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Observations on the Structure of the Nucleocapsids of some Paramyxoviruses

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Abstract

SUMMARY The intact particles and nucteocapsids of mumps, Sendai and measles viruses are of closely similar appearance, size and structure. The intact parti- cles are about 15o nm. in diameter. The filamentous nucleocapsids have a modal length of about ~. I/Am., and are constructed of subunits arranged as a single start helix of pitch 5.0 nm. for Sendai virus, and about 6"o nm. for mumps and measles viruses. A (helical) projection of the structure of the Sendai nucleocapsid calculated from an electron micrograph showed that the structural subunits are hour-glass-shaped and are arranged in the helix with their long axes inclined at an angle of about 6o ° to the long axis of the particle. There are probably J ~ or I3 subunits in each turn of the basic helix. Optical diffraction patterns of electron micrographs of mumps and measles nucleo- capsids show that they have closely similar structures.
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... 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]. ...
... 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. ...
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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.
... The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04.23.057810 doi: bioRxiv preprint viruses (e.g., 11,12 ). Furthermore, the sample in which specific SARS-CoV-2 target peptides can still be detected (20 pg sample) is estimated to contain 5 % viral proteins and this then represents 0.9 pg NCAP (20 amol, or 12E6 molecules) and would correspond to ~10,000 virus particles, under the assumption that all NCAP present is assembled into virus particles. ...
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The rapid, sensitive and specific diagnosis of SARS-CoV-2 by fast and unambiguous testing is widely recognized to be critical in responding the current outbreak. Since the current testing capacity by conventional PCR based methods is insufficient because of shortages of supplies such as RNA extraction kits and PCR reagents, alternative and/or complementary testing assays should be developed. Here, we exploit the potential of targeted mass spectrometry based proteomic technologies to solve the current issue of insufficient SARS-CoV-2 diagnostic testing capacity. We have assessed the limit of detection by parallel reaction monitoring (PRM) on an Orbitrap Eclipse mass spectrometer for target tryptic peptides of several SARS-CoV-2 proteins from a sample of virus infected Vero cells. For Nucleocapsid protein the limit of detection was found to be in the mid-attomole range (0.9 x 10-12 g), which would theoretically correspond to approximately 10,000 SARS-CoV-2 particles, under the assumption that all viral proteins are assembled in macromolecular virus particles. Whether or not this sensitivity is sufficient to play a role in SARS-CoV-2 detection in patient material such as swabs or body fluids largely depends on the amount of viral proteins present in such samples and is subject of further research. If yes, mass spectrometry based methods could serve as a complementary protein based diagnostic tool and further steps should be focused on sample preparation protocols and on improvements in sample throughput.
... Paramyxoviridae [Finch & Gibbs, 1970]. Chan et al., 2004;Houben et al., 2007;Jensen et al., 2008;Jensen et al., 2011;Communie et al., 2013;Habchi et al., 2011;Johansson et al., 2003]. ...
Thesis
Mumps is a highly contagious disease caused by the mumps virus. The prevention treatment (vaccine) against it is already in the routine use. However, recent outbreaks still remain uncontrollable. Therefore, it is important to understand the molecular mechanism of the mumps virus life cycle. This virus belongs to the family of Paramyxoviridae. Its genome, negative strand non-segmented RNA is protected by the nucleoprotein (N) by forming filamentous structures called nucleocapsids. N plays an important role in viral genome synthesis. Together with the polymerase and its cofactor phosphoprotein (P) they constitute the transcription-replication machinery. Both N and P contain folded and unfolded regions. Despite mumps virus common morphology with other paramyxovirus, there are some differences. It has been proposed that P is an antiparallel oligomer with two extremities on the one side being in interaction with the structural part of N (Ncore). The function of the disordered domain (Ntail) remains unclear, as it does not seem to bind to the C-terminal part of P, as is the case for other paramyxoviruses. The role of the disordered domains of P is also not known. In this project we revealed mechanisms of interaction between different regions of N and P and we explain how disordered regions of N and P are implicated in the regulation of the complex machinery of viral replication. We used the nuclear magnetic resonance which is the most powerful method to determine structure, dynamics and potential interaction partners, and therefore, function of disordered viral proteins.
... non-segmented, linear, negative-strand RNA genome, and its replication is vital for virus survival 52 and pathogenicity (Ruigrok et al., 2011). One remarkable character of negative-strand RNA 53 viruses is their genomes are enwrapped by the nucleoprotein (N), which results in the formation of 54 helical nucleocapsids (Finch and Gibbs, 1970;Heggeness et al., 1980;Longhi, 2009). During viral 55 RNA synthesis, the assembled nucleocapsid, rather than the naked RNA genome, is opened and 56 unveiled for the recognition of the viral RNA-dependent RNA polymerase (RdRp) and serves as 57 the template for both replication and transcription (Dochow et al., 2012;Emerson and Wagner, 58 1972; Emerson and Yu, 1975;Fearns et al., 1997;Perlman and Huang, 1973;Severin et al., 2016). ...
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Non-segmented negative-strand RNA viruses, such as Measles, Ebola and Newcastle disease viruses (NDV), encapsidate viral genomic RNAs into helical nucleocapsids which serve as the template for viral replication and transcription. Here, the clam-shaped nucleocapsid structure, where the NDV viral genome is sequestered, was determined at 4.8 Å resolution by cryo-electron microscopy. The clam-shaped structure is composed of two single-turn spirals packed in a back-to-back mode, and the tightly packed structure functions as a seed for nucleocapsid to assemble from both directions and grows into double-headed filaments with two separate RNA strings inside. Disruption of this structure by mutations on its loop interface yielded a single-headed unfunctional filament.
... MeV is a member of the Paramyxoviridae family that also includes a number of zoonoses, such as Nipah and Hendra viruses. Replication and transcription of paramyxoviral genomes by the viral polymerase (L) requires an RNA template that is encapsidated by thousands of nucleoproteins (N) in the ribonucleoprotein, forming long helical nucleocapsids (NCs) (1,2). Genome encapsidation is thought to protect the viral RNA from recognition and degradation by the innate immune system. ...
Article
Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5′) in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5′ and 3′ binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3′ end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.
... T he paramyxovirus family comprises a multitude of major human and animal respiratory pathogens, such as measles virus (MeV), canine distemper virus (CDV), the parainfluenzaviruses, mumps virus (MuV), and the recently emerged highly pathogenic Hendra and Nipah (NiV) viruses (1). Together with the rhabdo-, borna-, filo-, and pneumoviruses, the paramyxoviruses belong to the order Mononegavirales, which is characterized by nonsegmented negative-polarity RNA genomes that are encapsidated by viral nucleocapsid (N) proteins into helical ribonucleoprotein (RNP) complexes (2)(3)(4)(5). ...
Article
The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design.
... In addition to measles virus (MeV), an archetype member of the family and member of the morbillivirus genus, the paramyxoviruses comprise major respiratory pathogens, such as mumps virus (MuV), the parainfluenzaviruses (PIVs), Nipah (NiV) and Hendra (HeV) viruses, and canine distemper virus (CDV). Being part of the order mononegavirales, paramyxoviruses feature nonsegmented negativestranded genomes that are encapsidated by the viral N protein, resulting in the formation of helical N:RNA ribonucleoprotein assemblies (2)(3)(4)(5). Only the encapsidated RNA is recognized as a template by the viral RNA-dependent RNA-polymerase (RdRp) complex that is responsible for both transcription and replication of all viral RNA (6)(7)(8). ...
Article
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