The Nucleocapsid of Paramyxoviruses: Structure and Function of an Encapsidated Template

Viruses of the Paramyxoviridae family share a common and complex molecular machinery for transcribing and replicating their genomes. Their non-segmented, negative-strand RNA genome is encased in a tight homopolymer of viral nucleoproteins (N). This ribonucleoprotein complex, termed a nucleocapsid, is the template of the viral polymerase complex made of the large protein (L) and its co-factor, the phosphoprotein (P). This review summarizes the current knowledge on several aspects of paramyxovirus transcription and replication, including structural and functional data on (1) the architecture of the nucleocapsid (structure of the nucleoprotein, interprotomer contacts, interaction with RNA, and organization of the disordered C-terminal tail of N), (2) the encapsidation of the genomic RNAs (structure of the nucleoprotein in complex with its chaperon P and kinetics of RNA encapsidation in vitro), and (3) the use of the nucleocapsid as a template for the polymerase complex (release of the encased RNA and interaction network allowing the progress of the polymerase complex). Finally, this review presents models of paramyxovirus transcription and replication.


Introduction
Members of the Paramyxoviridae family are enveloped viruses with non-segmented negative-strand RNA genomes. These viruses share similar gene expression and genome replication mechanisms to the members of the other ten families of the Mononegavirales order. Paramyxoviruses are currently divided into 4 subfamilies, 17 genera, and 78 species (Figure 1) [1] (information as of November 2021, for an update see https://talk.ictvonline. org/taxonomy/). Paramyxoviruses are found in a broad range of animals, including mammals, fishes, reptiles, or birds and include several human and animal pathogens such as measles virus (MeV), mumps virus (MuV), canine distemper virus (CDV), and Newcastle disease virus (NDV). Some, such as Nipah virus (NiV) and Hendra virus (HeV), regularly emerge in human or animal populations where they cause deadly epidemics. Although vaccines exist against some paramyxoviruses, effective antiviral treatments for post-exposure prophylaxis are urgently needed [2,3].
Viral transcription and replication are both mediated by an RNA synthesis machinery composed of a viral polymerase complex and its encapsidated template. The polymerase complex consists of the large protein (L) and its co-factor the phosphoprotein (P) [4][5][6][7]. This complex uses a genome (or antigenome) embedded into a homopolymer of viral nucleoproteins (N) as a template [8][9][10]. The encapsidation of the RNA in this ribonucleoprotein structure, known as the nucleocapsid, prevents the annealing of positive and negative RNA strands into double-stranded RNA structures, as well as the folding of genomic RNA into secondary RNA structures, which helps to prevent recognition by innate immune receptors [11,12]. It also protects the genome from degradation by nucleases [13,14] and from being targeted by siRNA [15,16]. Viral transcription and replication are both mediated by an RNA synthesis machinery composed of a viral polymerase complex and its encapsidated template. The polymerase complex consists of the large protein (L) and its co-factor the phosphoprotein (P) [4][5][6][7]. This complex uses a genome (or antigenome) embedded into a homopolymer of viral nucleoproteins (N) as a template [8][9][10]. The encapsidation of the RNA in this ribonucleoprotein structure, known as the nucleocapsid, prevents the annealing of positive and negative RNA strands into double-stranded RNA structures, as well as the folding of genomic RNA into secondary RNA structures, which helps to prevent recognition by innate immune receptors [11,12]. It also protects the genome from degradation by nucleases [13,14] and from being targeted by siRNA [15,16].
The mechanisms used by the polymerase complex to access the encased RNA, progress along the nucleocapsid, and generate new encapsidated products with soluble nucleoprotein monomers are yet to be fully deciphered. However, in the past few years, the atomic structures of the nucleoprotein of a number of paramyxoviruses from different genera have been solved in either assembled [17][18][19][20][21][22][23] or non-assembled form [24][25][26][27] (Figure 1). Moreover, since the atomic structure of the polymerase complex of parainfluenza   The genome contains at least six conserved adjacent genes separated by intergenic regions (IR). These genes encode for the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the receptor-binding protein (RBP), and the large protein (L). The promoters located at the 3 end of the genome (transcription and replication) and the antigenome (replication only) are bipartite and made of two promoter elements (PE1 and PE2). The transcription of each gene starts on a "gene start" signal (GS, in yellow) and ends on a "gene end" signal (GE, orange). (B) Cartoon representation of the structure of the protein components of the RNA synthesis machinery of PIV5 (N, PDB: 4XJN; P NTD , PDB: 5WKN; P OD , P XD , and L, PDB: 6V85). Disordered regions are represented as dotted lines. (C) Schematic representation of viral transcription and replication. Encapsidated genomes are transcribed into a gradient of mRNAs. Replication of the genomes requires the production of encapsidated antigenome intermediates.

Components of the RNA Synthesis Machinery
The nucleoproteins of paramyxoviruses are made of a folded domain (N core ) followed by a long intrinsically disordered tail (N tail ) [34] ( Figure 2B). While N core is responsible for the oligomerization on the RNA and constitutes the main body of the nucleocapsid [35][36][37][38], N tail mediates the recruitment of the polymerase complex [34,39,40]. The nucleoproteins self-assemble on the genome due to their affinity for RNA and the interactions between adjacent protomers, as discussed later.
The L protein is a large, multi-domain protein, made of five globular domains linked together by flexible linkers [28,41] (for a review see [42,43]) ( Figure 2B). It carries all the enzymatic activities required for transcribing and replicating the genome: RNA synthesis [5,6], mRNA cap addition [44,45], guanine-N-7-methylation of the cap, and ribose 2 O-methylation of the first nucleotide [46,47]. Polyadenylation of the mRNA occurs when the polymerase stutters on a short poly(U) sequence located in a polyadenylation signal [48,49].

Transcription and Replication
Following fusion of viral and host cell membranes during entry, the polymerase complexes packaged in virions transcribe the viral genes into capped and polyadenylated mRNAs ( Figure 2C) [31]. The viral genes are separated by short non-transcribed intergenic regions and contain a gene start (GS) and a gene end (GE) signal at their 3 and 5 ends, respectively ( Figure 2A). The current model suggests that RNA synthesis starts at the 3 end of the genome where the polymerase recognizes the transcription promoter. The L protein first synthesizes the leader RNA, a short, uncapped RNA, which is released upon recognition of the first intergenic region. The polymerase then scans the genome to find the first GS signal and re-initiates RNA synthesis. The nascent RNA is capped and methylated by the L protein. Upon recognition of the GE signal, the polymerase adds a poly(A) tail, releases the mRNA, and scans the genome to find the next gene start signal. As the transcription re-initiation is not perfectly efficient, the polymerase generates a decreasing gradient of mRNAs ( Figure 2C).
Once the concentration of nucleoproteins reaches a certain threshold, the polymerase can switch from a transcription mode to a replication mode [76,77]. Although the accumulation of N proteins may not be the only trigger, artificially increasing the amount of N proteins advances the onset of replication [77,78]. Initiation of replication occurs when the nascent leader RNA is encapsidated by monomers of N and the polymerase ignores the transcription signals, instead synthesizing a full-length encapsidated copy of the genome (antigenome) [76,79]. This antigenome is then used as a template to generate full-length encapsidated copies of the viral genome ( Figure 2C). Viral transcription and replication take place in the cytoplasm in membraneless inclusion bodies. Viral genomes, together with N, P, and L proteins, are concentrated in these inclusions formed by liquid-liquid phase separations. The disordered regions of the N and Viruses 2021, 13, 2465 5 of 28 P proteins play a critical role in the formation of these liquid-like structures (for a review, see [80][81][82]).

