an ultraweak interaction in the intrinsically disordered ... · ω2-1h (ppm) ω 1-15 n (ppm) 118...

SC I ENCE ADVANCES | R E S EARCH ART I C L E

V IROLOGY

1Université Grenoble Alpes, CNRS, Commissariat à l’Energie Atomique et aux En-ergies Alternatives, Institut de Biologie Structurale, 38000 Grenoble, France. 2In-ternational Center for Infectiology Research, INSERM, U1111, Université ClaudeBernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Universitéde Lyon, Lyon, France.*Corresponding author. Email: [email protected] (R.W.H.R.); [email protected] (M.B.)

Milles et al., Sci. Adv. 2018;4 : eaat7778 22 August 2018

Copyright © 2018

The Authors, some

rights reserved;

exclusive licensee

American Association

for the Advancement

of Science. No claim to

originalU.S. Government

Works. Distributed

under a Creative

Commons Attribution

NonCommercial

License 4.0 (CC BY-NC).

Dow

nloaded

An ultraweak interaction in the intrinsically disorderedreplicationmachinery is essential formeasles virus functionSigrid Milles1, Malene Ringkjøbing Jensen1, Carine Lazert2, Serafima Guseva1,Stefaniia Ivashchenko1, Guillaume Communie1, Damien Maurin1, Denis Gerlier2,Rob W. H. Ruigrok1*, Martin Blackledge1*

Measles virus genome encapsidation is essential for viral replication and is controlled by the intrinsically disorderedphosphoprotein (P) maintaining the nucleoprotein in a monomeric form (N) before nucleocapsid assembly. Allparamyxoviruses harbor highly disordered amino-terminal domains (PNTD) that are hundreds of amino acids in lengthandwhose function remains unknown. Using nuclearmagnetic resonance (NMR) spectroscopy, we describe the struc-ture and dynamics of the 90-kDa N0PNTD complex, comprising 450 disordered amino acids, at atomic resolution. NMRrelaxation dispersion reveals the existence of an ultraweak N-interaction motif, hidden within the highly disorderedPNTD, that allows PNTD to rapidly associate and dissociate from a specific site on N while tightly bound at the aminoterminus, thereby hindering access to the surface of N.Mutation of this linearmotif quenches the long-range dynamiccoupling between the two interaction sites and completely abolishes viral transcription/replication in cell-basedminigenome assays comprising integral viral replicationmachinery. This description transforms our understanding ofintrinsic conformational disorder in paramyxoviral replication. The essential mechanism appears to be conservedacross Paramyxoviridae, opening unique new perspectives for drug development against this family of pathogens.

from
on A
ugust 22, 2020http://advances.sciencem

ag.org/
INTRODUCTION
Measles virus (MeV) is a nonsegmented negative-sense RNA virusbelonging to the family of Paramyxoviridae that includes a numberof emerging human pathogens with dangerously high mortality ratesfor which there is currently no treatment. In addition to the viral poly-merase (L), paramyxoviral replication machinery is composed of thenucleoprotein (N), which encapsidates the viral genome, and the tetra-meric phosphoprotein (P) (1–3), a cofactor of L that interacts with Nat different stages of the viral replication process.

Paramyxoviral P proteins are essential for replication and transcrip-tion, interacting with N at different stages of the viral cycle (4–9) andexhibiting very long intrinsically disordered N-terminal domains(PNTDs), whose function is not understood (10, 11). The first 40 aminoacids of P bind tightly to N that is maintained in a monomeric state(N0P) (12, 13) before encapsidation of the viral RNA to form the nu-cleocapsid (NC). X-ray structures of heterodimeric constructs ofN0P1–40 have been determined for MeV (14), Nipah virus (NiV)(15), parainfluenza virus 5 (16), and metapneumoviruses (17). In allcases, the flexible domains of N that are required for assembly intoNCs (18, 19), including the intrinsically disordered C-terminal domainof N (NTAIL) and most of the disordered domain of PNTD (more than400 amino acids inNiV and 300 amino acids inMeV), were removed tofacilitate crystallization. The functional N0P assembly, however, com-prises extensive disorder (more than 450 amino acids per heterodimer),whose role has never been studied at a molecular level. The presence ofthis level of disorder, in viruses whose genetic information is normallyso parsimoniously exploited, remains unexplained.

To investigate the role of the disordered PNTD, we designed anMeVN0P construct comprising integral N, which is fused to the entire 300–

amino acid PNTD (20). This approach allowed us to use solution nuclearmagnetic resonance (NMR), small-angle x-ray scattering (SAXS), en-semble simulation, and NMR exchange spectroscopy to investigatethe conformational behavior of the 90-kDaN0PNTD complex, includ-ing the intrinsically disordered regions (IDRs) PNTD, NARM, CARM, andNTAIL (in total ofmore than 450 amino acids), and to identify the role ofthese flexible domains in the molecular processes of replication andtranscription (see fig. S1 for domain description). The atomic resolutiondescription of such a highly dynamic complex presents significant chal-lenges for structural biology, requiring approaches that explicitly accountfor their time- and ensemble-dependent conformational heterogeneity(21–23) and the dynamic exchange between free and bound forms ofthe proteins (24–27). Here, we apply these approaches to describe thebehavior of PNTD alone and upon formation of the N0P complex.

Rather than containing apparently nonfunctional sequences, PNTDharbors a previously hidden linear motif that interacts ultraweakly butspecifically with the surface of N. The two N0PNTD binding sites areseparated by 150 highly dynamic residues and differ in affinity by ordersofmagnitude, allowing PNTD to transiently wrap around the surface ofN,exchanging rapidly between compact and more extended forms. Thislong-range allosteric coupling possibly enhances the chaperone functionby frustrating the interaction of NCORE (the folded domain of N) withRNAorhost factors.Cell-basedMeVminigenome studies combinedwithmutagenesis demonstrate that despite the inherently weak affinity of thenewly discovered interaction, this mechanism is essential for viral tran-scription and replication and therefore also for successful host infection.The interaction motif appears to be conserved within paramyxoviruses,providing a new target for treating these dangerous human diseases.

RESULTSNMR spectroscopy describes the conformational behaviorof PNTDWecharacterized the conformational behavior of PNTD at themolecularlevel using NMR spectroscopy (fig. S2). Protein backbone resonanceswere assigned, and representative multiconformational ensembles were

1 of 10

http://advances.sciencemag.org/


http://advances.sciencem

ag.org/D

ownloaded from

generated on the basis of 1H, 15N, and 13C (CO, Ca, and Cb) backbonechemical shifts, using a combination of flexible-meccano and the ge-netic algorithm ASTEROIDS (Fig. 1 and fig. S2) (28, 29). The resultingensemble faithfully predicted experimental ensemble-averaged residualdipolar couplings (RDCs) that are not used in the description (Fig. 1).This site-specific description of the conformational sampling of freePNTD reveals that a helices contained in the first 37 residues (a1 anda2), which are involved in formation of the N0P complex, appear astransient helical structures within the unbound protein, as previouslyobserved for vesicular stomatitis virus P (30). Two additional regionshave similar or higher propensities to form helices in the unboundPNTD: residues 87 to 93 (a3) and 189 to 198 (a4; Fig. 1A and fig. S2).Spin relaxation reveals increased rigidity in a2, a4, and the hydrophobicregion around position 110 that is implicated in the binding of STAT(signal transducers and activators of transcription) (31), while long,highly dynamic segments link a1/2 to a3 and a3 to a4 (Fig. 1).

