Crystal Structure of the Lassa Virus Nucleoprotein–RNA Complex Reveals a Gating Mechanism for RNA Binding

Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding

Kathryn M. Hastiea, Tong Liub, Sheng Lib, Liam B. Kinga, Nhi Ngoa, Michelle A. Zandonattia, Virgil L. Woods, Jr.b, Juan Carlos de la Torrea, and Erica Ollmann Saphirea,c,1

aDepartment of Immunology and Microbial Science, and cThe Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; and bDepartment of Medicine, University of California at San Diego, La Jolla, CA 92093

Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved October 17, 2011 (received for review May 26, 2011) Arenaviruses cause disease in industrialized and developing nations C-terminal domains (16). Here we present two structures of the alike. Among them, the hemorrhagic fever virus Lassa is responsible N-terminal domain of LASV NP, now in complex with ssRNA. for ∼300,000–500,000 infections/y in Western Africa. The arenavirus nucleoprotein (NP) forms the protein scaffold of the genomic ribo- Results nucleoprotein complexes and is critical for transcription and repli- Structure of LASV NP in Complex with RNA. The recombinant N- cation of the viral genome. Here, we present crystal structures of terminal domain of LASV NP (NP1–340) is monomeric in nature the RNA-binding domain of Lassa virus NP in complex with ssRNA. and is stably and irreversibly bound to ssRNA derived from the This structure shows, in contrast to the predicted model, that RNA expression host (SI Appendix, SI Materials and Methods, and Fig. binds in a deep, basic crevice located entirely within the N-terminal S1A). Crystals of NP1–340 belong to the space group P61, contain domain. Furthermore, the NP-ssRNA structures presented here, six molecules in the asymmetric unit, and diffract to 3 Å reso- combined with hydrogen-deuterium exchange/MS and functional lution. Treatment of NP1–340 with pepsin (NPpep) removes a studies, suggest a gating mechanism by which NP opens to accept flexible polypeptide corresponding to residues 126–143 (SI Ap- RNA. Directed mutagenesis and functional studies provide a unique pendix, Figs. S1B and S2). Crystals of NPpep belong to the space look into how the arenavirus NPs bind to and protect the viral ge- group P21212, contain one molecule in the asymmetric unit, and nome and also suggest the likely assembly by which viral ribonu- diffract to 1.8 Å resolution (SI Appendix, Table S1). cleoprotein complexes are organized. MICROBIOLOGY In the structure of NPpep, electron density is well defined for residues 8–112 and 163–339, indicating that residues 113–127 structural biology | virology and 146–162, which flank the pepsin-deleted region, are indeed disordered as suggested by hydrogen/deuterium exchange (DX)/ he arenavirus family has a worldwide distribution and con- MS (SI Appendix, Fig. S2A). The undigested NP1–340 contains an Ttains significant human pathogens such as Lassa (LASV), additional α-helix (α6) that corresponds to residues 130–144, as Machupo, Junin, Lujo (1, 2), and lymphyocytic choriomeningitis well as an additional two to three residues resolved on either side virus. Of these arenaviruses, LASV carries the largest disease of this helix depending on the peplomer. In each of the six copies burden, causing 300,000 to 500,000 infections per year in Western of NP1–340 in the asymmetric unit, α6 extends from the NP core Africa. It is also the hemorrhagic fever most frequently trans- in a different location and orientation (SI Appendix, Fig. S3). ported out of Africa to the United States and Europe (2–4). Consistent with