Overall Architecture
Nucleocapsids extracted from disrupted virions or infected cells form left-handed, rodlike helical structures with a herringbone appearance under an electron-microscope [107][108][109][110] ( Figure 3A). The coiling and rigidity of the helix depend on the conditions of the milieu, such as the ionic strength [111]. Indeed, while, at low salt concentration, the nucleocapsids are loose and flexible, and at high ionic strength, the helixes are tight and rigid with a length of about 1 µm and a diameter of 15-20 nm. The flexibility also varies from one virus to another, with the nucleocapsids of MuV being more flexible than the nucleocapsids of MeV, themselves more flexible than the nucleocapsids of SeV or PIV5 [109,112]. Purified intact nucleocapsids from the same virus, or even sections of a single nucleocapsid, can also adopt condensed conformations with different pitches ranging from 5.3 to 6.8 nm for SeV [110], 5.2 to 6.6 nm for MeV [113], or 5.8 to 6.7 nm for MuV [114] ( Figure 3C). A smaller pitch correlates with a more rigid and straight helix [112]. In intact MeV particles, the nucleocapsids have a higher pitch (7-8 nm), thus raising the question of whether a preferred pitch exists in physiological conditions and which value it adopts [115]. The average number of N subunits per helical turn is relatively conserved and varies from 12.3 to 13.4 [18,110,113,114]. Removal of the encapsidated RNA from MuV nucleocapsid increases the flexibility of the helix but does not affect its helical conformation, indicating that the nucleocapsid assembly is stable even without RNA [114].   Expression of the nucleoprotein in avian cells [118], mammalian cells [36], insect cells [119], or bacteria [120], induces the formation of nucleocapsid-like complexes (NCLC) containing cellular RNAs. These complexes can form helices [118,121], rings [17,122], or clamshaped structures [19,21,22] (Figure 3B-D). The last has been observed for NDV, SeV, and NiV, and is made of two helical turns interacting in a back-to-back manner ( Figure 3D). The detection of this structure for paramyxoviruses from different genera and its presence in nucleocapsids extracted from NDV and SeV virions, suggests it may be biologically relevant [19,21]. The clam-shaped structure has been proposed to play a role in seeding the assembly of double-headed helices, protecting the 5′ end of the genome from nucleases, and promoting the encapsidation of multiple nucleocapsids per virion [123][124][125].

Structure of the Nucleoprotein
The sequence and the organization of Ncore are well conserved across the Paramyxoviridae family, while the sequence and length of Ntail vary greatly ( Figure 4A). The structure of the N protein in an oligomeric, RNA-bound conformation (i.e., engaged in an NCLC) have been solved for PIV5 [17], MeV [18,20], NDV [19], cetacean morbillivirus (CeMV) [23], NiV [22], SeV [21], and MuV [126], while the structure of N in complex with Expression of the nucleoprotein in avian cells [118], mammalian cells [36], insect cells [119], or bacteria [120], induces the formation of nucleocapsid-like complexes (NCLC) containing cellular RNAs. These complexes can form helices [118,121], rings [17,122], or clam-shaped structures [19,21,22] (Figure 3B-D). The last has been observed for NDV, SeV, and NiV, and is made of two helical turns interacting in a back-to-back manner ( Figure 3D). The detection of this structure for paramyxoviruses from different genera and its presence in nucleocapsids extracted from NDV and SeV virions, suggests it may be biologically relevant [19,21]. The clam-shaped structure has been proposed to play a role in seeding the assembly of double-headed helices, protecting the 5 end of the genome from nucleases, and promoting the encapsidation of multiple nucleocapsids per virion [123][124][125].

Structure of the Nucleoprotein
The sequence and the organization of N core are well conserved across the Paramyxoviridae family, while the sequence and length of N tail vary greatly ( Figure 4A). The structure of the N protein in an oligomeric, RNA-bound conformation (i.e., engaged in an NCLC) have been solved for PIV5 [17], MeV [18,20], NDV [19], cetacean morbillivirus (CeMV) [23], NiV [22], SeV [21], and MuV [126], while the structure of N in complex with P NTD (i.e., N 0 -P complex) was solved for NiV [24], MeV [25], PIV5 [26], and hPIV3 [27] (Figure 1). PNTD (i.e., N 0 -P complex) was solved for NiV [24], MeV [25], PIV5 [26], and hPIV3 [27] ( Figure 1).  [71,73], NiV [74,127], and SeV Ncore consists of two globular domains, the N-terminal (NNTD) and C-terminal domains (NCTD), which form a positively charged cavity to accommodate the RNA ( Figure  4B,C). These domains are preceded and followed by an N-terminal and a C-terminal arm (NNTDarm and NCTDarm) connected by flexible linkers; these arms are responsible for the selfassembly of N protomers onto the RNA. The superposition of the structures of the N proteins of paramyxoviruses shows high conservation of the tertiary and secondary structures ( Figure 4D). The global architecture is also well conserved among members of the Mononegavirales order, since the N proteins of respiratory syncytial virus (RSV) [129,130] and human metapneumovirus [131] (two pneumoviruses), Ebola virus [132][133][134][135][136] and Marburg virus [137] (two filoviruses), Borna disease virus [138] (a bornavirus), vesicular stomatitis Indiana virus (VSIV) [139] and rabies virus [140] (two rhabdoviruses) have similar structures and modes of self-assembly (for a review see [141]) ( Figure 4E). N core consists of two globular domains, the N-terminal (N NTD ) and C-terminal domains (N CTD ), which form a positively charged cavity to accommodate the RNA ( Figure 4B,C). These domains are preceded and followed by an N-terminal and a C-terminal arm (N NTDarm and N CTDarm ) connected by flexible linkers; these arms are responsible for the self-assembly of N protomers onto the RNA. The superposition of the structures of the N proteins of paramyxoviruses shows high conservation of the tertiary and secondary structures ( Figure 4D). The global architecture is also well conserved among members of the Mononegavirales order, since the N proteins of respiratory syncytial virus (RSV) [129,130] and human metapneumovirus [131] (two pneumoviruses), Ebola virus [132][133][134][135][136] and Marburg virus [137] (two filoviruses), Borna disease virus [138] (a bornavirus), vesicular stomatitis Indiana virus (VSIV) [139] and rabies virus [140] (two rhabdoviruses) have similar structures and modes of self-assembly (for a review see [141]) ( Figure 4E).

