Structural and Functional Insights Into Alphavirus Polyprotein Processing and Pathogenesis

Structural and functional insights into alphavirus polyprotein processing and pathogenesis

Gyehwa Shina,1,2, Samantha A. Yosta,1, Matthew T. Millera, Elizabeth J. Elrodb, Arash Grakouib, and Joseph Marcotrigianoa,3

aCenter for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854; and bEmory Vaccine Center, Division of Microbiology and Immunology, Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322

Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved August 31, 2012 (received for review June 19, 2012) Alphaviruses, a group of positive-sense RNA viruses, are globally of two polyproteins (P123 or P1234). P1234 is expressed as a distributed arboviruses capable of causing rash, arthritis, encephali- read-through of an opal termination codon at the end of nsP3. tis, and death in humans. The viral replication machinery consists of These precursor polyproteins are cleaved by a protease within four nonstructural proteins (nsP1–4) produced as a single polypro- nsP2 (1, 4). After translation of P1234, cleavage at the P3/4 tein. Processing of the polyprotein occurs in a highly regulated junction occurs either in cis or trans, followed by the P1/2 junc- manner, with cleavage at the P2/3 junction influencing RNA tem- tion, which occurs in cis only (4, 5). Both P123+nsP4 and nsP1+ plate use during genome replication. Here, we report the structure P23+nsP4 preferentially synthesize negative strand viral RNA of P23 in a precleavage form. The proteins form an extensive in- (6, 7). The final cleavage event between P23 produces fully mature terface and nsP3 creates a ring structure that encircles nsP2. The nsPs and switches RNA template for synthesis of positive-sense P2/3 cleavage site is located at the base of a narrow cleft and is not genomic and subgenomic RNAs. The correlation between P23 readily accessible, suggesting a highly regulated cleavage. The cleavage and the switch from negative- to positive-sense RNA nsP2 protease active site is over 40 Å away from the P2/3 cleavage production is poorly understood. site, supporting a trans cleavage mechanism. nsP3 contains a pre- In mammalian cells, alphavirus infection inhibits host gene viously uncharacterized protein fold with a zinc-coordination site. expression, causes severe cytopathic effects (CPEs), disrupts the Known mutations in nsP2 that result in formation of noncyto- cellular response to IFN, and leads to cell death (8–11). Mature pathic viruses or a temperature sensitive phenotype cluster at nsP2 contains an amino-terminal RNA helicase domain, a central the nsP2/nsP3 interface. Structure-based mutations in nsP3 oppo- protease domain that catalyzes all cleavages between the non- site the location of the nsP2 noncytopathic mutations prevent ef- structural proteins, and an inactive RNA methyltransferase-like ficient cleavage of P23, affect RNA infectivity, and alter viral RNA (MT-like) domain (12). During infection, a portion of nsP2 production levels, highlighting the importance of the nsP2/nsP3 localizes to the nucleus (13, 14) and plays a role in shutting off interaction in pathogenesis. A potential RNA-binding surface, host-cell transcription and viral cytopathogenicity (9, 10). Two spanning both nsP2 and nsP3, is proposed based on the location genetically distinct groups of alphaviruses termed New World of ion-binding sites and adaptive mutations. These results offer and Old World have emerged because geographic isolation (8). unexpected insights into viral protein processing and pathogenesis In Old World alphaviruses, such as Sindbis virus (SINV) and that may be applicable to other polyprotein-encoding viruses such as Semliki Forest virus (SFV), transcriptional shutoff is nsP2-de- HIV, hepatitis C virus (HCV), and Dengue virus. pendent (15). Even in the context of a replicon lacking the structural proteins, the nsPs of SINV and SFV alone can effi- lphaviruses are small, enveloped RNA viruses that infect ciently shut off host transcription and induce severe CPE (16). Avarious vertebrates, including humans, horses, birds, and Mutations that cause alphaviruses to establish a persistent in- rodents (1). Transmission occurs via an invertebrate vector, fection with minimal or no CPE have been identified (16–20). A usually mosquitoes. In humans, typical alphavirus infection can key feature of these mutants is their inability to inhibit host-cell result in rash, arthritis, encephalitis, and