the previously reported RNA-free structures Arenaviruses have a bisegmented, negative-sense, single- of LASV NP (16, 19), NP1–340 has a compact, mostly α-helical stranded RNA genome with a unique ambisense coding strategy structure consisting of head and body regions that contain four that produces just four known proteins: a glycoprotein, a nucleo- and eight helices, respectively. The head region is formed by protein (NP), a matrix protein (Z), and a polymerase (L) (2). Of residues 8–24, 83–122, and 261–340, but the body is formed by these proteins, NP is the most abundant in an infected cell. NP residues 25–82 and 123–260 (Fig. 1A). The fragments released associates with L to form the ribonucleoprotein (RNP) core for through pepsin digestion are separate collinear sections that RNA replication and transcription (5) and the matrix protein Z each contain part of the head and part of the body (Fig. 1B). – for viral assembly (6 8). The arenavirus NP also plays an impor- Six RNA nucleotides, corresponding to bases 2–7, are resolved tant role in the suppression of the innate immune system (9–11). in NPpep (SI Appendix, Fig. S4 A and B). Bases 2–7 are resolved Genome and antigenome RNAs of negative-strand RNA in NP1–340 as well as bases 1 and 8, depending on the peplomer viruses (NSV) do not exist as naked RNA, but rather as a RNP (SI Appendix, Fig. S4 C and D). Bases 1–4 are nearly identical in complex in which the RNA is encapsidated by the viral nucleo- all NP peplomers, but bases 5–8 show small deviations in their protein. During replication of many negative-strand RNA viru- relative positions. Although NP was predicted to bind RNA ses, the nascent nucleoprotein (usually termed N) is bound by a between the N- and C-terminal domains, this RNA-bound polymerase cofactor (often a phosphoprotein, termed P), which structure indicates that instead, the RNA is bound by the N- prevents polymerization of N and nonspecific encapsidation of – 0 terminal domain in a deep, basic crevice that channels between host cell RNAs (12 15). The resulting complex is termed N -P, its head and body regions (Fig. 1C). in which N0 denotes RNA-free N. The arenavirus, orthomyx- ovirus (flu), and bunyavirus (Hanta, Rift Valley Fever) families (i.e., segmented NSV) do not encode an analogous P protein, Author contributions: K.M.H., V.L.W., J.C.d.l.T., and E.O.S. designed research; K.M.H., T.L., and the mechanism by which the nucleoprotein controls RNA S.L., L.B.K., N.N., and M.A.Z. performed research; K.M.H., T.L., V.L.W., J.C.d.l.T., and E.O.S. binding during virus infection is not yet understood. analyzed data; and K.M.H. and E.O.S. wrote the paper. The arenavirus nucleoprotein (termed NP instead of N) has The authors declare no conflict of interest. fl distinct N- and C-terminal domains connected by a exible linker This article is a PNAS Direct Submission. – (16 19). The C-terminal domain functions as an exonuclease Data deposition: The atomic coordinates and structure factors have been deposited in the (16, 17) specific for dsRNA (17) and linked to antagonism of Protein Data Bank, www.pdb.org (PDB ID codes 3T5N and 3T5Q). type I IFN (16, 17). A structure of LASV NP, in the absence of 1To whom correspondence should be addressed. E-mail: [email protected]. RNA, predicted the presence of a cap-binding site in the N- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. terminal domain and an RNA-binding site between the N- and 1073/pnas.1108515108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1108515108 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 C A C B C Head