The highly disordered N0P complex contains a hiddendynamic interaction site with NWe used NMR to investigate the N0PNTD complex by titrating 15N-labeled P1–304 with unlabeled P1–50N1–525 (Fig. 2). P1–304 is able todisplace P1–50 from the P1–50N1–525 complex, supporting the obser-vation that this complex can be purified from amixture of P1–304 andP1–50N1–525 (figs. S3 and S4A). In addition to decreased peak intensitiesin a1/2, known to engage in the N0P complex (13–15, 32), a 20-residuesequence is found to interact with N, comprising the a4 helical motif.Small local increases in relaxation rates were also observed, centered onaromatic residues F65 andY110 to Y113, suggesting the existence of ad-ditional transient hydrophobic interactions with N within the complex.

Exchange NMR reveals the detail of the additionalN interaction siteTo further investigate this additional interaction site, intermediatestoichiometric complexes were formed where 15N-labeled P1–304 is


in exchange between bound and unbound forms (Fig. 2). While thebound a1/2 region is in slow exchange, manifest through duplicationof the peaks on the edges of the helical region (fig. S4, B to D), theregion around a4 exhibits chemical exchange kinetics in the milli-second regime between free and bound PNTD, as evidenced by 15NCarr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (33)measured at substoichiometric ratios (50 mM P1–304 and 25 mMP1–50N1–525; fig. S5).

Using a shorter construct of PNTD (P140–304) that does not containthe N-terminal interaction site, the a4 site is shown to bind indepen-dently from a1/2 (Fig. 3 and fig. S5, C and D). This analysis revealsmuchmore detail concerning the exact nature of the interactionmotif,particularly in identifying an additional linear motif (181DVETA185,termed d) immediately preceding helix a4, which also participates inthe interaction (Fig. 3B). This motif exhibits no detectable intrinsic pro-pensity or secondary structure in the free form of PNTD.

15N relaxationdispersion experiments measured at two magnetic field strengths(600 and 950 MHz) allowed us to estimate the intrinsic dissociationconstant (Kd) for the combined da4 interaction with N0P as 614 ±218 mM (Fig. 3D). Both a4 and d motifs have the same effective Kd

and exchange kinetics, indicating that they participate in a concertedinteraction with N0P. NMR titration studies using the N-terminalregion of PNTD, without the da4 motif (P1–160), show that the transienthydrophobic interaction sites centered on residues 65 and 110 are stillpresent even in the absence of the second binding motif (fig. S6).

Identification of a conserved interaction motif within PNTDhelix a4

To delineate the core interaction site involved in da4 binding to N, wecompared the sequences and secondary structure predictions of PNTDfrom different paramyxoviruses, revealing that a helix resembling a4 ispredicted throughout the family (Fig. 4A). A comparison of 259 non-redundant, curated PNTDMeV sequences also shows clear conservationof sequence identity in a1, a3, and the da4motif (Fig. 4B), including two

on August 22, 2020

α R

α1/2 α3 α4

α1/2

α3

α4

A D

B

C

Fig. 1. P1–304 structuraldynamics. (A) PopulationofaR conformationofP1–304 as calculated fromanASTEROIDSensemblebasedonchemical shifts andSAXS (graybars) comparedto those expected from a statistical coil ensemble (flexible-meccano, black lines). (B) NHN RDCs (

1DNH-N) of P1–304 obtained from alignment in a liquid crystal made from polyethyleneglycol (PEG) and 1-hexanol (gray bars). Red line shows NHN RDCs back-calculated from the ASTEROIDS ensemble selected against chemical shifts only. (C) Heteronuclear {1H}-15NOverhauser effects (nOes) of P1–304,measured at a 1H frequencyof 600MHzand25°C. (D) Representationof thedifferential flexibility along the sequenceof PNTD. The image shows thecolor andwidthof the ribbonas a functionof theheteronuclearnOe in (C) (thick, red ribboncorresponds tomore flexible regions; thin, blue ribbon to less flexible regions). Annotationsindicate the presence of helical elements. The protein is highly dynamic, and the actual conformation is not representative of the ensemble of conformers.

2 of 10



http://advances.sciencemag.

Dow

nloaded from

ω2-1H (ppm)

ω1-

15N

(pp

m)

118

119

120

121

122

123

8.6 8.4 8.2 8.0 7.8

V182

M33

S40N42

W36

I29I39I190

A35E38

I16

E31I24

E186N11

E14

Q5

E46D181

P1–304 N+ N1–525

D

ω1-

15N

(pp

m)

0.6

0.2

1.0

1.4

I/I 0

I/I0

0.6

0.2

1.0

1.4

100 300Sequence

B

C

108

4

0

6

2

12

R2 (

s–1)

A E

20050 250150

125

120

115

110

P1–304NP1–304

P1–50N1–525

1–525

15

Fig. 2. P1–304 interaction with N. (A) R2 of P1–304 (gray bars) and P1–304N1–525 (red lines) as purified from a Superdex 200 column (fig. S3) measured at a 1H frequency of950 MHz. (B) Interaction profile of P1–304 with P1–50N1–525. Titration admixtures included 25 (gray), 50 (red), 100 (green), and 150 mM (blue) final concentration of P1–50N1–525

and P1–304 at a final concentration of 50 mM. Shown are normalized peak intensities (I/I0). (C) Interaction profile of P1–304,HELL→AAAA with P1–50N1–525. Concentrations andcolors are the same as in (B). (D) 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum of P1–304 in absence (blue) and presence (red) of P1–50N1–525. ppm, partsper million. (E) 1H-15N HSQC spectra of P1–304 (red), P1–50N1–525 (green), and P1–304N1–525 (blue).

on August 22, 2020

org/

νCPMG (Hz)

24

8

16

24

8

16

R2,

eff (

s–1)

24

8

16

24

16

32

10008006004002000

DS180

S180

L193

L193

950

600

950

600

Sequence

ΔR2,

eff (

s–1)

ΔR1ρ

(s–1

)I/I

0

δ α4

A

B

C

Fig. 3. da4 interactionwithN. (A) Intensity ratios of 15N P140–304 peaks extracted from 1H-15N HSQC spectra in the presence of 5% (gray bars), 10% (red), or 20% (blue)P1–50N1–525 with respect to the unbound P140–304. Concentration of P140–304 remained constant at 200 mM throughout the titration. (B) R1r of P140–304 alone (gray bars)and with 20% P1–50N1–525 (blue) recorded at 16.5 T. (C) CPMG relaxation dispersion of 15N P140–304 (200 mM) in the presence of 20% P1–50N1–525: DR2,eff was determined at 14 Tas the difference in effective R2 at CPMG frequencies of 31 and 1000 Hz. (D) CPMG relaxation dispersion of 15N P140–304 (200 mM) in the presence of 20% P50N525 at 22.3 Tand 14 T. Data from eight sites throughout d and a4 were fitted simultaneously, assuming a two-site exchange process giving kex = 624 ± 88 s and population, 4.7 ± 0.5%.Example sites from residues S180 (d) and L193 (a4) are shown.