death. Within the past transcription (10, 15). The best-characterized noncytopathic decade, infection by Chikungunya virus (CHIKV), a member of mutations map to nsP2 proline 726 (P726) within the MT-like the genus alphavirus, caused massive epidemics in Africa and domain (10, 16, 17, 21, 22). The P726 mutation has been shown Southeast Asia, with positive cases reported in Europe and the to attenuate the cytotoxic effect caused by the expression of wild- United States, raising the possibility of an emerging threat (2). type SINV nsP2 protein alone (10). Noncytopathic mutations of Additionally, CHIKV often presents with nonclassic symptoms nsP2 P726 also reduce viral RNA replication levels and are unable that go undiagnosed, making global pathogenicity difficult to to shut off host transcription and translation processes, showing monitor (3). The alphavirus genome consists of single-stranded, positive-sense RNA of ∼9–11 kb with a 5′ cap structure and 3′ polyadenosine tail. Author contributions: G.S., S.A.Y., and J.M. designed research; G.S., S.A.Y., and J.M. per- fi ′ formed research; G.S., S.A.Y., E.J.E., A.G., and J.M. contributed new reagents/analytic The genome contains two cistrons. The rst, located in the 5 two- tools; G.S., S.A.Y., M.T.M., and J.M. analyzed data; and G.S., S.A.Y., M.T.M., and J.M. wrote thirds of the genome, encodes the viral replication machinery, the paper. termed nonstructural proteins (nsPs), whereas the structural The authors declare no conflict of interest. proteins that form virus particles are encoded in the second This article is a PNAS Direct Submission. cistron. The genomic RNA is used as an mRNA for the trans- Freely available online through the PNAS open access option. lation of the nsPs and as a template for the synthesis of com- Data deposition: The atomic coordinates and structure factors have been deposited in the plementary negative-sense RNA. The negative-sense strand is a Protein Data Bank, www.pdb.org (PDB ID code 4GUA). template for subgenomic RNA containing the second cistron 1G.S. and S.A.Y. contributed equally to this work. and progeny, genomic RNA. Because genomic and subgenomic 2Present address: Science and Engineering Campus, Korea University, Seoul, Republic of RNAs are made in vast excess, the negative-sense RNA strand is Korea, 136-701. considered a replication intermediate. 3To whom correspondence should be addressed. E-mail: [email protected]. The alphavirus replication machinery is composed of four This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nonstructural proteins (nsP1 to -4), which are expressed as one 1073/pnas.1210418109/-/DCSupplemental.

16534–16539 | PNAS | October 9, 2012 | vol. 109 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1210418109 Downloaded by guest on September 30, 2021 the overall importance of nsP2 to viral replication, as well as host-cell interaction (11, 16). The function(s) of nsP3 in alphavirus replication remains un- resolved. Based on sequence conservation, nsP3 is organized into three domains: an amino-terminal macro or X domain (23), a central alphavirus-specific region, and a hypervariable-sequence carboxyl terminus. Macro domains have been shown to bind ADP ribose (24) with certain viral macro domains also retaining ADP ribose-1′′ phosphatase activity (25, 26). Recent work in SFV shows that residues just after the macro domain of nsP3 play a role in positioning of the P23 cleavage site (27). Additionally, the carboxyl-terminal region of nsP3 contains numerous phosphorylation sites targeted by yet unknown cellular kinases (28, 29). Here, we present the structure of an uncleaved P23 precursor protein spanning the nsP2 protease domain through to the central, zinc-binding domain (ZBD) of nsP3 from SINV (Fig. 1A). The P23 cleavage site is located in a narrow cleft formed between nsP2 and nsP3 that is inaccessible for proteolysis. Surprisingly, all of the previously reported nsP2 noncytopathic mutants lie at the interface between nsP2 and nsP3. Muta- genesis of residues in nsP3 opposite of nsP2 P726 showed inefficient cleavage of P23, delayed onset of pathogenesis and host gene shutoff, and altered viral RNA synthesis. Because P23pro-zbd copurified with large amounts of RNA, we propose a potential RNA-binding surface that extends across both nsP2 and nsP3 and hypothesize that cleaving the P2/3 junction may alter the RNA-binding surface, thereby contributing to template switching by the replication machinery. Our