113-162 180ºº N 113-1277 N

5 14 8- 12128-145

146-162 Body NP1-340 NPpep

C D

Y308 R329 Y213 Ar3 K309 Ar8 R300 Ar3 Cr7 Ar8 Ur4 R323 Ur1 Ur6 Ur1 Cr5 K253 Ur2

F176 T178 N240 S247 W164 T216 S237

Fig. 1. Structure and RNA binding of the N-terminal domain of LASV NP. (A) Cartoon representation of NP1–340, colored by head (tan) and body (blue) regions of LASV NP1–340. Residues 113–127 are disordered. (B)NPpep colored by 21-kDa N-terminal (yellow) and 14-kDa C-terminal sections that result from pepsin digestion (teal). Note that the head and body regions are made by interweaving of the N- and C-terminal sections of polypeptide. Residues 112–126 and 144–166 are disordered, and residues 124–143 were removed by digestion with pepsin. In A and B, for clarity, only the protein portion of the complex is

illustrated. (C) Electrostatic surface potential of NPpep calculated using APBS (34) shows a deep basic groove through which the single-stranded RNA channels. The tunnel is between 6 and 16 Å wide and at position Ar3, is recessed 10 Å from the protein surface. Positive surface is colored blue; negative surface is

colored red with limits ± 10 kbT/ec.(D) The side chains of positively charged and other polar residues that interact with the phosphate backbone and bases of the single-stranded RNA are labeled. The eight nucleotides are colored from red (3′; Left) to blue (5′; Right). Residues and secondary structural elements of the head domain are colored tan; residues and secondary structural elements of the body domain are colored blue. Although many of the residues are nonspeciﬁc and have thus been built as uridine or cytosine, residues 3 and 8 are clearly purines and have been built as adenosine.

