Structure of the Rift Valley Fever Virus Nucleocapsid Protein Reveals Another Architecture for RNA Encapsidation

Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation

Donald D. Raymonda,b, Mary E. Piperc, Sonja R. Gerrardc,d, and Janet L. Smitha,b,1

aLife Sciences Institute, bDepartment of Biological Chemistry, cCellular and Molecular Biology Program, and dDepartment of Epidemiology, University of Michigan, Ann Arbor, MI 48109

Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved May 19, 2010 (received for review February 17, 2010)

Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus negative-sense RNA segments, termed small (S), medium (M), Phlebovirus, family Bunyaviridae) that infects livestock and humans and large (L) (2). It belongs to the Phlebovirus genus in the Bunya- and is endemic to sub-Saharan Africa. Like all negative-sense viridae family. As with all negative-sense RNA viruses, the genome viruses, the segmented RNA genome of RVFV is encapsidated by is bound, or encapsidated, by N. The N of RVFV is a 27-kDa pro- a nucleocapsid protein (N). The 1.93-Å crystal structure of RVFV N tein encoded by the S segment. Encapsidation of RVFV genomic and electron micrographs of ribonucleoprotein (RNP) reveal an RNA, as with all negative-sense RNA viruses, plays an essential encapsidated genome of substantially different organization than role in multiple steps within the replicative cycle including tran- in other negative-sense RNA virus families. The RNP polymer, scription and replication by the RNA-directed RNA polymerase viewed in electron micrographs of both virus RNP and RNP reconsti- (RdRp) and packaging of genome into virions (3). RVFV N is tuted from purified N with a defined RNA, has an extended structure thought to interact with the viral RdRp because N is essential without helical symmetry. N-RNA species of ∼100-kDa apparent to replication and transcription (4). N also plays a role in virus molecular weight and heterogeneous composition were obtained assembly through interactions with the viral envelope glycopro- G G by exhaustive ribonuclease treatment of virus RNP,by recombinant teins ( N and C) (5). expression of N, and by reconstitution from purified N and an RNA Bunyavirus N binds single-stranded RNA (ssRNA) nonspeci- oligomer.RNA-free N, obtained by denaturation and refolding, has a fically (6, 7), although some N may have a preference for specific novel all-helical fold that is compact and well ordered at both the N viral RNA sequences (8–10). Studies on some animal viruses and C termini. Unlike N of other negative-sense RNA viruses, RVFV N within the Bunyaviridae family found that encapsidated RNA has no positively charged surface cleft for RNA binding and no is resistant to disruption by high salt and Ribonuclease treatment protruding termini or loops to stabilize a defined N-RNA oligomer (8, 10, 11). Despite the common property of tight, nonspecific or RNP helix. A potential protein interaction site was identified in binding to single-stranded RNA, homology of N within the a conserved hydrophobic pocket. The nonhelical appearance of Bunyaviridae family is not apparent from sequence data, because phlebovirus RNP, the heterogeneous ∼100-kDa N-RNA multimer, N from different genera appear unrelated. However, within a and the N fold differ substantially from the RNP and N of other genus, the N are clearly homologous. When RVFV N is compared negative-sense RNA virus families and provide valuable insights across the Phlebovirus genus, the amino acid identity generally into the structure of the encapsidated phlebovirus genome. ranges from 50% to 59% and is 36% for Uukuniemi virus, the most divergent clade within the Phlebovirus genus. The high ribonucleoprotein ∣ viral protein ∣ segmented genome ∣ degree of sequence identity indicates that the phlebovirus N have negative-sense RNA virus similar structures and form similar RNPs. Additionally, the phlebovirus N are distantly related to the N of the Tenuivirus, a genus NA viruses cause a myriad of human and animal diseases, of negative-sense RNA viral plant pathogens with worldwide Rincluding measles, poliomyelitis, rabies, the common cold, distribution (12). Otherwise, the phlebovirus N appear unrelated dengue fever, and Rift Valley fever. Despite tremendous diversity to N of other negative-sense RNA viruses. among RNA viruses, all must package a protected RNA genome Structures are available for N from several negative-sense into virus particles. RNA viruses protect their genome in one of RNA viruses, including influenza A virus (FLUA) (13), rabies two ways, providing either a protein shell or a protein coat for the virus (RABV) (14), human respiratory syncytial virus (HRSV) genome (1). The process is generally known by the term encap- (15), vesicular stomatitis virus (VSV) (16), and Borna disease virus (BDV) (17). For some of these, ribonucleoprotein (RNP)