Interactions between Protomers
Each N protomer forms tight connections with its neighbors via its two arms protruding on each side of the N protein ( Figure 5A). On one side, the N NTDarm of the N i protomer forms an alpha helix that binds a hydrophobic groove on the back of the N CTD of the N i-1 Viruses 2021, 13, 2465 8 of 28 protomer ( Figure 5B(i)). On the other side, the N CTDarm folds in two alpha helixes interacting with the top of the N CTD of the N i+1 protomer ( Figure 5B(ii)). A third tight connection has been described between an extended loop of the N NTD of the N i protomer that enters into a hole formed by the N NTD and N NTDarm of the N i+1 protomer ( Figure 5B(iii)) [21,126]. In addition to these interactions, there are large contact areas between adjacent protomers with numerous additional bonds [23].

Interactions Between Protomers
Each N protomer forms tight connections with its neighbors via its two arms protruding on each side of the N protein ( Figure 5A). On one side, the NNTDarm of the Ni protomer forms an alpha helix that binds a hydrophobic groove on the back of the NCTD of the Ni-1 protomer ( Figure 5B(i)). On the other side, the NCTDarm folds in two alpha helixes interacting with the top of the NCTD of the Ni+1 protomer ( Figure 5B(ii)). A third tight connection has been described between an extended loop of the NNTD of the Ni protomer that enters into a hole formed by the NNTD and NNTDarm of the Ni+1 protomer ( Figure 5B(iii)) [21,126]. In addition to these interactions, there are large contact areas between adjacent protomers with numerous additional bonds [23]. These N-N interactions curve the N oligomer so that the RNA is exposed on the outside of the helix (or ring) and all the tight inter-protomer connections are hidden inside the helix ( Figure 5C) [17,18,117]. This conformation is similar to that observed in the nucleocapsids of filoviruses [134] and pneumoviruses [130], but opposite to the one found in rhabdoviruses [142,143] where the RNA is located in the interior of the helical nucleocapsid.

Interactions with RNA
The genomic RNA is inserted in a cavity formed by the NNTD and the NCTD ( Figure  6A,B). Each N protomer binds six nucleotides in agreement with the rule of six [17,18]. The RNA is mainly stabilized by interactions between positively charged residues and the RNA backbone, thus explaining the low sequence specificity of encapsidation ( Figure 6A). The RNA adopts a regular conformation with a three-base stack oriented outwards to the solvent (bases "out") and the following three-base stack facing toward the protein (bases "in") ( Figure 6B). These N-N interactions curve the N oligomer so that the RNA is exposed on the outside of the helix (or ring) and all the tight inter-protomer connections are hidden inside the helix ( Figure 5C) [17,18,117]. This conformation is similar to that observed in the nucleocapsids of filoviruses [134] and pneumoviruses [130], but opposite to the one found in rhabdoviruses [142,143] where the RNA is located in the interior of the helical nucleocapsid.

Interactions with RNA
The genomic RNA is inserted in a cavity formed by the N NTD and the N CTD ( Figure 6A,B). Each N protomer binds six nucleotides in agreement with the rule of six [17,18]. The RNA is mainly stabilized by interactions between positively charged residues and the RNA backbone, thus explaining the low sequence specificity of encapsidation ( Figure 6A). The RNA adopts a regular conformation with a three-base stack oriented outwards to the solvent (bases "out") and the following three-base stack facing toward the protein (bases "in") ( Figure 6B). Using recombinant N proteins fused to a cleavable PNTD, NCLCs were generated in vitro after cleavage of the PNTD and incubation with either a six-nucleotide-long poly(A) RNA or an RNA corresponding to the first six nucleotides of the viral genome [20,23]. For both MeV and CeMV, NCLCs adopt a helical conformation with the RNA hexamer in the same phase register. These structures suggest that the first two nucleotides of the viral genome point toward the solvent and that the 3′ end of the genome is accessible to the viral polymerase ( Figure 6B). This register is in agreement with data showing that cytidines at positions 1 and 6, which are both in "out" positions, are more sensitive to chemical modifications [144].
Paramyxoviruses have bipartite promoters [145][146][147][148] (for a review see [30]). The first promoter element (PE1) contains at least the first twelve nucleotides of the genome ( Figure  2A). The second promoter element (PE2) is composed of three successive hexamer sequences: 3′-CNNNNN-5′ located at positions 14 to 16 (in the hexamer register) for orthoparamyxoviruses [146], and 3′-NNNNCG-5′ at position 13 to 15 for rubulaviruses [147] and avulaviruses [148]. According to the RNA register described for MeV and the average of 13 N per turn for native nucleocapsids, PE2 is located in the successive turn of the helical nucleocapsid just under PE1 in the helical conformation of the nucleocapsid ( Figure  6C). Moreover, the conserved nucleotides of the two types of PE2 are facing the solvent, thus reinforcing the idea that these bases could be sensed by the polymerase complex and participate in promoting RNA synthesis.
The side chain of the residue Q202 of the nucleoproteins of PIV5 and MeV interacts with the base of the nucleotide in position six, and thus the first nucleotide of the promoter [17,20] (Figure 6A). In a cell-based minigenome assay, mutation Q202A of the N protein of human parainfluenza virus 2 allows the transcription and replication of the minigenome, irrespectively of whether its length is a multiple of six and the presence of the PE2 Using recombinant N proteins fused to a cleavable P NTD , NCLCs were generated in vitro after cleavage of the P NTD and incubation with either a six-nucleotide-long poly(A) RNA or an RNA corresponding to the first six nucleotides of the viral genome [20,23]. For both MeV and CeMV, NCLCs adopt a helical conformation with the RNA hexamer in the same phase register. These structures suggest that the first two nucleotides of the viral genome point toward the solvent and that the 3 end of the genome is accessible to the viral polymerase ( Figure 6B). This register is in agreement with data showing that cytidines at positions 1 and 6, which are both in "out" positions, are more sensitive to chemical modifications [144].
Paramyxoviruses have bipartite promoters [145][146][147][148] (for a review see [30]). The first promoter element (PE1) contains at least the first twelve nucleotides of the genome (Figure 2A). The second promoter element (PE2) is composed of three successive hexamer sequences: 3 -CNNNNN-5 located at positions 14 to 16 (in the hexamer register) for orthoparamyxoviruses [146], and 3 -NNNNCG-5 at position 13 to 15 for rubulaviruses [147] and avulaviruses [148]. According to the RNA register described for MeV and the average of 13 N per turn for native nucleocapsids, PE2 is located in the successive turn of the helical nucleocapsid just under PE1 in the helical conformation of the nucleocapsid ( Figure 6C). Moreover, the conserved nucleotides of the two types of PE2 are facing the solvent, thus reinforcing the idea that these bases could be sensed by the polymerase complex and participate in promoting RNA synthesis.
The side chain of the residue Q202 of the nucleoproteins of PIV5 and MeV interacts with the base of the nucleotide in position six, and thus the first nucleotide of the promoter [17,20] (Figure 6A). In a cell-based minigenome assay, mutation Q202A of the N protein of human parainfluenza virus 2 allows the transcription and replication of the minigenome, irrespectively of whether its length is a multiple of six and the presence of the PE2 element [149,150]. The authors suggest that residue Q202 may stabilize the first nucleotide of the genome, thus inhibiting the initiation of RNA synthesis by the viral polymerase. PE2 would then promote RNA synthesis initiation by stabilizing the polymerase complex on the promoter. Since the function of PE2 depends on its phase register, the requirement of PE2 would ensure the conservation of the rule of six [149,150].
In addition to its impact on the promoters, the phase of the RNA bases also affects the transcription signals. Indeed, during transcription, the P messenger RNA of paramyxoviruses can be edited by the viral polymerase which adds one or several extra G residues by stuttering along a short stretch of C residues [151]. Incremental modifications of the phase register of the editing site modify the efficiency of the editing and the number of residues added [144]. Moreover, within each genus, the gene start signals are found in a few preferred phases, suggesting the phasing of the gene start signal may also affect the efficiency of the re-initiation of transcription at gene junctions [33]. Overall, these results show that the imprint of the N protomers on the RNA influences how the viral polymerase uses its template.