Although each NP-RNA monomer binds random RNA from visualized in the basic crevice (16). Notably, the location of each the expression host, position 3 in each RNA strand is clearly a of these nucleotides is essentially equivalent to that of Ur2 in the purine (SI Appendix, Fig. S4 A and B), and is anchored between ssRNA-bound structure, suggesting that the soaked-in dUTP and R300 and Y308. In addition, when visible, position 8 in all but TTP may not be mimicking cap, but rather, are reflecting the one RNA strand can also be built as purine. Hence, although the positions of individual nucleotides within the RNA strand (SI overall binding of RNA by NP is thought to be nonspecificin Appendix, Fig. S5). Indeed, there is as yet no direct evidence that nature, NP may have some unexpected, partial sequence speci- LASV NP binds cap and our attempts to bind various NPs to ficity. However, whether LASV prefers this motif in the actual cap-conjugated agarose beads were unsuccessful (SI Appendix, genome, or simply in static binding of ssRNA in the expression Fig. S6). Furthermore, recent biochemical analysis of several host, is as yet unclear. residues previously proposed to be involved in cap-binding dem- An extensive network of interactions anchors the RNA onstrates that mutation of these residues results in defects in phosphate backbone and the rest of the bases to the protein (Fig. antigenome synthesis rather than defects in mRNA levels (19). 1D). Bases 2–4 demonstrate the highest level of interaction with Hence, the binding of ssRNA observed here combined with the the nucleoprotein through key residues on α12, α14, and η2. positional homology of the individual, soaked-in nucleotides These α-helices contain all of the arginine and lysine residues indicates that this site is likely not a binding site for cap, but responsible for binding the RNA backbone, as well as Y308, rather is a binding site for the viral genome. which stacks against Ar3, forming a strong π-interaction with the The RNA-free structure also includes the C-terminal domain six-membered ring of this base. RNA residues 1 and 5–8 show of NP. In this context, LASV NP forms a trimer with N- and C- more modest interactions, primarily through a hydrogen-bond terminal domains oriented in a head-to-tail fashion, forming an network between the phosphate backbone and several threonine RNA-free ring of NP monomers. In the RNA-free structure, and serine residues of NP. helix α5 is extended by 10 residues (residues 112–122) and both the extended helix α5 and helix α6 lie across the RNA-binding RNA Binding Is Controlled Through a Gating Mechanism. The pre- crevice, occluding access to RNA. The C-terminal domain viously determined RNA-free structure of LASV NP suggested interacts with these helices and the loop connecting them to that the basic crevice in the N-terminal domain was responsible stabilize this “closed,” trimeric form of RNA-free NP (SI Ap- for binding to the m7GTP cap of mRNA (16). Attempts to pendix, Fig. S7). In contrast, in the RNA-containing NP1–340 cocrystallize NP with an m7GTP analog were unsuccessful, but structure, residues 112–122 are mobile and disordered. As a re- the single nucleotides dUTP and dTTP could be soaked in and sult, helix α5 is shorter and terminates before the crevice. In

