Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3

Uncoupling of Protease

trans

-Cleavage

and Helicase Activities in Pestivirus NS3

Fengwei Zheng,a_{Guoliang Lu,}b_{Ling Li,}a_{Peng Gong,}b_{Zishu Pan}a

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, Chinaa_{; Key}

Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, Chinab

ABSTRACT The nonstructural protein NS3 from the Flaviviridae family is a multi-functional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ⬃2,200-Å2 _{intramolecular} protease-helicase interface comprising three clusters of interactions, representing a “closed” global conformation related to the NS3-NS4A cis-cleavage event. Although this con-formation is incompatible with proteasetrans-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to per-turb this conformation impaired both the helicase activities in vitro and virus pro-duction in vivo. Our work reveals important features of protease-helicase coordina-tion in pestivirus NS3 and provides a key basis for how different conformacoordina-tional states may explicitly contribute to certain functions of this natural protease-helicase fusion protein.

IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replica-tion and transcripreplica-tion by unwinding structured RNA. Being naturally fused to a pro-tease participating in viral polyprotein processing, the NS3 helicases encoded by the

Flaviviridaefamily viruses are unique. Therefore, how these two enzyme modules coor-dinate in a single polypeptide is of particular interest. Here we report a previously un-identified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis-cleavage event and is optimal for the function of helicase. This work provides an important basis to un-derstand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different conformational states.

KEYWORDS pestivirus, NS3, protease, helicase, crystal structure

P

(type species:Yellow fever virus[YFV]),Hepacivirus(only species:Hepatitis C virus[HCV]), and Pegivirus (type species: Pegivirus A) genera. The Pestivirus genus encompasses

Bovine viral diarrhea virus(BVDV),Classical swine fever virus (CSFV), andBorder disease virus (BDV) (1). Pestiviruses are single-stranded, positive-sense RNA viruses with ge-nome length of⬃12.3 kb. The viral RNA genome contains one large open reading frame (ORF) encoding a polyprotein of⬃4,000 amino acids. The polyprotein is co- and posttranslationally processed into at least 12 mature proteins by viral and host pro-teases throughcis(intramolecular) or trans(intermolecular) mechanisms (2–5). Core, Erns_{, E1, and E2 are the structural proteins that become part of the mature virion (6, 7),} and the eight nonstructural proteins Npro_{, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B} are involved in polyprotein processing, viral genome replication, and virus

morpho-Received29 June 2017Accepted7 August 2017

Accepted manuscript posted online23 August 2017

CitationZheng F, Lu G, Li L, Gong P, Pan Z. 2017. Uncoupling of proteasetrans-cleavage and helicase activities in pestivirus NS3. J Virol 91:e01094-17.https://doi.org/10.1128/JVI .01094-17.

EditorJulie K. Pfeiffer, University of Texas Southwestern Medical Center

Copyright© 2017 American Society for Microbiology.All Rights Reserved.

Address correspondence to Peng Gong, [email protected], or Zishu Pan, [email protected].

F.Z. and G.L. contributed equally to this article.

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genesis (8–13) (Fig. 1A). Generally considered, cis-cleavage events are essential to generate structurally independent proteases that may carry outtrans-cleavages more efficiently than proteases in the form of polyprotein. Limitations are also applied to

cis-cleavages, as the intramolecular recognition of the cleavage site requires the protease protein to adopt certain conformations that may not always achievable.

Similar to its counterparts in otherFlaviviridaeviruses, the⬃680-residue pestivirus NS3 is a multifunctional enzyme and a natural fusion of an N-terminal chymotrypsin-like serine protease and a C-terminal nucleotide triphosphatase (NTPase)/helicase (12, 14–17). AllFlaviviridaeNS3 proteases require a segment of another viral protein (NS4A for pestiviruses and HCV and NS2B for flaviviruses) as the structurally integrated essential cofactor (hereinafter termed protease cofactor segment [PCS]) to fulfill the protease function (15, 18, 19), and for pestiviruses the protease is responsible for the cleavage of all the linkages in the NS3-NS4A-NS4B-NS5A-NS5B region of the viral polyprotein (20, 21). The NS3-NS4APCS_{protease contains a typical dual}␤_{-barrel core} observed in the chymotrypsin-like serine proteases, and the PCS participates in the folding of both the N- and C-terminal barrels (N-barrel and C-barrel) (22). The primary pestivirus NS3 cleavage sites are Leu/Ser, Leu/Ala, and Leu/Asn (23), which differ from the Cys/X sites in HCV (24) and the dibasic sites in flaviviruses (25). Recently, two minor autocleavage sites (Leu192/Met193and Leu159/Lys160) within the NS3 protease module were identified, and altering either of the cleavage sites by Leu deletion or mutation greatly inhibited RNA replication in a CSFV replicon system and resulted in a loss of genome RNA infectivity (10). The C-terminal two-thirds ofFlaviviridaeNS3 is a super-family 2 (SF2) DExH/D-box RNA helicase (26) with 3=-to-5=translocation directionality, featuring two conserved RecA-like domains (D1 and D2) possessing the NTPase activity and a third domain (D3) that is important for helicase function (27–29). HCV and flavivirus NS3 proteins have been found to unwind both RNA and DNA substratesin vitro, while pestivirus NS3 has yet been found to unwind only RNA (30–33). The recently reported CSFV helicase crystal structure revealed that D3 contains two insertions not present in NS3 from HCV or flaviviruses, and as a result, the putative single-stranded-RNA (sssingle-stranded-RNA) binding groove (ssRBG) between D2 and D3 appears to be narrower than those observed in otherFlaviviridaeNS3 structures (28).

Cross talk between the protease and helicase modules has been reported for

Flaviviridae, with the majority of data suggesting stimulatory effects (17, 28, 34–38). However, the available crystal structures of full-length NS3 of Flaviviridae provide limited information on the mechanism of its autoregulation (8, 27, 29, 37, 39). Three different conformations were observed in the flavivirus full-length NS3 crystal struc-tures, none of which defined extensive or conserved interactions between protease and helicase modules (8, 27, 37). The consensus conformation observed in the HCV full-length NS3 structure defines a protease-helicase interface mostly involving helicase D3 (29, 39, 40). In these HCV structures, the C terminus of NS3 helicase is buried in its protease active site, providing information valuable to the understanding of NS3-NS4A

cis-cleavage. The conformational diversity observed in availableFlaviviridaeNS3 struc-tures suggests that each genus may have evolved distinct autoregulation mechanisms. Very recently, a full-length CSFV NS3 structure revealed two global conformations that both have very limited intramolecular interactions between the protease and helicase modules (22).

In this study, we crystallized the full-length CSFV NS3 with its NS4A PCS covalently tethered to its N terminus through a flexible linker and solved its crystal structure at a resolution of 2.35 Å. The structure provides structural insights into pestivirus NS3-NS4A

cis-cleavage and protease substrate recognition, allowing us to find evolutionary linkages between pestivirus NS3 protease and its hepacivirus and flavivirus counter-parts. More importantly, the structure reveals a previously unidentified intramolecular interface between the protease and helicase modules that may play key roles for the function of both enzymes.In vitrohelicase assays and virological data further validated the functional importance of this interface and its relevance to the RNA unwinding function of the helicase. The structural and functional analyses in this work pave a way

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FIG 1Crystal structure of the full-length CSFV NS3 with its NS4APCS_{. (A) Schematic diagrams of the CSFV polyprotein (top)} and the NS3/NS4APCS_{construct (middle), with the structure of the protease (bottom left) and helicase (bottom right)} modules observed in the full-length NS3 crystal structure. Dashed curves represent regions not resolved in the structure; residue numbers are indicated where necessary. ssRBG, putative single-stranded RNA binding groove. (B) Comparison of three global conformations of full-length CSFV NS3. The conformation observed in the current study represents a “closed” status with the C terminus (indicated by the blue sphere) buried intramolecularly in the protease active site (structure on the left). Both conformations observed in the recently reported structure exhibit relatively “open” statuses with limited intramolecular protease-helicase interactions (structures on the right). For each open conformation, the protease of a neighboring NS3 that binds the C-terminal fused NS4A residues 1 to 8 (in gray) is shown by a dashed circle. The indicated solvent-accessible interface areas were calculated based on the total surface area occluded by the intramolecular protease-helicase interactions. (C) Comparison of global conformation ofFlaviviridaeNS3. The helicase D1 of each structure was used for THESEUS superpositioning. Coloring scheme for all panels: the PCS in light blue, protease in chocolate, D1 in yellow, D2 in green, and D3 in cyan. The␣-carbon of the C terminus in each NS3 protein in panels B and C is shown as a blue sphere. The ssRNA in the HCV structure is shown in purple. PDB entries used were 5WX1 (CSFV closed [this work]), 5LKL (22) (CSFV open), 3O8C (39) (HCV), 2VBC (27) and 2WHX (37) (Dengue virus[DENV]), and 2WV9 (8) (Murray Valley encephalitis virus[MVEV]).

