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