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1108515108 Hastie et al. Downloaded by guest on September 25, 2021 addition, helix α6 is rotated away from the crevice. Furthermore, ability of a panel of NPs with mutations to the RNA-binding the loop connecting α5 and α6 (residues 232–243) is shifted crevice to promote expression of a CAT reporter gene within a outward in the presence of RNA and appears to form a “gate” LASV minigenome (MG) (Fig. 3). Mutations to residues that (Fig. 2). When in the “open” conformation, several residues in contact RNA bases 1–4 essentially abrogated MG transcriptional this loop interact with the RNA backbone and sugar moieties. activity compared with WT NP. Mutations to residues that However, in the closed conformation, this loop would clash with contact RNA bases 5–8 show less pronounced effects. S247A, bound RNA. Thus, it appears the trimeric form of Lassa NP which contacts base 5, and Y213A, which contacts base 8, result would be unable to bind to RNA. in ∼40% the transcriptional activity of WT, but mutations to To determine if the trimeric ring arrangement causes the C- residues that contact bases 6 and 7 have no effect on MG tran- terminal domain to stabilize α5 and α6 in the closed form of scription. Overall, expression levels of NP with these mutations is LASV NP, we made the mutations S45R and K189E to residues similar to that of WT and all of these mutants are recognized by that lie at the NP–NP interface in the trimeric structure (SI conformational anti-Lassa NP antibodies (SI Appendix, Fig. S10). Appendix, Fig. S8A). Correspondingly, although WT NP elutes Hence, loss of MG activity is likely a result of loss of RNA from a size-exclusion column as a monomer, trimer, and hex- binding rather than defects in protein expression or structural fi amer, mutant NPS45R,K189E elutes as a dimer and tetramer (SI integrity, con rming the importance of the observed RNA- Appendix, Fig. S8B). To further investigate the potential struc- binding site to NP function. tural changes resulting from this change in oligomeric state, we per- In contrast, mutation of W164 or F176 to alanine also prevents formedDX/MSanalysisonboththeWTandmutantNPS45R,K189E. MG activity, but the resulting mutant NPs are not recognized WT NP demonstrates low-moderate exchange rates for α5, α6, by conformational anti-NP antibodies. These residues form a and their connecting loop (SI Appendix, Fig. S9A). In contrast, hydrophobic pocket adjacent to bases 1 and 2. Although it was previously proposed that these residues formed a portion of the NPS45R,K189E shows very high exchange rates for this region, cap-binding pocket (16), it is possible that these residues are similar to those rates of NP1–340 from which the C-terminal domain is absent (SI Appendix, Figs. S9B and S2A, respectively). instead important for the structural integrity of the protein. Furthermore, residues of the C-terminal domain that interact Other mutations to residues that lie within a deep pocket of the with either the N-terminal domain of the same NP (α17), or the RNA-binding crevice (R17, Y209, E266, and Y319), yet do not N-terminal domain of the next NP in the ring (α22, and β10 and directly contact the RNA itself, also prevent CAT expression. β11), also show alterations in the amide hydrogen exchange rates These residues coordinate two water molecules and appear to be when mutated. Additionally, although recombinant WT NP ex- important for the structural integrity of NP as overall expression MICROBIOLOGY levels of these mutants are greatly reduced compared with WT hibits a A260/280 ratio of 0.95, NPS45R,K189E exhibits a ratio of 1.3, suggesting that more RNA is bound to mutant NP in which NP, and the E266A and Y319A mutants are not recognized by the trimeric interface is disrupted than to WT NP. Taken to- conformational anti-NP antibodies. Finally, a mutation to pro- gether, these results indicate that the trimeric form of NP, as line of G243, a residue that lies at the base of the RNA gate, also eliminates MG-derived CAT expression. It is likely that mutation previously crystallized, constitutes and stabilizes a closed con- fl formation. In order for RNA to bind, NP must undergo struc- of this exible glycine residue to a more rigid proline locks the tural changes that may ultimately result in reorganization of gate in a closed position, preventing RNA from binding. G243P the oligomer. NP expresses to levels similar to WT NP and G243P is recognized by conformational anti-NP antibodies. Hence, the experi- Residues Within the RNA-Binding Pocket and at NP Interfaces Are mentally visualized RNA-binding site and the gate that opens it Important for Transcription and Replication. To characterize the are both critical for MG replication and transcription. role of residues involved in binding of RNA, we examined the We also sought to characterize the importance of interactions between the N- and C-terminal domains of a single NP, and of one NP with its neighboring NP in RNA synthesis (Fig. 3). At the interface between N- and C-terminal domains, some mutations block activity and others have little effect. Notably, it is those RNA-free RNA-bound residues that are involved in protein-protein hydrogen bonds between the two domains that appear important for MG activity, but other basic residues that are not involved in protein-protein hydrogen bonds do not seem important. Specifically, K110A, extended R115A, R118A, and W331A mutations prevent or greatly reduce helix 5 CAT expression. Each of the basic residues K110, R115, and 112 R118 form hydrogen bonds with neighboring residues or the RNA gate shifted helix 6 carbonyl backbone, but W331 stacks against the aliphatic chain of K110. Although a K110A mutation blocks CAT activity, 122 a K110E mutation in which the basic Lys is replaced by an acidic Glu unexpectedly doubles CAT expression to 200% of WT. A K110E/S111R double mutation has ∼40% of WT CAT expression but a K110E/S111E double-mutation has no MG activity, suggesting the additional importance of residue 111. In contrast, mutations to other basic residues within the N–C interface that do not specifically form salt-bridges or hydrogen bonds to the other protein domain (K167A, R556A, and R561A) show only a modest reduction in CAT expression (65–75% the Fig. 2. Comparison of the RNA-free and -containing structures of the LASV NP activity of WT). These results suggest that interaction between N-terminal domain. RNA binding seems to be regulated through conformational changes that open and close the RNA-binding pocket. In the RNA-free confor- the N- and C-terminal domains is important for replication and mation (gray), the RNA gate is closed. Helix α5 is extended across the RNA- transcription but do not necessarily rule out the possibility of binding pocket and α6 is positioned on top of the pocket, preventing RNA from a secondary RNA-binding site between the two NP domains. binding. When RNA is bound (blue), residues 112–122 are disordered, α6 shifts Other sites, beyond specific RNA contacts are also important away from the pocket, and the RNA gate opens to accommodate the ssRNA. for NP function. Outside of a single NP peplomer, mutations

