Human metapneumovirus (HMPV for short) is a major cause of infections of the airways and lungs, particularly in children, elderly individuals and people with weakened immune systems. As for all viruses, HMPV cannot survive on its own. Instead, it must invade and hijack cells in order to replicate its own genetic material and form new viruses. In HMPV, this genetic information is in the form of a strand of RNA, and is protected by a shell-like structure called a nucleocapsid. Drugs that disrupt the nucleocapsid may therefore help to kill the viruses and treat the illnesses that they cause.

Nucleocapsids are built out of many copies of a protein called nucleoprotein, which binds to a strand of RNA. However, viral nucleocapsids can only be built from nucleoproteins that are bound to viral RNA. Potentially, nucleoproteins could instead bind to RNA belonging to the cells that HMPV infects and they would then be trapped in a dead-end state. To prevent this type of unproductive binding, before the nucleocapsid is formed the nucleoprotein is kept unassembled with the help of another protein called the polymerase cofactor. However, it was not clear exactly how the polymerase cofactor helps to maintain this unassembled state.

Using techniques called cryo-electron microscopy and X-ray crystallography, Renner et al. studied the structures formed when nucleoproteins are either bound to RNA or are unassembled and bind to the polymerase cofactor. Comparing these structures revealed that RNA normally binds to a specific cleft in the nucleoprotein. However, when nucleoprotein is bound to the polymerase cofactor a portion of the nucleoprotein folds into this cleft instead, blocking the insertion of RNA. This prevents the nucleoprotein from associating with the wrong RNA, allowing the nucleoprotein to remain in an unassembled state until it is needed for the virus.

Renner et al. also found that the interactions between the nucleoprotein and the polymerase cofactor of HMPV occur at sites that are also found in several other related viruses, such as Ebola. Targeting this common region could therefore be a good strategy for developing new antiviral drugs.

Viruses possessing a non-segmented, single-strand, negative-sense RNA genome are the causative agents of many serious human illnesses. Notable members belonging to this group of viruses (Mononegavirales) include measles, rabies, Ebola, respiratory syncytial virus (RSV) and Human metapneumovirus (HMPV). HMPV (Paramyxoviridae, subfamily Pneumovirinae) is a leading cause of serious respiratory tract infections in children, the elderly, and immunocompromised individuals (Boivin et al., 2003; Osterhaus and Fouchier, 2003; van den Hoogen et al., 2001). In all members of the Mononegavirales, the RNA genome is packaged in the form of a nucleocapsid, a ribonucleoprotein complex consisting of polymerized viral nucleoproteins (N) and RNA (Ruigrok et al., 2011). Besides protecting the viral genome from host nucleases, the nucleocapsid serves as the template for transcription by the viral RNA-dependent RNA polymerase L. Nucleocapsid assembly necessitates a pool of monomeric, RNA-free N, termed N⁰, which is kept in an unassembled state through an interaction with an N-terminal portion of the polymerase cofactor P, until delivered to the sites of viral RNA synthesis (Ruigrok et al., 2011; Curran et al., 1995; Mavrakis et al., 2006). The P protein is a multifunctional, modular protein containing large intrinsically disordered regions and is found to be tetrameric in HMPV (Leyrat et al., 2013). In addition, P binds to the nucleocapsid via its C–terminus, and mediates the attachment of the RNA-dependent RNA polymerase L. Furthermore in pneumoviruses, P recruits the processivity factor M2-1 (Leyrat et al., 2014). A great deal of effort has been spent on understanding the functions of P and recent crystal structures of P bound to N proteins (N⁰-P) from vesicular stomatitis virus (VSV), Ebola virus, Nipah virus, and measles virus have highlighted its role in preventing assembly of N by blocking the C-terminal and N-terminal extensions of N (CTD-arm and NTD-arm) which facilitate N oligomerization (Leyrat et al., 2011; Guryanov et al., 2015; Leung et al., 2015; Yabukarski et al., 2014). However, there is still paucity in our understanding of the molecular details behind the proposed mechanisms, specifically regarding how P-bound N is released, attaches to the nucleocapsid and is loaded with RNA. To address these questions in the mechanism of N-chaperoning by P and nucleocapsid assembly we performed a structural analysis of assembled and unassembled N from HMPV. Our structure of N⁰-P reveals a conformational change, in which the negatively charged CTD-arm of N occupies the positively charged RNA binding site via specific and conserved interactions. Together with our RNA-bound structure of N these data imply a mechanism of how the growth of nucleocapsid filaments is coordinated in HMPV and related viruses.