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to the further understanding of how this natural protease-helicase fusion protein works in harmony and how its different conformational states may play distinct roles to achieve versatile functions.

RESULTS

Protein crystallization and structure determination. Fusing the PCS to the N terminus of theFlaviviridaeNS3 has been a valid strategy to construct fully functional NS3 (10, 41). We therefore made a CSFV NS3/NS4APCS_{protein with NS4A residues 21 to} 57 covalently attached to the N terminus of NS3 through a GSGS tetrapeptide linker and purified the protein to homogeneity. For protein crystallization, an S163A mutation of NS3 was introduced to minimize the protease activities that may lead to autocleav-age. Single crystals of NS3/NS4APCS_{were obtained both in the initial screening and} subsequent reproduction and optimization trials. The structure was solved by molec-ular replacement using three separate search models (protease, helicase D1-D3, and helicase D2) modified from CSFV NS3 helicase and full-length HCV NS3 structures (28, 29). The final model has Rwork/Rfree values of 0.196/0.250 and a resolution of 2.35 Å (Table 1). The structure is relatively complete, with 660 out of 683 residues in NS3 and 31 out of 37 residues in NS4APCS_{present in the final model, and the majority of the} unresolved residues are within the linker regions either between the protease and helicase or between helicase domains D1 and D2 (Fig. 1A).

Global architecture and conformation of the full-length CSFV NS3. The full-length NS3 structure could be divided into a single-domain protease module and a helicase module comprising three globular domains—D1, D2, and D3—that are similar in size (Fig. 1A). The protease module consisting of NS3 residues 1 to 197 and NS4A residues 23 to 53 contains a typical dual␤-barrel core and is connected to the helicase through an extended linker (residues 198 to 207, largely unresolved) (Fig. 1A). The global arrangement of the helicase domains is consistent with the recently reported

TABLE 1X-ray diffraction data collection and structure refinement statistics for the crystal structure determined in this study

Parameter Value(s) for crystal structure

Data collectiona

Space group P42212

Cell dimensions

a,b,c(Å) 111.1, 111.1, 139.0

␣,␤,␥(°) 90, 90, 90

Resolution (Å)b _{60.0–2.35 (2.43–2.35)}

Rmerge 0.088 (0.52)

I/␴I 15.3 (3.7)

Completeness (%) 97.1 (98.5)

Redundancy 6.1 (6.1)

Refinement

Resolution (Å) 60.0–2.35

No. of reflections 35,913

Rwork/Rfreec(%) 19.6/25.0

No. of atoms

Protein 5,326

Ligand/ion/water 0/0/226

B-factorse_(Å2₎

Protein 53.3

Ligand/ion/water //49.1

RMSD

Bond length (Å) 0.008

Bond angle (°) 1.100

Ramachandran statisticsd _{89.0/10.7/0.0/0.3}

a_{One crystal (PDB code5WX1) was used for data collection.} b_{Values in parentheses are for the highest-resolution shell.} c_{Five percent of data are taken for theR}

freeset.

d_{Values are percentages and are for most favored, additionally allowed, generously allowed, and disallowed} regions in Ramachandran plots, respectively.

e_{B-factor is given byB}

i⫽82Ui2, whereUiis the mean displacement of atomi.

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helicase-alone structures of CSFV and other helicase-containingFlaviviridaestructures (28, 42, 43), with D1, D2, and D3 arranged in a clockwise manner if taking a front view by looking into the putative ssRBG (Fig. 1A). In SF2 helicases, relative and repetitive motions between the NTPase domains (D1 and D2 in NS3) in accordance with other domains (D3 in NS3) are believed to couple with processive translocation on nucleic acids (8, 26). The Flaviviridae NS3 contains a unique extended anti-parallel ␤-type structural element (residues 473 to 492 in CSFV NS3, termed “D2-D3 bridge” here) within D2, serving as a bridge to mediate domain motions through its extensive contacts with D3 (Fig. 1A). The D2-D3 bridge in HCV NS3 also harbors one of the “bookend” residues (V432, the other being W501 in D3) that are observed to sandwich a 5-nucleotide ssRNA stretch in an NS3-ssRNA complex crystal structure (39). Based on the NS3 translocation directionality and the relative placement of the D2-D3 bridge and the ssRBG, the bridge likely serves as a steric module at the ssRNA and double-stranded RNA (dsRNA) junction (ss-ds junction) (Fig. 1A and C).

Among the information provided by the full-length CSFV NS3 structure, the relative placement of the protease and helicase modules and the interactions between them are of particular interest. It turned out that NS3 adopted a relatively compact or “closed” conformation with the protease approaching the “back” of the helicase, forming intramolecular interactions mainly with helicase D3 (Fig. 1B, closed). This global ar-rangement is analogous to that of the HCV full-length NS3 structures but is quite different from those observed in the flaviviruses (Fig. 1C) (8, 27, 29, 40). Our CSFV NS3 structure and the HCV NS3 structure both represent a postcleavage state in the NS3-NS4A cis-cleavage event (Fig. 1C, the two structures on the left), with the C-terminal residues L683 and T631 occupying the S1 pocket of the protease active site, respectively. It is worth mentioning that the capture of thecis-cleavage-related con-formation is not a consequence of any protease cleavage event, since the NS3/NS4APCS construct used to yield the crystal contains the P-side NS3 residues but not the P=-side NS4A residues. In the recently reported full-length CSFV NS3 crystal structure (22), the protein adopted two relatively open conformations, both having very limited intramo-lecular protease-helicase interactions (Fig. 1B, open). Note that these open conforma-tions were derived from an NS3 construct having a C-terminal fusion of NS4A residues 1 to 8 (Fig. 1B, open), and this C-terminal fusion was found buried in the protease active site of a neighboring NS3 molecule, with the NS4A L8 residue occupying the protease S1 pocket, mimicking a post-trans-cleavage situation. We should note that the differ-ences at the C termini of the CSFV NS3 constructs likely play roles critical to the different global conformations and different cleavage modes captured by crystallography. De-spite the major differences in NS3 global conformation, the protease and helicase modules are very similar, with average root mean square deviation (RMSD) values for superimposable␣-carbon atoms (95% coverage for both the protease and helicase) of 0.7 Å and 1.1 Å, respectively.

Comparison of the construction modes of theFlaviviridaeNS3 protease and its PCS.The construction mode of the CSFV NS3 protease module and its NS4A PCS better resembles that observed in the flavivirus NS3/NS2BPCS_{structures (18, 44) than that of} the HCV structure (45) (Fig. 2A and B). In such an arrangement, the N-terminal stretch of the PCS (NS4A residues 23 to 34 [PCSN]) is integrated into the N-barrel of the protease (Fig. 1A and 2A) and the C-terminal stretch (residues 35 to 53, [PCSC]) wraps around the C-barrel (Fig. 1A and 2A). For flaviviruses, the functions of corresponding PCSNand PCSChave been delineated, with the PCSNplaying structural roles and PCSC participating in protease substrate binding (18, 44) (Fig. 2A, bottom). Consistent with the structural observations in the current study, both PCSNand PCSCare required for pestivirus protease function (15). While the PCSCof flavivirus can reach the substrate binding cleft, the PCSCof CSFV may make only structural contributions to the C-barrel (Fig. 2A, compare the CSFV and WNV structures). In contrast, HCV requires only a 12-residue NS4A segment (CSFV PCSNequivalent) for protease function, and this agrees with available crystal structures with the PCS primarily participating in the folding of only the N-barrel (29, 45, 46) (Fig. 2A and D).