sidation, but functionally and structurally encapsidation takes a BIOCHEMISTRY variety of forms. Most positive-sense RNA and double-stranded complexes have been visualized by crystallography or electron RNA viruses place their genome within a protein shell, known as microscopy (FLUA) (18), (RABV) (14), (HRSV) (15), (VSV) a capsid. By contrast, the negative-sense RNA viruses encapsi- (16). The crystallized RNP oligomers are high-order ring struc- date their genome by coating the length of the RNA with a nu- tures formed by specific contacts of loops or chain termini of cleocapsid protein (N). Although capsid and N all bind RNA, the neighboring N subunits. In some viruses, the number of subunits resulting RNA-protein complexes differ, and it is not possible to in the ring matches the helical repeat of the RNP polymers (15). make generalizations about the proteins involved in encapsida-

tion across all RNA virus families. Author contributions: D.D.R., M.E.P., S.R.G., and J.L.S. designed research; D.D.R., M.E.P., Rift Valley fever is a mosquito- and aerosol-borne disease of and S.R.G. performed research; D.D.R., M.E.P., S.R.G., and J.L.S. analyzed data; and livestock and humans in sub-Saharan Africa and is caused by Rift D.D.R., M.E.P., S.R.G., and J.L.S. wrote the paper. Valleyfever virus (RVFV).Rift Valleyfever in domestic ruminants The authors declare no conflict of interest. results in abortion and high rates of mortality, especially among This article is a PNAS Direct Submission. young animals (2). In humans, Rift Valley fever is typically a Data deposition: The coordinates and structure factors for the crystal structure have been self-limited febrile illness, although severe disease, such as hemor- deposited in the Protein Data Bank, www.rcsb.org (PDB ID code 3LYF). rhagic fever and encephalitis, occurs in a small percentage of cases 1To whom correspondence should be addressed. E-mail: [email protected]. (2). No effective therapeutics exist for treating Rift Valley fever. This article contains supporting information online at www.pnas.org/lookup/suppl/ RVFVhas a membrane envelope and a genome composed of three doi:10.1073/pnas.1001760107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1001760107 PNAS ∣ June 29, 2010 ∣ vol. 107 ∣ no. 26 ∣ 11769–11774 Downloaded by guest on September 24, 2021 None of the structurally characterized N is from the Bunyaviridae A B family and none has detectable homology with the phlebovirus N. DSP DSP RVFV RNPs N N-RNA Early electron micrographs of encapsidated bunyavirus genomes reveal a noncondensed, macrocircular form that appears to lack * 250 kDa * 250 kDa 170 kDa symmetry (19, 20). Nevertheless, negative-sense RNA viruses are * 130 kDa * 130 kDa assumed to have condensed helical structures based on micro- * 95 kDa graphs of RNP from several virus families (15, 21–24). 72 kDa * 95 kDa Here we report the 1.93-Å crystal structure of recombinant 72 kDa RVFV N and views of two forms of RVFV RNP by electron 55 kDa * 55 kDa microscopy. N has a novel protein fold that differs substantially * * from N of other negative-sense RNA viruses. The refolded, 43 kDa recombinant N forms stable multimeric N-RNA complexes of 36 kDa similar appearance to N-RNA multimers released from virus 36 kDa 34 kDa RNP by exhaustive ribonuclease treatment. The N-RNA multi- 28 kDa – 26 kDa mer is heterogeneous with 4 7 N subunits and has an apparent 28 kDa molecular weight of 100 kDa. Authentic virus RNP and RNP