results provide a better understanding of the mechanism of viral polyprotein processing and pathogenesis. Results Alphavirus P23pro-zbd Structure. The SINV P23 precursor protein extending from the nsP2 protease domain to the nsP3 central domain was shown to be fully intact by SDS/PAGE and mass spectrometry despite the presence of a competent nsP2 protease BIOCHEMISTRY active site (Fig. S1 A and B). Sequence alignment of the central domain of nsP3 shows numerous absolutely conserved cysteine pro-zbd – Fig. 1. Overview of the alphavirus replication machinery and P23 residues (Fig. S2), suggesting the presence of a metal ion binding structure. (A) Schematic representation of the alphavirus replication ma- site. Various purified preparations of the middle region of nsP3 chinery with emphasis on domain organization of nsP2 and nsP3. The were analyzed by quantitative X-ray fluorescence for common crystallization construct consists of four domains: protease (blue) and transition metals found in metalloproteins (30, 31) and shown to methyltransferase-like (teal) domains of nsP2 and the macro (yellow) and have one zinc ion per molecule. Crystals of P23pro-zbd diffracted zinc-binding (red) domain of nsP3, encompassing amino acids 1011–1675 of to 2.85-Å resolution and the structure was determined by mo- the P1234 polyprotein. Amino acid numbering provided from the amino pro terminus of nsP2 and nsP3 are also noted in parentheses. (B–D)Ribbondia- lecular replacement with structures of nsP2 (12) and nsP3 pro-zbd macro domain (32) (Table S1). The resulting electron density gram of P23 colored according to A. A gray sphere denotes the position of the zinc ion. The P2/3 cleavage site, nsP2 protease active site, and ADP allowed for an unambiguous trace of the entire polypeptide ribose–binding site are labeled with an arrow, asterisk (*), and filled circle, chain, including the previously uncharacterized nsP3 central re- respectively. (C) Ribbon diagram of P23 rotated 90° about a vertical axis gion containing the zinc-binding site. The location of the zinc ion from the view in B.(D) Ribbon diagram of P23 rotated 180° about a vertical was confirmed by anomalous difference maps (Table S1 and axis from the view in B. Box shows location of the view in E.(E) Previously see Fig. S4B). described nsP3 temperature-sensitive mutant ts7 (F312S) highlighted in SINV P23pro-zbd shows a highly compact structure, with the green with the immediately surrounding amino acids located in the macro, four domains (protease and MT-like from nsP2; macro and ZBD ZBD, and MT-like domains. nsP2 residues are shown in italics. (F and G) The from nsP3) arranged with each domain occupying a vertex of a P2/3 cleavage site is located at the base of a narrow cleft formed by the MT- rectangle (Fig. 1 B–D). The polypeptide chain progresses around like and macro domains. Three amino acids on either side of the scissile bond (arrow) are shown in stick representation. The view in F is similar to that in B, the perimeter of the rectangle such that the nsP2 protease and whereas that in G is rotated 90° about a horizontal axis from F, looking nsP3 ZBD are adjacent to one another. The carboxyl terminus of directly into the cleft. the P23pro-zbd construct is pointing toward the protease active site (Fig. 1 B–D). A previously described SINV mutant (ts7) defective in RNA synthesis at nonpermissive temperatures contains a F312 and nsP3 is ordered, permitting modeling of the P2/3 cleavage to serine mutation in nsP3 (33). This residue is located in the hy- site. The P2/3 cleavage site is located at the base of a narrow drophobic core and surrounded by residues from the macro, ZBD, cleft about 11–13 Å wide formed by the MT-like domain of nsP2 and MT-like domains (Fig. 1E). Mutation of this residue would and the macro domain of nsP3 (Fig. 1 F and G). The cleavage site likely destabilize the core and disrupt domain interaction, giving is 40 Å away from the nsP2 protease active site, supporting a − rise to the RNA phenotype at the nonpermissive temperature. trans cleavage mechanism (5, 34, 35). Although the 6 aa sur- In a previously described nsP2pro structure, the six carboxyl- rounding the scissile bond are solvent-exposed, attempts to position terminal amino acids were disordered (12). In the P23pro-zbd the protease domain onto the P2/3 cleavage site using the pro- structure presented here, the entire polypeptide between nsP2 posed model (36) resulted in numerous steric clashes.

