The N-Terminal Domain of the Arenavirus L Protein Is an RNA Endonuclease Essential in mRNA Transcription

doi:10.1371/journal.ppat.1001038

PLoS Pathog. 2010 September; 6(9): e1001038.

Published online 2010 September 16. doi: 10.1371/journal.ppat.1001038

PMCID: PMC2940758

Copyright Morin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The N-Terminal Domain of the Arenavirus L Protein Is an RNA Endonuclease Essential in mRNA Transcription

Benjamin Morin,¹ Bruno Coutard,¹ Michaela Lelke,² François Ferron,¹ Romy Kerber,² Saïd Jamal,¹ Antoine Frangeul,¹ Cécile Baronti,³ Rémi Charrel,³ Xavier de Lamballerie,³ Clemens Vonrhein,⁴ Julien Lescar,^1,⁵ Gérard Bricogne,⁴ Stephan Günther,² and Bruno Canard¹^*

¹Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, Marseille, France

²Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany

³Unité des Virus Emergents UMR190, Université de la Méditerranée & Institut de Recherche pour le Développement, Marseille, France

⁴Global Phasing Ltd., Cambridge, United Kingdom

⁵School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

Michael J. Buchmeier, Editor

University of California Irvine, United States of America

* E-mail: bruno.canard/at/afmb.univ-mrs.fr

Conceived and designed the experiments: B. Morin, R. Charrel, S. Günther, B. Canard. Performed the experiments: B. Morin, B. Coutard, M. Lelke, F. Ferron, R. Kerber, S. Jamal, A. Frangeul, C. Baronti, R. Charrel, X. de Lamballerie, J. Lescar. Analyzed the data: B. Morin, B. Coutard, M. Lelke, F. Ferron, R. Kerber, C. Vonrhein, G. Bricogne, S. Günther, B. Canard. Contributed reagents/materials/analysis tools: G. Bricogne, B. Canard. Wrote the paper: B. Morin, J. Lescar, B. Canard. Coordinated the study: B. Canard.

Received April 7, 2010; Accepted July 12, 2010.

See "Bunyaviridae RNA Polymerases (L-Protein) Have an N-Terminal, Influenza-Like Endonuclease Domain, Essential for Viral Cap-Dependent Transcription" , e1001101.

This article has been cited by other articles in PMC.

Abstract

Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.

Author Summary

The Arenaviridae virus family includes several life-threatening human pathogens that cause meningitis or hemorrhagic fever. These RNA viruses replicate and transcribe their genome using an RNA synthesis machinery for which no structural data currently exist. They synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism thought to involve the large L protein, which carries RNA-dependent RNA polymerase signature sequences. Here, we report the crystal structure and functional characterization of an isolated N-terminal domain of the L protein (NL1) from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures and mutagenesis studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. Reverse genetic studies show that mutation of active site residues selectively abolish transcription, not replication. We show that this endonuclease domain is conserved and active across the virus families: Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.

Introduction

The Arenaviridae family includes 22 viral species into a single genus Arenavirus, with new species awaiting classification [1], [2]. They cause chronic and asymptomatic infections in rodents, and occasional transmission to man may result in life-threatening meningitis and/or hemorrhagic fever. Lymphocytic choriomeningitis virus (LCMV) is the prototypic species and first arenavirus isolated in 1933. Because its natural host is the common house mouse (Mus musculus), LCMV is the only known arenavirus presumably exhibiting a worldwide distribution. LCMV is a human pathogen of significant clinical relevance, causing central nervous system disease, congenital malformation, choriomeningitis, and systemic and highly fatal infection in immuno-compromised, organt transplant recipient patients [3], [4], [5], [6]. Humans are generally infected through the respiratory tract after exposure to aerosols, or by direct contact with infectious material.