Hastie et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 A RNA-binding crevice N-C interface NP-NP interface

R17 D437 R329 K167 Y213 Y319 Y308 K189 E266 R300 R118 K309 Y209 K253 N240 R115 S45 R323 R566 T178 W331 S111 G243 R55 S247 F176 K110 R52 W164 T216 S237 R561 D500 K56

B Percent Activity Compared to WT

Contact RNA bases 1-4 Contact RNA in RNA RNA bases 5-8 binding gate crevice N-C interface NP-NP interface

RNA binding crevice

Fig. 3. RNA binding, N–C interface, and NP–NP interface residues and their roles in viral RNA transcription. (A) Locations of mutations made to residues in three functional sites in the full-length NP: within the RNA-binding crevice, at the N–C interface, which is between the N- and C-terminal domains of one NP monomer, and at the NP–NP interface between two different NP monomers. (B) Ability of this panel of NP mutants to transcribe a CAT reporter gene in a LASV MG assay. The large array of mutations made in the RNA-binding crevice is subdivided into those mutations that contact RNA bases 1–4, those mutations that contact bases 5–8, others deep in the crevice, and a key glycine at the base of the RNA-binding gate.

that prevent NP–NP interactions (S45R, K189E, R55A/K56E, 28). Furthermore, LASV NP could have some partial sequence D437R, D500R) also eliminate CAT expression. Hence, NP–NP specificity or preferences, but no specificity was observed in N- association is critical for replication and transcription, perhaps to RNA complexes of the nonsegmented viruses RSV, RABV, allow processive movement of the polymerase L. and VSV. Comparison of the RNA-free and RNA-containing structures Discussion and accompanying DX/MS analysis of LASV NP indicate that Crystal structures of the nucleoprotein-RNA complexes from RNA binding is controlled through a gating mechanism of con- the nonsegmented negative-strand RNA viruses rabies (RABV), formational changes within the N-terminal domain. Taken to- vesicular stomatitis (VSV), and respiratory synsyctial viruses gether with mutational analysis of LASV NP, these results suggest (RSV) demonstrate that RNA binds nonspecifically in a basic a possible model for how the RNP might be organized. In this groove between the N- and C-terminal domains of N (20–22). In model, the trimeric form of NP could represent “NP0,” in which contrast, for LASV, a segmented RNA virus, RNA binds within trimerization performs the equivalent function of the P protein the compact N-terminal domain. of other negative-strand viruses, and therefore prevents RNA Structures of nucleoproteins from other segmented viruses binding (Fig. 4 A and B). Upon RNA binding, which could be (influenza and Rift Valley fever) have been determined in the triggered by an as-yet identified factor or perhaps the viral ge- absence of RNA (23–25), but this work represents a unique nome itself, the C-terminal domain of NP rotates slightly away structure of an NP from a segmented virus in complex with RNA. from its position in the RNA-free trimer, allowing helices α5 and Comparison of the structures reveals some organizational fea- α6 to open away from the RNA-binding crevice (Fig. 4C). With tures that appear to be in common among the NP and RNP RNA bound, NP will no longer be able to form the trimeric ring complexes of these segmented viruses. The Ns of influenza virus and instead may form another arrangement such as a linear and Rift Valley fever virus and the N-terminal domain of LASV chain of NP molecules that line the ssRNA, with NP–NP inter- NP are compact, rather than more extended like the Ns of the actions mediated through the N-terminal portion of one NP and nonsegmented RABV, VSV, RSV, and Borna disease virus. the C-terminal portion of another (Fig. 4D). Such an assembly Furthermore, electron microscopy of RNPs of the segmented would create a continuous head-to-tail polymer attached to the Pichinde arenavirus, influenza, and Rift Valley fever viruses RNA, in which only the RNA bound within the crevice of the N- demonstrates the RNP complexes of segmented viruses are less terminal domain is resistant to RNase attack. We indeed note helical in nature than those of the nonsegmented viruses (26– that only the portion of RNA specifically bound by each N-

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1108515108 Hastie et al. Downloaded by guest on September 25, 2021 NP-NP interface NP-NP interface A N-C interface B N-C interface RNA-binding RNA-binding pocket pocket

C + ssRNA C-terminal domain shifts unknown away from crevice cofactor? to allow RNA to bind

NP-NP interface D N-C interface

3’ 5’ MICROBIOLOGY

Fig. 4. A model for arenavirus RNP organization. (A) Organization of the trimeric, RNA-free LASV NP (Protein Data Bank ID code 3MWP). (B) The N-terminal domain of LASV NP colored by electrostatic surface potential and the C-terminal domain modeled as a cartoon (green) show the closed form of the NP structure. In this conformation, the RNA-binding crevice is not available to accept ssRNA. (C) To bind the viral genome, the C-terminal domain must shift away from the RNA-binding crevice to allow RNA to enter. This shift could be initiated by binding of NP by an as yet unidentiﬁed cofactor or perhaps the viral genome itself. (D) When bound to ssRNA, the trimer of NP will not form. Instead, monomers of NP line the ssRNA backbone. Each N-terminal domain of NP interacts with the adjacent C-terminal domain of a neighboring NP.