Shin et al. PNAS | October 9, 2012 | vol. 109 | no. 41 | 16535 Downloaded by guest on September 30, 2021 ZBD in nsP3 Is Essential for Viral Replication. After the P2/3 cleavage site, the polypeptide chain continues into the nsP3 macro domain. The ADP ribose–binding site within the macro domain is solvent-exposed and points away from the other four domains. The polyprotein exits the last helix of the macro domain adjacent to the P2/3 cleavage site and continues into a stretch of ∼40 aa that lacks secondary structure but forms a twist (Fig. S3). The conserved nsP3 central domain contains an antiparallel α-helical bundle, two parallel β-strands, and a previously unknown zinc- coordination site (Fig. S4B). The zinc ion is coordinated by C263, C265, C288, and C306 of nsP3. C263 and C265 are located in the loop between the last two α-helices, whereas C288 and C306 are at the carboxyl termini of the two parallel β-strands (Fig. S3C). Each of the zinc coordinating cysteines is invariant among all alphavirus sequences (Fig. S2). Structural comparison of the nsP3 ZBD against all known folds in the Protein Data Bank using PDBe Fold (37) and Dali servers (38) failed to produce any statistically significant hits, and no metalloprotein folds were identified. The spatial arrangement of secondary structural elements that contribute to nsP3 zinc-binding site, i.e., two cysteines in a loop between alpha helices and the other two cysteines at the end of two parallel β-strands, represents a structural scaffold for zinc-ion coordination (39). Site-directed mutagenesis was performed to examine the functionality of the four conserved cysteine residues involved in zinc coordination in nsP3 (Fig. S4A). All four SINV mutants (C263A, C265A, C288A, and C306A) failed to produce pro- ductive infection in BHK cells, as evaluated by infectious center and plaque assays (Fig. S4C). A conserved cysteine residue in nsP3 (C247) unrelated to the zinc-binding site served as a control and C247A mutant produced infectious particles to near wild-type levels. Zinc-coordination mutants failed to express the non- Fig. 2. nsP2 and nsP3 interface and the location of nsP2 noncytopathic structural polyprotein and replicate virus, thereby supporting the mutants. (A) Solvent-accessible surface of nsP3. (B and C) Surface of nsP3 (B) structural role of the zinc ion (Fig. S4D). or nsP2 (C) colored for electrostatic potential at ±5 kT/e; blue (basic), red The coordination site forms a distorted tetragon due to the (acidic), and white (neutral) with ribbon diagram of nsP2 (B)ornsP3(C). (D) presence of two highly conserved serine residues (S289 and S290) Molecular surface of nsP2 highlighting (white) the location of nsP2 non- below the zinc atom (Fig. S2). Mutation of these serine residues cytopathic mutations. The numbering corresponds to SINV nsP2 sequence. did not alter viral replication levels. Furthermore, a double (E) Location of P726 in nsP2, highlighting interactions with the nsP3 linker. serine to alanine mutant (S289A+S290A) resulted in decreased A portion of the last helix in the nsP3 macro domain is shown in yellow. (F) Surface of nsP2 P726 and surrounding area colored for electrostatic po- viral RNA infectivity, but overall viral titers were comparable to ± C tential at 4 kT/e with nsP3 linker region in stick format. Residues labeled in wild type (Fig. S4 ). italics are located in nsP2. Arrow indicates P2/3 cleavage site. Domain col- oring in each panel is identical to Fig. 1. Importance of P23 Interface for Polyprotein Processing and Viral Pathogenesis. The interface between the macro and ZBD of nsP3 has a buried surface area of 570 Å2, whereas the linker cytopathogenicity and the level of RNA replication with the size connecting the two domains is extended, making nsP3 into a of the mutant side chain. Molecular modeling analyses demon- ring-like structure with an inner diameter of 15–18 Å (Fig. 2A). strated that mutating P726 to Phe, Tyr, Leu, Arg, or Gln resulted Together, nsP2 and nsP3 share an extensive interface with 3,000 in numerous steric clashes with nsP3, whereas Ser, Thr, Ala, and Å2 of buried surface area. nsP3 encircles the MT-like domain of Val had fewer conflicts. Moreover, the P726G mutation could nsP2, with residue R781 of nsP2 protruding through the opening destabilize the α9-β11 loop, causing it to interfere with P23 (Fig. 2C). The interface between nsP2 and nsP3 is charged, with interaction. Attempts to