reconstituted from refolded N and defined RNA have a similar 1 2 34105129876 11 13 nonhelical appearance and similar ribonuclease resistance. Fig. 1. Similar multimer complexes of authentic virus RNPs and purified Results recombinant N bound to RNA. (A) Viral RNP. Purified RVFV RNPs were cross-linked with 0.0, 5.0, or 20.0 mM DSP and analyzed by immuno-blot. Protein Oligomeric State in Solution. Purification of recombinant N Asterisks indicate predominant cross-linked species. Molecular weight (recN) under native conditions, including exhaustive ribonuclease markers are in the rightmost lane. (B) Recombinant N. N or N bound to U25 treatment, resulted in a discrete complex of the protein and single-stranded RNA (N-RNA) was cross-linked using 0.0, 1.0, 5.0, 10.0, or Escherichia coli nucleic acid as determined by the ratio of absor- 20.0 mM DSP, separated by SDS/PAGE, and visualized with colloidal Coomassie bances at 260 nm and 280 nm (Fig. S1 A and B). The protein could stain. The dominant cross-linked species are indicated by asterisks. not be separated from nucleic acid under native conditions using high salt concentrations and pH extremes. The recombinant Electron Microscopy of Authentic Virus RNPs and Reconstituted RNPs. N-RNA multimer had an apparent mass of 100 kDa by size-exclu- We compared RNPs isolated from virus with RNPs reconstituted sion chromatography. Nucleic acid was extracted from the recom- from refolded N and a large single-stranded RNA. Freshly pre- binant RVFV N by denaturation and then treated with either pared samples (Fig. S2) were viewed by negative-stain electron deoxyribonuclease or ribonuclease. The nucleic acid was sensitive microscopy before and after overnight ribonuclease digestion only to ribonuclease treatment, demonstrating that N was bound at room temperature. Both authentic virus RNP and reconsti- to RNA. The formation of a nonspecific ribonucleoprotein (RNP) tuted RNP were ribonuclease-resistant and had a remarkably complex between recombinant RNA-binding proteins and E. coli similar string-like appearance (Fig. 2A and Fig. S3). No helical RNA is not uncommon (6, 15). Crystal structures of RABV (14), symmetry was apparent in either sample. The appearance of phle- VSV (16), and HRSV (15) RNPs were solved from RNPs bound to bovirus RNP is strikingly different from images of similarly pre- E. coli RNA, but no crystals were obtained using the recombinant pared RNP from other negative-sense RNA viruses, which have RVFVRNPs. We therefore used denaturation to obtain RNA-free obvious helical symmetry (15, 21–24). recN for crystallization. After purification from RNA and refolding, N was predominantly a monomer of apparent molecular mass Heterogenous Multimeric N-RNA Complexes. Aggressive ribonu- 21 kDa, with about 10% as a dimer (Fig. S1C). clease digestion (3 d at 37 °C) released a multimer from the virus RNP (Fig. 2B and Fig. S4A). In electron micrographs, this particle Cross-Linking of Authentic Virus RNPs and Recombinant N with RNA. is similar in appearance to the ribonuclease-resistant, recombinant We tested whether the refolded recN could interact with RNA N-RNA multimer purified from E. coli (Fig. 2C and Fig. S4B) and similarly to N in viral RNPs. Purified viral RNPs, refolded recN, to multimers reconstituted from refolded N and defined RNA and reconstituted N-RNA multimer (recN-RNA) were cross- oligomers (Fig. 2D and Fig. S4 C and D). Remarkably, the multi- linked with a homobifunctional amine-reactive cross-linker and mers are of similar size distribution (10–12 nm diameter) regard- then separated by electrophoresis under denaturing conditions less of the source of RNA. The N-RNA multimers from all (Fig. 1). In the absence of cross-linker (Fig. 1A, lane 1; Fig. 1B, sources appear heterogeneous, with 4–7 bright objects per particle lanes 4 and 9), N from all samples migrated as a monomer. When (Fig. 2 B–D). The heterogeneity of the recombinant N-RNA viral RNPs were exposed to increasing amounts of cross-linker, multimer explains its inability to crystallize. The recombinant the monomer band decreased in intensity and four higher mole- and reconstituted multimers have an apparent molecular weight cular weight complexes appeared (Fig. 1A, lanes 2 and 3). Cross- of 90–100 kDa by size-exclusion chromatography (Fig. S5). Thus, linking of RNA-free, refolded recN resulted in predominant we conclude that the N-RNA multimers contain 4–7 N subunits monomer and minor dimer species (Fig. 1B, lanes 5–8), consis- and an unknown amount of RNA. tent with the behavior of recN in size-exclusion chromatography The stoichiometry of N and RNA in a reconstituted N-RNA (Fig. S1C). In contrast, when recN-RNA was cross-linked, many multimer was estimated using known N and RNA extinction species of higher molecular weight were observed (Fig. 1B, lanes coefficients at 260 nm and 280 nm. We used a short RNA decamer 10–13). The number of N within the dominant cross-linked in order to enhance the contribution of protein to the total absor- species created from recN-RNA and virus RNPs (Fig. 1B, lane bance, which was dominated by RNA. The reconstituted N-RNA10 11 and Fig. 1A, lane 3) was estimated to be 2, 4, 6, and 10 based multimer had similar behavior upon gel filtration (Fig. S5) and on an apparent molecular weight of 25 kDa for N. Thus, refolded similar appearance in electron micrographs (Fig. S4) to the recN behaves similarly to viral N in its ability to bind RNA and to N-RNA25 multimer. The purified, reconstituted N-RNA10 multi- form multimeric complexes. The cross-linked species formed by mer had an A260∕A280 ratio of 0.94, indicating an average stoichio- both viral RNPs and recN-RNA appear primarily as multiples of metry of 5∶1 N∶RNA10, in good agreement with the gel filtration two, consistent with a previously reported dimeric association of data. This result indicates that each bright spot on multimer images N (25). is due to one N subunit.