Arenaviruses are enveloped viruses with a bisegmented negative single-strand RNA genome. Each RNA segment, called large (L; ~7.2 kb) and short (S; ~3.5 kb), contains two open reading frames in mutually opposite orientations and use an ambisense coding strategy to direct the synthesis of two polypeptides [7]. Between the two open reading frames of each segment resides a non-coding intergenic region (IGR), composed of a sequence predicted to form a stable hairpin structure [8]. The S RNA encodes the viral nucleoprotein (NP; ~63 kDa) and glycoprotein precursor (GPC; ~75 kDa), whereas the L RNA encodes a small RING finger protein (Z; ~11 kDa) and a large protein (L; ~250 kDa) which is the viral RNA-dependent RNA polymerase (RdRp). The two RNA genomes are encapsidated by the NP, which is the most abundant protein in virions and infected cells, and act as templates for two fundamentally different processes, RNA replication and transcription. During RNA replication, the L protein first binds to the 3′-end of RNA templates and reads them from end to end to direct the synthesis of encapsidated full-length anti-genomes. During transcription, the RdRp stops RNA synthesis at a pause site located near the IGR [7]. The newly synthesized mRNA molecules have a non-polyadenylated 3′-end with a heterogeneous sequence mapped within the predicted hairpin in the IGR [9]. Furthermore, non template-directed sequences have been identified at the 5′-end of the subgenomic mRNA [10]. These sequences are variable in length [9], [10], [11] and terminate with a 5′-cap structure, which suggests the presence of a cap-snatching mechanism for arenaviruses. In this process, originally described for influenza viruses [12], [13] and bunyaviruses [14], the viral RdRp binds cellular mRNAs caps and ‘steals’ them using an endonuclease activity, located in the influenza PA subunit [15], [16], and presumably in L protein of bunyaviruses. These short capped RNAs are then used as primers for mRNA synthesis. The arenavirus L protein is an essential element in genome replication and transcription [17]. It is the largest viral protein composed of approximately 2200 amino-acid (aa) residues, and sequence analysis using homologous proteins led to the prediction of several conserved domains [18], [19]. A biological function can be inferred for the L3 domain containing conserved and typical RdRp signature sequence motifs [19], [20]. For Tacaribe virus, both domains L1 and L3 interact with the Z protein [21]. By analogy with influenza and bunyaviruses, the L protein may also carry activities and domains responsible for a cap-snatching mechanism that would account for the sequence diversity found at the 5′-end of RNA transcripts. The expression and purification of such a large viral polymerase is problematic and has not been documented.

We report here the first crystal structure of an Arenaviridae L protein domain at 2.13 Å resolution, that of the N-terminus domain of the LCMV L protein. We show that this domain is able to bind nucleotides, with a preference for UTP, and RNA. Structural comparison with the N-terminal part of the influenza virus PA protein characterizes unambiguously the domain as an endonuclease. Sequence and secondary structure analysis of L proteins from various Bunyaviridae family members predict that their N-terminal end carries a similar endonuclease activity, that we demonstrate for Toscana virus (TOSV) (genus Phlebovirus, family Bunyaviridae). Activity assays and mutagenesis show that the arenavirus endonuclease exhibits sequence-specificity with a preference for uracil-containing substrates. Lastly, reverse genetics studies correlate expression of endonuclease activity with the selective production of mRNA, making the N-terminus domain of the L protein a likely candidate to be involved in the cap-snatching mechanism of arenaviruses.

Results

Delineation of an Arenavirus L Protein Domain and its Crystal Structure

Based on aa sequence conservation across arenaviruses and on the presence of a potential nucleotide-binding site, we designed cDNA constructs encoding aa residues 1 to ~250 for the N-terminal end of four arenavirus (Pirital virus (PIRV), Lassa fever virus (LASV), Parana virus (PARV), and LCMV) L proteins. All four domains were expressed as soluble recombinant proteins. We observed a self-limited proteolysis of the Parana arenavirus N-terminus L domain which prompted us to refine boundaries into a shorter 196 aa form, hereafter named “NL1”, fully included in the previously predicted arenavirus L1 domain (1–250 aa) [19]. The construct was expressed in E.coli and purified, but yielded crystals diffracting to 8 Å. However, the homologous 196 residues domain of LCMV yielded well-diffracting crystals. The atomic structure of NL1 was first determined by the SAD technique with seleno-methionylated crystals that diffracted to 3.4 Å. The structure was refined using a native data set at 2.13 Å resolution (Table 1). Two NL1 molecules are present within the asymmetric unit. Residues 1–191 are visible for one molecule whilst only 1–175 could be modelled for the other NL1 molecule owing to high mobility of the C-terminal end of helix α7.

Table 1

Data Collection and Refinement Statistics.

The LCMV NL1 Domain Exhibits a Type II Endonuclease Fold

The LCMV NL1 monomer structure has approximate dimensions of 59 Å ×37 Å ×27 Å. It features four mixed β-strands forming a twisted plane surrounded by seven α-helices (Figure 1A). The two anti-parallel strands β1 and β2 are connected by helix α4, whereas the two parallel strands β3 and β4 are connected by the long helix α5. These two helices run parallel to the central β-sheet and are disposed at the same side of the latter. On the opposite side of the β-sheet, helix α3 is surrounded at its extremity by two N-terminal (α1 and α2) and C-terminal helices (α6 and α7). A search for similar protein folds using the DALI server [22] returned the PA N-terminal domain structure that was recently identified as a type II endonuclease domain [15], [16]. The structural match with published molecular structures of the influenza PA N-terminal domains (PA_N) returns a Z-score of 5.7 and an r.s.m.d. of 3.9 Å for 121 superposed aa (PDB code 3EBJ) and Z-score 5.2, r.m.s.d. 4 Å for 122 aa (PDB code 2W69). As was the case for PA_N, other type II endonuclease proteins are also recovered: the Tt1808 hypothetical protein from Thermus Thermophilus HB88 (PDB code 1WDJ, Z-score 3.8, r.m.s.d. 3.4 Å for 81 aa), and the restriction endonuclease SdaI (PDB code 2IXS, Z-score 3.6, r.m.s.d. 6.3 Å for 104 aa).