Biochemical studies of the nucleocapsid building block N are complicated by the fact that N proteins have a strong tendency to irreversibly oligomerize and bind host nucleic acids immediately upon recombinant expression (Gutsche et al., 2015; Tawar et al., 2009). One technique to mitigate this problem is to truncate regions of N that facilitate oligomerization (Yabukarski et al., 2014). To stabilize monomeric full-length N⁰ we fused the N-terminal domain of P to N, a strategy that has seen success with nucleoproteins from other viruses (Guryanov et al., 2015; Kirchdoerfer et al., 2015). We obtained crystals of RNA-free HMPV N in a monomeric state and bound to a P peptide at 1.9 Å resolution by adding trace amounts of trypsin (Dong et al., 2007) to prune flexible loops and promote crystallization (Figure 1—figure supplement 1 and Table 1). In the structure, the P peptide is firmly nestled into a hydrophobic surface of the C-terminal domain of N (CTD) primarily composed of α-helices αC1 and αC2 (Figure 1A,B). Ile9, Leu10 and Phe11 of P occupy key positions and insert into this hydrophobic groove (Figure 1B). Unlike the N⁰-P structure recently reported for measles (Guryanov et al., 2015), we find that the linker connecting N and P in our chimeric construct has been cleaved prior to crystal growth. The P peptide wraps around the CTD and residues 12–28 form an alpha helix that lies atop N (Figure 1B). This helix is initiated at Gly12 and pinned to the CTD through an aromatic side-to-face interaction of Phe23 with Tyr354 of N, both residues belonging to the so-called mir motif which is conserved within Pneumovirinae (Karlin and Belshaw, 2012). This result is consistent with an earlier study, in which alanine mutations of the corresponding residues in respiratory syncytial virus resulted in a drop of polymerase activity by more than 75% in a minireplicon system (Galloux et al., 2015).

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Alignment of Paramyxoviridae N sequences revealed that many hydrophobic residues lining the P-binding surface of αC1 through αC2 are shared within the family (Figure 1C). For all known N⁰-P complexes (Leyrat et al., 2011; Leung et al., 2015; Yabukarski et al., 2014), P binds to the CTD of N (Figure 1D). Interestingly, although the specific interaction sites diverge (Figure 1D, indicated by white and black arrows), a sub-region of the CTD (Figure 1D, indicated by dotted circle) is bound by P in all structures, indicating that it is widely conserved throughout Mononegavirales.

To provide a rationale for the molecular switching between the monomeric, P-bound state and the assembled, RNA-bound state, a direct comparison at the atomic level is necessary. To this end, we purified and crystallized assembled HMPV N in the form of a decameric N-RNA ring (Figure 2—figure supplement 1 and Table 1). By exploiting the ten-fold non-crystallographic symmetry in the rings, we were able to obtain excellent electron density maps at 4.2-Å resolution (Figure 2—figure supplement 2A–C) and build a reliable model (Karplus and Diederichs, 2012) (Figure 2—figure supplement 2D). Assembled HMPV decameric N-RNA rings are ~0.5 MDa in molecular mass and 160 Å in diameter and 70 Å in height (Figure 2A). The observed RNA binding mode is similar to that seen in the related RSV N-RNA structure (Tawar et al., 2009). The RNA wraps around the N ring and wedges tightly in the cleft between the NTD and CTD of N, which is lined by positively charged residues (Figure 2A and Figure 2—figure supplement 3). In members of the Paramyxovirinae, the number of nucleotides in the viral genome is required to be a multiple of six (Calain and Roux, 1993) and the structural basis for this so-called rule of six has been elucidated recently (Gutsche et al., 2015). In members of the Pneumovirinae, however, this rule is not observed (Tawar et al., 2009). Our structure further highlights this difference; with each N subunit contacting seven RNA nucleotides (Figure 2—figure supplement 2C and Figure 2—figure supplement 3B).