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FIG 2Comparison of theFlaviviridaeNS3 proteases. (A) Stereo-pair images of the CSFV, HCV, andWest Nile virus(WNV) NS3 protease structures. The PCS in each structure is shown in blue. The side chains of the catalytic triad and the peptide-like inhibitor in the WNV active site are shown as sticks. Dashed lines

(Continued on next page)

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Structural implications ofcis-cleavage at the NS3-NS4A junction by pesti- and hepacivirus NS3.As mentioned above, both the HCV and the “closed” CSFV full-length NS3 structures were captured in an NS3-NS4Acis-cleavage-related conformation, hav-ing the NS3 C terminus integrated into the protease active site, a situation not yet observed in any flavivirus NS3 structures (Fig. 1C and 2C) (29, 39, 40). Note that all

Flaviviridaehelicase D1s and D3s are anchored by extensive interactions, not allowing large-scale domain motions relative to each other. Thus, the placement of the C terminus in D3 relative to the protease-helicase linker adjacent to D1 likely has significant impact on whether and how a cis-cleavage event could occur at the NS3-NS4A junction. While the HCV and CSFV NS3 have the C terminus and the linker both on the backside of the helicase, the flavivirus NS3 rather has the linker in the back and the C terminus in front of the helicase and adjacent to the ssRBG (Fig. 1B and C). These differences suggest that the NS3-NS4Acis-cleavage in flaviviruses may be difficult to achieve and, if indeed occurring, is likely incompatible with its helicase function by occluding RNA binding in the putative ssRBG. Indeed, NS3cis-cleavage in flavivirus was evident only at the NS2B-NS3 junction instead (47).

Substrate specificity and characteristic structural details of the pestivirus NS3 protease.With a conformation captured related to thecis-cleavage mode, the closed-conformation full-length CSFV NS3 structure also provides information for protease substrate specificity. The S1 pocket of the active site to accommodate the P1 residue (i.e., the immediate N-terminal residue of the cleavage site [48]) is relatively deep and small, with a primarily hydrophobic environment, similar to the S1 pocket of HCV NS3 (Fig. 2C, top and middle). In contrast, the flavivirus NS3 P1 site is relatively flat and spacious but not as hydrophobic (Fig. 2C, bottom). This comparison provides the structural basis for substrate specificity at the S1 pocket, with pestivirus and hepacivirus NS3 preferring leucine and threonine/cysteine, respectively, with small side chains (20, 45), and flavivirus NS3 taking lysine/arginine, with long amphipathic side chains with polar heads (49) (Fig. 2C).

The full-length CSFV NS3 structure also provides some genus-specific structural details for its protease. The N-terminal 14 residues (␤1 in Fig. 2) intertwined with NS4A residues 29 to 41 (␤1=) to form a long anti-parallel ␤-type structural element that bridges the N- and C-barrels of the protease. This is consistent with the observation that pestivirus NS3 protease activity was sensitive to a deletion at the N-terminal region of NS4APCS_{(10). As comparisons, the hepacivirus NS3 N terminus forms a shorter} anti-parallel structure with its PCSN, while the flavivirus NS3 does not have any structural equivalent in available crystal structures. The second hallmark of the pestivirus NS3 protease is a clamp-like structure (residues 122 to 129, corresponding to the␤9-␤10 loop; residues 146 to 151) within the C-barrel that grips residues 42 to 49 of the PCSC, taking a relay from the N-terminal 14 residues and further integrating the cofactor into the protease body (Fig. 2A). Compared to the HCV NS3 protease, which does not have a PCSCto interact with, CSFV NS3 essentially has two insertions, eight and six residues each (Fig. 2D), to build such a clamp. In flavivirus NS3, the path of PCSCis quite different and a␤-type region (residues 108 to 118) corresponding to␤10 of CSFV NS3 plays a primary role in interacting with the PCSC(Fig. 2A, B, and D).

FIG 2Legend (Continued)

indicate unresolved regions. The protease structures were superimposed using THESEUS and shown separately, and the N-barrel dominated the superposi-tioning due to high conservation. For an evaluation of structural similarity, the pairwise RMSD values between the CSFV structure and the HCV/WNV structures are 1.8 Å (60% coverage)/1.9 Å (53% coverage) if using the least-square superpositioning method. (B) Topology of the CSFV NS3 protease with its PCS.␤-Strand labeling in panels A and B is based on secondary-structure assignment in panel D. (C) Stereo-pair images of the CSFV, HCV, and WNV NS3 protease active sites. The protease (pink) is shown by a combination of loop and surface representations. The C termini (cyan) of CSFV/HCV NS3 and the peptide-like inhibitor (gray) in the WNV structure that occupy the protease active sites are shown as sticks. Side chains of the catalytic triad are shown as sticks. Key side chains in the P1 residue binding pocket (i.e., the S1 pocket) are shown as balls and sticks. In the open-conformation CSFV structure, the C-terminal fusion of NS4A residues 1 to 8 is similarly fed into the protease active site intrans(gray ribbons with spheres indicating NS3 L683 and NS4A L8, overlaid onto the CSFV structure by superposing the protease module). (D) Sequence alignment ofFlaviviridaeNS3 protease-PCS with secondary-structure assignments. Helices and␤-strands are shown as springs and arrows, respectively. White characters with a red background indicate identical residues; red characters within blue boxes indicate residues with similar side chain properties. Red stars, catalytic triad; yellow bars, two insertions (corresponding to the “clamp” in panel A) in the CSFV protease compared to the HCV structure; magenta triangle, two leucine residues corresponding to the internal cleavage sites. PDB entries used include 5WX1 (CSFV, this work), 2FM2 (75) (HCV), and 2FP7 (44) (WNV).

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The intramolecular interface between protease and helicase in CSFV NS3 featured by three clusters of interactions.With the contribution from the protease-helicase linker (residues 198 to 207) excluded, the intramolecular interface formed between NS3 protease and helicase occludes a solvent-accessible surface area of 2,243 Å2_{in total, about 27% larger than that observed in the full-length HCV NS3 structure} (29) (Fig. 1B and C and 3). On the protease side, both the N- and C-barrels participate in the interface, while on the helicase side, D3 and D2-D3 linker are the primary contributors. The interface includes a mixture of charged, hydrophilic, and hydrophobic interactions that can be defined into three clusters (Fig. 3). The first cluster involves the entire P-side of the protease active site and the C-terminal tail (residues 679 to 683) of FIG 3Intramolecular interface between protease and helicase in the CSFV NS3/NS4APCS_{crystal structure.} (A) Three clusters of interactions comprise the protease-helicase interface. Top panels (for visualization of global distribution of interface interactions only) show the distribution of interactions, with the left panel viewing from the helicase (only key side chains are shown) to the protease (surface representation) and the right panel viewing from the protease (only key side chains are shown) to the helicase (surface representation). Bottom panels (for nonstereo visualization of interface interaction details) show clusters 1, 2, and 3. (B and C) Stereo-pair images of the protease-helicase interface viewing from two different angles with 3,500-K composite SA omit electron density map (contoured at 1.2␴; see Materials and Methods for a detailed description) of the helicase (B) or the protease (C) overlaid. Key side chains or peptide backbones are shown as sticks. Key hydrogen bonding or charge interactions are indicated by dashed lines. The coloring scheme is as in Fig. 1A.