purify P23pro-zbd with a P726S or the nsP2 surface being mostly basic, whereas nsP3 is generally P726L mutation resulted in poorly soluble and aggregated acidic (Fig. 2 B and C). protein, supporting the importance of this position. Several groups have identified mutations in the carboxyl-ter- To investigate how destabilizing P23 interface may contribute to minal portion of nsP2 that result in noncytopathic virus and changes in viral pathogenesis and replication, site-directed muta- persistent infection (16–20). These mutations reduce viral RNA genesis to the corresponding surface of SINV nsP3 in close prox- replication levels with minimal effects on host gene expression imity to nsP2 P726 were created (Fig. 2E). The side chains of V162 and viral yields. All of the noncytopathic mutations map to the and L165 of nsP3 are ∼4.6 and 3.3 Å away from nsP2 P726 side surface of nsP2 at the nsP3 interface (Fig. 2D). The mutations chain, respectively (Fig. 2F). V162 and L165 of nsP3 were mutated follow the encircling path made by the macro to ZBD linker. to one of the following: Ala, Asp, Glu, Phe, Arg, or Trp. nsP2 P726 Mutagenesis of P726 in SINV to amino acids with large side to Ser, Gly, or Leu mutants were used for comparison. RNA in- chains (Phe, Tyr, Leu, Arg, and Gln) caused a dramatic decrease fectivity measured by infectious center assay on BHK cells were in RNA replication and were noncytopathic, whereas substitutions reduced in P726L, L165D, L165E, and L165R mutants compared with small side chains (Ala or Val) behaved like wild-type virus, with the wild-type SINV (Fig. 3A). P726L mutation in nsp2 re- with Gly, Ser, and Thr substitutions giving an intermediate effect duced RNA infectivity to only 2% of wild-type levels, whereas the (16). P726 is located in the loop connecting α9 with β11 and three nsP3 L165 mutants averaged an RNA infectivity at 8% of makes direct contact with nsP3 linker just after the macro do- wild type. These four mutants also displayed a small size plaque main (Fig. 2E). There is a striking, direct correlation between phenotype in infectious center assay (Fig. S5 A–D). In addition,

16536 | www.pnas.org/cgi/doi/10.1073/pnas.1210418109 Shin et al. Downloaded by guest on September 30, 2021 reached wild-type levels of virus titers. All V162 mutants behaved similarly to wild type in both RNA infectivity and viral titer and were not pursued further. None of the nsP3 mutants displayed a noncytopathic phenotype in the TSG/puromycin N-acetyl- tranferase (PAC) replicon background (16), although onset of CPE in L165 to D, E, and R mutants was delayed. Radiolabeling experiments were performed to evaluate both viral and host protein and RNA production over the course of infection and to analyze efficiency of host-cell transcriptional and translational shutoff caused by SINV nsP3 mutations. Wild- type SINV is able to halt host translation completely within 6 h postinfection, whereas P726S and P726G mutants were unable to do so even up to 12 h postinfection, as indicated by persistent actin production (Fig. S6). nsP2 P726L and the four selected nsP3 L165 mutants (Asp, Glu, Arg, Trp) were able to shut off host translation, although at a slightly delayed time postinfection compared with wild type (Fig. S6). Tritium-labeled uridine was used to track viral and host RNA production over the course of infection. In the presence of actinomycin D, both genomic and subgenomic viral RNA production can be clearly observed in wild-type SINV starting as early as 2 h postinfection and remaining stable until 12 h (Fig. 3C). Levels of subgenomic and genomic RNA in P726G and P726S mutants are reduced compared with wild type. nsP2 P726L and the four nsP3 L165 mutants exhibit a slow initial rate of transcription, especially of genomic RNA; however, these mutants reach wild-type levels by 4 h postinfection. Thereafter, the levels of RNA replication in the L165 mutants continue to increase, and even sur- pass, wild-type RNA replication, especially subgenomic RNA (Fig. 3C). RNA labeling in the absence of actinomycin D showed complete inhibition of host cellular transcription by 8 h postinfection as demonstrated by decreasing 28S and 18S cellular rRNA (Fig. 3D). P726G and P726S mutants exhibited a lack of host-cell transcriptional shutoff with rRNA persisting up to 12 h postinfection. The nsP2 P726L mutant and the four nsP3 L165 mutants showed a delay in host transcription inhibition but BIOCHEMISTRY