Milles et al., Sci. Adv. 2018;4 : eaat7778 22 August 2018 3 of 10



consecutive bulky hydrophobic residues (isoleucine, leucine, or valine).On the basis of these comparisons, the central interaction motif,191HELL194 was mutated (HELL→AAAA). This mutant no longer in-teracts with N through da4 (Fig. 2C and fig. S7), although the helicalpropensity remains intact (fig. S7C). Comparison of MeV P sequencesalso underlines the strongly conserved acidic nature of the long dynamicloop connecting residues 125 and 170 (comprising 10 conserved acidicside chains and no conserved basic side chains).

The da4 motif induces a more compact conformation of N0PWe investigated the influence of the HELL→AAAA mutation on theoverall dimensions of the P1–304N1–525 complex using SAXS. Ensemble


analysis based on SAXS and NMR data from wild-type (WT) and mu-tated forms reveals that the P1–304N1–525 ensemble requires a bimodaldistribution of radii of gyration (fig. S8, A to D), likely due to interac-tions between da4 and N. Mutation of the HELL motif leads to signif-icant quenching of the population of more compact conformations, inagreement with the abrogation of binding around da4. The presence ofthe 125–amino acid disordered NTAIL domain potentially masks theimpact of compaction of N0P on SAXS data. We therefore carriedout a similar analysis on N1–405P1–304 (in the absence of NTAIL) furtheremphasizing the effect of da4, exhibiting average radii of gyration ofWTand HELL→AAAA forms of N1–405P1–304 of 45 ± 6 and 58 ± 8 Å, re-spectively (fig. S8, E and F).

on August 22, 2020


Dow

nloaded from

α1 α2 α3

α4δ

176

326205

210

363239

α4δ

102

101

100

NLR

P P (HELL→ AAAA)

P1–303 (HELL→ AAAA)

1,E+02

1,E+03

1,E+04

1,E+05

1,E+06

Firefly NanoLuc Firefly NanoLuc

Ø P HELL→AAAA 106

105

104

103

(+) (–)Minigenomepolarity

Gene #(reporter)

RLU

P1–303 P39–303

A

B

C D

Fig. 4. Conservation and functional impact of the da4motif. (A) Alignment of phosphoproteins fromdifferent related viruses (respective UniProt identifiers are shownon theleft, andmorbilliviruses are shown above thedashed line). Full PNTD sequences alignedwithMUSCLE (35). Positions of the helical elementspredicted using PSIPRED (57) are shownas boxes (red, negatively charged; blue, positively charged). (B) Conservation ofMeV sequences of PNTD. Comparison of 259nonredundant sequences curated inGenbank (60). Theposition of a helices, additional interaction sites d and a, STAT interaction site, and the RNA editing site defining the start position of the unique domain of V are indicated. Imageobtained using WebLogo software (http://weblogo.berkeley.edu/logo.cgi). (C and D) Functional impact of HELL→AAAA mutation in cellulo. (C) Ability of MeV P constructs toassociate with MeV N protein in cultured human cells, as determined by Gaussia luciferase–based protein complementation assay. Gray zone indicates the threshold NLR(normalized luminescence ratio) value. (D) Inability of HELL→AAAA Pmutant to support the expression of two reporter genes from either a (+) or (−) strandMeVminigenomewhen coexpressed with MeV N and L proteins using a reverse genetic assay [relative light units (RLU): luciferase reporter activity]. NanoLuc, nanoluciferase.

4 of 10

http://weblogo.berkeley.edu/logo.cgi



http://advD

ownloaded from

Functional relevance of the HELL interaction site in cellulaThe functional importance of the da4 N

0P interaction site for viralfunction was determined by measuring polymerase replication in vivousing cell-based MeV genome replication assays exploiting reversegenetics with a dual-luciferase reporter (9). In these assays, the viralpolymerase function relies on simultaneously providing a host cell withthe antigenomic or genomic RNA, N, P, and the polymerase (L), andpolymerase-mediated transcription is determined by the quantificationof the luciferase activities. Irrespective of whether antigenomic or ge-nomic RNA was provided, luciferase activity observed with PWT wascompletely suppressed when using PHELL→AAAA (Fig. 4D). All P var-iants were expressed (fig. S9A), and N:P binding and P oligomerizationwas unaffected by mutation (Fig. 4C and fig. S9B), indicating that theabrogation of transcription/replication results unambiguously from theabsence of the da4 motif.

da4 binds N on its N-terminal lobeAlthough NCORE backbone resonances are not observed in 1H-15Ncorrelation spectra of N0P, a truncated construct comprising the N-terminal lobe NNTD (34) was used to identify the interaction site(fig. S10, A and B) that is located in a contiguous region spanning res-idues 96 to 127, situated on the opposite side of N to the known N0Pbinding site. The HELL→AAAA mutant of P140–304 did not interactwith P1–50N1–525 (fig. S7), and the interaction profile of P140–304 closelyresembles that obtained from P1–50N1–525, confirming that NNTD

contains the full interaction site for da4 (Fig. 5).

on August 22, 2020

ances.sciencemag.org/

DISCUSSIONParamyxoviral phosphoproteins are essential for viral replication andtranscription, acting as essential cofactors for the polymerase complex.The architecture of paramyxoviral P proteins is highly conserved overParamyxoviridae, comprising a tetrameric coiled-coil domain and along intrinsically disordered PNTD, whose length varies between 215and 470 amino acids. Although the first 40 N-terminal amino acidsare known to bind N0, the role of the remainder of PNTD has remainedunknown, and no rationale exists for the conserved requirement ofthese long unfolded chains in all paramyxoviral P proteins.

We used NMR spectroscopy to investigate the conformational be-havior ofMeVPNTD in its free state and todescribe the structural, dynamic,and kinetic behavior of this highly disordered 90-kDa complex. Al-though unfolded, PNTD exhibits transient helical propensities in theN-terminal N binding site (a1/2), around residues 87 to 93 (a3) and189 to 198 (a4), linked by long, highly dynamic segments (Fig. 1).

In addition to the knownN-terminal binding concerning the first40 amino acids of PNTD, a 20-residue sequence, comprising the a4191HELL194motif, preceded by the additional unstructuredmotif d, alsointeracts with N. The HELL interaction motif, as well as the helix itself,appears to be conserved within morbilliviruses and representativeparamyxoviruses, in particular the two bulky hydrophobic residuesat the C terminus of the motif (Fig. 4A). The distance between the a1/2and putative da4 binding sites is, however, highly variable among dif-ferent viruses—for example,NiVPNTD is considerably longer (470 aminoacids) than in MeV and is predicted to exhibit helical propensity atpositions 340 to 348, containing the sequence RELL. Despite near-negligible identity among PNTD from different paramyxoviruses, theMUSCLE sequence alignment algorithm (35) successfully aligns theseconserved motifs, even when comparing entire PNTD sequences of verydifferent lengths. A number of physical characteristics are also con-


served; for example, the interaction motif is preceded by a negativelycharged region (4 acidic residues out of 12 in MeV PNTD) and followedby a positively charged region (4 basic residues out of 14; Fig. 4B).Similarly, analysis of nonredundant MeV sequences reveals that da4 isone of only four strongly conserved linear sequences in the 300–aminoacid PNTD (Fig. 4B).

The da4:NCORE interaction occurs independently of the presence ofa1/2, as demonstrated by a truncation mutant of PNTD. Using NMR re-laxation dispersion, we have investigated the kinetics of the interaction,revealing that the intrinsic interaction strength is ultraweak, with a Kd

around 600 mM. The effective affinity within the PNTD:NCORE complexis expected to be significantly higher because of the elevated effectivepopulation of da4:NCORE that is maintained by the much higher-affinitya1/2:NCORE interaction. The a1/2:NCORE interaction is in slow exchangeon the NMR time scale and has sufficiently slow dissociation rates thatthe complex can be copurified on a size-exclusion column.