Figure 1

NL1 structure and comparisons with the influenza PA_N structure.

The β-sheet forms a negatively charged cavity creating a binding site for divalent cations, whilst above that cavity, the C-terminal end of helix α5 forms a positively charged patch and a concave surface that is likely to accommodate the RNA substrate (Figure 1B, arrow). The PA protein constitutes one subunit that associates with PB1 and PB2 to form the heterotrimeric influenza virus polymerase. Its N-terminal domain PA_N hosts the RNA cap-snatching endonuclease activity [15], [16]. Both NL1 and PA_N share a similar core structure. Except for the absence of a fifth β-strand in NL1, all other secondary structure elements are conserved (Figure 1C) and the overall topology of these two structures is very similar (Figure 1D), albeit with interesting differences in the vicinity of the PA_N active site (discussed below). At the aa sequence level, NL1 shares the conserved active site sequence motif characteristic of type II endonucleases: PD…(D/E)XK. In NL1, the corresponding residues are P88, D89…E102, and either K115 or K122 (Figure S1A, B). The identity of the distal lysine is not certain since it is found at different positions in the primary sequence, as is the case for influenza virus. The influenza PA_N domain was crystallized either in the presence of magnesium or manganese ions in the active site which comprises five conserved catalytic residues: H41, E80, D108, E119 and K134. A structural superimposition of the arenavirus NL1 and influenza PA_N active sites shows that the side-chains of three evolutionary-conserved residues within arenaviruses (P88, D89 and E102) closely superimpose with P107, D108 and E119 of the influenza virus PA_N protein, pointing to a common function for these residues (Figure 2A and Figure S1B). Upon superimposition with PA_N, one Mn²⁺ ion needed for the enzymatic reaction coordinated by D108 in the PA_N active site, falls at right distances to be coordinated by the carboxylate side-chains of D89 and E102. NL1 was crystallized without metal ions and a water molecule is found close to the position that should be occupied by the divalent metal. Interestingly, no close structural match is found neither for H41 nor K134 of the influenza virus PA_N. This points to differences between the two active sites since His41 was proposed to play a catalytic role in the influenza PA_N. However, we note that another possible contributor could be NL1 C103 main-chain carbonyl as it superimposes quite well with PA_N I120 main-chain carbonyl (Figure 2B). The triad made of K115, D119, and K122 in NL1 is spatially equivalent to K134 in PA_N. In summary, despite no aa sequence homology, the active site structures of the influenza PA_N and LCMV NL1 domains are clearly related but not identical (Figure 1C, ,2),2), strongly suggesting that these two domains exhibit closely related enzymatic activities (see below).

Figure 2

The endonuclease active site.

The NL1 Endonuclease Fold is Conserved Amongst Bunyaviridae

In addition to Arenaviridae and Orthomyxoviridae, Bunyaviridae is the other family of virus to possess a segmented negative-strand RNA genome. It contains four genera of animal viruses (Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus) and one genus of plant virus (Tospovirus) [23]. Although the genomic organisation differs between these three virus families, Bunyaviridae are also thought to use a cap-snatching mechanism to prime mRNA synthesis [24]. Arenaviruses, and Bunyaviridae share a conserved RdRp motif within their large L protein, as well as a conserved N-terminus domain [18]. Amino-acid sequence alignments, assisted by secondary structured prediction, of the N-terminal part of LCMV and Bunyaviridae L protein reveal that the latter also possesses the conserved active site motifs characteristic of type II endonucleases (Figure S2A). However, we could identify the catalytic motif within the L protein N-terminal end for only four out of the five bunyavirus genera: Orthobunyavirus, Phlebovirus, Hantavirus and Tospovirus. The L protein of Nairovirus is much larger (~4000 aa) than the L protein of other members of the Bunyaviridae family (~2200 aa). The putative endonuclease catalytic motif was located after aa ~700, the N-terminal of Nairovirus L protein being assigned as a so-called OTU-like domain [25].

Secondary structure predictions were used to draw the topology diagram of the NL1-like domain for each genera (Figure S2B). As expected from the sequence alignment, each genus seems to share a β-sheet with a variable number of β-strands. Furthermore, the PD catalytic motifs are in each case located in a loop before a β-strand, as expected. The PUMV, HLCV and RVFV NL1-like domains are more closely related to LCMV NL1 than are the TOMV and CCGV. The TOMV NL1-like domain contains 6 β-strands and shares the PD motif just upstream the first β-strand, whereas it is just upstream the second β-strand in the case of NL1 and PA_N. Finally, the structural organization of the putative CCGV endonuclease domain seems to diverge even further from the others. Indeed, whereas the conserved lysine is shared by the same helix for all the domains, that of Nairovirus may be located at the end of the β4 strand (Figure S2B). Thus we conclude that the endonuclease motif is conserved across four animal virus genera Orthobunyavirus, Phlebovirus, Nairovirus and Hantavirus.