eventually achieved complete shutoff 12 h postinfection (Fig. 3D). Given that residues R159 and E163 of SFV nsP3 have been shown to affect P2/3 cleavage (27), we assayed the effect of the SINV nsP3 mutations on cleavage activity. nsP3 linker region mutants L165D and L165E revealed drastic inefficiency in P23 cleavage that was not seen in L165W and L165R mutants (Fig. 3 E and F and Fig. S7). The presence of uncleaved P23 in the nsP3 L165D and L165E mutants started 6 h postinfection and persisted up to 12 h, whereas P726 mutants, as well as nsP3 L165R and nsP3 L165W, showed sub- stantially lower amounts of uncleaved P23. Western blots probed with anti-nsP3 antibodies were used to confirm the presence of Fig. 3. In vivo effects of nsP3 linker region mutations. (A and B) Viral RNA uncleaved P23 (Fig. 3F). nsP3 appears as a smear, indicative infectivity [infectious units (IU)/μg RNA] as measured by infectious center of alternate phosphorylation states and possibly other post- assay (A) and viral titers [plaque-forming units (pfu)/mL] (B) 24 h after translational modifications (40). The nsP2 P726 mutants may also electroporation of BHK cells, as determined by plaque assay over two or accumulate precleavage forms but at very low concentrations that more independent experiments. Error bars represent the SEM. (C and D)Cells were infected with wild-type or mutant SINV at a multiplicity of infection do not build up over time, as in the case of the nsP3 linker mutants. (MOI) of 10. At indicated time points postinfection, RNAs were labeled for 3 h in the presence (C) and absence (D) of actinomycin D. Total RNAs were Discussion pro-zbd harvested and analyzed by denaturing agarose gel electrophoresis. The The structure of alphavirus precursor polyprotein P23 pre- position of genomic (G), subgenomic (SG), 28S, and 18S RNAs are labeled. M sented here reveals the arrangement of four domains (protease, indicates mock-infected cells labeled for 3 h. (E and F) Analysis of P23 MT-like, macro, and ZBD) in a precleavage form. Based on the cleavage of nsP2 P726 and nsP3 L165 mutants in vivo. Total protein was structure of P23pro-zbd, several inferences can be made about the harvested from wild-type and indicated mutant SINV-infected BHK cells at mechanism of alphavirus polyprotein cleavage. First, the car- indicated hours postinfection and analyzed by Western blot using anti-nsP2 boxyl terminus of the P23pro-zbd structure is pointing toward the (E) and anti-nsP3 (F) antibodies. nsP2 protease active site about 40–45 Å away. It is possible that the large, flexible carboxyl region of nsP3 could span the distance, placing the P3/4 junction in the protease active site for cis L165A, L165D, L165E, and L165R mutants produced lower viral cleavage (34). Second, the 40 Å distance between the P2/3 titers at 24 h after electroporation, only reaching an average of cleavage site and the nsP2 protease active site supports the current 34% of wild-type levels (Fig. 3B). Of the nsP3 mutants generated, trans model of cleavage (5, 34, 35). Third, the inaccessibility of L165D had the lowest titers, only producing 26% compared with the P23 cleavage site itself indicates that access is tightly regulated. wild-type titers. All others, including all three P726 mutants, It is possible the activator sequence present in the extreme

Shin et al. PNAS | October 9, 2012 | vol. 109 | no. 41 | 16537 Downloaded by guest on September 30, 2021 amino terminus of nsP2, which becomes exposed after cleavage MT-like domain facilitates separation of nsP2 and nsP3 post- at the P1/2 site, may be responsible for this regulation (5). cleavage, which is triggered by binding viral proteins or RNA to the During the course of alphavirus infection, a portion of nsP2 is concave side of the nsP2 MT-like β-sheet. localized to the nucleus, whereas nsP3 remains cytoplasmic (13, 14, Previously described noncytopathic mutations at the surface 41). At the moment, it is unclear how nsP2 would dissociate post- of nsP2 contacting the nsP3 linker region appear by molecular cleavage from the extensive, encircling grasp of nsP3. Comparison modeling to destabilize P23 interaction, decreasing efficiency of the of the domains in P23pro-zbd with the isolated structures of nsP2pro RNA replication complex during virus infection. Altering the cor- (12) and nsP3 macro domain (32) demonstrated that the protease responding nsP3 residue (L165), which contacts nsP2 P726, did not