The binding site of da4 on N (residues 96 to 127) is located at theextremity of its N-terminal lobe, which is the most evolutionarilyvariable part of NCORE within paramyxoviruses and the Morbillivirusgenus (15). A single-chain antibody that binds to this region of NCORE

from vesicular stomatitis virus, a related member of the Rhabdoviridaefamily, is a potent inhibitor of virus transcription and replication (36),while a homologous region in respiratory syncitial virus binds the Cterminus of the P protein and is the target for inhibitory small-moleculedevelopment (37). In MeV, the affected binding surface on N, situatedat the opposite end of NCORE to the a1/2 binding site, comprises anexposed b-strand (Fig. 5), adjacent to a highly dynamic surface loopthat is devoid of any secondary structure in solution. This subdomainexhibits conformational heterogeneity between known structures,adopting a helix in crystalline N0P (14) [and in the homologous NiVstructure (15)] and a surface loop in the cryo–electron microscopyNC structure (18). A recent study identified this domain as insertion-tolerant, allowing the molecular recognition element of NTAIL to begrafted into the domain while maintaining viral replication (38).

On the basis of our observations, we conclude that, contrary to cur-rent understanding, whereby the N0P complex comprises a single N:Pbinding site that is sufficient for chaperone activity,MeVPNTD interactswith monomeric N via two distinct linear regions, separated by 150amino acids, andwith affinities that differ bymany orders ofmagnitude.The presence of two N binding sites on PNTD helps to rationalize thepresence of long intrinsically disordered PNTD domains (11) throughoutthe paramyxoviral family. The length of the long dynamic linker con-necting the two interaction sites provides sufficient degrees of freedomto sample very different conformations and, in particular, to allow forsignificant conformational disorder within the complex (Fig. 5), evenwhen both sites are occupied. The relative weakness of the da4 interac-tion, therefore, leads to rapid exchange between more voluminous freeand more compact bound forms of P40–300, while a1/2 remains bound.When simultaneously bound to N, the a1/2 and da4 binding sites arepositioned at opposite extremities of the NCORE structure, so that theconformational fluctuations of the acidic loop between the binding siteson PNTD are able to maximally frustrate access to the surface of N, po-tentially inhibiting interaction with RNA and consequent self-assemblywith other N monomers as well as interactions with host proteins thatmay play a role in the immune response (39).

The da4 interaction is abrogated bymutation of four central residues(191HELL194) of the a4 motif, although its helical propensity is main-tained under alanine mutation, demonstrating that its helical naturealone is not sufficient to ensure interaction.Mutation ofa4 also removes

5 of 10



on August 22, 2020


Dow

nloaded from

the more compact, enwrapped form of N0P from the equilibrium asshown from small-angle scattering. We demonstrated the importanceof the da4N

0P interaction site for viral functionbymeasuringpolymerasereplication in vivo using recombinant minigenome assays. Only the N-genomic RNANC can be used as a template, so that the observed lack ofreporter gene expression from the genomic (−) stranded RNA demon-strates that the HELL→AAAA mutation prevents assembly of N andRNA into a functional NC and subsequent transcription. The possibilitythat HELL→AAAA specifically targets replication and not transcrip-tion is supported by the previous demonstration that a PNTD-deletedP protein remains active in transcription in the closely related Sendaiparamyxovirus (40).

Despite its weak affinity, the da4 interaction is therefore essential forviral function, reinforcing recent observations of the importance of ultra-


weak interactions for intrinsically disordered protein function, asillustrated for example in the case of linear motifs present in the nuclearpore complex (41). In the case of N0P, the combination of distinct affi-nities on PNTD, regulated by its highly disordered nature, coordinatesallosteric coupling between the two interaction sites linkingopposite endsof NCORE. The bipartite N:PNTD interactions may also facilitate NCassembly or provide the molecular basis of formation of organelles thatstimulate viral replication (42). We note that the observations made herein the context of PNTD are potentially equally valid for the genome-editedviral proteinV (43–46) that comprises PNTD until the editing site (residue229), followed by a folded zinc finger domain, which is unique to V.

In summary, an atomic resolution description of the structure anddynamics of the highly disordered 90-kDa N0P complex of MeV, com-prising 450 intrinsically disordered amino acids, reveals the presence of

1.0

0.0

I/I0

160 180 200 220 240 260 280 300140

α3

α4

α1

α2

α3

4

α1

α2

α4

NTAILPNTD

NCORE

A

B

C

D

E F

Acidic loop

δ

Fig. 5. N-da4 binding site and N0P model. (A) Ratio of peak intensities from 1H-15N HSQC spectra of free 15N P140–304 and15N P140–304 bound to unlabeled N1-261 (gray bars)

superimposedwith an intensity ratio of 15N P140–304 unbound andbound to P1–50N1–525. (B) Transverse relaxation of15N13C2DN1–261. (C) Secondary structurepropensities (SSPs) for

N1–261. Red bars indicate interaction regions with P140–304. (D)1H-15N HSQC spectra of 15N, 2DN1–261 alone (red) and in the presence of 50 mM (blue) and 170 mM (cyan) unlabeled

P140–304. (E) Cartoon of interaction of P1–304 (yellow and blue) with NCORE (gray). Red surface denotes the interaction region on N. Dashed red line indicates the dynamic region ofNCORE. IDRs aremarked as dotted lines. Yellow indicates transient helices. (F) Representation of conformational space available to P1–304 (blue)whenboth a1/2 (yellow spheres) andda4 (yellow spheres) are bound to NCORE (gray surface; red shading shows the a4 interaction region on NCORE). All conformations shown have both interaction sites in proximity tothe respective N binding sites. Gray indicates NTAIL.

6 of 10



a hitherto unidentified N interaction site, 150 amino acids distant froma1/2 that is essential for viral function. The newly identified motifexhibits an intrinsic affinity that is remarkably low but neverthelessspecific, constraining the unfolded chain to rapidly interchange betweendifferent states while bound at the N-terminal site, thereby affordingprotection against N-RNA,N-N, or N–host factor interactions.We dis-cover that this linearmotif is essential forMeV replication and transcrip-tion, opening up new perspectives for drug development against theseincreasingly important humanpathogens. Paramyxoviruses share similarreplication machinery with filoviruses, including Ebola, suggestingthat the discovery of this essential allosteric mechanism may have yetfurther-reaching consequences for human health.