Position and Influence of N tail within the Nucleocapsid
N tail belongs to the group of pre-molten globules within the class of intrinsically disordered proteins [34,57,59]. Although N core is sufficient to generate the helical core of the nucleocapsid [35,36,38], N tail influences the conformation of the helix. Indeed, treatment of the nucleocapsid or NCLC by trypsin cleaves off N tail , reduces the pitch, and tightens the helix [21,34,113,117,121] (Figure 3B). Moreover, while the expression of the full-length N protein of MuV generates NCLCs in a ring conformation, the cleavage of N tail generates helical structures [126].
In agreement with its disordered nature, N tail is not visible by electron microscopy [34,113,126]. Nuclear magnetic resonance spectroscopy and small-angle scattering showed that N tail escapes from the nucleocapsid core toward the exterior between two helical turns [152] ( Figure 7A,B), in agreement with the location of epitopes recognized by antibodies targeting N tail [153]. While the first 50 residues of N tail are spatially constrained by the helical turns of the nucleocapsid core, the C-terminal residues have high angular freedom [75,127,152]. This model is further supported by the recently solved structure of the NCLCs formed by SeV N proteins [21]. After cleavage of most of N tail , the nucleocapsids were found to become more rigid and allowed a resolution of 2.9 Å. The atomic model was reconstituted up to residue 414, thus including the first thirteen residues of the tail (aa 402-414). The residues bind the surface of N CTD and point toward the exterior of the nucleocapsid core ( Figure 7A).  [149,150]. The authors suggest that residue Q202 may stabilize the first nucleotide of the genome, thus inhibiting the initiation of RNA synthesis by the viral polymerase. PE2 would then promote RNA synthesis initiation by stabilizing the polymerase complex on the promoter. Since the function of PE2 depends on its phase register, the requirement of PE2 would ensure the conservation of the rule of six [149,150].
In addition to its impact on the promoters, the phase of the RNA bases also affects the transcription signals. Indeed, during transcription, the P messenger RNA of paramyxoviruses can be edited by the viral polymerase which adds one or several extra G residues by stuttering along a short stretch of C residues [151]. Incremental modifications of the phase register of the editing site modify the efficiency of the editing and the number of residues added [144]. Moreover, within each genus, the gene start signals are found in a few preferred phases, suggesting the phasing of the gene start signal may also affect the efficiency of the re-initiation of transcription at gene junctions [33]. Overall, these results show that the imprint of the N protomers on the RNA influences how the viral polymerase uses its template.

Position and Influence of Ntail within the Nucleocapsid
Ntail belongs to the group of pre-molten globules within the class of intrinsically disordered proteins [34,57,59]. Although Ncore is sufficient to generate the helical core of the nucleocapsid [35,36,38], Ntail influences the conformation of the helix. Indeed, treatment of the nucleocapsid or NCLC by trypsin cleaves off Ntail, reduces the pitch, and tightens the helix [21,34,113,117,121] (Figure 3B). Moreover, while the expression of the full-length N protein of MuV generates NCLCs in a ring conformation, the cleavage of Ntail generates helical structures [126].
In agreement with its disordered nature, Ntail is not visible by electron microscopy [34,113,126]. Nuclear magnetic resonance spectroscopy and small-angle scattering showed that Ntail escapes from the nucleocapsid core toward the exterior between two helical turns [152] (Figure 7A,B), in agreement with the location of epitopes recognized by antibodies targeting Ntail [153]. While the first 50 residues of Ntail are spatially constrained by the helical turns of the nucleocapsid core, the C-terminal residues have high angular freedom [75,127,152]. This model is further supported by the recently solved structure of the NCLCs formed by SeV N proteins [21]. After cleavage of most of Ntail, the nucleocapsids were found to become more rigid and allowed a resolution of 2.9 Å. The atomic model was reconstituted up to residue 414, thus including the first thirteen residues of the tail (aa 402-414). The residues bind the surface of NCTD and point toward the exterior of the nucleocapsid core ( Figure 7A).  N tail is thought to mediate the contact between the nucleocapsid and its environment. While N tail is not required for the encapsidation of the RNA, it is essential for RNA synthesis [37,154]. Indeed, N tail contains an α-helical molecular recognition element (α-MoRE) which folds upon binding to the C-terminal domain of the phosphoprotein (P XD ) and thus participates in the recruitment of the polymerase complex on its template [39,40,71,[73][74][75]127,128,153,[155][156][157] (Figure 7C). In addition to its interaction with P XD , N tail also binds the matrix protein, thus promoting the packaging of the nucleocapsids into viral particles [158][159][160][161][162]. N tail also mediates the interaction with several cellular factors [61], including heat shock protein 70 (hsp70) [154,163,164] and peroxiredoxin 1 [165].