11770 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001760107 Raymond et al. Downloaded by guest on September 24, 2021 nearly identical copies of the dimer (RSMD of 0.48 Å for 476 Cα atoms) and because a dimeric species was detected in solution (Fig. 1 and Fig. S1B). The dimer is formed by contacts of residues in helices α1, α7, and α8. The side chain of Trp125 (α7–α8 loop) is buried in the hydrophobic, water-free dimer interface where it contacts Met1, Gln5, Ile9, and Trp125 in the second monomer (Fig. 3D). Ala12, Val120, Val121, Glu124, and Thr131 also form intersubunit van der Waals contacts. The small dimer interface 2 (502 Å buried surface area per monomer) is consistent with the low proportion of dimeric species in solution (Fig. S1C), nevertheless the dimer is expected to predominate at the high protein concentration in crystals.

Comparison with N of Other Negative-Sense RNA Viruses. Given the rapid rate of virus divergence, we anticipated that the phlebovirus N might resemble the N of other negative-sense RNA viruses even though the sequences are dissimilar. Two different folds for N have been reported, one for FLUA (13) in the family Ortho- myxoviridae, and the other for four viruses in the order Monone- gavirales [RABV (14), VSV (16), HRSV (15), and BDV (17)]. However, the phlebovirus N fold differs from both these other N folds. Thus at least three different folds exist for N of the negative-sense RNA viruses. Intriguingly, all three folds are predominantly helical and are bilobed. However, the phlebovirus N has a more compact structure. RNA binds in a deep, positively charged cleft between the two lobes of N from both the Monone- gavirales and FLUA (14–18). Phlebovirus N lacks a cleft between the N and C lobes (Fig. 3). Another important difference is the lack of protrusions in phlebovirus N. The N and C termini of N of the Mononegavirales protrude from the subunit, as does an extended loop in the N of FLUA. These protrusions contact other N subunits and are important to the structure of the RNP (14–18). Fig. 2. Electron micrographs of RNP. (A) Authentic virus RNP. Freshly prepared virus RNP has an overall string-like appearance and lacks helical Conservation of Phlebovirus N. Among the highly conserved phle- – symmetry. The inset shows an enlarged region. (B D) Four representative bovirus N (Fig. S7), a total 66 invariant residues map primarily classifications of virus (B), recombinant (C), and reconstituted (D) N-RNA multimers. (Scale bars, 11 nm.) to the core of the structure where they are important for conservation of the overall fold (Fig. S8A). Residues in the dimer interface are not strictly conserved, but a dimer contact appears feasible in Crystal Structure of N. Recombinant, RNA-free RVFV N was all phlebovirus N because compensatory sequence changes accom- crystallized and the structure was solved by multiwavelength modate the size and hydophobicity of the residue corresponding to anomalous diffraction from the selenomethionyl (SeMet) protein Trp125. The structure is consistent with published mutagenesis (TableS1). The crystals contained four N polypeptides in the asym- P1 data suggesting that the N terminus of RVFV N is required for metric unit of space group , affording four independent views of dimer formation (25). However, the conserved residues tested the structure. The four copies of the N polypeptide are nearly iden- in the previous study (Tyr4, Phe11) point away from the dimer tical with root-mean-square-deviations (RMSD) of 0.46 Å for 238 interface toward the inside of the monomer where