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helicase D3. Compared to the helicase-alone structure (28), the tail shifts its secondary structure from helical to ␤-type and forms antiparallel␤ interactions with protease ␤13b, integrating itself into the protease C-barrel and poising the P1 position L683 in the S1 pocket (Fig. 3A and C). The second cluster includes a triple salt bridge charge network and a “hydrophobic cap” at its edge (Fig. 3A and B). The salt bridges of E141:K183 in the protease C-barrel and R551:E571 in the helicase D3 are in close proximity, allowing an additional salt bridge formed between E141 and R551. These interactions are further stabilized by the hydrophobic interactions between two glycine residues in the␤10-␤11 turn (residues 139 to 140) adjacent to E141 and the aromatic ring of Y555 from helicase D3 (Fig. 3A and B). The third cluster involves the protease ␤7b-␤8 hairpin of the N-barrel, a loop in helicase D3 (residues 559 to 563), and the D2-D3 linker (Fig. 3A and C). In this cluster, the side chains of T96/N93/S525/E561 and M95/T96/E561 participate in a hydrogen bonding network and hydrophobic interac-tions, respectively (Fig. 3A and C). Due to the limited number of viral species in the

Pestivirus genus, the conservation evaluation of the interface interactions cannot be readily achieved. However, the structural details of the interface strongly indicate its functional relevance not only in the aforementioned protease cis-cleavage at the NS3-NS4A junction but also in other regulations between the two enzymes, including the allosteric regulation of helicase function through protease-helicase interactions.

Perturbation into the protease-helicase interface inhibited in vitro helicase activities but not ATPase activities.To assess the significance of the protease-helicase interactions to virus production and, in particular, to the function of helicase, we designed three sets of mutants according to the three clusters of interface interactions observed in our NS3/NS4APCS _{structure (Fig. 3 and Table 2), both in the context of} recombinant NS3 protein and full-length virus genome. Similar to what was done in the structural study, the S163A mutation was introduced into NS3/NS4APCS_{; here this} protease-defective protein construct is referred as the wild-type (WT) NS3 in the enzymatic charac-terizations described below.

A traditional helicase assay was used to assess the RNA unwinding activity of the WT NS3 and its variants under a saturating ATP concentration (28). A partially single-stranded RNA (pssRNA) substrate, T40:R20, was constructed by annealing an unlabeled 40-mer template strand (T40) and a fluorescently labeled 20-mer release strand (R20) (see Materials and Methods). The percentage of the unwound release strand to the total

TABLE 2Structure-based design of the CSFV NS3 protease-helicase interface mutations

Namea _Mutation(s)

Deleted

residues Descriptions/notes

WT NAb _NA

Set 1 C-terminal tail (interacting with protease active site)

TAIL_Δ2 NA 680–681 Keep P1 (G682) and P2 (L683) residues

TAIL_Δ4 NA 675–678 Remove one helix turn, keep the␤-type region

680–683

Set 2 Salt bridges and hydrophobic cap

CEX_1 R551E, E571R NA Charge exchange within helicase

CEX_2 E141K, K183E NA Charge exchange within protease

CAP_1 Y555F NA Keep hydrophobic interactions with G139/G140

CAP_2 Y555A NA Reduce hydrophobic interactions with G139/G140

Set 3 Hydrogen bonding and hydrophobic network

HB_1 N93A NA Disrupt a hydrogen bond

HB_2 N93D NA Interfere with the hydrogen bonding network

HB_3 S525A NA Disrupt a hydrogen bond

HB_4 E561D NA Shorten the side chain but preserve the charge

a_{The WT enzyme contains the NS4A}PCS_{(residues 21 to 57) and the full-length NS3 as the N- and C-terminal} regions, respectively. The S163A mutation was introduced to abolish the autocleavage activity of the protease in all protein constructs listed here. Mutation sets 1, 2, and 3 are according to interface interaction clusters 1, 2, and 3.

b_{NA, nonapplicable.}

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amount of release strand was calculated based on the fluorescent signals detected from native polyacrylamide gel electrophoresis (PAGE) resolving free R20 from T40:R20 complex and was used to assess the helicase activity of NS3 and its variants. WT NS3 unwound 81% of the R20 under the conditions tested, while an NS3 construct con-taining only the helicase module (NS3Hel) unwound only 20% of the substrate (Fig. 4A, lanes 4 and 5, and Table 3). This is consistent with a previous report that the removal

FIG 4Helicase and ATPase activities of CSFV NS3/NS4APCS_{and its variants. (A) The helicase unwinding activity was measured using a} T40:R20 pssRNA construct. Lane 1, annealed T40:R20 in the absence of helicase (the upper band represents the annealed form); lane 2, heat-denatured T40:R20 (the lower band represents the released R20). The negative control (NC), helicase-only, and WT constructs (lanes 3 to 5) were compared to three sets of mutants (lanes 6 to 15). The mean unwound percentages (Table 3) are shown underneath the gels. Note that lanes 1 to 9 and lanes 10 to 15 were run on two different gels. (B) ATP hydrolysis was indirectly measured through the detection of phosphate production. The initial reaction rates, shown as the averages from three independent experiments with standard deviations (SDs) under different ATP concentrations, were fitted to a standard Michaelis-Menten curve. The same set of WT data was included in all three panels for a comparison with the data from three sets of interface mutants.

TABLE 3Enzymatic and virological characterizations of the intramolecular protease-helicase interface in CSFV NS3a

Constructb

Helicase activity (%)c

ATPase activity

Viral antigen

Virus titer (log10TCID50/ml)

Plaque diam (pixel)

Kmapp

(␮M)

kcat (sⴚ1₎

WT NS3 80.9⫾3.8d _41.2⫾_{2.8 4.3}⫾_0.1 ⫹ _6.6⫾_{0.3 52.0}⫾_5.8

NS3Hel 20.0⫾3.3 36.7⫾3.6 4.3⫾0.1 NTe _{NT NT}

NCf _{4.1⫾1.3 ND}g _{ND ⫺ NT NT}

TAIL_Δ2 51.1⫾2.0 35.4⫾3.0 4.7⫾0.1 ⫺ ND NT

TAIL_Δ4 41.2⫾3.7 114.2⫾6.4 4.0⫾0.1 ⫺ ND NT

CEX_1 83.4⫾4.4 36.0⫾3.1 5.0⫾0.1 ⫺ ND NT

CEX_2 63.6⫾4.1 28.3⫾3.3 4.8⫾0.2 ⫺ ND NT

CAP_1 85.8⫾1.6 39.4⫾2.4 4.3⫾0.1 ⫹ 4.6⫾0.1 41.0⫾5.5

CAP_2 78.5⫾5.0 28.7⫾2.9 4.7⫾0.1 ⫹ 1.2⫾0.4 34.8⫾6.0

HB_1 84.0⫾4.0 35.9⫾3.5 4.6⫾0.1 ⫹ 5.7⫾0.2 40.4⫾9.0

HB_2 36.8⫾5.9 46.9⫾2.5 3.6⫾0.1 ⫹ 4.5⫾0.2 38.6⫾4.6

HB_3 82.0⫾1.1 50.1⫾3.2 4.4⫾0.1 ⫹ 6.4⫾0.1 50.4⫾6.1

HB_4 63.9⫾3.9 39.8⫾4.1 4.5⫾0.1 ⫹ 6.1⫾0.3 46.3⫾5.2

a_{Values are means⫾SDs.}

b_{The S163A mutation introduced into the protein constructs were not present in virus variants.} c_{The helicase activity is evaluated by the percentage value of the unwound release strand (R20).} d_{The WT helicase unwinding data were based on 10 measurements.}