on August 22, 2020


Dow

nloaded from

MATERIALS AND METHODSCloning, protein expression, and purificationN0P constructs comprising full-length PNTD were generated by purify-ing a heterodimeric N1–525P1–304 complex with a TEV (tobacco etchvirus) cleavage site between the two proteins, similar to the productionof the shorter P1–50N1–525 described previously (20), or bymixing TEV-cleaved P1–50N1–525 with P1–304 at equal concentrations and subsequentpurification of the P1–304N1–525 complex by size exclusion chromatog-raphy (SEC; fig. S3). TEV cleavage was performed before a final SEC(Superdex 200, GE Healthcare) in NMR buffer [50 mMNa-phosphate(pH 6), 150 mM NaCl, and 2 mM dithiothreitol (DTT)]. P1–304 wascloned into pET41c(+) between the Nde I and Xho I cleavage sites,where the Xho I site was ligated with a cleaved Sal I site of the insert,yielding a construct with a C-terminal 8His-tag. P1–304 was expressedinEscherichia coliRosettaTM (lDE3)/pRARE (Novagen) overnight at20°C after induction at an optical density of 0.6 with 1 mM isopropyl-b-D-thiogalactopyranoside. Cells were lysed by sonication and subjectedto standard Ni purification in 20 mM tris (pH 8) and 150 mM NaCl.The protein was eluted from the beads in 20 mM tris (pH 8), 150 mMNaCl, and 400mM imidazole and was then concentrated and subjectedto SEC (Superdex 200) in NMR buffer. The HELL mutation was in-serted into P by site-directed mutagenesis, and P1–304,HELL→AAAA wasexpressed and purified the same way. Shorter P constructs (P1–100,P1–160, P140–304, and N1–261) were cloned into pet41c(+) between Nde Iand Xho I sites and were subjected to the same expression and puri-fication procedure; the final SEC stepwas conducted on a Superdex 75(GE Healthcare) column. N1–525P1–304 was cloned in two steps, firstinserting N1–525-TEVsite between the Nde I and Not I cleavage sitesof pET41c(+), followed by insertion of P1–304 between the Not I andXho I sites into the resulting vector. The Xho I site was ligated with aSal I site of the P1–304 insert. Expression of unlabeled protein was per-formed in LB medium. Protein labeled for NMR (15N and 13C) wasexpressed under the same conditions in M9 minimal medium. Fordeuterated protein (N1–261), the M9 medium was made in D2O.

NMR experimentsUnless otherwise noted, all experiments were acquired in NMR buffer[50 mM Na-phosphate (pH 6), 150 mM NaCl, and 2 mM DTT] at25°C. The spectral assignments of the different 13C- and 15N-labeledP constructs were obtained using sets of triple resonance experimentscorrelating Ca, Cb, and CO resonances at a 1H frequency of 600 MHz.N1–261 was assigned as a

13C-, 15N-, and 2D-labeled sample using band-selective excitation short transient–transverse relaxation-optimizedspectroscopy (BEST-TROSY) triple-resonance experiments recordedat a 1H frequency of 700 MHz (47). The spectra were processed with


NMRPipe (48), and automatic assignment was performed with theprogram MARS (49) and manually verified. Secondary chemicalshifts were calculated using the random coil values from refDB (50).For the measurement of RDCs, 13C, 15N-labeled P1–304 was aligned inpolyethylene glycol (PEG) and 1-hexanol, yielding a D2O splitting of25 Hz (51). RDCs were measured using BEST-type HNCO experi-ments that allow for spin-couplingmeasurements in the 13C dimensionat a 1H frequency of 800 MHz (52).

Interactions between different proteins were performed as describedin the main text and figures. Peak intensities (I) and 1H as well as 15Nchemical shifts were extracted from 1H-15N HSQC or 1H-15N TROSYspectra (for N1–261). Combined chemical shift differences (DCS) werecalculated as

DCS ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðd1H ⋅ 6:5Þ2 þ ðd15NÞ2

qð1Þ

15N R1, R1r, {1H}-15N heteronuclear Overhauser effects (nOes) were

acquired at different fields, as indicated in the figure legends. 15N R1relaxation rates were obtained by sampling the decay of magnetizationat 5 to 10 delays between 0 and 1.71 s. R1r relaxation rates were mea-sured using 5 to 10 delay times between 0.001 and 0.25 s. The spin-lockfield was 1500 Hz, and R2 was calculated from R1 and R1r, consideringthe resonance offset (53).

15N relaxation dispersion was measured at a P1–304 concentration of50 mM in the presence of 25 mMunlabeled P1–50N1–525 at 600MHz andusing 14 points at CPMG frequencies between 31 and 1000 Hz using aconstant-time relaxation of 32 ms (33). 15N relaxation dispersion ofP140–304 was measured at a concentration of 200 mM in the presenceof 40 mM P1–50N1–525 at

1H frequencies of 600 and 950 MHz underotherwise the same conditions as for P1–304. Data recorded at both fieldswere fit together using ChemEx (https://github.com/gbouvignies/chemex) and a two-state exchange model. The Kd value was cal-culated from the concentrations of P140–304 and P1–50N1–525 as well asthe fraction of bound P140–304 (pb), as obtained from the fit andassuming a 1:1 binding stoichiometry. The error of the Kd value wasestimated by error propagation from the fitting error of pb and as-suming a 5% error in concentration determination of both proteins.Nonoverlapped residues 180, 181, 182, 184, 185, 193, 196, and 199covering both d and a4 were included in the fit.

Small-angle x-ray scatteringSAXS experimentswere performed onP1–304N1–525 complexes purified,as described above. Samples were adjusted to three different concentra-tions between 0.25 and 3.5 mg/ml and were measured at 20°C on theBM29 beamline at the European Synchrotron Radiation Facility(ESRF), Grenoble, France. Scattering was recorded at a wavelength of0.992 Å, and samples were exposed 10 times for 2 s. All frames wereanalyzed for radiation damage and excluded if necessary. All otherframes of sample and buffer were averaged respectively, and bufferscattering curves were subtracted from the scattering curves of thesamples.

Ensemble description of P1–304 and P1–304N1–525

A statistical coil ensemble of P1–304 comprising 10,000 structureswas generated using flexible-meccano. Two hundred conformationsthat best described the experimentally obtained N, HN, Ca, Cb, andCO chemical shifts were selected from the ensemble using the geneticalgorithm ASTEROIDS. A new ensemble of 8500 conformers was

7 of 10

https://github.com/gbouvignies/chemex

https://github.com/gbouvignies/chemex



on August 22, 2020


Dow

nloaded from

generated on the basis of theF andY angles of the selected conformers,supplemented with 1500 conformers from the initial statistical coil en-semble (28, 29). This new ensemble was subjected to another round ofASTEROIDS selection, and the iteration step was repeated eight timesuntil the ensemble converged with respect to the chemical shifts. En-semble averaged chemical shifts were calculated using SPARTA (54),and SAXS curves were obtained using CRYSOL (55). Another roundof ASTEROIDS was used to include, in addition to chemical shifts,the SAXS curves into the selection.

To build a conformational model of P1–304N1–525 and N1–405P1–304,the structure ofNCORE and the first 50 residues of P in the complexwereadapted from the x-ray structure of MeV (14) and NiV (15) N0P. TheN-terminal flexible arm of N, NTAIL, and PNTD were subsequently builtonto the structure using flexible-meccano with the helical sampling ofthe NTAIL molecular recognition element, as previously described (56),as well as the F and Y angles of the last selection of P1–304 to describePNTD within the complex. The order by which the different chains werebuilt onto the folded structure did not make a difference for the finalensemble of 100,000 conformers. ASTEROIDS selections with sizes of5 to 50 conformers were then selected on the basis of the SAXS scatter-ing curve. The same procedure was applied to describe the scatteringcurve of P1–304,HELL→AAAAN1–525 and P1–304,HELL→AAAAN1–405. Toillustrate the interaction between the HELL motif within P1–304 andN, 200 conformers were filtered that fulfill a distance constraint of 5 Åbetween the P1–304 HELL motif and its binding site on N (Fig. 5F).