NL1 is a Mn²⁺-Dependent RNA Endonuclease

Recent crystal structures of complexes of PA_N with three different nucleoside monophosphates show that PA_N binds nucleotides [26]. The ability of NL1 to bind nucleotides was investigated using UV-crosslink experiments. We observe that NL1 binds NTPs, preferably UTP and GTP, whereas ATP and CTP show a weaker association (Figure 3A). The PA_N structures were determined in complex with ATP, CTP and UTP but not GTP [26] whereas NL1 bind GTP in a stronger fashion than ATP or CTP. The crystal structure relatedness to the endonuclease fold would suggest that the NL1 domain is able to bind RNA rather than nucleotides. We tested RNA binding by NL1, and found that indeed, NL1 binds RNA (Figure 3B). The band shift assay is also suggestive that the RNA substrate is cleaved under the assay conditions, as judged by degradation products at the bottom of the gel under the labeled RNA oligo (Figure 3B). Therefore, we surmise that nucleotide binding properties observed here reflect the ability of NL1 to bind RNA with some sequence specificity in the cap-snatching pathway (see below).

Figure 3

Nucleotide and RNA binding assays of LCMV NL1 domain.

Several synthetic RNA oligonucleotides were used to characterize the endonuclease activity (Figure 4). NL1 is able to cleave ssRNA having no stable secondary structure at specific sites indicating a preference for the presence of uracil (Figure 4A, B), and adenosine to a lesser extent. Likewise, a moderately stable RNA hairpin containing uracil (ΔG

−3.4 kcal/mole) is cleaved down to a 14/15-mer product whereas a stable (ΔG

−14.7 kcal/mole) RNA hairpin devoid of uracil remains unattacked even in its single stranded regions (Figure 4A, B). PolyU RNA is cleaved randomly down to a 8-mer product with a better efficiency than polyA, whereas polyC is not a substrate for NL1 (not shown). A 5′-terminal nucleoside uracil or adenosine 5′-monophosphate is also cleaved and the 5′-monophosphate RNA end apparently competes for internal cleavage. A 5′-capped RNA of 264 nucleotides in length also acts as a substrate. It is cleaved at several specific positions indicated by the sequential appearance of band products over time (Figure 4B). This indicates that the cap structure does not seem to be a direct RNA binding determinant. A Phlebovirus (Toscana) virus endonuclease domain was prepared according to bio-informatic predictions described above. Its endonuclease activity was compared to both that of arenavirus NL1 and the influenza H5N1 endonuclease [16]. The enzymes were equally active using short RNA substrates, although it is apparent that sequence-specific cleavage is different for each enzyme: the influenza enzymes prefers cleavage at puric sites, Toscana virus and LCMV enzymes prefer adenosine- and uracil-containing sites (Figure 4B). NL1 is ~90-fold more active in the presence of Mn²⁺ than Mg²⁺, and shows background activity with Ca²⁺ and Zn²⁺(Figure 4C and not shown). The Mn²⁺ ion has also a significant stabilizing effect as judged by thermostability studies, whereas Zn²⁺ has a deleterious effect.

Figure 4

Endonuclease activity of NL1.

Mutagenesis analysis of most residues identified as part of the active site (Figure 2A) impaired the endonuclease activity. The most drastic effect was observed for D119, but residual activity was scored for E51, D89, and less for E102 (Figure 4D). As these three residues might coordinate metal ions as proposed above, defective metal-binding due to a point mutation might be compensated by the presence of the remaining two adjacent acidic residues. A double mutant D89A/E102A shows further reduced but not abolished activity. Mutations K115A and K122A generated strongly altered activity, but the similar level of residual activity does not allow the identification of which lysine is predominant in catalysis.

The Endonuclease Activity is Essential for RNA Transcription, not Replication

The effect of 33 mutations in L1 on virus RNA and protein expression was studied in a cell-based mini-replicon system. The LCMV L protein mediates the synthesis of two RNA species: first, capped mRNA terminating within the intergenic region, and second, antigenomic RNA being a full-length copy of the genomic RNA template [9], [27]. This dual role in RNA synthesis is recapitulated in the mini-replicon system. It contains all trans-acting factors (L protein and NP) required for transcription and replication of a genome analogue containing Renilla luciferase as a reporter gene (mini-genome). Reporter gene expression was measured in luciferase assay (Table 2), while RNA synthesis was measured in Northern blot (Figure 5), in which luciferase mRNA and antigenome can easily be distinguished due to their size difference. Wild-type (WT) L protein led to expression of high levels of Renilla luciferase (2–3 log units signal-to-noise ratio) as well as Renilla luciferase mRNA and antigenome in a ratio of about 1 [ratio]

1. Expression of mutant L protein was verified by immunoblotting (Figure S3).

Table 2

Functional Analysis of L Protein Mutants in LCMV Mini-Replicon System.

Figure 5

Mutational analysis of the L protein in the context of the LCMV replicon system.