e_{NT, not tested.} f_{NC, negative control.} g_{ND, not detected.}

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of the protease module significantly inhibited the helicase activity of pestivirus NS3 (28). The NS3 “Walker A” K232A mutant designed to abolish helicase activity was used as a negative control (NC) (17). As expected, the K232A mutant exhibited a very low level of unwinding activity (Fig. 4A, lane 3 [4% unwound]). Among the 10 interface mutants, 5 retained a WT level of unwinding activity and the other 5 showed reduced activity, with the latter 5 having unwound percentages ranging from 37 to 64% (Fig. 4A; WT levels are shown in lanes 8, 10, 11, 12, and 14 and reduced levels are shown in lanes 6, 7, 9, 13, and 15). Both of the C-terminal deletion mutants in the first set (TAIL_Δ2, deletion of residues 680 and 681 [Δ680 – 681]; TAIL_Δ4, Δ675– 678) exhibited a rela-tively high level of inhibition (Fig. 4A, lanes 6 and 7 [51% and 41% unwound]). For the second set, only the charge exchange mutant on the protease side (CEX_2, E141K/ K183E) has a moderate inhibitory effect (Fig. 4A, lane 9 [64% unwound]). Two out of four mutants in the third set (HB_2, N93D, and HB_4, E561D) showed apparent effects (Fig. 4A, lanes 13 [37% unwound] and 15 [64% unwound]). Although the precise mechanisms of how each specific mutation alters the helicase activity cannot be judged with the current data, these observations collectively indicate that perturbations into the protease-helicase interface, in general, downregulate helicase activityin vitro, demon-strating the direct and positive contribution of the protease to helicase function and the functional importance of the closed conformation observed in our CSFV NS3/ NS4APCS_{crystal structure. Among the three clusters of interface interactions, the second} cluster, involving the triple salt bridges and the Y555:(G139/G140) hydrophobic inter-actions, have relatively lower impact on helicase activity. We further assessed the ATPase activity of CSFV NS3 and its variants using a malachite green-based ATPase assay (43, 51). Except for the active-site mutant K232A (NC) losing the specific affinity to ATP and showing a linear relationship between initial hydrolysis rates and ATP concentrations, all the NS3 constructs hadkcatvalues in the range of 3.6 to 5.0 s⫺1(Fig. 4B and Table 3). All NS3 constructs had apparentKm(Km

app_{) (ATP) values in the range}

of 28 to 50␮M except that the set 1 TAIL_Δ4 mutant had aKmapp(ATP) value of 114

␮M, suggesting a moderate reduction in ATP substrate affinity for this mutant (Fig. 4B and Table 3). These data indicated that the interface mutations do not significantly affect the ATP hydrolysis. Therefore, the overall inhibitory effect on helicase unwinding brought by the interface mutations probably was achieved through the perturbation of the interface but not through the impairment of ATP hydrolysis.

Perturbation into the protease-helicase interface inhibited CSFV replication.To investigate the effect of the mutations at the protease-helicase interface of NS3 on infectious virus production, the 10 interface mutations were each introduced into an infectious cDNA clone pSM (derived from the CSFV Shimen strain) (52, 53). Infectious RNAs were transcribedin vitrofrom each cDNA construct and subsequently used to transfect PK-15 cells. The lowercase “v” as the prefix of each construct is used to differentiate the virus constructs from the protein constructs used in the enzymatic characterizations. Recovery of infectious virus in transfected cells was detected by immunofluorescence assay (IFA) with an anti-NS3 antibody (17) at 72 h posttransfection (p.t.). PK-15 cells transfected with construct transcripts according to the vWT NS3, set 2 “CAP” mutants vCAP_1/2 (Y555F and Y555A), and set 3 mutants vHB_1/2/3/4 (N93A, N93D, S525A, and E561D) were positive for viral antigen detection (Table 3). In contrast, no evidence of virus production was observed upon transfection of transcripts with set 1 mutants vTAIL_Δ2/Δ4 (Δ680 – 681 and Δ675– 678) and set 2 charge exchange mutants vCEX_1/2 (R551E/E571R and E141K/K183E). The related virus titration data showed that compared with the vWT, the vHB_3/4 mutants exhibited similar virus titers, while the vHB_1/2 and vCAP_1/2 mutations significantly impaired infectious virus production (Fig. 5A). Consistent with viral antigen detection, the infectious virus was not detected for the vTAIL deletion and vCEX mutant constructs (Fig. 5A and Table 3). Compared with vWT CSFV, only the vHB_3 mutant virus exhibited a similar plaque diameter, while all other interface mutants had apparently reduced plaque sizes (Fig. 5B and C). Collectively, both the virological and the helicase unwinding data demonstrated the

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importance of the protease-helicase interface interactions. The different extents of effect brought by a certain mutation in different assays likely indicate that the closed conformation of NS3 may not only contribute to helicase function but also be relevant to other processes in the CSFV life cycle.

DISCUSSION

On the mechanism of NS3 helicase regulation by its fusion partner protease.It is quite common that multidomain and multifunctional enzymes utilize autoinhibition or autostimulation mechanisms through intramolecular interactions to fulfill their roles under certain circumstances. As an example, the bacterial transcription-repair coupling factor Mfd contains an N-terminal part (MfdN) that is important in recruiting nucleotide excision repair (NER) protein UvrA and a C-terminal ATP-dependent DNA translocase (MfdC), and the DNA translocase function is strongly repressed through conserved intramolecular interactions between MfdNand MfdC(54, 55). This autoinhibitory effect forms the basis for Mfd to turn on its translocase activity only upon interaction with an RNA polymerase trapped by DNA damage. In contrast, our structural and functional data suggest that the helicase function of CSFV NS3 is rather stimulated through the intramolecular interactions between the protease and helicase. Taking the fact that the ATPase properties were not much affected by the NS3 interface mutations, we propose that the stimulatory effect is likely achieved through interactions between the protease and RNA substrate of the helicase in the closed-conformation state. In the HCV NS3 crystal structure in complex with a single-stranded RNA (ssRNA), the 5= end of the ssRNA “hits” the D2-D3 bridge (39). Thus, the folded RNA with the unwound 5=region most likely resides on the backside of the helicase, across the D2-D3 bridge (see a hypothetical model in Fig. 6A). In our full-length CSFV NS3/NS4APCS _{structure, the} protease is also on the backside of helicase and its N-barrel contains a positively charged surface patch involving NS3 residues R50, K74, and K94 and NS4APCS_residue H24 (Fig. 6). This surface patch, in a relay fashion, is spatially connected to one

FIG 5Characterization of infectious WT CSFV and its variants bearing mutations probing the protease-helicase interface. (A) Infectious virus production. Titers are shown as the averages from three independent experiments, with SDs. (B) Plaque formation. (C) Plaque size comparison. Average plaque diameters (calculated based on 18 to 34 plaques for each construct as indicated by the number above each column) are shown, with SDs.*,P⬍0.05;**,P⬍0.01. The lowercase “v” as the prefix of each construct is used to differentiate the virus constructs from the protein constructs in the enzymatic characterizations.

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positively charged patch (residues R476, R478, K491, and K492) in the D2-D3 bridge and then another (residues K416, K417, K419, and K421) in a D3␣-helix (assigned as␣7 in the CSFV helicase-only structural work [28]) (Fig. 6A). The electrostatic potential analysis indicates that these three surface patches form a continuous and extended positive-potential groove (Fig. 6B). This observation is reminiscent of that of the coronavirus nsp10-nsp16 complex, in which nsp10 may stimulate the RNA capping process by extending the positively charged RNA binding groove in nsp16 methyltransferase (56). For a better understanding of how NS3 protease possibly facilitates the helicase function through RNA binding, we generated a model of the full-length NS3 in complex with a partially single-stranded RNA (pssRNA) taken from a rhinovirus polymerase elongation complex (57) using the ssRNA from the HCV NS3 for guidance (Fig. 6A). The model has the 3=ssRNA bound in the putative ssRBG in the front and the downstream folded RNA on the backside approaching the protease. The 5=end of the downstream RNA points in the direction of the aforementioned positively charged surface patch in the protease N-barrel where the unwound RNA in the 5=direction (absent in the model) could bind. We should note that this is a crude model expressing the basic concept of how the protease module might facilitate the helicase function through its interactions with the downstream RNA but not predicting any interaction details. In superfamily 1 (SF1) helicase PcrA, a set of crystal structures, including that of a PcrA:pssDNA complex, provides the basis of how the PcrA ATPase domains 1A and 1B coordinate between each other for ATP hydrolysis-driven conformational changes leading to an inchworm-ing motion along the sinchworm-ingle-stranded region of the nucleic acids, and how domain D2B forms the interactions with the dsDNA downstream (58). However, the SF2 pestivirus NS3 is quite different from the SF1 helicases with respect to the relative placement of accessory domains (D3 and protease in NS3; D1B and D2B in PcrA) relative to the ATPase domains (D1 and D2 in NS3; D1A and D2A in PcrA). Therefore, the construction