they form α C atoms. The refined structure is complete with the exception of stabilizing contacts in the hydrophobic core of the protein. The – – residues 16 19 and 28 30. In these regions, each of the four observed loss of dimer formation of Tyr4Gly and Phe11Gly (25) polypeptides lacks density for one, four or seven amino acids. is likely due to destabilization of helix α1 (residues 3–10) and, RVFV N has a compact, helical fold consisting of N-terminal indirectly, the dimer interface. and C-terminal lobes of approximately equal size, connected by a α – linker helix ( 7, residues 112 121) (Fig. 3 A and B). Both the N Mutagenesis of the Dimer Interface and C-Terminal Salt Bridge. The BIOCHEMISTRY lobe (α1–α6, residues 1–111) and the C lobe (α8–α12, residues structure suggested that Trp125 is critical for dimer formation 122–245) have a central helix (α3 and α9) surrounded by four because of the hydrophobic contacts it makes with Met1, Ile9, or five other helices. Despite these similarities, the topologies and Trp125 of the opposing N monomer. Additionally, a salt differ and the N and C lobes cannot be superimposed. We exam- bridge between the C-terminal carboxyl group of Ala245 and ined the structural database for proteins with folds similar to the Arg178 side chain, which is Arg or Lys in all phlebovirus RVFV N. Remarkably, the folds of both the N and C lobes appear N, may be important for overall structural integrity. To test the to be novel. No structure similar to either lobe was identified in significance of these interactions, three mutant N alleles were searches with the servers Dali (26) and VAST (27). generated, Trp125Ala, Arg178Gln, and Arg178Glu, and the func- The crystallized protein includes the full natural sequence tionality of each was analyzed in a cell-based RdRp transcription (Met1-Ala245) without additional residues. Both chain termini assay in which RdRp and N were expressed from separate plas- are well ordered (Fig. S6). Met1 makes intra- and intermolecular mids. When RdRp and N were both present and functional, a contacts with hydrophobic residues in helix α1 (residues 3–10). luciferase mRNA from a recombinant S segment was transcribed The C-terminal α-carboxyl of Ala245 forms a salt bridge with (28). The Trp125Ala allele was severely compromised and activity the Arg178 side chain. Neither of the chain termini nor any loops was only 4% of the wild type allele (Table S2), suggesting that protrude from the protein. Trp125, and perhaps an N dimer, is essential for transcription. RVFV N crystallized as a symmetric dimer (Fig. 3C). This may If the salt bridge of Arg178 with the C terminus is critical, then be a natural dimer because the crystal contains two independent, the Arg178Gln allele should retain more function than the

Raymond et al. PNAS ∣ June 29, 2010 ∣ vol. 107 ∣ no. 26 ∣ 11771 Downloaded by guest on September 24, 2021 Fig. 3. Structure of RVFV N. (A) Polypeptide fold. The stereo ribbon diagram is colored as a rainbow from blue at the N terminus to red at the C terminus with loops in gray. Helix α7, (vertical) in the center of the image, links the N lobe at the left and the C lobe at the right. (B) Diagram of helical secondary structure in the RVFN polypeptide. Colors are matched to A.(C) RVFV N dimer. In this view along the dimer axis, monomers are in green and cyan and the twofold axis is indicated by an ellipse. (D) Details of the dimer interface. The subunits are colored as in C, side chains with dimer contacts are shown in stick form in the stereo view. Hydrogen bonds are shown as dashed lines.