Identification of the HELL interaction site bysequence analysisVarious paramyxovirus phosphoproteins were aligned in a multiplesequence alignment using MUSCLE (35). Although sequence con-servation in IDRs is weak, the algorithm identified a HELL-likemotif within all sequences that coincided with the position of pre-dicted helices of all viruses using the program PSIPRED (57).

In cellula analysesThe expression of P protein constructs was verified by Western blotusing a polyclonal rabbit MeV P protein as detailed (9). The abilityof P and domains of P to associate with N protein in cellula was testedby the Gaussia-based protein complementation assay, as detailed else-where (8, 9, 20). Briefly, plasmids encoding P protein fused to eitherthe C terminus or N terminus of the N-terminal glu1 domain, and Nprotein fused to the C terminus of glu2 domain by polymerase chainreaction and In-Fusion (Clontech) recombination. Glu1 and glu2fusion constructs were transfected in human embryonic kidney–293Tcells (fromCelluloNetBioBankBB-0033-00072, SFRBioSciences),andGaussia luciferase activity wasmeasured after 2 days of culture. Theresults were expressed in normalized luminescence ratio as described(58). Each test was carried out three times with each condition appliedin triplicate.

Minigenome assay was performed in BSR-T7 cells, which con-stitutively express the T7 polymerase (59), as detailed previously (9).The minigenome included the leader and trailer sequences separatedby a first gene encoding the firefly luciferase and a second gene encodingthe Gaussia luciferase, the expression of which relies on the edition ofthe gene transcript. The two genes are separated by an intergenic junc-tion elongated by 324 nucleotides (9). Briefly, plasmids encoding forMeVminigenomeRNAof (+) or (−) polarity, N, L, and P protein underthe control of T7 promoter were transfected together in BSR-T7 cells.Two days later, the expression of the two-luciferase reporter gene was


recorded by luminescencemeasurement in the presence of their specificsubstrate. Each test was performed three times with each condition per-formed in triplicate.

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/8/eaat7778/DC1Fig. S1. Scheme showing the different domains of N and P proteins referred to in the maintext.Fig. S2. NMR of P1–304.Fig. S3. Purification of P1–304N1–525 complexes.Fig. S4. Interaction of N with a1/2 of P.Fig. S5. Analysis of P interaction dynamics with N1–525.Fig. S6. Interaction of P1–160 with N.Fig. S7. P140–304 interaction with P1–50N1–525.Fig. S8. Defining the conformational ensemble of P1–304N1–525 from SAXS curves.Fig. S9A. Expression of MeV P protein constructs used for functional studies.Fig. S9B. Functional assays using MeV minireplicon.Fig. S10. NMR spectroscopy of N-terminal domain of NCORE in interaction with P1–304.

REFERENCES AND NOTES1. N. Tarbouriech, J. Curran, R. W. H. Ruigrok, W. P. Burmeister, Tetrameric coiled coil domain

of Sendai virus phosphoprotein. Nat. Struct. Biol. 7, 777–781 (2000).2. G. Communie, T. Crépin, D. Maurin, M. R. Jensen, M. Blackledge, R. W. Ruigrok, Structure

of the tetramerization domain of measles virus phosphoprotein. J. Virol. 87, 7166–7169(2013).

3. J. F. Bruhn K. C. Barnett, J. Bibby, J. M. Thomas, R. M. Keegan, D. J. Rigden, Z. A. Bornholdt,E. O. Saphire, Crystal structure of the Nipah virus phosphoprotein tetramerizationdomain. J. Virol. 88, 758–762 (2014).

4. S. Longhi, V. Receveur-Bréchot, D. Karlin, K. Johansson, H. Darbon, D. Bhella, R. Yeo,S. Finet, B. Canard, The C-terminal domain of the measles virus nucleoprotein isintrinsically disordered and folds upon binding to the C-terminal moiety of thephosphoprotein. J. Biol. Chem. 278, 18638–18648 (2003).

5. K. Houben, D. Marion, N. Tarbouriech, R. W. H. Ruigrok, L. Blanchard, Interactionof the C-terminal domains of sendai virus N and P proteins: Comparison ofpolymerase-nucleocapsid interactions within the paramyxovirus family. J. Virol. 81,6807–6816 (2007).

6. S. Gely, D. F. Lowry, C. Bernard, M. R. Jensen, M. Blackledge, S. Costanzo, J. M. Bourhis,H. Darbon, G. Daughdrill, S. Longhi, Solution structure of the C-terminal X domainof the measles virus phosphoprotein and interaction with the intrinsically disorderedC-terminal domain of the nucleoprotein. J. Mol. Recognit. 23, 435–447 (2010).

7. G. Communie, J. Habchi, F. Yabukarski, D. Blocquel, R. Schneider, N. Tarbouriech,N. Papageorgiou, R. W. Ruigrok, M. Jamin, M. R. Jensen, S. Longhi, M. Blackledge, Atomicresolution description of the interaction between the nucleoprotein and phosphoproteinof Hendra virus. PLOS Pathog. 9, e1003631 (2013).

8. J. Brunel, D. Chopy, M. Dosnon, L.-M. Bloyet, P. Devaux, E. Urzua, R. Cattaneo, S. Longhi,D. Gerlier, Sequence of events in measles virus replication: Role of phosphoprotein-nucleocapsid interactions. J. Virol. 88, 10851–10863 (2014).

9. L.-M. Bloyet, J. Brunel, M. Dosnon, V. Hamon, J. Erales, A. Gruet, C. Lazert, C. Bignon,P. Roche, S. Longhi, D. Gerlier, Modulation of re-initiation of measles virus transcriptionat intergenic regions by PXD to NTAIL binding strength. PLOS Pathog. 12, e1006058(2016).

10. J. Habchi, S. Longhi, Structural disorder within paramyxovirus nucleoproteins andphosphoproteins. Mol. Biosyst. 8, 69–81 (2012).

11. G. Communie, R. W. Ruigrok, M. R. Jensen, M. Blackledge, Intrinsically disorderedproteins implicated in paramyxoviral replication machinery. Curr. Opin. Virol. 5, 72–81(2014).

12. M. Huber, R. Cattaneo, P. Spielhofer, C. Orvell, E. Norrby, M. Messerli, J. C. Perriard,M. A. Billeter, Measles virus phosphoprotein retains the nucleocapsid protein in thecytoplasm. Virology 185, 299–308 (1991).

13. J. Curran, J. B. Marq, D. Kolakofsky, An N-terminal domain of the sendai paramyxovirusP protein acts as a chaperone for the Np protein during the nascent chain assemblystep of genome replication. J. Virol. 69, 849–855 (1995).

14. S. G. Guryanov, L. Liljeroos, P. Kasaragod, T. Kajander, S. J. Butcher, Crystal structure of themeasles virus nucleoprotein core in complex with an N-terminal region ofphosphoprotein. J. Virol. 90, 2849–2857 (2015).

15. F. Yabukarski, Structure of Nipah virus unassembled nucleoprotein in complex with itsviral chaperone. Nat. Struct. Mol. Biol. 21, 754–759 (2014).

8 of 10

http://advances.sciencemag.org/cgi/content/full/4/8/eaat7778/DC1

http://advances.sciencemag.org/cgi/content/full/4/8/eaat7778/DC1



on August 22, 2020


Dow

nloaded from

16. M. Aggarwal, G. P. Leser, C. A. Kors, R. A. Lamb, Structure of the paramyxovirusparainfluenza virus 5 nucleoprotein in complex with an amino-terminal peptide of thephosphoprotein. J. Virol. 92, e01304-17 (2018).