FIG 6Hypothetic RNA unwinding model of pestivirus NS3. (A) A pssRNA (purple) was modeled into the full-length NS3 structure as guided by the HCV NS3 helicase:ssRNA (blue) complex crystal structure (PDB entry 3O8C [39]). The downstream RNA may approach the backside of the helicase and the protease where three positively charged surface patches (␣-carbons as blue spheres) could assist RNA binding. (B) The electrostatic potential analysis of the full-length NS3 structure shows a continuous groove (circled by green dashed lines) with high positive potential (blue). Scale bar:⫺3 kT/e to 3 kT/e, where k is Boltzman constant, T is temperature in Kelvin, and e is the charge of an electron. (C) Comparison of the superfamily 2 (SF2) CSFV NS3 model with the SF1 PcrA structure in complex with a pssDNA (blue) (PDB entry 3PJR [58]). The two proteins were manually oriented based on the structural conservation of the␤-sheet in NS3 D1 and PcrA D1A. The coloring scheme for NS3 is as in Fig. 1A. The PcrA coloring scheme is as follows: D1A is yellow, D1B is orange, D2A is light green, and D2B is dark green. ADP (modeled from PDB entry 3O8D [39]) and ATP molecules are shown as sticks in the NS3 and PcrA structures,

respectively.

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mode of PcrA:pssDNA cannot be readily applied to the unresolved NS3:pssRNA com-plex. Obtaining a high-resolution NS3 structure in complex with pssRNA is likely the ultimate validation to the proposal of protease stimulation in helicase function through RNA binding, while characterizing the helicase function by mutagenesis targeting the positively charged surface patches could test our general hypothesis of the contribu-tion of these residues to the interaccontribu-tions with the downstream RNA.

Relevance of pestivirus NS3 conformation states with various protease cleav-age events. Polyprotein processing is a common feature shared by many positive-strand RNA viruses, and cleavages at the viral protease termini likely play a critical role to generate free proteases or modulate protease activity to facilitate subsequent cleavage events. For the Flaviviridae family, the PCS of NS3 is either within the N-terminal neighboring NS2B (Flavivirusgenus) or within the C-terminal neighboring NS4A ( Hepa-civirusandPestivirusgenera). Therefore, cleavage at the corresponding NS3 terminus to free the cofactor segment-containing protein may be essential for the life cycle of the

Flaviviridaeviruses. These critical cleavage events in theory could occur through intra-or intermolecular (i.e.,cisortrans) mechanisms. Biochemical evidence ofcis-cleavages at the hepaci- and pestivirus NS3-NS4A and flavivirus NS2B-NS3 has been reported for various virus systems (10, 15, 47, 59). The capture of the post-cis-cleavage state in HCV (29) and in CSFV (this study) has provided valid evidence to further support the existence ofcis-cleavage at the NS3-NS4A junction for theHepacivirusandPestivirus

genera. These data collectively suggest that theFlaviviridaeNS3 protease likely takes the intramolecular mechanism to cleave its junction with the PCS. The cleavage at the other end is rather through other mechanisms. For hepaci- and pestiviruses, the NS2-NS3 junction is cleaved by NS2 protease. For flaviviruses, the NS3-NS4A junction is probably achieved through NS3trans-cleavage, as thecismechanism is not supported by either biochemical or structural data (8, 27, 60, 61) (Fig. 1C). We should note that the

cis-cleavage-related closed conformation of pestivirus NS3 is not compatible with

trans-cleavage, since the protease active site is partially occupied by the NS3 C terminus (Fig. 7, top). However, thistrans-cleavage-defective state is optimal for helicase function as supported by our biochemical data. When pestivirus NS3 switches to the open-conformation state, it is capable of proteasetrans-cleavage but its helicase function is downregulated (Fig. 7, bottom). We suggest that pestivirus NS3 utilizes this conforma-tional switching to fine-tune its protease and helicase activities, and the apparent coupling of NS3-NS4Acis-cleavage and RNA unwinding may well be a consequence of coevolution of these two processes.

Internal cleavages within NS3 have been reported for variousFlaviviridae(10, 47, 62). The cleavage sites were mapped to Leu159/Lys160 and Leu192/Met193 of CSFV NS3, both within the protease module, resulting in an inactive and an active protease FIG 7Working model describing the relationship between the pestivirus NS3 conformational states and the protease/helicase activities. (Top) the closed conformation captured in the current study is related to NS3-NS4A proteasecis-cleavage event and is beneficial to the helicase RNA unwinding activity. However, this conformation is defective in proteasetrans-cleavage. (Bottom) The open conformation is capable of proteasetrans-cleavage, but the helicase RNA unwinding activity is impaired without the intramolecular protease-helicase interactions observed in the closed conformation.

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fragment, respectively (10). Although the cleavage at these internal sites is not as efficient as that at the canonical Leu/Ser, Leu/Ala, and Leu/Asn sites, mutations at either residue 159 or 192 sites greatly affect virus production, implying the functional impor-tance of these internal sites. Very interestingly, both Leu159 and Leu192 are part of the active-site S1 pocket to accommodate the leucine side chain of the P1 residue (Leu683) of the cleavage substrate in our NS3/NS4APCS_{structure (Fig. 2C and D and 3).} Conse-quently, cleavage at these sites may be achieved intramolecularly through reorganiza-tion of the protease active site or intermolecularly. While the CSFV NS3 internal cleavages may be essential in controlling the overall polyprotein processing, the removal of the majority of the protease module may be detrimental to the helicase function, as suggested by previous studies (17, 28) and this work. The low cleavage efficiency at these internal sites therefore may reflect a balance of requirements in polyprotein processing and RNA genome replication.

In summary, by solving the closed-conformation crystal structure of the full-length CSFV NS3 in complex with its NS4APCS_{, we improved the understanding of the} pestivirus NS3 proteasecis-cleavage at the NS3-NS4A junction of the viral polyprotein. Based on the observation of the intramolecular interface between protease and heli-case in this structure, we obtained evidence that key protease-heliheli-case interactions are beneficial to the helicase functionin vitroand are important for virus production. Our work provides an important basis for how this closed-conformation state may explicitly contribute to proteasecis-cleavage and helicase RNA unwinding processes and how the conformational diversity of pestivirus NS3 is essential to fulfill its versatile and distinct functions.

MATERIALS AND METHODS

Plasmid construction and protein expression.The full-length NS3 (NS3) and its NS4A protease cofactor segment (NS4APCS_{; residues 21 to 57) were amplified from the CSFV infectious clone pSM} (Shimen strain) (52, 53) and cloned into the pET28a vector according to a previously reported strategy to yield the pET28a-NS3/NS4APCS_{plasmid expressing the N-terminal NS4A}PCS_{and the C-terminal NS3} connected through a GSGS tetrapeptide linker (10). To minimize the autocleavage activity of the expressed product, an NS3 S163A mutation was introduced to generate pET28a-NS3S163A/NS4APCSusing the QuikChange mutagenesis method (63). The plasmid expressing the helicase-only construct (NS3Hel; residues 204 to 683) was made using pSM as the template and pET28a as the vector. N- and C-terminal histidine tags were added to NS4APCS_-NS3

S163A and NS3Hel, respectively. All 10 protease-helicase interface mutants and the helicase “Walker A” mutant (K232A, referred as the negative control [NC] in helicase and ATPase assays) were constructed using pET28a_NS4APCS_-NS3

S163Aas the template and the QuikChange mutagenesis method (Table 2). All mutations were confirmed by sequencing data.