Arg178Glu allele, and this was the observed result (Table S2). identity (36–59%) assure that all phlebovirus N possess the The activity of the Arg178Gln and Arg178Glu alleles was 25% RVFV N fold and also suggest that all phlebovirus N bind and 7% of wild type, respectively. All alleles expressed protein RNA similarly. The fold may also exist in N throughout the at a level similar to wild type and all appeared capable of forming Bunyaviridae family. higher molecular weight complexes with RNA (Fig. S9). This work establishes that RNP organization in the Phlebo- viruses is different than in other negative-sense RNA viruses. Interaction Sites. We considered whether the RVFV N has an Crystal structures or reconstructions from electron cryomicro- obvious RNA-binding surface. The N lobe has a higher calculated scopy have been reported for RNPs from four negative-sense isoelectric point (pI of 9.4 vs. 8.3) and a more positively charged RNA viruses (Mononegavirales and Orthomyxoviruses) (14–16, surface (Fig. 4A) than the C lobe. We compared RVFV N with 18). In all cases, RNA binds nonspecifically in an electropositive structure-based homology models of N from other clades within cleft between the lobes of the N subunit. RNP oligomers from the Phlebovirus genus. Based on the high sequence identity, N these viruses have a similar architecture in which RNA binds from all phleboviruses are expected to bind RNA similarly. There around either the outside or inside of a ring of 9–11 N subunits. is a general trend of greater positive charge on the N lobe than on In all cases, protrusions from the N subunits make specific con- the C lobe, but the structures lack a common conserved basic tacts with adjacent subunits to maintain the ring structure. In surface. We also mapped sequence conservation onto the RVFV some cases, the number of subunits in the oligomer ring matches N surface (Fig. 4B). The most strongly conserved surface is a the helical repeat of the polymeric RNP, which is apparent in hydrophobic pocket at the junction of the N and C lobes formed electron micrographs. For HRSV, each N subunit also interacts by a loop (residues 27–35) together with the C-terminal half of with other N subunits in the preceding or following turns of the α10 and the five succeeding amino acids (residues 198–210) helical nucleocapsid (15). (Fig. 4B and Fig. S8). Residues 27–35 are among the most mobile In contrast to the RNPs of Mononegavirales and Orthomyxo- regions of the N structure (Fig. S8B). The combination of mobi- viruses, no helical structure was apparent in any electron micro- lity, conservation, and hydrophobicity suggest that this site may be graphs of RVFV authentic or reconstituted RNP (Fig. 2A and involved in a conserved protein-protein interaction. Fig. S3). Our results are similar to early electron micrographs of bunyavirus RNP, which lacked helical symmetry and also sug- Discussion gested that bunyavirus RNPs form large macrocircles (20), prob- The structure of RVFV N reveals an addition to nature’s reper- ably due to pairing of 10–15 complementary bases at the 3′ and 5′ toire of RNA-binding proteins (Fig. 3). High levels of sequence ends of each genomic segment (29). RVFV RNP macrocircles

11772 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001760107 Raymond et al. Downloaded by guest on September 24, 2021 A oligomer, it may be a hexamer because our cross-linking data showed a preponderance of species containing multiples of two N subunits (Fig. 1). Overall, the organization of phlebovirus RNP appears less symmetric and with few specific protein–protein interactions compared to the helical RNP of the Mononegavirales 180˚ and Orthomyxoviruses. The evolutionary path to phlebovirus RNP is not common with the path to helical RNP of some other negative-sense RNA viruses. The unusual N-RNA multimer and perhaps the nonhelical RNP may be a general property of the Bunyaviridae. The observed RVFV N-RNA multimer species are similar to the reported

