbreak was first reported in South and Central America and the Caribbean, it has spread to both North America and Asia1. Preliminary reports have now estimated as many as 1.3 million cases of infection in this widespread epidemic2. Initially it was thought that the ZIKV only causes mild symptoms such as fever, myalgia, and rash; recent studies have revealed that ZIKV can also cause severe neurological pathologies such as neonatal microcephaly and Guillain-Barre syndrome3. In response to the spreading epidemic of ZIKV infection, the World Health Organization has declared a Public Health Emergency of International Concern4. ZIKV is a mosquito-borne flavivirus, whose NS3 protease (NS3pro) is required for proteolytic processing of its polyprotein to enable viral replication5, thus making NS3pro an attractive therapeutic target for inhibiting viral proliferation. In the past, successful structure-based drug development against NS3pro of the hepatitis C virus (HCV) has generated a series of United States Food and Drug Administration (FDA)-approved inhibitors with potent efficacy for viral eradication6. Flavivirus NS3pro exists predominantly in an inactive state; however, upon association with the NS2B viral protein, its enzymatic activity increases by 3 300-6 600-fold7. The recent structure of ZIKV NS2B-NS3pro in complex with a boronate inhibitor has provided some important insights for drug design8. However, it remains still largely unknown the exact molecular mechanism of ZIKV NS3pro activation by NS2B, which requires a structure of the apo NS2B-NS3pro complex. An improved understanding of the mechanism of NS3pro activation can lead to identification of novel hot spots that can be targeted in rational drug design.

We designed a single-chain fusion protein of NS2B and NS3pro by connecting the 45-residue core region (residues 48-92) of ZIKV NS2B to the N-terminus of NS3pro (residues 1-170) via a glycine-serine linker (G4SG4) (Supplementary information, Figure S1A). The amino acid sequences of NS2B and NS3pro were derived from the Z1106033 ZIKV strain (NCBI ID: KU312312) isolated from Suriname9 (Supplementary information, Figure S1B). The NS2B-NS3pro fusion protein was purified from E. coli, and gel filtration analysis revealed that majority of this complex existed in a monomeric form at various protein concentrations (Supplementary information, Figure S1C). NS2B-NS3pro can be activated readily by adding a widely used synthetic substrate AC-LKKR-AMC10. Using Michaelis-Menten kinetics we determined that the Km and kcat are 85.3 ± 2.1 μM and 1.149 ± 0.069 s−1, respectively (Supplementary information, Figure S1D), which are comparable to the dengue virus (DENV) and the West Nile virus (WNV) NS2B-NS3pros11,12, but different from ZIKV NS2B-NS3 (R95A/R29G), which has an increased activity8. To discover potential inhibitors of ZIKV, we screened small molecule compounds against ZIKV NS2B-NS3pro. Aprotinin13, a competitive serine protease inhibitor used to reduce bleeding during complex surgery, was found to be a potent inhibitor of ZIKV NS2B-NS3pro with a Ki of 361 ± 19 nM (Supplementary information, Figure S1E). This finding suggests that ZIKV NS2B-NS3pro may contain a canonical catalytic site of a serine protease that can serve as a drug target.

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