17. M. Renner, M. Bertinelli, C. Leyrat, G. C. Paesen, L. F. Saraiva de Oliveira, J. T. Huiskonen,J. M. Grimes, Nucleocapsid assembly in pneumoviruses is regulated by conformationalswitching of the N protein. eLife 5, e12627 (2016).

18. I. Gutsche, A. Desfosses, G. Effantin, W. L. Ling, M. Haupt, R. W. H. Ruigrok, C. Sachse,G. Schoehn, Near-atomic cryo-EM structure of the helical measles virus nucleocapsid.Science 348, 704–707 (2015).

19. M. Alayyoubi, G. P. Leser, C. A. Kors, R. A. Lamb, Structure of the paramyxovirusparainfluenza virus 5 nucleoprotein-RNA complex. Proc. Natl. Acad. Sci. U.S.A. 112,E1792–E1799 (2015).

20. S. Milles, M. R. Jensen, G. Communie, D. Maurin, G. Schoehn, R. W. Ruigrok, M. Blackledge,Self-assembly of measles virus nucleocapsid-like particles: Kinetics and RNA sequencedependence. Angew. Chem. Int. Ed. 55, 9356–9360 (2016).

21. G. Nodet, L. Salmon, V. Ozenne, S. Meier, M. R. Jensen, M. Blackledge, Quantitativedescription of backbone conformational sampling of unfolded proteins at amino acidresolution from NMR residual dipolar couplings. J. Am. Chem. Soc. 131, 17908–17918(2009).

22. M. R. Jensen, M. Zweckstetter, J. R. Huang, M. Blackledge, Exploring free-energylandscapes of intrinsically disordered proteins at atomic resolution using NMRspectroscopy. Chem. Rev. 114, 6632–6660 (2014).

23. A. Abyzov, N. Salvi, R. Schneider, D. Maurin, R. W. Ruigrok, M. R. Jensen, M. Blackledge,Identification of dynamic modes in an intrinsically disordered protein usingtemperature-dependent NMR relaxation. J. Am. Chem. Soc. 138, 6240–6251 (2016).

24. K. Sugase, H. J. Dyson, P. E. Wright, Mechanism of coupled folding and binding of anintrinsically disordered protein. Nature 447, 1021–1025 (2007).

25. R. Schneider, D. Maurin, G. Communie, J. Kragelj, D. F. Hansen, R. W. H. Ruigrok,M. R. Jensen, M. Blackledge, Visualizing the molecular recognition trajectory of anintrinsically disordered protein using multinuclear relaxation dispersion NMR.J. Am. Chem. Soc. 137, 1220–1229 (2015).

26. J. Kragelj, A. Palencia, M. H. Nanao, D. Maurin, G. Bouvignies, M. Blackledge, M. R. Jensen,Structure and dynamics of the MKK7-JNK signaling complex. Proc. Natl. Acad. Sci. U.S.A.112, 3409–3414 (2015).

27. E. Delaforge, J. Kragelj, L. Tengo, A. Palencia, S. Milles, G. Bouvignies, N. Salvi,M. Blackledge, M. R. Jensen, Deciphering the dynamic interaction profile of anintrinsically disordered protein by NMR exchange spectroscopy. J. Am. Chem. Soc. 140,1148–1158 (2018).

28. V. Ozenne, F. Bauer, L. Salmon, J. R. Huang, M. R. Jensen, S. Segard, P. Bernadó,C. Charavay, M. Blackledge, Flexible-meccano: A tool for the generation of explicitensemble descriptions of intrinsically disordered proteins and their associatedexperimental observables. Bioinformatics 28, 1463–1470 (2012).

29. M. R. Jensen, L. Salmon, G. Nodet, M. Blackledge, Defining conformational ensembles ofintrinsically disordered and partially folded proteins directly from chemical shifts. J. Am.Chem. Soc. 132, 1270–1272 (2010).

30. C. Leyrat, M. R. Jensen, E. A. Ribeiro Jr., F. C. Gérard, R. W. Ruigrok, M. Blackledge,M. Jamin, The N0-binding region of the vesicular stomatitis virus phosphoprotein isglobally disordered but contains transient a-helices. Protein Sci. 20, 542–556(2011).

31. P. Devaux, V. von Messling, W. Songsungthong, C. Springfeld, R. Cattaneo, Tyrosine 110 inthe measles virus phosphoprotein is required to block STAT1 phosphorylation. Virology360, 72–83 (2007).

32. M. Mavrakis, S. Méhouas, E. Réal, F. Iseni, D. Blondel, N. Tordo, R. W. Ruigrok, Rabies viruschaperone: Identification of the phosphoprotein peptide that keeps nucleoproteinsoluble and free from non-specific RNA. Virology 349, 422–429 (2006).

33. D. F. Hansen, P. Vallurupalli, L. E. Kay, An improved 15N relaxation dispersion experimentfor the measurement of millisecond time-scale dynamics in proteins. J. Phys. Chem. B112, 5898–5904 (2008).

34. N. Pereira, C. Cardone, S. Lassoued, M. Galloux, J. Fix, N. Assrir, E. Lescop, F. Bontems,J. F. Eléouët, C. Sizun, New insights into structural disorder in human respiratory syncytialvirus phosphoprotein and implications for binding of protein partners. J. Biol. Chem. 292,2120–2131 (2017).

35. R. C. Edgar, MUSCLE: Multiple sequence alignment with high accuracy and highthroughput. Nucleic Acids Res. 32, 1792–1797 (2004).

36. L. Hanke, F. I. Schmidt, K. E. Knockenhauer, B. Morin, S. P. J. Whelan, T. U. Schwartz,H. L. Ploegh, Vesicular stomatitis virus N protein-specific single-domain antibodyfragments inhibit replication. EMBO Rep. 18, 1027–1037 (2017).

37. M. Ouizougun-Oubari, N. Pereira, B. Tarus, M. Galloux, S. Lassoued, J. Fix, M. A. Tortorici,S. Hoos, B Baron, P. England, D. Desmaële, P. Couvreur, F. Bontems, F. A. Rey, J. F. Eléouët,C. Sizun, A. Slama-Schwok, S. Duquerroy, A druggable pocket at the nucleocapsid/phosphoprotein interaction site of human respiratory syncytial virus. J. Virol. 89,11129–11143 (2015).


38. R. M. Cox, S. A. Krumm, V. D. Thakkar, M. Sohn, R. K. Plemper, The structurally disorderedparamyxovirus nucleocapsid protein tail domain is a regulator of the mRNA transcriptiongradient. Sci. Adv. 3, e1602350 (2017).

39. S. Delpeut, R. S. Noyce, R. W. Siu, C. D. Richardson, Host factors and measles virusreplication. Curr. Opin. Virol. 2, 767–777 (2012).

40. J. Curran, Reexamination of the Sendai virus P protein domains required for RNAsynthesis: A possible supplemental role for the P protein. Virology 221, 130–140(1996).

41. S. Milles, D. Mercadante, I. V. Aramburu, M. R. Jensen, N. Banterle, C. Koehler,S. Tyagi, J. Clarke, S. L. Shammas, M. Blackledge, F. Gräter, E. A. Lemke, Plasticity of anultrafast interaction between nucleoporins and nuclear transport receptors. Cell163, 734–745 (2015).