N C C 109-kDa recombinant RNP from Bunyamwera virus (6). Bunyam- wera virus and RVFV belong to different genera within family Bunyaviridae and their N are not obviously similar at the amino acid level. B The N lobe of RVFV N was identified as a potential RNA interaction site because it is more positively charged than the C lobe in N from all phlebovirus clades. In whatever manner N binds RNA, it is expected to engage the phosphate backbone because the multimer is so highly ribonuclease resistant. The electron micrographs do not permit assignment of RNA to a specific location in the multimer particles. The relevance of the N dimer to the RNP is unclear. Based on the size of multimers in electron micrographs (Fig. 2 and Fig. S4) and their gel filtration profile (Fig. S5), each bright spot in the multimer images must be a monomer and not a dimer of N. A Variable Conserved precedent for different oligomer species for RNA-free and Fig. 4. Properties of the RVFV N surface. (A) Electrostatic surface potential. RNA-bound N exists for FLUA where the free protein is a trimer The surface potential from −20 kT in red to þ20 kT in blue is shown for and the RNP is a nonamer (13, 18). We probed the RVFV N the front and back of the RVFV N monomer. The ribbon diagrams below dimer interface by site-directed mutagenesis at Trp125 but found show the positions of the N-terminal (blue) and C-terminal (green) lobes cross-linked RNA complexes similar to the wild type (Fig. S9). and the linker (pink). The image at left is in the same orientation as (Fig. 3A). However, the Ala substitution revealed an important role for (B) Conserved hydrophobic pocket. The most conserved surface of N is at the Trp125 in replication (Table S2). This could be due to any number top relative to A. In this view, the N-terminal domain is at left and coloring is by conservation among phlebovirus N, as indicated. of molecular interactions, most obviously with the RdRp. The most highly conserved surface of phlebovirus N is a hydrophobic pocket at the interface of the N- and C-terminal lobes were seen in some of our images. Aggressive ribonuclease treat- (Fig. 4 and Fig. S8). The conservation in this region suggests an ment of RVFV RNP released an N-RNA multimer that appears important function that is common to phleboviruses, and the heterogeneous in composition and has an average diameter of hydrophobicity of the surface suggests that it is not a site for 11 nm (Fig. 2B and Fig. S4A). Multimers of nearly identical RNA binding but rather is for interaction with a viral or (uniden- appearance to those from virus RNP were observed both for tified) host protein. Among potential viral protein partners, the ribonuclease-treated recombinant RNP and for RNP reconstituted with small RNAs (Fig. 2 C and D and Fig. S4). The hetero- RdRp is an obvious possibility because N is required for transcrip- geneity of these minimal particles was surprising but is consistent tion and replication by the RdRp (4). However, several lines of with the lack of helical symmetry in the RNP. The N crystal struc- evidence suggest that an envelope glycoprotein may be the target ture is also consistent with the lack of helical symmetry. The of the conserved hydrophobic pocket on N. highly compact phlebovirus N has no protruding loops or termini Packaging of RNPs into virions occurs at a site of virus assembly on the Golgi membrane (30). The cytoplasmic tail of the RVFV that could link it to other N molecules in a superstructure like the G rings of 9–11 subunits observed for the Mononegavirales and envelope glycoprotein N was shown recently to recruit the encap- Orthomyxoviruses (14–16, 18), although we cannot rule out the sidated genome to the Golgi membrane prior to virion assembly G possibility of major conformational changes to N upon RNA (31). Moreover, three regions within the N tail of the Uukuniemi BIOCHEMISTRY binding. We observed no large superstructure for recombinant virus were shown to be important for nucleoprotein binding to the RVFV N in solution, unlike the recombinant rings purified for glycoproteins (5). Genome recruitment is expected to be similar in N from RABV (14), HRSV (15), VSV (16), and FLUA (18). all phleboviruses, thus the conserved hydrophobic pocket of N is a G All N-RNA multimers that we could test by gel filtration had candidate N binding site. This hypothesis is consistent with the a similar apparent molecular weight of ∼100 kDa (Fig. S5). ability of bunyaviruses, both in nature and in vitro, to undergo Our working model for the structure of phlebovirus RNP is that reassortment in which progeny have genomic segments that derive N binds cooperatively to RNA. However, the cooperativity is from more than one parental virus (32–34). Reassortment requires limited to 4–7 N subunits based on the similar size and appearance promiscuity in the interaction of N with genomic RNAs from of multimers from viral, recombinant and reconstituted RNP and heterologous viruses and in protein-protein interactions necessary their similar behavior upon gel filtration (Fig. 2 and Figs. S4 and for assembling virions. All characterized reassortant bunyaviruses S5). The RNP lacks a strong helical structure, and N-N contacts isolated in nature are M segment reassortants (35, 36), demon- are too weak for ribonuclease treatment to release a specific strating that the envelope glycoproteins, which are encoded by N-RNA oligomer from virus RNP. Instead of a tightly associated, the M segment, are capable of interacting with heterologous G symmetric N-RNA oligomer, limited protein–protein interactions RNPs. The hydrophobic character of some regions of the N-tail, may lead to oligomers of 4–7 N monomers bound to RNA, result- as well as Pro and Trpresidues within it, are conserved among phle- ing in the observed mixture of multimer species upon exhaustive boviruses (37) and could function in protein–protein interactions G RNase treatment. If there is a weakly associated fundamental with N. Whether the conserved pocket of N interacts with the N