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Similar to N proteins from other members of Mononegavirales (Tawar et al., 2009; Alayyoubi et al., 2015; Albertini et al., 2006; Green et al., 2006), the NTD- and CTD-arms grasp the neighbouring protomers, thus facilitating assembly of polymeric N (Figure 2B). The NTD-arm packs against the flank of the previous protomer (Figure 2B, the NTD-arm of N_i+1 packs against N_i). The CTD-arm in turn latches onto the top of the CTD of the next protomer (Figure 2B, CTD-arm of N_i-1 latches onto CTD of N_i). We observed that the binding site of the P peptide overlaps with the binding sites of the NTD- and CTD-arms (Figure 2C). Our structures thus provide conclusive evidence that P hampers subdomain exchange between adjacent proteins in Pneumovirinae. This mechanism has also been proposed for a range of viruses thoughout Mononegavirales (Leyrat et al., 2011; Guryanov et al., 2015; Yabukarski et al., 2014; Alayyoubi et al., 2015) and there is mounting evidence that it may be universal throughout the entire viral order.

A hinge-like motion has been proposed by which N alternates between an open, RNA-free conformation (N⁰) and a closed RNA-bound (N-RNA) conformation (Guryanov et al., 2015; Yabukarski et al., 2014). Comparison of these two states for HMPV reveals a rigid body movement of the NTD relative to the CTD (Figure 2D). The conformational change rotates the NTD towards the CTD by 10°, the interface between the two domains acting as a hinge. At the interface, hinge residues Thr257 and Ala254 play a particularly crucial role. In the open, RNA-free state the hinge is maintained in a helical conformation by stabilization of Ala254 through the side chain of Thr257 and an additional backbone interaction with Thr175 (Figure 2E). Upon RNA binding, Thr257 contacts the backbone of a nucleotide instead of stabilizing Ala254 (Figure 2F). In addition, the loop containing Thr157 retracts to sterically accommodate the RNA chain. Having lost the stabilizing contacts of Thr257 and Thr157, the helical hinge region around Ala254 unravels and becomes flexible (Figure 2F, indicated by white arrow), allowing the relative domain motions of NTD and CTD. Furthermore, we propose that Tyr252 is important in facilitating the hinge motion. Tyr252 is positioned just before the pivot point and packs tightly against αC3 (Figure 2—figure supplement 4A). An aromatic residue at this position is found packing against the same helix in most known structures of N (Figure 2—figure supplement 4B–G). Transition from the RNA-free to RNA-bound state induces a rotation of αC3, exerting upwards pressure on Tyr252 that is conferred onto the NTD (Figure 2—figure supplement 4A). Intriguingly, in structures of Paramyxovirinae N, which obey the rule-of-six, this aromatic is flipped in the opposite direction (Figure 2—figure supplement 4H,I) and contacts RNA (Gutsche et al., 2015), suggesting a similar coupling of RNA-binding and hinge-motion in these viruses.