Expression of NS3S163A/NS4APCSand its variants and NS3Hel was performed according to previously reported methods for expressingFlaviviridaereplication proteins (17, 28). Briefly, the plasmids were transformed intoEscherichia coliBL21-CodonPlus(DE3)-RIL strain and then the bacteria were grown at 37°C in terrific broth (TB) medium containing 50 ␮g/ml of kanamycin (KAN50) and 25 ␮g/ml of chloramphenicol (CHL25) until the optical density at 600 nm (OD600) reached 0.6 to 0.8. Isopropyl-␤-D -1-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.5 mM, and the cells were grown for an additional 4 h at 25°C before harvesting.

Protein purification.Purification of NS3S163A/NS4APCSand its variants and NS3Hel was performed according to methods for purifying flavivirus replication proteins (64, 65). The cells were resuspended in lysis buffer (150 mM Na2SO4, 50 mM Tris [pH 8.0], 10 mM imidazole, 0.02% [wt/vol] NaN3, 20% [vol/vol] glycerol) and were lysed by passage through an AH-2010 homogenizer (ATS Engineering Ltd.) at 14,500 lb/in2_{. The lysate was clarified by centrifugation for 60 min at 17,000 rpm in a Fiberlite F21-8x50y rotor} (Thermo Scientific). The clarified lysate was loaded onto a HisTrap HP column (GE Healthcare), and the target protein was eluted with elution buffer (300 mM imidazole, 50 mM Tris [pH 8.0], 150 mM Na2SO4, 20% [vol/vol] glycerol, and 0.02% [wt/vol] NaN3). For all full-length proteins, fractions containing the target protein were pooled and diluted to reduce the Na2SO4concentration to 38 mM prior to loading onto a HiTrap SP HP column (GE Healthcare) and elution with a linear gradient to 300 mM Na2SO4in 25 mM morpholineethanesulfonic acid (MES; pH 6.0), 20% (vol/vol) glycerol, and 0.02% (wt/vol) NaN3. The pooled fractions were concentrated and run over a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 150 mM NaCl, 5 mM Tris 7.5, 10% (vol/vol) glycerol, and 0.02% (wt/vol) NaN3. Pooled fractions were supplemented with Tris-(2-carboxylethyl)phosphine (TCEP) at a final concentration of 5 mM, concentrated, flash frozen in liquid nitrogen, and stored as aliquots at⫺80°C. The molar extinction coefficient of each purified protein was calculated with the ExPASy ProtParam tool (http://web.expasy .org/protparam/) based on its amino acid sequence. The typical yield is 4 to 5 mg of pure protein per liter of bacterial culture. For NS3Hel, a HiTrap Q column was used in the second chromatography and Tris (pH 7.5) was used as the buffering agent.

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Protein crystallization, data collection, and structure determination.Octahedron-shaped crystals of NS3S163A/NS4APCSwere obtained by sitting-drop vapor diffusion at 16°C using 10 mg/ml of protein, and the best-diffracting crystal yielding the final structure appeared between 3 and 4 weeks. Typically, a volume of 0.4 to 1␮l of protein was mixed with an equal volume of 2.1 MDL-malic acid (pH 7.0) in the crystallization drop. Crystals were directly flash cooled and stored in liquid nitrogen prior to data collection. The X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U1 (wavelength, 0.9793 Å; temperature, 100 K), and 360° of data was collected in 0.75° oscillation steps. Reflections were integrated, merged, and scaled using HKL2000 (66). The initial structure solution was obtained using the molecular replacement (MR) program PHASER (67). Three separate ensembles were used for the MR search: the core␤-strands of the protease region derived from a full-length HCV NS3 structure (PDB entry 1CU1), D1/D3 of a CSFV NS3 helicase structure (PDB entry 4CBG), and D2 of the same CSFV helicase structure (28, 29). Manual model building and structure refinement were done using Coot and Phenix, respectively (68, 69). Four translation, libration, and screw motion (TLS) groups were applied in the last round of Phenix refinement: protease, helicase D1, helicase D2 with the tip of the D2-D3 bridge excluded, and helicase D3 combined with the tip of D2-D3 bridge. The 3,500-K composite simulated-annealing omit 2Fo-Fcelectron density maps were generated using Crystallography and NMR System (CNS) (70). In such a process, the crystallographic asymmetric unit was divided into small boxes, each containing a fraction not exceeding 5% of the entire model. The omit map corresponding to each box was then calculated by excluding the model in that box. The composite map was finally made by stitching the omit map from all the boxes. The Ramachandran statistics for the final models are 89.0% and 0.3% for most favored and disallowed regions. Unless otherwise indicated, all protein superimpositions were done using the maximum likelihood-based structure superpositioning program THESEUS (71), which downweights variable regions. In the case of the open- and closed-conformation pestivirus NS3, the superposition was dominated by the helicase module and allowed better visualization of the relative placement of the protease and helicase modules. Wherever necessary, RMSD values calculated based on traditional least-square method were also provided to evaluate structural similarity.

Helicase assays.The helicase unwinding pssRNA substrate T40:R20 was prepared by annealing the template strand (T40, transcribed by T7 RNA polymerase; sequence, 5=-GGGCCAAUCAUGCAUACGAGAA UGAACUAACCUCGUAUAC-3=) and the release strand labeled at the 3= end with 6-carboxy-tetra-methylrhodamineN-succinimidyl ester (6-TAMRASE) (R20, chemically synthesized by GenScript; sequence, 5=-UAUCUCGUAUGCAUGAUUGG-3=; the underlined region in R20 was designed to form an 18-bp duplex with the underlined region in T40) in an annealing buffer (25 mM HEPES [pH 7.5], 500 mM NaCl) at a 2.5:1 molar ratio. The mixture was incubated at 95°C for 5 min and was then slowly cooled down to 25°C in a rate of 0.01°C/s (Tgradient thermocycler; Biometra). A typical 20-␮l unwinding reaction mixture contains 8 U of RNasin (RiboLock; Thermo Scientific), 50 mM morpholinepropanesulfonic acid (MOPS)-NaOH (pH 7.0), 5 mM ATP, 2.5 mM MgCl2, 1 mM dithiothreitol (DTT), 0.5% Tween 20, 0.1 mg/ml of bovine serum albumin (BSA), 10 nM T40:R20 (according to the concentration of the R20), 100 nM unlabeled release strand (competitive strand), and 25 nM NS3 (protein/pssRNA substrate molar ratio⫽2.5:1). For simplicity, we use “NS3” to represent all variants of NS3S163A/NS4APCS-derived proteins when describing allin vitroenzymatic assays. The unwinding reaction proceeded at 37°C for 30 min and then was terminated by addition of 2.2␮l of a 10⫻loading buffer (50 mM Tris [pH 7.5], 50 mM EDTA, 1% [wt/vol] SDS, 50% [vol/vol] glycerol, 0.1% [wt/vol] xylene cyanol). RNAs in the quenched reaction mixtures were resolved by 12% nondena-turing polyacrylamide gel electrophoresis. The fluorescent signal of 6-TAMRASE-labeled R20 was detected with a Typhoon 9200 imager (GE Healthcare) with an excitation wavelength of 532 nm and a 580-nm emission filter. The band intensities were quantified by ImageJ (http://imagej.nih.gov/ij), and the unwound percentages were calculated based on the intensity fraction of the released R20. Unless otherwise indicated, each unwinding reaction was performed five times; the mean unwound percentage and its standard deviation are listed in Table 3.

ATPase assays.The ATPase activity was measured by using a malachite green-based method as described previously (17, 28). The reactions were carried out in a 96-well plate (Nunc; Immuno-plate F96 MaxiSorp). For each reaction, a 90-␮l mixture containing all components except for the ATP substrate was preincubated for 5 min at 37°C, and the reaction was initiated by addition of a 10-␮l ATP solution to yield a final reaction mixture containing 10 nM NS3, 50 mM Tris (pH 7.5), 2.5 mM MgCl2, 50 mM NaCl, and 5 to 500␮M ATP and proceeded for 15 min at 37°C. The malachite green mixture (water/0.081% [wt/vol] malachite green/5.7% [wt/vol] ammonium molybdate in 6 M HCl ratio⫽3:2:1 [vol:vol:vol]) was added at the 15-min time point, and the absorbance was measured at 630 nm immediately on a Multiskan MK3 microplate reader (Thermo Fisher Scientific). Initial ATPase catalytic rates were determined based on the slope of the initial absorbance change and the reference standard curve of absorbance versus phosphate concentration determined independently. The observed ATP hydrolysis rates at various ATP concentrations were fitted to Michaelis-Menten kinetics to yield the ATPase parameters (Kmappand

kcat).