terminal domain is resistant to RNases, with intervening nucleic interactions, and the role, if any, that L plays in mediating NP– acid easily clipped. A continuous head-to-tail polymer of NP NP interactions are unknown. However, DX/MS of NP, purified would presumably also help the polymerase move along the in the absence of L, does support the existence and importance template by transiently displacing NP (to copy the template), and of NP–NP interactions. repositioning NP on the template once the polymerase has The tight sequestering of RNA and likely conformational passed. An alternative model in which the RNA binds both in- change needed for replication and transcription makes NP an side the N-terminal crevice and between the N- and C-terminal excellent target for small molecules that either prevent NP domains is also possible as this interface does contain several binding of RNA or that stabilize the RNP to prevent conforma- basic residues. However, the majority of these basic residues are tional changes induced by the polymerase. The arenavirus NPs involved in hydrogen bonds to the other NP domain. Mutation are well conserved, with a mean 73% similarity overall calculated of other basic residues at this interface that are not involved in for six family members spanning both the Old and New World protein–protein interactions results in only modest effects on groups (SI Appendix, Fig. S11A). Importantly, each residue that replication and transcription, suggesting that NP likely binds makes contact with the RNA is 100% conserved across the family RNA without the strict requirement of basic residues between (SI Appendix, Fig. S11B). The depth and intimate interactions the N- and C-terminal domains. by which the purine at position 3 packs in between Y308 and How is the RNA contained in the RNP read by polymerase R300, coupled with the conservation of these two amino acids during RNA synthesis? The narrow crevice through which across the arenavirus family, makes this region a particularly at- ssRNA tunnels through the N-terminal domain of NP and the tractive target for inhibitors of arenavirus replication. nuclease-resistance conferred by NP binding together suggest The work presented here also provides a structural template that the bound portion of RNA is not available to form duplexes by which we may determine the precise role of specific amino during replication. Furthermore, the depth at which the ssRNA acids of NP in their functional interactions with the arenavirus is bound likely renders it unable to form a base pair with a polymerase and explore the relative roles of NP assemblies in the complementary strand when in complex with NP. A scenario that viral life cycle, in this and other families of pathogens that fits with our crystallographic, DX/MS, and functional data is threaten human health. a viral polymerase-induced conformational change within NP Materials and Methods that transiently exposes the RNA for replication and transcription. The ability of mutants that block NP–NP interactions to Crystallization of NP1–340 and NPpep. Crystals of NP1–340 and NPpep were obtained by hanging drop vapor diffusion using a 1:1 ratio of well solution also block viral RNA synthesis suggests that the polymerase may to protein at 10–12 mg/mL. Data for NP1–340 were collected at the Stanford need to interact with the N- and C-terminal domains of adjacent Synchrotron Radiation Lightsource, Beamline 12.2. Data for native and

NP molecules simultaneously to induce this conformational SeMet-derivatized NPpep crystals were at the Advanced Light Source, change or to move along the RNP. Residues involved in NP–L Beamline 8.3.1.

Hastie et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 Structure Determination. Structure determination of NPpep using SIRAS (29) corresponding LASV-NP mutants, as previously described (32, 33). At 60 h was carried out by AutoRickshaw (30). One copy of NPpep was found in the posttransfection, cell lysates were prepared and assessed for levels of CAT asymmetric unit. Molecular replacement for the structure of the complete, protein using a CAT ELISA kit. undigested NP1–340 was performed with PHENIX (31) using just the protein portion of the model derived from the 1.8Å NPpep structure. Six copies of ACKNOWLEDGMENTS. We thank Dr. James Robinson (Tulane University) for NP1–340 were found in the asymmetric unit. the gift of conformational human antibodies against Lassa virus nucleoprotein; Xiaoping Dai for assistance with data processing; and Beamlines 12.2 of m7GTP Pull Downs. Two hundred micrograms of each protein were incubated the Stanford Synchrotron Radiation Lightsource (Menlo Park, CA) and 8.3.1 with 15-μL m7GTP-conjugated agarose beads for 4 h at room temperature. of the Advanced Light Source (Berkeley, CA) for data collection. This study Beads were washed three times with 50 mM Tris pH 8, 300 mM NaCl. Bound was supported in part by Viral Hemorrhagic Fever Research Consortium protein was eluted with SDS/PAGE loading buffer. Samples were analyzed and Contract HHSN272200900049C Solicitation Number BAA-NIAID-DAIT- by Western blot. NIHAI2008031 (to V.L.W. and E.O.S.); and National Institutes of Health Grants GM093325, GM020501, GM066170, NS070899, and RR029388 (to LASV MG Transcription Assay. The 293T cells were cotransfected with a plas- V.L.W.), AI077719 and AI047140 (to J.C.d.l.T.); an Investigators in Pathogen- mid encoding a LASV MG under control of the T7 RNA polymerase, pol-II esis of Infectious Diseases Award from the Burroughs Wellcome Fund (to expression plasmids directing expression of the virus L and T7RP, and the E.O.S.); and The Skaggs Institute for Chemical Biology (E.O.S.).

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