Raymond et al. PNAS ∣ June 29, 2010 ∣ vol. 107 ∣ no. 26 ∣ 11773 Downloaded by guest on September 24, 2021 cytoplasmic tail, with the RdRp, or with a host protein, it has oligomer for 30 min at a ratio of 6∶1 recN∶RNA. The sample was then run potential as a drug target because it is conserved in phleboviruses. on an S200 size-exclusion column to separate N-RNA from RNA-free recN. The structure and characterization of phlebovirus N and RNP The recN and N-RNA were dialyzed against PBS to remove the Tris storage buf- reveal another paradigm for encapsidation of the genome of fer prior to incubation with DSP. Purified recN, N-RNA, or purified viral RNPs (vRNP) were cross-linked by incubating 30 μg of recN at a concentration of negative sense RNA viruses, provide a platform for further 1 ∕ μ studies of virus pathogenicity, and suggest a potential site for mg mL, or purified vRNPs with 0.0, 1.0, 5.0, 10.0, or 20.0 M dithiobis[succi- nimidyl propionate] (Pierce) for 15 min at room temperature. The cross-linking development of effective antiviral therapeutics. was quenched by addition of Tris pH 6.7 to a final concentration of 100 mM. Materials and Methods Protein complexes were analyzed by SDS/PAGE followed by either colloidal Coomassie stain or western blot using a polyclonal rabbit anti-N antibody. Detailed methods are given in SI Text.

Electron Microscopy. All samples were absorbed onto a carbon-coated grid Production and Purification of Recombinant N. The expression construct for N and stained with 0.75% uranyl formate using standard protocols. A Morgag- encoded an N-terminal His6 tag fused to SUMO fused to N. The expression ni 268(D) transmission electron microscope equipped with a mounted Orius plasmid was transformed into E. coli, and the protein was purified from the SC200W CCD camera was used for imaging at room temperature. soluble fraction of the cell lysate. His6-SUMO-N was bound to a metal-affinity column, unfolded in 8 M urea, washed with several column volumes of 8 M 1 × 105 urea to remove RNA, refolded in a linear gradient of 8 M to 0 M urea, and RVFV transcription assay. BSR-T7/5 cells were plated at cells/well in 12-well culture plates. After 24 h, cells were transfected using 2 μg∕mL TransIT eluted with an imidazole gradient. The His6-SUMO was cleaved from the μ Δ Δ refolded protein with SUMO protease (38), a kind gift of C. Lima, Memorial LT1 (Mirus Corporation) and plasmids in the ratio 0.25 g pSTrRVFV-S N ∶ ∶0 50 μ μ Sloan-Kettering Cancer Center. The cleavage product, N with its full natural NSs hRLuc . g pN: 0.75 g pRdRp. At 48 h posttransfection, the cells were sequence, was purified by a second step of metal-affinity chromatography harvested and analyzed by luciferase assay and western blot. followed by gel filtration (Fig. S1). ACKNOWLEDGMENTS. We thank Georgios Skiniotis, Min Su, and Justin Crystallography. The crystal structure was solved by Se-MAD using the SeMet Schilling (Life Sciences Institute, Univ. Michigan) for advice and assistance protein. Crystals in space group P1 contain four N polypeptides per unit cell with electron microscopy, and Katrin Karbstein and Crystal Young (Dept. with cell constants a ¼ 67.1 Å, b ¼ 69.6 Å, c ¼ 80.6 Å, α ¼ 78.4°, β ¼ 69.7°, Chemistry, Univ. Michigan) for the 684-mer RNA sample. The research was γ ¼ 60.9° for the 1.93-Å dataset from a crystal of wild-type N. The final model supported by National Institutes of Health (NIH) Grant P01-AI055672 to J.L.S., by NIH Regional Center of Excellence for Bio-defense and Emerging is of excellent crystallographic and stereochemical quality with R ¼ 0.211, Infectious Diseases Grant U54-AI057153 to S.R.G., by the Rackham Graduate R ¼ 0.254, RMSD ¼ 0.013 Å and 99.6% of residues in allowed regions free bonds School at the University of Michigan to S.R.G., and by NIH Training Grants of the Ramachandran plot. T32-GM008270 in Molecular Biophysics to D.D.R. and T32-GM007315 in Cell and Molecular Biology to M.E.P. The GM/CA beamlines are supported Cross-Linking. For cross-linking, a reconstituted RNP (N-RNA) was generated by by the NIH Institute of General Medical Sciences and National Cancer incubating refolded recombinant N (recN) with a 25-nucleotide polyU RNA Institute, and the APS by the United States Department of Energy.

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