Structure of the N 0 -P Complex
The N-terminal domain of the phosphoprotein interacts with the monomeric, RNAfree form of the N protein (referred to as N 0 ), thus forming the so-called N 0 -P complex [55,166]. This interaction prevents the formation of NCLCs on cellular RNAs and recruits the N proteins to the encapsidation site where N 0 -P complexes are used as the substrate for the encapsidation of genomes and antigenomes [7,55,56].
The first 50 residues of P (herein referred to as P NTD ) of paramyxoviruses contain a conserved α-MoRE [57,59,62,167] that adopts an alpha-helical structure upon binding to N core ( Figure 8). The structures of the N 0 -P NTD complex of NiV [24], MeV [25], PIV5 [26], and hPIV3 [27] were solved by X-ray crystallography ( Figure 8A-D). To crystallize the complex in a monomeric and RNA-free state, the N NTDarm and N CTDarm were removed (with the exception of MeV in which the N CTDarm was included). For all three viruses, P NTD binds to N CTD in the groove occupied by the N NTDarm of the adjacent N i+1 protomer in the nucleocapsid conformation ( Figure 8B). The interactions between the groove and the P NTD or the N NTDarm are both of hydrophobic nature. For MeV and NiV, the first alpha-helix is followed by a second alpha-helix segment that binds the top of the N CTD in place of the N CTDarm of the adjacent N i-1 protomer ( Figure 8B). Contrary to MeV, hPIV3, and NiV, the P NTD of PIV5 extends on its N-terminus and reaches the RNA cavity, potentially directly inhibiting binding to the RNA ( Figure 8C). Inhibition of the binding of the arms of the adjacent N protomers by P NTD is a conserved feature among mononegaviruses [131,137,[168][169][170][171][172].
For MeV, NiV, and PIV5, the structures of both the RNA-free N core in complex with P NTD and the RNA-bound N core engaged in an NCLC (N NUC ) have been solved. The overall architecture of N core is conserved between the N 0 conformation (or "open" conformation) and the N NUC conformation (or "closed" conformation) ( Figure 8D). Although individually the N NTD and N CTD have very similar structures, their relative orientation differs ( Figure 8D-F). Indeed, the kinked alpha-helix located between the N NTD and the N CTD acts as a hinge and allows the rotation of the N CTD ( Figure 8F). This rotation finalizes the formation of the RNA cavity. While NiV P NTD has been suggested to maintain the N protein in the N 0 conformation by rigidifying the structure [24], modeling and molecular simulations suggest that the PIV5 N protein preferably adopts the N 0 conformation in the absence of ligand (P NTD or RNA) [26]. The interactions between the RNA backbone and residues from both the N NTD and N CTD most likely participate in the stabilization of the N NUC conformation, even in the absence of adjacent protomers.
In addition to P NTD , another ultra-weak interaction was detected between N core and a fragment of the long, disordered region of P located between P NTD and P OD [173]. This fragment contains a short peptide (δ) with no apparent secondary structure and a transient short alpha-helix (α4), which both interact with the bottom of N NTD ( Figure 8G). The sequence of α4 has some level of conservation among morbilliviruses and henipaviruses, and the introduction of mutations in this sequence inhibits gene expression in a minigenome assay. The homologous sequence in the NiV P protein also adopts a transient alpha-helix structure in solution [62,174]. Thus, this ultra-weak interaction may be conserved and essential for maintaining the N protein in the N 0 state [173]. The interaction between the α-MoRE of N tail (N α-MoRE ) and P XD may also further stabilize the N 0 -P complex [175]. Viruses 2021, 13, x FOR PEER REVIEW 12 of 29 For MeV, NiV, and PIV5, the structures of both the RNA-free Ncore in complex with PNTD and the RNA-bound Ncore engaged in an NCLC (N NUC ) have been solved. The overall architecture of Ncore is conserved between the N 0 conformation (or "open" conformation) and the N NUC conformation (or "closed" conformation) ( Figure 8D). Although individually the NNTD and NCTD have very similar structures, their relative orientation differs (Figure 8D-F). Indeed, the kinked alpha-helix located between the NNTD and the NCTD acts as a hinge and allows the rotation of the NCTD ( Figure 8F). This rotation finalizes the formation of the RNA cavity. While NiV PNTD has been suggested to maintain the N protein in the N 0 conformation by rigidifying the structure [24], modeling and molecular simula-

Encapsidation of the RNA
The fusion of full-length N protein (or N core ) to a cleavable P NTD allows the production of soluble N 0 -P complexes [20,23,176]. Incubation of these complexes with RNA triggers the formation of NCLCs in vitro. This experimental setup revealed that the encapsidation of the RNA is sequence-specific and that the N protein of MeV preferably encapsidates a poly(A) hexamer (but not poly(U)), as well as an RNA corresponding to the first six nucleotides of the genome. Similar specificity was observed for other mononegaviruses, suggesting that the N protein has a higher affinity for poly(A) RNA and the first nucleotides of the corresponding genome [177][178][179][180]. The formation of long helical NCLCs from short RNA hexamers indicates that a continuous RNA molecule is not required for the assembly of N proteins. The kinetics of the encapsidation was analyzed by real-time nuclear magnetic resonance and fluorescence spectroscopy and revealed a two-step mechanism. The first step is rapid and could correspond to the binding of N monomers to the RNA, while the second step consists of the assembly of RNA-bound N proteins [176].
Finally, the local concentration of N 0 -P complexes seems critical to efficient encapsidation of the RNA and generation of NCLCs. Indeed, the formation of droplets formed by liquid-liquid phase separation increases both the local concentration of N 0 -P complexes and the rate of formation of NCLCs in vitro [181]. For VSIV (Rhabdoviridae family), although dispensable [182], P OD enhances genome replication possibly by increasing the local concentration of N 0 at the site of encapsidation [183].

The Nucleocapsid as a Template
Unlike the distantly related members of the Articulavirales and Bunyavirales orders for which the polymerase can directly bind the extremity of the viral genomes, the viral polymerase of mononegaviruses cannot interact with its nucleocapsid template without its cofactor P [43]. Moreover, the genomes of mononegaviruses are deeply encased in helical nucleocapsids, thus raising the question of how the polymerase accesses the RNA.