and macro domains were highly similar with a root mean square produce virus with the same noncytopathic phenotype. In contrast deviation (rmsd) of <0.6 Å, whereas the MT-like domains have an to the nsP2 P726G and P726S mutants, nsP3 L165 mutants had rmsd of 1.3 Å for similar α carbon. A superposition of the nsP2 enhanced RNA replication levels, indicating the apparent stabili- structures shows the discrepancy between nsP2pro and P23pro-zbd is zation of the replication complex. Despite the large, bulky nature of limited to specific regions within the MT-like domain (Fig. 4 A and a tryptophan substitution at L165, the virus remained similar in B). α-Helices α8, α10, and α11, as well as β-strands β11, β12, and phenotype to wild type. The L165D and L165E mutants, however, β13, between the two structures superimpose well with an rmsd of exhibited the most extreme phenotype in terms of reduced RNA <1 Å, although the β13-α10 loop moves 5 Å because of inter- infectivity and inefficient P23 cleavage. The surface of nsP2 sur- actions with the nsP3 linker. In addition, the nsP3 linker interacts rounding P726 is both basic and hydrophobic (Fig. 2F), suggesting with α9, causing the helix to rotate 8° with the carboxyl-terminal that the P23 interface may be stabilized by the introduced acidic end of the helix moving 3.8 Å to form a single continuous helix amino acid at L165 through interaction with the surface of nsP2. This with α8. This movement of the α9 helix causes the β-sheet to be- stabilization could cause the interface to become “locked in,” fl come more concave. The distortion of the MT-like domain in the preventing the exibility needed for conformational change and, P23pro-zbd becomes more pronounced the further the secondary thus, disallowing nsP2 access to the P2/3 cleavage site. structures are from α9, with α7 and β9 moving about 4 Å from The buildup of uncleaved P23 is evidence that the nsP3 linker their corresponding positions in nsP2pro. Perhaps movement of the region, which encircles nsP2, may function in positioning and recognition of the P2/3 cleavage site. Given the nsP3 linker residues and nsP2 P726 are within reasonable distance to the P2/3 cleavage site (21 and 17 Å from Cα of L165 or P726 to the cleavage site, respectively), it is plausible that mutation of the nsP3 linker region shifts local structural features, altering recognition of the cleavage site (Fig. 2F). A previously described E163R mutation in SFV nsP3 that altered P2/3 cleavage is completely solvent-exposed (Fig. 2E) (27). These results may indicate that the nsP3 linker residues surrounding nsP2 P726 are recognized by the nsP2 protease or another factor necessary for P2/3 cleavage. Cleavage between P2/3 dictates template use by the alphavirus replication machinery. During purification, P23pro-zbd bound extensive amounts of bacterial RNA, which could only be removed by high concentrations of salt (Fig. S1 A and C). The electrostatic potential of the P23pro-zbd structure identified a large basic surface that centers on the zinc-binding site of nsP3 and extends to nsP2 (Fig. 4C). Many zinc metalloproteins are involved in nucleic acid–binding and gene regulation (42); therefore, the location of a basic patch in close proximity to the zinc-binding site suggests a potential role in RNA binding. Several molecules of sulfate and 2-(N-morpholino)ethane sulfonic acid (MES), used in crystallization, were bound to P23pro-zbd along the basic surface, which may indicate potential binding sites for the phosphate groups of nucleic acids (Fig. 4 C–E). In fact, a molecule of MES occupies the ADP ribose–binding site in each of the three macro domains in P23pro-zbd in the asymmetric unit, as well as in the macroH2A1.1 structure (Fig. 4E) (43). The alphavirus genome contains conserved sequence elements (CSEs) that serve as pro- moters for production of positive- and negative-sense genomic RNA, as well as subgenomic RNA. Mutations in a 51-nt-long CSE in the nsP1-coding region of SINV disrupted the RNA structure without affecting the coding sequence and demonstrated reduced replication in mosquito cells but not in mammalian cells (44). Adaptive mutations (E118K of nsP2 and T286I Fig. 4. (A and B) Superposition of the MT-like domains of P23pro-zbd (teal) of nsP3) were shown to restore replication of the mutated CSE. and nsP2pro (gray) (12). The nsP3 linker (for simplicity only residues V162- T286 is 2 aa before one of the zinc-coordinating cysteine residues W178 are shown) connecting the macro domain and ZBD is shown in red. The (C288) (Fig. 4F) and is located on the basic surface of the ZBD. view in B is rotated 90° about a horizontal axis from A.