The phenotype of mutants E41A, E41Q, K44A, S54A, C60A, T108S, F116A, D142N, and W155A is similar to that of wild-type. Mutants E179A, E179Q, E182A, E182Q, and Y183A also express luciferase and RNA at high level, but the steady-state level of mRNA relative to that of antigenome is reduced by about 50%. Mutants F104A, R106A, F112A, K115A, D142A, R144A, R161A, R185A neither express Renilla luciferase nor any RNA species, indicating that global functions of L protein are affected.

The most interesting phenotype is observed with mutants D89A, D89N, E102A, E102N, D119A, D119N, K122A, D129A, and D129N. They synthesize antigenome close or equal to wild-type level, but are defective in mRNA and, thus, reporter gene expression (Figure 5 and Table 2, shown in boldface). A similar phenotype is seen with mutants E51A and E51Q, though associated with reduced antigenome level. These data indicate that residues E51, D89, E102, D119, K122, and D129A are essential for viral mRNA synthesis, but not required for expression of uncapped RNA species. With the exception of the D129 residue located at the surface of the protein remote from the endonuclease active site, it is remarkable that these transcription-null mutants form the catalytic site (Figure 2) and match precisely those of the PD…(D/E)XK endonuclease type II signature sequence.

Discussion

The structural and functional results presented here show that the LCMV NL1 domain is an RNA endonuclease. The uncoupling of RNA replication from transcription and selective disappearance of mRNA when NL1 active site residues are mutated strongly suggests that this activity is involved in cap-snatching.

The identification of the arenavirus endonuclease is in line with the recent discovery of the PA_N endonuclease domain of influenza virus. Whereas the active site of influenza virus features a cluster of three acidic residues, the active site of arenavirus contains four acidic residues (E51, D89, E102 and D119), as well as two important lysine residues K115 and K122 neighboring D119 (Figure 2A). The NL1 active site resembles but is clearly distinct from that of influenza PA_N. Indeed, there is no histidine in the catalytic center, and the arenavirus NL1 nuclease has some specific features both upstream and downstream of the PD signature sequence. We define the arenavirus endonuclease motif as E-X₃₈-P-D-X_(11,13)-E-X₁₂-K-X₃-D-X₂-K. The most obvious difference with the only known related RNA endonuclease, that of influenza virus PA_N, is a divergence upstream the PD motif in structural elements carrying the E51 residue (Figure 1C), and the presence of a triad K…D…K at the distal side of the latter signature sequence (Figure 2A). Contrary to PA_N which shares a conserved and essential histidine involved in the binding of both the metal ion and a nucleotide onto helix α3 [15], [16], [26], NL1 does not possess this conserved histidine residue. Instead, NL1 has a glutamic acid residue E51, which might reflect a different nucleobase specificity as detected in our nuclease assays (Figure 4). Likewise, residues downstream the PD motifs are distinct from the consensus sequence, and differently organized into a triad including two lysines. The presence of water molecules and previous structural models for influenza PA_N allows to propose putative positions of metal ions, coordinated by D89 and E102.

The first step in the general mechanism for phosphodiester hydrolysis is the preparation of the attacking nucleophile by deprotonation, usually involving a general base deprotonating a water molecule. Lysine is often considered as this general base candidate in endonucleases but is not strictly conserved [28], [29]. Here, there are no indications against D119 being this general base. Alternately, it could well be either lysine K115 or K122. Both are oriented towards the active site, and they could well have their pKa lowered by D119 in order to initiate the reaction. Reverse genetic studies provide evidence for K122, not K115. Indeed, mRNA production is selectively abolished and clearly uncoupled from RNA synthesis in the case of K122A mutant, while the K115A mutant was completely defective preventing interpretation of its role in the endonuclease catalytic site. Although it is not known if uncapped mRNAs are synthesized and degraded for the transcription-null mutants, the most plausible scenario is that primer shortage prevents significant capped mRNA synthesis. Overall, the replicon data presented here closely match those obtained on the closely related Lassa arenavirus using a similar replicon system [30]. Arenaviruses may thus use two clearly independent and distinct RNA synthesis priming mechanisms: one is dependent on an active endonuclease carried by the N-terminus of the L protein, and the other might be linked to the observation that an extra G residue is found at the 5′-end of arenavirus genomes and antigenomes. The latter G bases would thus reflect a yet-uncharacterized priming mechanism unrelated to the U/A cleavage sequence preference of NL1.

NL1 also binds nucleotides, but the NTP binding site should differ from that of PA_N. Indeed, the influenza PA_N histidine 41 is involved in binding the nucleobase of the presumed incoming RNA substrate. The NL1 endonuclease does not share the same sequence specificity, and E51 is positioned at a spatially equivalent position.

The cap structure does not seem to be a direct RNA binding determinant (Figure 4B), as endonucleolytic cleavage is not directed to cleavage sites preferentially in the vicinity of the cap. We thus infer that an independent cap-binding site way exist elsewhere in viral proteins to bind and select cellular mRNAs, a possibility reminiscent of influenza for which PA carries the endonuclease activity and PB2 the cap binding site [15], [16], [31].