42. J. Nikolic, R. Le Bars, Z. Lama, N. Scrima, C. Lagaudrière-Gesbert, Y. Gaudin, D. Blondel,Negri bodies are viral factories with properties of liquid organelles. Nat. Commun. 8,58 (2017).

43. S. M. Thomas, R. A. Lamb, R. G. Paterson, Two mRNAs that differ by two nontemplatednucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5.Cell 54, 891–902 (1988).

44. R. Cattaneo, K. Kaelin, K. Baczko, M. A. Billeter, Measles virus editing provides anadditional cysteine-rich protein. Cell 56, 759–764 (1989).

45. J. Curran, R. Boeck, D. Kolakofsky, The Sendai virus P gene expresses both an essentialprotein and an inhibitor of RNA synthesis by shuffling modules via mRNA editing.EMBO J. 10, 3079–3085 (1991).

46. B. Precious, D. F. Young, A. Bermingham, R. Fearns, M. Ryan, R. E. Randall, Inducibleexpression of the P, V, and NP genes of the paramyxovirus simian virus 5 incell lines and an examination of NP-P and NP-V interactions. J. Virol. 69, 8001–8010(1995).

47. Z. Solyom, M. Schwarten, L. Geist, R. Konrat, D. Willbold, B. Brutscher, BEST-TROSYexperiments for time-efficient sequential resonance assignment of large disorderedproteins. J. Biomol. NMR 55, 311–321 (2013).

48. F. Delaglio, S. Grzesiek, G. W. Vuister, G. Zhu, J. Pfeifer, A. Bax, NMRPipe: Amultidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6,277–293 (1995).

49. Y.-S. Jung, M. Zweckstetter, Mars - Robust automatic backbone assignment of proteins.J. Biomol. NMR 30, 11–23 (2004).

50. H. Zhang, S. Neal, D. S. Wishart, RefDB: A database of uniformly referenced proteinchemical shifts. J. Biomol. NMR 25, 173–195 (2003).

51. M. Rückert, G. Otting, Alignment of biological macromolecules in novel nonionicliquid crystalline media for NMR experiments. J. Am. Chem. Soc. 122, 7793–7797(2000).

52. R. M. Rasia, E. Lescop, J. F. Palatnik, J. Boisbouvier, B. Brutscher, Rapid measurement ofresidual dipolar couplings for fast fold elucidation of proteins. J. Biomol. NMR 51, 369–378(2011).

53. M. Akke, A. G. Palmer, Monitoring macromolecular motions on microsecond tomillisecond time scales by R1r−R1 constant relaxation time NMR spectroscopy.J. Am. Chem. Soc. 118, 911–912 (1996).

54. Y. Shen, A. Bax, Protein backbone chemical shifts predicted from searching a database fortorsion angle and sequence homology. J. Biomol. NMR 38, 289–302 (2007).

55. D. Svergun, C. Barberato, M. H. J. Koch, CRYSOL – A program to evaluate x-ray solutionscattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28,768–773 (1995).

56. M. R. Jensen, G. Communie, E. A. Ribeiro Jr., N. Martinez, A. Desfosses, L. Salmon, L. Mollica,F. Gabel, M. Jamin, S. Longhi, R. W. H. Ruigrok, M. Blackledge, Intrinsic disorder in measlesvirus nucleocapsids. Proc. Natl. Acad. Sci. U.S.A. 108, 9839–9844 (2011).

57. L. J. McGuffin, K. Bryson, D. T. Jones, The PSIPRED protein structure prediction server.Bioinformatics 16, 404–405 (2000).

58. P. Cassonnet, C. Rolloy, G. Neveu, P.-O. Vidalain, T. Chantier, J. Pellet, L. Jones, M. Muller,C. Demeret, G. Gaud, F. Vuillier, V. Lotteau, F. Tangy, M. Favre, Y. Jacob, Benchmarking aluciferase complementation assay for detecting protein complexes. Nat. Methods 8,990–992 (2011).

59. S. Finke, K.-K. Conzelmann, Virus promoters determine interference by defective RNAs:Selective amplification of mini-RNA vectors and rescue from cDNA by a 3′ copy-backambisense rabies virus. J. Virol. 73, 3818–3825 (1999).

60. K. Clark, I. Karsch-Mizrachi, D. J. Lipman, J. Ostell, E. W. Sayers, GenBank. Nucleic Acids Res.44, D67–D72 (2016).

AcknowledgmentsFunding: We thank GRAL (ANR-10-LABX-49-01) and Finovi, the Grenoble Instruct–ERIC Center(ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INSB-05-02)within the Grenoble Partnership for Structural Biology and the ESRF. S.M. acknowledges theEuropean Molecular Biology Organization for a long-term fellowship (ALTF 468-2014) and

9 of 10



the European Commission (EMBOCOFUND2012, GA-2012-600394) via Marie Curie Action.Author contributions: S.M., M.R.J., D.G., R.W.H.R., and M.B. conceived and designed theproject and wrote the manuscript. S.M., G.C., S.I., S.G., D.M., M.R.J., C.L., and D.G. carried outexperiments. All authors analyzed data, discussed results, and commented on the manuscript.Competing interests: The authors declare that they have no competing interests. Dataand materials availability: All data needed to evaluate the conclusions in the paper arepresent in the paper and/or the Supplementary Materials. Additional data related to this papermay be requested from authors.


Submitted 3 April 2018Accepted 18 July 2018Published 22 August 201810.1126/sciadv.aat7778

Citation: S. Milles, M. R. Jensen, C. Lazert, S. Guseva, S. Ivashchenko, G. Communie, D. Maurin,D. Gerlier, R. W. H. Ruigrok, M. Blackledge, An ultraweak interaction in the intrinsically disorderedreplication machinery is essential for measles virus function. Sci. Adv. 4, eaat7778 (2018).

10 of 10

on August 22, 2020


Dow

nloaded from


measles virus functionAn ultraweak interaction in the intrinsically disordered replication machinery is essential for

Damien Maurin, Denis Gerlier, Rob W. H. Ruigrok and Martin BlackledgeSigrid Milles, Malene Ringkjøbing Jensen, Carine Lazert, Serafima Guseva, Stefaniia Ivashchenko, Guillaume Communie,

DOI: 10.1126/sciadv.aat7778 (8), eaat7778.4Sci Adv

ARTICLE TOOLS http://advances.sciencemag.org/content/4/8/eaat7778

MATERIALSSUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2018/08/20/4.8.eaat7778.DC1

REFERENCES

http://advances.sciencemag.org/content/4/8/eaat7778#BIBLThis article cites 60 articles, 17 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.Science AdvancesYork Avenue NW, Washington, DC 20005. The title (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 NewScience Advances

License 4.0 (CC BY-NC).Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of

on August 22, 2020


Dow

nloaded from

http://advances.sciencemag.org/content/4/8/eaat7778

http://advances.sciencemag.org/content/suppl/2018/08/20/4.8.eaat7778.DC1

http://advances.sciencemag.org/content/4/8/eaat7778#BIBL

http://www.sciencemag.org/help/reprints-and-permissions

http://www.sciencemag.org/about/terms-service


an ultraweak interaction in the intrinsically disordered ... · ω2-1h (ppm) ω 1-15 n (ppm) 118...

Documents