Superimposition of eZiPro5 and bZiPro6 crystal structures.

A. eZiPro (PDB ID GJ4), NS3pro and NS2B are marked with light and dark blue, respectively. Peptide fragment TGKR (bound to eZiPro) shown in green. B. bZiPro (PDB ID 5GPI), NS3pro and NS2B are marked with magenta and grey, respectively. Peptide fragment KKGE (bound to bZiPro) shown in orange. C. Superposition of A and B. Catalytic residues marked in red. Note that protease catalytic center is occupied in both crystal structures. (PDF)</p

Modeling ZIKV NS3pro-NS3hel mutual orientation.

Models of NS2B-NS3pro in the open conformation and NS3hel built using three different crystal structural templates demonstrate the flexibility of the linker between NS3pro and NS3hel. (A) 2WV9 structure template from MEV virus (PDB ID: 2WV9); (B) 2WHX structures structure template from DENV4 (PDB ID: 2WHX); (C) 2WZQ structure template from DENV4 (PDB ID: 2WZQ). (D) A homology-built model oriented to juxtapose the patch of positively charged residues in NS3hel domain and the positively charged forks in the open conformation of NS2B-NS3pro. The N- and C-terminal helices of NS2B (grey and sky-blue, respectively) are oriented to be inserted in the ER membrane denoted by the dashed line.</p

Allosteric inhibitors of ZIKV NS2B-NS3pro interfere with RNA binding.

(A) Small allosteric inhibitors of ZIKV NS2B-NS3pro block ssRNA binding. Fluorescent polarization (%) using FAM labeled ssRNA (20 poly-rU). Each condition is compared to protease alone, * p<0.05, ** p<0.05, *** p<0.0005, **** p<0.0005 two-tailed unpaired t-test with Welch’s correction. All inhibitors were assayed at 10 μM. (B) IC50 for inhibition of ZIKV, WNV, and DENV2 NS2B-NS3pro proteolysis by the corresponding inhibitors above (in A). (C) Crystal structure of ZIKV NS2B-NS3-Mut7 protease with the NSC86314 inhibitor (PDB 7M1V).</p

Models of the RNA-NS2B-NS3pro-NS3hel complex.

The N- and C-terminal helices of NS2B (grey and green, respectively) are inserted in the ER membrane (grey mesh) modeled to scale using a fragment of the membrane (PDB ID: 2MLR). Similar orientations of NS2B-NS3pro with respect to NS3hel in (A) closed (PDB ID: 5LC0), (B) open (model based on WNV PDB ID: 2GGV), and (C) super-open (PDB ID: 7M1V) conformations. Position of the protease active site of NS2B-NS3pro in closed conformation is marked by yellow star. RNA strand in B is modeled to span a positively charged surface of NS3hel contiguous with positively charged forks in the open conformation of NS2B-NS3pro. Shorter RNA strands associated only with NS3hel domain is shown in A and C reflecting the lack of apparent RNA binding structures in the closed and super-open conformations of NS2B-NS3pro.</p

Structural and functional alterations in ZIKV NS2B-NS3pro preclude ssRNA binding.

(A) Modifications of NS2B-NS3pro (see S1 Fig for details) that prevent efficient binding of FAM-labeled ssRNA (20 poly-rU). (B) Inhibition of NS2B-NS3pro catalytic activity by aprotinin (~0.15 μM) and WRPK3 (covalent inhibitor) blocks ssRNA binding. Each condition is compared to protease NS2B-NS3pro, *** p<0.0005, two-tailed unpaired t-test with Welch’s correction.</p

Volumes and position of the cavities at the NS3pro and NS3hel interface.

Positions of the cavities at the interface of the best aligned NS3pro and NS3hel to accommodate single stranded RNA (same as Fig 8D). Colored meshed areas mark the positions of cavities.</p

Modeling directionality of RNA proceasing by ZIKV NS3hel.

(A) Panel represent a structure of ZIKV NS3hel with model build and AMBER force field minimized RNA fragment. On the left side of the molecule there is unprocessed dsRNA, while on the right side there is a leading strand of ssRNA after splitting and detaching the other strand. The ssRNA movement is facilitated by a patch of positively charged residues (right side of NS3hel–blue color). (B) Panel shows crystal structure (PDB id: 5GJB) of ZIKV NS3hel with short fragment of ssRNA (orange) and tentative positions of ZIKV RNA knots (PDB id: 5TPY). The modeling explains why RNA is processed from 3’-end toward 5’-end. Unwinding the knot is much more difficult if it is done via pulling its 5’-end (right side knot), due to stronger interaction with other nucleotides, then via pulling with its 3’-end (left side knot).</p

The open conformation of ZIKV NS2B-NS3pro is uniquely suitable to bind RNA.

Closed (PDB ID: 5LC0), open (model based on WNV PDB ID: 2GGV) and super-open (PDB ID: 7M1V) conformation of ZIKV NS2B-NS3. The open conformation is shown with RNA inserted into the fork-like structures composed of positively charged amino acids (marked blue). One fork is close to 3’-end and another is located in the middle of the RNA strand. The structures are oriented so that RLLG loop of NS3pro is facing down. For the closed conformation a peptide-based substrate (RKADI, green ball and stick model) in the protease active center is modeled on the related structure of WNV NS2B-NS3pro + aprotinin (PDB ID: 2IJO).</p

ssRNA inhibits the proteolytic activity of NS2B-NS3pro.

Inhibition of ZIKV NS2B-NS3pro proteolytic activity by ssRNA (20-rA, left) and ssDNA (20-dA, right).</p

A “reverse inchworm” model of the helicase-protease structure-activity cycle for ZIKV RNA-NS2B-NS3pro-NS3hel complex.

(A) NS2B-NS3pro is in the closed conformation (likely induced by a substrate), unable to bind RNA. Yellow star denotes the protease active center facing the ER membrane; (B) NS2B-NS3pro is in the open conformation (proteolytically inactive), binds RNA via 2 positively charged fork-like structures; (C) NS2B-NS3pro is switched to the open conformation with bound RNA, the linker between NS3pro and NS3hel domains is maximally elongated; (D) NS2B-NS3pro is in the super-open conformation unable to bind RNA; the dissociated RNA strand is cartooned in grey. The pink circular arrows indicate the cycle of NS2B-NS3pro conformational changes involved in dsRNA unwinding by NS3hel. (E) A cartoon of the reverse inchworm model showing the dissociated helicase and the protease activities of NS2B-NS3pro. The green “ATP” arrow indicates the direction of NS3hel movement unwinding dsRNA driven by ATP. GKRSS denotes a fragment of ZIKV polypeptide cleaved by ZIKV Ns2B-NS3pro between Arg (R) and Ser (S). All models built to scale based on the crystal structures and energy minimization (see text for details).</p