Recruitment and Progression of the Polymerase Complex
Since P XD mediates the interaction of L with the nucleocapsid and the polymerase complex is made of the L protein and a tetramer of P proteins, the polymerase complex possesses four potential nucleocapsid-binding sites. This architecture is the basis of the socalled "cartwheeling" model in which the polymerase complex progresses on its template due to cycles of association/dissociation between P XD and the nucleocapsid [184,185].
The P XD of paramyxoviruses adopts a three-helix bundle structure [71][72][73]75,186,187]. P XD binds to the α-MoRE of N tail [73][74][75]127,128], the L protein [28,188,189], and N core [68,114,190,191] (Figure 9). P XD has a prism-like shape with three faces: α1 + α2 form face 1 (F1), α2 + α3 form face 2 (F2), and α3 + α1 form face 3 (F3) ( Figure 9A). Each interaction is mediated by a different face, with N α-MoRE interacting with F2 [73], the L protein with F3 [28,189], and N core with F1 [191] (Figure 9). is mediated by a different face, with Nα-MoRE interacting with F2 [73], the L protein with F3 [28,189], and Ncore with F1 [191] (Figure 9).  Amino acids shown as red spheres correspond to CDV N residues that once mutated restore the growth of a recombinant CDV bearing deletions in the NCDR [191]. The amino acids corresponding to the residues of NiV identified as important for the interaction with PXD are shown as pink spheres [192]. The interaction of PXD with Nα-MoRE is weak, with a constant of dissociation (KD) in the µM range [74,128,157,193]. While the disordered Nα-MoRE folds into an alpha-helix upon binding to PXD, the flanking regions of Ntail remain disordered [74,75,127,187,[194][195][196][197]. This phenomenon, called fuzziness [198], affects the interaction between Ntail and its partners. Indeed, the long-disordered region upstream of Nα-MoRE slows the disorder-to-order transition of Nα-MoRE upon binding to PXD [199,200]. Accordingly, the deletion of the disordered region upstream MeV, NiV, and HeV Nα-MoRE increases the affinity for PXD (and also HSP70 for MeV) [199]. The dampening effect of the disordered regions does not directly depend on the sequence, but rather on a combination of length and disorder. Although not directly involved, the poorly conserved disordered region of Ntail upstream the Nα-MoRE affects the interaction with PXD [199][200][201]. Amino acids shown as red spheres correspond to CDV N residues that once mutated restore the growth of a recombinant CDV bearing deletions in the N CDR [191]. The amino acids corresponding to the residues of NiV identified as important for the interaction with P XD are shown as pink spheres [192]. The interaction of P XD with N α-MoRE is weak, with a constant of dissociation (K D ) in the µM range [74,128,157,193]. While the disordered N α-MoRE folds into an alpha-helix upon binding to P XD , the flanking regions of N tail remain disordered [74,75,127,187,[194][195][196][197]. This phenomenon, called fuzziness [198], affects the interaction between N tail and its partners. Indeed, the long-disordered region upstream of N α-MoRE slows the disorder-toorder transition of N α-MoRE upon binding to P XD [199,200]. Accordingly, the deletion of the disordered region upstream MeV, NiV, and HeV N α-MoRE increases the affinity for P XD (and also HSP70 for MeV) [199]. The dampening effect of the disordered regions does not directly depend on the sequence, but rather on a combination of length and disorder. Although not directly involved, the poorly conserved disordered region of N tail upstream the N α-MoRE affects the interaction with P XD [199][200][201].
The low affinity between P XD and N α-MoRE permits efficient progression of the polymerase complex on the nucleocapsid. Indeed, modifications of the binding strength between MeV P XD and N α-MoRE alter the activity of the polymerase [202][203][204]. Indeed, there is a positive correlation between the P XD /N α-MoRE interaction strength and the efficiency of the re-initiation of the transcription at gene junctions [204] (Figure 2A). This effect is further enhanced when the polymerase crosses long non-transcribed intergenic regions, suggesting the P XD /N α-MoRE interaction keeps the polymerase in an active state when crossing the gap between transcription units.
Although the P XD /N α-MoRE interaction regulates the activity of the polymerase, it is not strictly required for RNA synthesis. Indeed, while the truncation of MeV N from N α-MoRE to its C-terminus abrogates gene expression in a minigenome assay, further deletion of the 43 upstream residues restores activity [205] (Figure 10). This suggests that the central disordered region (N CDR ) upstream of the N α-MoRE has a negative effect on the activity of the polymerase and the binding of P XD to N α-MoRE counteracts this effect. Accordingly, deletions in the N CDR of CDV and NiV increase activity [206], and when MeV N α-MoRE is relocated to a variable region of N NTD , removing N CDR enhances gene expression [207] (Figure 10). However, a recombinant MeV harboring N α-MoRE on N core and devoid of N CDR produces more polycistronic mRNAs. Similarly, two recombinant CDV with deletions in N CDR produce more polycistronic mRNA and show an altered RNA synthesis balance with enhanced transcription over replication [206]. Therefore, the central disordered region of N tail inhibits RNA synthesis, and P XD counterbalances this negative effect. This interplay would finely tune the polymerase's activity and ensure the quality of the generated mRNAs by improving the recognition of the signals at the intergenic junctions.
tween MeV PXD and Nα-MoRE alter the activity of the polymerase [202][203][204]. Indeed, there is a positive correlation between the PXD/Nα-MoRE interaction strength and the efficiency of the re-initiation of the transcription at gene junctions [204] (Figure 2A). This effect is further enhanced when the polymerase crosses long non-transcribed intergenic regions, suggesting the PXD/Nα-MoRE interaction keeps the polymerase in an active state when crossing the gap between transcription units.
Although the PXD/Nα-MoRE interaction regulates the activity of the polymerase, it is not strictly required for RNA synthesis. Indeed, while the truncation of MeV N from Nα-MoRE to its C-terminus abrogates gene expression in a minigenome assay, further deletion of the 43 upstream residues restores activity [205] (Figure 10). This suggests that the central disordered region (NCDR) upstream of the Nα-MoRE has a negative effect on the activity of the polymerase and the binding of PXD to Nα-MoRE counteracts this effect. Accordingly, deletions in the NCDR of CDV and NiV increase activity [206], and when MeV Nα-MoRE is relocated to a variable region of NNTD, removing NCDR enhances gene expression [207] ( Figure  10). However, a recombinant MeV harboring Nα-MoRE on Ncore and devoid of NCDR produces more polycistronic mRNAs. Similarly, two recombinant CDV with deletions in NCDR produce more polycistronic mRNA and show an altered RNA synthesis balance with enhanced transcription over replication [206]. Therefore, the central disordered region of Ntail inhibits RNA synthesis, and PXD counterbalances this negative effect. This interplay would finely tune the polymerase's activity and ensure the quality of the generated mRNAs by improving the recognition of the signals at the intergenic junctions. Figure 10. Summary of experimental data on the effect of Ntail on the polymerase's activity. Schematic representation of recombinant N proteins analyzed in minigenome assays. The levels of gene expression are normalized to the level of the wild-type protein (first protein of each set). Activity levels: 0-10%: "-"; 50-75%: "+++"; 75-100%: "++++"; >100%: "+++++". For mutants (6) and (7) Nα-MoRE is inserted in a loop of NNTD at residue 138. Mutants (4), (5), (6), (9), and (11) contain internal deletion shown with a "^". Activity levels are inferred from the references indicated on the right.
In addition to interacting with the α-MoRE, PXD of CDV was also shown to directly interact with Ncore [191]. This interaction is mediated by an acidic loop of NNTD (residues 146 to 161) and requires the presence of Nα-MoRE in complex with PXD ( Figure 9C-E). Accordingly, mutations in the homologous region of NiV Ncore were previously shown to inhibit the P-N interaction [192]. After several passages of a recombinant CDV expressing a Ntail with a large deletion in NCDR, compensatory mutations appeared in PXD and NNTD that decreased the affinity between the two domains. Thus, NCDR may inhibit the interaction of PXD with Ncore. Modeling and molecular docking predict that the PXD/Nα-MoRE complex binds between two N protomers. On one side the F1 of PXD binds to the acidic loop Figure 10. Summary of experimental data on the effect of N tail on the polymerase's activity. Schematic representation of recombinant N proteins analyzed in minigenome assays. The levels of gene expression are normalized to the level of the wild-type protein (first protein of each set). Activity levels: 0-10%: "-"; 50-75%: "+++"; 75-100%: "++++"; >100%: "+++++". For mutants (6) and (7) N α-MoRE is inserted in a loop of N NTD at residue 138. Mutants (4), (5), (6), (9), and (11) contain internal deletion shown with a "ˆ". Activity levels are inferred from the references indicated on the right.
In addition to interacting with the α-MoRE, P XD of CDV was also shown to directly interact with N core [191]. This interaction is mediated by an acidic loop of N NTD (residues 146 to 161) and requires the presence of N α-MoRE in complex with P XD (Figure 9C-E). Accordingly, mutations in the homologous region of NiV N core were previously shown to inhibit the P-N interaction [192]. After several passages of a recombinant CDV expressing a N tail with a large deletion in N CDR , compensatory mutations appeared in P XD and N NTD that decreased the affinity between the two domains. Thus, N CDR may inhibit the interaction of P XD with N core . Modeling and molecular docking predict that the P XD /N α-MoRE complex binds between two N protomers. On one side the F1 of P XD binds to the acidic loop of one protomer and on the other side, N α-MoRE interacts with a different loop (residues 133 to 142) of the N NTD of the other protomer ( Figure 9D,E). The requirement of N α-MoRE for this interaction suggests P XD first binds N α-MoRE and then N core . A similar interaction with the acidic loop may explain the singular behavior of MuV P XD , which can bind N core in the absence of N tail [68,190]. This atypical phenotype may be linked to the lower stability of the MuV P XD tertiary structure in solution and to a folding-upon-binding mechanism [186,190]. The binding of CDV P XD to N NTD also agrees well with the density map of MuV nucleocapsids bound to P XD [114]. The P protein of the respiratory syncytial virus (Pneumoviridae family) also binds the nucleocapsid on the same location on N NTD , suggesting an ancestral mode of interaction between the polymerase complex and its template [191,208,209]. P binds the L protein via the C-terminal part of the P OD and the third face of the P XD of one P protomer [28,188,189] (Figure 9F). Using different combinations of mutants in a minigenome assay, it was shown that at least one P XD of the tetramer must have the ability to bind the L protein and one different P XD must be able to bind N α-MoRE [189]. The P XD that binds L does not have to be able to bind N α-MoRE and it is unclear whether P XD can simultaneously bind L and N α-MoRE . It is also unknown whether the interaction between L and P XD is stable or undergoes rounds of association/dissociation during RNA synthesis.
Of note, some cellular factors influence the progression of the polymerase complex. For instance, hsp70 [163] and peroxiredoxin 1 [165] both compete with P XD for the binding to N tail and enhance RNA synthesis.
Taken together, these results show that P XD is at the core of an interaction network, which is more complex than initially anticipated. P XD participates in a finely regulated dance allowing the progression of the polymerase complex onto the nucleocapsid and the optimal recognition of the transcription signals.