(C and D) Potential T286 is 6.1 Å away from a sulfate ion that is coordinated by three RNA-binding surface of P23pro-zbd. The location of sulfate and MES ligands are pro-zbd highly conserved amino acids in nsP3: Y267, R273, and R276 represented by spheres. Surface of P23 is colored for electrostatic po- F tential at ±5 kT/e. D is a 180° rotation about the vertical axis from the view in (Fig. 4 ). Taken together, these data strongly implicate this sur- panel C.(E) Ribbon diagram of P23pro-zbd in the identical orientation as in C. face as a potential RNA-binding site. (F) ZBD of nsP3 showing the four zinc-coordinating cysteines, T286, a sulfate Many important human viral pathogens encode a polyprotein ion represented by spheres bound to Y267, R273, and R276, and surrounding that is cleaved by viral or cellular proteases. Given the wide residues. The close-up view corresponds to the box in E. prevalence of regulated polyprotein processing seen in a number

16538 | www.pnas.org/cgi/doi/10.1073/pnas.1210418109 Shin et al. Downloaded by guest on September 30, 2021 of viral families, there is lack of structural information regarding xylitol, and 5% (vol/vol) trimethylamine N-oxide and then flash-cooled in liquid the precursor forms. Currently, alphavirus P23pro-zbd and polio- nitrogen. Diffraction data were collected at the zinc, K absorption edge (1.2824 Å) virus 3CD (45) are the only structures of polyprotein precursor to 2.85 Å resolution at beam line X25 at Brookhaven National Synchrotron Light from a viral replication machinery. Both alphavirus P23pro-zbd Source (Table S1). Phases were determined by molecular replacement using the pro and poliovirus 3CD structures contain the protease domain re- nsP2 (12) and nsP3 macro structures (32). The final model contains 3 molecules pro-zbd sponsible for cleaving the precursor, which is thought to proceed by of P23 , 3 zinc ions, 31 sulfate ions, and 5 molecules of MES. Atomic coor- a trans mechanism. However, 3CD has no intramolecular con- dinates have been deposited in the Protein Data Bank under PDB ID code 4GUA. tacts and a solvent accessible cleavage site that is regulated by intermolecular contacts, whereas P23pro-zbd has an extensive in- Virological Studies. Infectious center and plaque assays were performed terface and an inaccessible cleavage site. The results presented similarly to those described in ref. 46. Protein and RNA radiolabeling were performed as described in ref. 9. Harvested protein from BHK-J cells at time here further expand our current understanding of viral poly- points postinfection were used in Western blotting experiments, and mem- protein processing, replication, and cytopathogenesis, which may branes were probed with anti-nsP2, anti-nsP3, and anti-GAPDH antibodies. be applicable to other positive-sense RNA viruses. ACKNOWLEDGMENTS. We thank J. Bonanno for assistance with the data Materials and Methods processing; C. Rice and M. MacDonald for providing BHK-J cells, Sindbis virus Complete materials and methods are provided in SI Materials and Methods. DNA, and nsP3 antibody; J. Millonig and P. Matteson for the GAPDH antibody; and A. Basant, J. Chiu, F. Jiang, A. Khan, C. Rice, A. Stock, V. Stollar, and Structure Determination. P23pro-zbd of the SINV strain Toto1101 was expressed J. Whidby for insightful discussions. S.A.Y. was supported by the NIH-NIAID training Grant 2T32AI007403-18. This study was supported by National in Escherichia coli as a fusion with GST. The protein was purified by sequential fi Institutes of Health Grants AI080659 (to J.M.) and DK083356 and AI070101 (to chromatography over GST af nity, hydroxyapatite, and heparin columns. A.G.), New Jersey Commission on Cancer Research Grant 10-1962-CCR-EO (to pro-zbd Crystals of P23 were grown by vapor diffusion against 2 M ammonium J.M.), and Yerkes Research Center Base Grant RR-00165 (to A.G). We wish to sulfate and 0.1 M MES (pH 6.5). Crystals were transferred to a cryoprotectant express our profound gratitude to the late Dr. Aaron Shatkin for his unwavering solution containing 2 M ammonium sulfate, 0.1 M MES (pH 6.5), 35% (vol/vol) support, sage advice, and insightful discussions.

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