The most profound changes between assembled and unassembled states, however, involve the CTD-arm of N, a region that has been little characterized in pneumoviruses. In the polymeric, RNA-bound state of N (N-RNA) the CTD-arm flips upwards and latches onto the next protomer, whilst in the monomeric state (N⁰) it packs down against the core of N (Figure 3A). The downward, monomeric conformation is stabilized by specific salt-bridges linking the CTD-arm with the core of N (Figure 3B). In this position the negatively charged CTD-arm folds into the positively charged RNA binding cleft, occupying it and directly blocking the binding of RNA (Figure 3A). It is interesting to note, that whilst the CTD-arm blocks the RNA site in HMPV, it is the P peptide that inserts itself there in VSV (Leyrat et al., 2011). Because this is not observed in paramyxoviral N⁰-P complexes (Guryanov et al., 2015; Yabukarski et al., 2014) we hypothesize that, in Rhabdoviridae, a different strategy has evolved to block off the RNA binding cleft. The question arises how the interactions that hold the downwards-positioned CTD-arm in place are broken when assembly of N-RNA necessitates it flipping into the upwards position. In the RNA-free state, Arg260 and Trp261 contact Glu375, while Arg186 forms a salt-bridge with Asp373 of the CTD-arm (Figure 3B). In the assembled, RNA-bound state these interactions are broken, with Arg186 and Trp261 now positioning RNA nucleotides in the cleft, whilst Arg260 instead fastens onto the NTD-arm of the neighbouring N_i+1 (Figure 3C). The shift from initial stabilization of the inhibitory (downwards) CTD-arm conformation to stabilization of bound RNA and neighbouring N subunit implies that attachment of a new N protomer and insertion of nascent RNA occur concomitantly. This makes sense in the context of viral replication sites, where tetrameric P proteins act as molecular chaperones attaching to the nucleocapsid template, polymerase and free N⁰, leading to high local concentrations of nucleoprotein and RNA.

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Based on the comparison of our N-RNA and N⁰-P structures we suggest a model for nucleocapsid growth (Figure 3D). Upon delivery of fresh N⁰-P to the growth site, addition of the next N protomer (N_i+1) to the filament necessitates that the CTD-arm of the terminal N_i unbinds and flips upwards (Figure 3D, indicated by dotted arrow), latching onto N_i+1 and displacing P. In our model, this is driven by the formation of new interactions to the NTD- and CTD arms and, importantly, the concerted insertion of nascent RNA into the RNA binding cleft of N_i, with the CTD-arm switching into the upward conformation. In this model the growth of the filament is reminiscent of a zipper closing up with one row of teeth corresponding to nascent viral RNA and the other to newly delivered N subunits which interdigitate in a fluid, concerted motion. The notion that concerted RNA insertion is required for the hand-over of N subunits from P lends additional specificity to the nucleocapsid polymerization reaction.

We hypothesized that the role of the CTD-arm in inhibiting premature RNA binding may be conserved and therefore compared sequences throughout Paramyxoviridae (Figure 3E). We find a semi-conserved LGLT-motif within the CTD-arms which is followed by a stretch of residues with helical propensity. The beginning of this stretch preferentially features negatively charged residues at positions equivalent to HMPV which may in turn pack against the complementary charges of the RNA binding cleft. Indeed, analysis of structures of more distantly related members of Mononegavirales shows that these negatively charged residues are topologically conserved and that a switch to the downward conformation would position these residues into the RNA binding cleft (Figure 3—figure supplement 1).

In conclusion, the reported structures of a paramyxoviral N protein reveal two distinct conformational states, N bound either to the polymerase cofactor P or to RNA. A direct comparison of these two structures provides a molecular level rationale for how nucleocapsid assembly is controlled through P by sterically blocking the binding sites of the NTD- and CTD-arms. In addition, this work elucidates a key role of the CTD-arm in hindering premature RNA insertion into the binding cleft, thus presenting a mechanistic explanation of how premature RNA uptake is directly inhibited in Paramyxoviridae. Peptides of the N⁰-binding region of P have previously been shown to inhibit replication activity in RSV (Galloux et al., 2015), Nipah virus (Yabukarski et al., 2014), and rabies virus (Castel et al., 2009). The characterization of P-binding surfaces on N proteins is therefore of biomedical importance as these surfaces constitute genuine targets for the development of antivirals.

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Author details

1. Max Renner

   Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   MR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Mattia Bertinelli

   Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   MB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Cédric Leyrat

   Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   CL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Guido C Paesen

   Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   GCP, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Laura Freitas Saraiva de Oliveira

   Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   LFSdO, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Juha T Huiskonen

   Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   JTH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

7. Jonathan M Grimes

   1. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
   2. Diamond Light Source, Didcot, United Kingdom

   Contribution

   JMG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   jonathan@strubi.ox.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-9698-0389

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