Access to the RNA
The viral genome is encased between the N NTD and N CTD of each N protomers, with only half of its bases facing the solvent. Thus, copying of the genome requires significant conformational rearrangements to allow the RNA to reach the catalytic site of the polymerase.
First, in the straight helical conformation of purified nucleocapsids, the RNA is covered by the N protomers of the adjacent helical turn, leaving an insufficient empty space above the RNA to accommodate the large polymerase complex ( Figure 5C). Therefore, a local modification of the conformation of the helical nucleocapsid must take place. Nucleocapsids are flexible and can adopt different conformations [21,[109][110][111]. Such uncoiling and bending could enable the polymerase complex to reach the RNA cavity. Although N tail affects the rigidity and the pitch of the helical nucleocapsids [21,113,117,121], the addition of monomeric P XD to intact nucleocapsids of HeV or MuV does not affect the coiling of the helix [75,114]. For HeV, the addition of P XD does not make the RNA more accessible to nucleases either. Similarly, the binding of the C-terminal domain of VSIV P to NCLC does not significantly modify the structure of the N proteins [210]. This suggests that either the binding of P XD does not participate in the uncoiling of the nucleocapsid, or the oligomerization of P XD is required. Some cellular factors may also contribute to the uncoiling of the nucleocapsid, such as hsp70, which binds MeV N tail [154,163], stimulates MeV and CDV polymerase activity [154,211], and seems to favor the production of "light" nucleocapsids (versus "dense" nucleocapsids) of CDV [212,213]. Conversely, the P NTD of MuV can uncoil the helical nucleocapsid and increase gene expression in a minigenome assay [68,114]. Similar to the binding of MuV P XD to N core , the atypical behavior of MuV P NTD could reflect a possibly common uncoiling mechanism.
Second, the RNA cavity of the N proteins must open to release the RNA upon the passage of the polymerase. A first model proposes that the N protomers undergo a large conformational rearrangement reverting from the N NUC to the N 0 conformation [24,214]. Molecular dynamics simulations show that in absence of ligands (RNA or P NTD ), the N protein of PIV5, initially in the N NUC conformation, adopts a conformation similar to the N 0 conformation. This suggests that the N 0 conformation is the most stable in the absence of RNA and adjacent N proteins [26]. However, the removal of the RNA from nucleocapsidlike structures of MuV or VSIV does not significantly change the relative orientation of N NTD and N CTD [179,215]. Moreover, when the simulations are made on a short RNAfree nucleocapsid of three N proteins of PIV5, only the conformations of the N i+1 and N i protomers slightly change toward the N 0 conformation, with the conformation of the N i protomer being modified to a lower extent. Thus, the conformational rearrangement from the N NUC to the N 0 conformation may partially take place only at the 3 end of the genome to facilitate the release of the RNA terminus and the engagement of the polymerase.
Another model proposes that a local conformational change of the α-helix 7 and the upstream flexible loop is enough to release the RNA [215] ( Figure 8E). Indeed, this helix, which is located at the surface of the protein, constitutes most of the bottom part of the RNA cavity and contains several amino acids implicated in RNA stabilization. Moreover, when the N NTD of MeV N NUC and N 0 are superimposed, the loop-α7 region is the only fragment that does not properly align ( Figure 8E). Thus, in addition to the rotation of the N NTD , the transition from the N 0 to the N NUC conformation also implies a local rearrangement of this region. Mutations in this helix affect gene expression, which is in agreement with a potential role in releasing the RNA during RNA synthesis [215]. P NTD has also been proposed to participate in the release of the RNA. Due to the tight N-N interactions and the high stability of the nucleocapsid, it seems unlikely that during the progression of the polymerase P NTD would bind the N protomers in an N 0 -P-like manner and sequentially "open" the N proteins. However, the N NTDarm -binding groove of the last N protomer of the nucleocapsid (or the first protomer on the 3 terminus) is not occupied and may therefore be engaged by P NTD to help to release the RNA. Due to the binding register of the first six nucleotides of the genome and the rule of six, the terminal 3 nucleotides are positioned such that they are exposed to the solvent and are therefore more accessible [20]. Therefore, the binding of P NTD only to the terminal N protomer may be sufficient for the polymerase to access the RNA. However, for SeV and hPIV3, the deletion of the P NTD abolishes genome replication but not transcription in vitro, thus suggesting that the potential role of P NTD in releasing the RNA is not essential (at least for members of the Respirovirus genus) [216,217].