Structure and sequence alignment studies show that the N-terminal endonuclease domain of the L protein is also conserved in the Bunyaviridae family, although the Nairovirus endonuclease domain is not located into the N-terminal end of the protein. These findings were confirmed by the endonuclease activity of the N-terminal end of the L protein of TOSV (Figure 4B). Thus, we provide evidence that all three segmented negative single-strand RNA virus species share an endonuclease domain probably involved in the cap-snatching process during the viral life cycle. These data raise the question of a possible common ancestor for these viruses. Indeed, these three virus families use a cap-snatching mechanism involving binding and cleavage of cellular mRNA caps subsequently used by a large primer-dependent RNA-dependent RNA polymerase. It seems more plausible that the L gene has evolved by divergence over time, rather than by multiple acquisitions of several activities converging into a common structure, at least in the case of the endonuclease. Furthermore, our study raises the interesting possibility that other activities involved in RNA replication/transcrition might be discovered by comparative analysis of Orthomyxoviridae PB1, PB2, PA and Arenaviridae/Bunyaviridae L proteins.

To our knowledge, a single crystal structure of a functional arenavirus protein is currently available, that of the Machupo virus glycoprotein GP1 in complex with its human receptor, TfR1 [32]. Our results provide an arenavirus L domain structure, with a role consistent with the hypothesis of a cap-snatching mechanism suggested for arenaviruses [9], [10]. The strategy used here to produce individually active domains might be useful to further characterize the Arenaviridae/Bunyaviridae large L protein which had so far resisted all biochemical characterization attempts.

The influenza, Arenaviridae and Bunyaviridae endonucleases are so far the only three examples of RNA endonucleases similar to type II DNA restriction endonucleases. The presence of such an endonuclease suggests that it could serve as a fruitful target for antiviral strategies against these two families, since such kind of inhibitors have been reported in the case of the influenza virus [33], [34], [35].

Materials and Methods

Cloning, Expression and Purification of LCMV NL1 Domain

The LCMV NL1 cDNA (Armstrong strain, aa 1 to 196) was cloned into pDest14 with a N-terminus hexa-histidine tag and expressed in E.coli Rosetta (DE3) pLysS (Novagen), at 17°C in 2YT medium overnight after induction with 500 µM IPTG. Cell pellets from harvested cultures were resuspended in 50 mM Tris buffer, pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1% Triton, 5% Glycerol. Lysozyme (0.25 mg/ml), PMSF (1 mM), DNase I (2 µg/ml), and EDTA free protease cocktail (Roche) were added before sonication. IMAC chromatography of clarified lysates was performed on a 5 ml His prep column (Akta Xpress FPLC system, GE Healthcare) eluted with imidazole. Size exclusion chromatography was performed on preparative Superdex 200 column (GE Healthcare) pre-equilibrated in 10 mM Imidazole, pH 8.0, 50 mM NaCl, 2 mM DTT. Protein was concentrated (28 mg/ml) using a centrifugal concentrator. For enzymatic studies, WT and mutants were express in the E.coli BL21 star strain (Invitrogen) and further purified on HiTrap Q sepharose 1 ml column (GE Healthcare) to remove E. coli RNase contaminants. Proteins eluted in a linear gradient from 50 mM to 1 M NaCl in 10 mM Hepes buffer, pH 7.5, 2 mM DTT. A synthetic gene of the H5N1 PA_N endonuclease was designed as described [16]. The Toscana virus (strain France AR_2005, aa 2 to 233) cDNA was obtained from infected cell cultures. Both ORFs were cloned as a N-terminal Thioredoxin-Hexahistidine fusion in pETG20A. The tag was cleaved using TEV protease before a final gel filtration.

Crystallization

Crystals grew in LiSO₄ 250 mM, citrate 50 mM, isopropanol 5.5%, using the hanging drop vapor diffusion method in Linbro plates by mixing 1 µl of protein solution with 1 µl of reservoir solution. Crystals were cryoprotected by dipping in a solution containing 65% of crystallization buffer and 35% of a buffer made of size exclusion chromatography buffer/glycerol (50/50). Crystals were cryo-cooled in liquid N₂. The crystals belong to space group C222₁ and have two molecules per asymmetric unit. Despite repeated attempts, crystal soaked into the above buffer supplemented with various concentrations of MnCl₂ yielded crystals diffracting to >4 Å.

Data Collection and Structure Determination

Diffraction intensities were recorded on the ID14-4 beamline at the European Synchrotron Radiation facility, Grenoble, France. Data were processed and integrated with MOSFLM [36]. Scaling and merging of the intensities was performed with SCALA and programs from the Collaborative Computational Project, No. 4 (CCP4) suite [37]. The structure was determined using SAD data from one selenomethionylated protein crystal diffracting to 3.4 Å resolution with SHARP/autoSHARP, followed by density modification with SOLOMON and DM. An initial model was built using BUCCANEER and completed in COOT, followed by refinement using BUSTER (see Text S1). Details of structure determination are given as supplemental material. Data from a native crystals diffracting to a 2.13-Å resolution were collected on an ADSC QUANTUM 315r at a wavelength of 0.9835 Å. The structure was refined with BUSTER and COOT using this data set (Table 1) [38]. The atomic coordinates have been deposited at the PDB (3JSB).