Virus rescue, titration, and plaque assays. To investigate the effect of NS3 protease-helicase interface mutations on infectious virus production, the aforementioned three sets of mutations were introduced into CSFV cDNA clone pSM using the QuikChange mutagenesis method. All mutations were confirmed by sequencing data. The virus rescue was carried out as previously described (53, 72). Briefly, 1␮g ofin vitroT7 RNA polymerase-transcribed viral RNAs was transfected into PK-15 cells (porcine kidney 15 cell line, obtained from the China Center for Type Culture Collection, Wuhan, China) by using Lipofectamine 2000 (Invitrogen). After incubation at 37°C for 72 h, the infectious virus production was monitored by indirect immunofluorescence (IF) assay with NS3-specific antibody and a secondary

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antibody, Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (IgG) (73). For virus character-ization, the cell cultures were incubated at 37°C for 72 h, and then the culture supernatant was harvested and clarified by centrifugation for subsequent assays. The titers of rescued virus were calculated by IF staining with anti-NS3 antibody in 96-well plates using the Reed-Muench method (74) and expressed as tissue culture infectious doses (50% endpoint) (TCID50) per milliliter. The plaque formation assay was conducted as previously described (72). Briefly, PK-15 cell monolayers in 24-well plates (95% confluent) were infected with 200 to 400 TCID50s of viruses. The cells were overlaid with 1.5% methylcellulose and incubated at 37°C for 96 h. The cells were then fixed with 50% (vol/vol) methanol-acetone and stained by immunohistochemistry (IHC) using anti-NS3 specific antibody and two-step IHC detection reagent (ZSGB-BIO). Unpaired Student’sttest was used to evaluate the significance of difference between the WT and each mutant virus with respect to the virus titer and plaque diameter values.

Accession number(s).The atomic coordinates and structure factor files have been deposited in the Protein Data Bank with accession code5WX1.

ACKNOWLEDGMENTS

We thank Bo Shu for X-ray diffraction data collection, Liu Deng for laboratory assistance, synchrotron SSRF (beamlines BL17U, BL19U1, and BL18U1, Shanghai, China) for access to beamlines, and the Core Facility and Technical Support of the Wuhan Institute of Virology for access to instruments.

This work was supported by the National Key Basic Research Program of China (2013CB911100), the National Natural Science Foundation of China (31272585, 31570152, and 31670154), the National Key Basic Research and Development Program of China (2016YFC1200400), the Open Research Fund Program of Wuhan National Bio-Safety Level 4 Laboratory of Chinese Academy of Sciences (NBL2017009), the Open Research Fund Program of the Key Laboratory of Special Pathogens and Biosafety, Chinese Academy of Sciences (2015SPCAS003), and the “One-Three-Five” Strategic Programs, Wuhan Institute of Virology, Chinese Academy of Sciences (Y605191SA1).

P.G. and Z.P. conceived the research; F.Z. and G.L. performed the crystallographic experiments; F.Z. performed the enzymatic experiments; L.L. performed the virological experiments; P.G., G.L., and F.Z. analyzed the crystallographic data; F.Z., Z.P., and P.G. analyzed the enzymatic data; and L.L. and Z.P. analyzed the virological data. All authors contributed to the writing of the paper.

REFERENCES

1. Lindenbach BD, Thiel H-J, Rice CM. 2007. Flaviviridae: the viruses and their replication, p 1101–1152. In Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE (ed), Fields virology, 5th ed, vol 1. Lippincott, Williams & Wilkins, Philadelphia, PA.

2. Bintintan I, Meyers G. 2010. A new type of signal peptidase cleavage site identified in an RNA virus polyprotein. J Biol Chem 285:8572– 8584.

https://doi.org/10.1074/jbc.M109.083394.

3. Harada T, Tautz N, Thiel HJ. 2000. E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J Virol 74:9498 –9506.https://doi.org/10.1128/JVI.74.20.9498-9506.2000. 4. Lackner T, Muller A, Pankraz A, Becher P, Thiel HJ, Gorbalenya AE, Tautz

N. 2004. Temporal modulation of an autoprotease is crucial for replica-tion and pathogenicity of an RNA virus. J Virol 78:10765–10775.https:// doi.org/10.1128/JVI.78.19.10765-10775.2004.

5. Lamp B, Riedel C, Roman-Sosa G, Heimann M, Jacobi S, Becher P, Thiel HJ, Rumenapf T. 2011. Biosynthesis of classical swine fever virus non-structural proteins. J Virol 85:3607–3620.https://doi.org/10.1128/JVI .02206-10.

6. Thiel HJ, Stark R, Weiland E, Rumenapf T, Meyers G. 1991. Hog cholera virus: molecular composition of virions from a pestivirus. J Virol 65: 4705– 4712.

7. Weiland F, Weiland E, Unger G, Saalmuller A, Thiel HJ. 1999. Localization of pestiviral envelope proteins E(rns) and E2 at the cell surface and on isolated particles. J Gen Virol 80(Part 5):1157–1165.

8. Assenberg R, Mastrangelo E, Walter TS, Verma A, Milani M, Owens RJ, Stuart DI, Grimes JM, Mancini EJ. 2009. Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for poly-protein processing and viral replication. J Virol 83:12895–12906.https:// doi.org/10.1128/JVI.00942-09.

9. Gladue DP, Holinka LG, Largo E, Fernandez Sainz I, Carrillo C, O’Donnell V, Baker-Branstetter R, Lu Z, Ambroggio X, Risatti GR, Nieva JL, Borca MV.

2012. Classical swine fever virus p7 protein is a viroporin involved in virulence in swine. J Virol 86:6778 – 6791. https://doi.org/10.1128/JVI .00560-12.

10. Lamp B, Riedel C, Wentz E, Tortorici MA, Rumenapf T. 2013. Autocatalytic cleavage within classical swine fever virus NS3 leads to a functional separation of protease and helicase. J Virol 87:11872–11883.https://doi .org/10.1128/JVI.00754-13.

11. Moser C, Stettler P, Tratschin JD, Hofmann MA. 1999. Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus. J Virol 73:7787–7794.

12. Moulin HR, Seuberlich T, Bauhofer O, Bennett LC, Tratschin JD, Hofmann MA, Ruggli N. 2007. Nonstructural proteins NS2-3 and NS4A of classical swine fever virus: essential features for infectious particle formation. Virology 365:376 –389.https://doi.org/10.1016/j.virol.2007.03.056. 13. Tratschin JD, Moser C, Ruggli N, Hofmann MA. 1998. Classical swine fever

virus leader proteinase Npro is not required for viral replication in cell culture. J Virol 72:7681–7684.

14. Gu B, Liu C, Lin-Goerke J, Maley DR, Gutshall LL, Feltenberger CA, Del Vecchio AM. 2000. The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication. J Virol 74:1794 –1800.https://doi.org/10.1128/JVI.74.4 .1794-1800.2000.

15. Tautz N, Kaiser A, Thiel HJ. 2000. NS3 serine protease of bovine viral diarrhea virus: characterization of active site residues, NS4A cofactor domain, and protease-cofactor interactions. Virology 273:351–363.

https://doi.org/10.1006/viro.2000.0425.

16. Wen G, Chen C, Luo X, Wang Y, Zhang C, Pan Z. 2007. Identification and characterization of the NTPase activity of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) expressed in bacteria. Arch Virol 152:1565–1573.https://doi.org/10.1007/s00705-007-0969-2.

17. Wen G, Xue J, Shen Y, Zhang C, Pan Z. 2009. Characterization of classical

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