Transcription
Upon fusion of viral and cellular membranes and the subsequent release of the nucleocapsid in the cytoplasm, the polymerase complexes previously packaged in the virion initiate primary transcription. Given that the nucleocapsid adopts a helical conformation, the two elements of the bipartite promoter are exposed on the same side. P XD binds N tail and recruits the polymerase complex on the nucleocapsid ( Figure 11A). When the polymerase reaches the 3 end of the nucleocapsid, it is somehow stabilized by the second element of the bi-partite promoter (PE2). The phase register of the encapsidation exposes the 3 end of the genome to the solvent. The release of the RNA from the first N protein may be enhanced by the lodging of one P NTD into the empty N NTDarm -binding groove of the first protomer ( Figure 11B). A conformational change of the first two N protomers from the N NUC to the N 0 conformation may also assist the release. When the polymerase complex passes on an N protomer, a transient local conformational change of the helix α7 of N NTD and the preceding loop allows the release of the RNA ( Figure 11C).
The progression of the polymerase along the genome induces a local conformational change of the helical nucleocapsid ( Figure 11D). A dynamic dance is orchestrated by P XD , which periodically binds the α-MoRE of N tail and N core . The fuzziness of N tail inhibits the passage of the polymerase complex, which uses P XD to "clear the way". First, P XD binds N α-MoRE which folds upon binding into an alpha-helix ( Figure 11E). Second, the P XD /N α-MoRE complex binds the N core of the same protomer or an adjacent protomer (model 1), thus stabilizing the N tail , decreasing the fuzziness, and facilitating the progression of the polymerase ( Figure 11F,G). As the binding of the P XD /N α-MoRE complex to the N NTD of N protomer from the same helical turn would potentially further block the way (Figure 11F), the complex could alternatively bind the N NTD of a protomer from the previous helical turn (model 2). In this alternative model, the anchoring of the N tail to the previous helical turn would further remove it from the path of the polymerase ( Figure 11H-K).
This dynamic interaction network regulates the speed of the RNA synthesis and ensures an optimal recognition of the transcription signals at the intergenic regions (IR) (Figure 2A). Indeed, upon the recognition of the first IR, the polymerase stops the synthesis of the leader RNA and scans the genome to find the next gene start signal (GS). Recognition of the GS triggers the re-initiation of RNA synthesis and capping of the mRNA. If recognized, the gene end signal triggers the synthesis of the poly(A) tail (for more details on the mechanisms of RNA synthesis, see [218]). The efficiency with which the polymerase crosses the IR and scans the genome depends on P XD and its interactions with the nucleocapsid. It is yet to be deciphered if the P XD that binds the L protein can simultaneously bind N tail and if it periodically associates with and dissociates from L.

Replication
After new viral proteins have been synthesized, the polymerase complex switches from a transcriptase mode to a replicase mode. The details of this switch are not fully understood, but the accumulation of the encapsidation substrate, the N 0 -P complex, plays a key role in this process. In the replicase mode, the polymerase complex encapsidates the nascent RNA and does not recognize any transcription signals. As the N protein has a higher affinity for an RNA with the sequence of the 5 end of the genome, the first six nucleotides of the nascent genome outcompete P NTD for the binding to N ( Figure 11L,M). For the following N protomers, P NTD is outcompeted by both the RNA and the N CTDarm of the previous N protomer ( Figure 11N). This double competition allows the continuous encapsidation of any RNA sequences, even with a poor affinity for N, such as poly(U) sequences.

Conclusions and Perspectives
In recent years, several significant advances have been made which add to our understanding of the structure and mechanisms of the molecular machinery underlying RNA synthesis of paramyxoviruses. We now have in hand structural information on the N 0 -P complex and the nucleocapsid of three and seven viruses, respectively. The determination of the phase register of the encapsidated RNA further reveals the mechanisms underlying the rule of six. Moreover, the deciphering of the rich interaction network connecting the different components of the ribonucleoprotein complex gives us a good overview of the dynamics of the progression of the polymerase complex. Finally, the resolution of the atomic structure of the polymerase complex of PIV5 provides a new tool and opens new opportunities to further elucidate the molecular details of the RNA synthesis.
Except for MuV and its atypical features (antiparallel coiled-coil for P OD , unstable P XD in solution, interaction between P XD and N core in the absence of N tail , and uncoiling of the nucleocapsid by P NTD ), the molecular machinery and the mechanisms at play during RNA synthesis appear to be well conserved among paramyxoviruses. The exposure of the RNA on the outside of the helical nucleocapsid, the binding site of the C-terminal domain of P to the N NTD , the coiled-coil structure of P OD , and the interaction sites between P and L suggest these mechanisms are shared with pneumoviruses and filoviruses but are quite different for rhabdoviruses.
Despite these new insights, several questions remain unanswered. The helical nucleocapsid must uncoil to allow the progression of the polymerase. What factors induce this conformational change? It is also still unclear how the nucleoproteins release the RNA and what signals trigger this release. Although structural information is available on the individual components, there is little insight into the organization or the structure of the polymerase complex on the nucleocapsid while synthesizing RNA. In addition, how does the second element of the bipartite promoter enhance initiation? How does the polymerase complex add nucleoproteins to the nascent RNA? How does the phosphoprotein orchestrate its interactions with N tail , N core , and L? How do other viral proteins such as the V, C, and M proteins interact with the ribonucleoprotein complex and regulate RNA synthesis?
Answering these questions should bring significant insight into the mechanisms at play and assist the development of safe and potent vaccine vectors, oncolytic viruses, and antiviral therapeutics. With the momentum provided by the recent advances and the constant improvement of powerful tools such as cryo-electron microscopy, there is no doubt our understanding of the viral molecular machinery of paramyxoviruses will continue to rapidly improve.
Funding: This work was supported by NIH grant AI059371 to Sean P.J. Whelan.