Sequence Retrieval

A PHI-BLAST search using the sequence corresponding to the L1 domain and the signature of the Arenaviridae endonuclease motif i.e. P-D-_x(11,13)-E-_x(12)-K-_x(3)-D-_x(2)-K ; was performed against non-redundant databases [39]. After 3 iterations, Batai and Kairi viruses both belonging to Orthomyxoviridae, appears in the section with an E-Value below threshold. A fourth iteration including these two sequences allows retrieving the entire family of orthomyxoviruses, with E-value comprised between 3e⁻¹⁸ and 2e⁻⁴.

A standard CDD search from the sequence of Tensaw virus allows retrieving all the L of the Bunyaviridae family hitting the pfam 04196 [40].

Sequence Comparison

A multiple sequence alignment of the N-terminal end of the L protein from LCMV, HLCV, BUNV, HANV, PUMV, RVFV, TOSV, TOMV, WTMV, CCGV, DUGV, was first performed with the T-coffee algorithm (http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi). Using the secondary structure prediction of the endonuclease domain of L proteins, the putative conserved active site residues were identified and placed correctly in the alignment.

UV-Crosslink Experiments

7 µg of purified protein were incubated for 15 min at 25°C, with 0.5 µl of the various α-³²P NTP (0.4 µCi/µl) in 10 µl of reaction buffer containing 10 mM Imidazole, pH 8.0, 50 mM NaCl, 2 mM DTT. The reaction mixtures were then exposed to UV light (254 nm) for 6 min at 5 mm distance. The crosslinked species were separated in a 15% polyacrylamide denaturing gel and visualized by autoradiography using photo-stimulated plates and a Fujilmager (Fuji).

RNA Binding Experiments

The RNA 5′-AUUUUGUUUUUAAUAUUUC-3′ (Ambion) was [³²P] 5′-end labeled, and 0.4 µM of radiolabelled RNA was incubated 20 min at 25°C without and with 1.4 µg, 4.2 µg and 7 µg of protein in 10 µl of 10 mM Imidazole, pH 8.0, 50 mM NaCl, 2 mM DTT. Reaction mixtures was analyzed by PAGE and visualized by autoradiography.

Ion Binding Assays

Titration curves with CaCl₂, MnCl₂, MgCl₂ and ZnCl₂ were performed at 1 mg/ml protein in gel filtration buffer using thermal shift assay. Technical details can be be found in [41].

Endonuclease Assays

Endonuclease activity was assayed using 4 different heteromeric RNA substrates: an unstructured 19 mer as described above, a 21 mer stable hairpin (5′-UGAGGCCCGGAAACCGGGGCC-3′ (Ambion), ΔG

−14.7 Kcal/mole), a 22 mer moderately stable hairpin (5′- CGCAGUUAGCUCCUAAUCGCCC-3′ (Ambion), ΔG

−3.4 Kcal/mole), and a long 264 mer RNA corresponding to the SARS-CoV 5′-genome sequence. The latter was radiolabelled with a cap structure at its 5′-end using the ScriptCap m7G Capping System (Epicentre Biotechnologies) with [α³²P]GTP. Endonuclease assays were carried out using 3.3 µM of radio-labeled RNA in a buffer containing 40 mM Tris-base, pH 7.5, 100 mM NaCl, 10 mM β-Mercaptoethanol and 2 mM MnCl₂. Reactions were initiated by the addition of 1 µM of protein and incubated at 37°C, and stopped by the addition of EDTA/formamide. Reactions products were analyzed using denaturing polyacrylamide gel electrophoresis (20% polyacrylamide, 7 M urea in TTE buffer (89 mM Tris, 28 mM taurine, 0.5 mM EDTA) and analyzed by autoradiography.

Mutagenesis and Reverse Genetics Assays Using a LCMV Mini-Replicon System

The LCMV replicon system is based on strain Armstrong clone 13 and has been established in analogy to the Lassa virus replicon described previously [42]. BSR T7/5 cells constitutively expressing T7 RNA polymerase [43] were transiently transfected with T7 promoter-driven expression constructs for L protein, nucleoprotein (NP), mini-genome (MG) containing Renilla luciferase reporter gene, and firefly luciferase as a transfection control. L protein mutants were generated as described [44]. One day after transfection, total RNA was prepared for Northern blotting and cell lysate was assayed for firefly and Renilla luciferase activity. Renilla luciferase levels were normalised with firefly luciferase levels resulting in standardized relative light units (sRLU). Northern blot was performed using an antisense ³²P-labeled riboprobe targeting the Renilla luciferase gene. Autoradiography was quantified on a PhosphorImager (Amersham Biosciences). To verify protein expression, hemagglutinin (HA)-tagged L protein was expressed in BSR T7/5 cells inoculated with modified vaccinia virus Ankara expressing T7 RNA polymerase (MVA- T7) [45] and detected in immunoblot using anti-HA antibody.

Supporting Information

Figure S1

Sequence Alignment of Viral Endonuclease Domains. A, Structure-based sequence alignment of NL1 with the two influenza PAN (3EBJ, 2W69), Tt1808 (1WDJ) and SdaI (2IXS), showing the structurally-conserved endonuclease motif (Highlighted in red). B, Sequence alignment of arenavirus NL1: Lymphocytic choriomeningitis virus (LCMV), Dandenong virus (DANV), Mopeia virus (MOPV), Morogoro virus (MORV), Mobala virus (MOBV), Ippy virus (IPPV), Lassa virus (LASV), Lujo Virus (LUJV), Parana virus (PARV), Pichinde virus (PICV), Allpahuayo virus (ALLV), Chapare virus (CHAV), Tamiami virus (TAMV), Whitewater Arroyo virus (WWAV), Bear Canyon virus (BCNV), Flexal virus (FLEV), Pirital virus (PIRV), Amapari virus (AMAV), Guanarito virus (GTOV), Cupixi virus (CPXV), Machupo virus (MACV), Junin virus (JUNV), Tacaribe virus (TCRV), Sabia virus (SABV), Oliveros virus (OLVV), Latino virus (LATV). Residues in a solid red background are strictly conserved. The blue point indicates the key active site residues.

(4.29 MB TIF)

Click here for additional data file.^{(4.0M, tif)}

Figure S2

Conservation of NL1 Between Arenaviruses and Bunyaviruses. A, Sequence alignment showing endonuclease motif (highlighted in red) of the NL1 domain from LCMV and the five genus of the Bunyaviridae families. Each genera is represented by two viruses: Orthobunyavirus: Human La Cross Virus (HLCV) and Bunyamwera virus (BUNV), Hantavirus: Hantaan virus (HANV) and Puumala virus (PUMV), Phlebovirus: Rift valley fever virus (RVFV) and Toscana virus (TOSV), Tospovirus: Tomato virus (TOMV) and Watermelon silver mottle virus (WTMV), Nairovirus: Crimean-Congo hemorrhagic fever virus (CCGV) and Dugbe virus (DUGV). Position of the start of the motif is labelled in blue for each virus. B, Based on secondary structure predictions topology diagrams were drawn for the NL1 domains from PUMV, RVFV, TOMV, HANV, CCGV. Colours are the same as in Figure 1D. N-terminal and C-terminal position are labelled with the aa position. The two red dots, the black dot and the green dot indicate the PD, E/D, and K residues, respectively, from the key active site. Principal conserved secondary structures are labelled as for LCMV NL1 domain.

(8.97 MB TIF)

Click here for additional data file.^{(8.5M, tif)}

Figure S3

Verification of L protein expression by immunoblotting. L protein used for analysis in replicon assay was tagged with HA tag, expressed under T7 promoter control in cells inoculated with modified vaccinia virus Ankara expressing T7 RNA polymerase (MVA-T7), and detected in immunoblot using anti-HA antibody. MVA, cells inoculated with MVA-T7 but not transfected; neg. ctrl., L mutant containing a mutation in the catalytic site of the RdRp.

(1.13 MB TIF)

Click here for additional data file.^{(1.0M, tif)}

Text S1

Supplementary Methods.

(0.08 MB DOC)

Click here for additional data file.^{(74K, doc)}

Acknowledgments

We thank Joëlle Boretto, Violaine Lantez, Karen Dalle for excellent technical assistance in preparing cDNAs and proteins used throughout, Lukas Flatz and Daniel Pinschewer for providing L and NP gene plasmids to facilitate construction of the LCMV mini-replicon system, Boris Klempa for helpful discussion and support on Bunyavirus work, Rolf Hilgenfeld and Hugues Tolou for sharing information and brainstorming at the initial phase of the project.

Footnotes

The authors have declared that no competing interests exist.

Toscana virus and H5N1 influenza endonuclease domains were produced as part of the European Virus Archive (EVA) project (European FP7 Capacities Project no 228292, http://www.european-virus-archive.com/). This work was supported in part by the VIZIER integrated project (LSHG-CT-2004-511960) of the European Union 6th Framework Programme (FP6), the Direction Générale de l'Armement (DGA 09cà402), the Fondation pour la Recherche Médicale (FRM-Aide aux équipes), the German Research Foundation grant GU 883/1-1, and a grant from the “Vereinigung der Freunde des Tropeninstituts Hamburg e.V.” to ML. JL was supported by grant 08/1/22/19/589 from BMRC and an ATIP from CNRS. We gratefully acknowledge the ID14-4 beamline staff and equipment at the European Syncrotron Radiation facility, Grenoble, France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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