Helical Nucleocapsid Research Articles

Ribonucleocapsid assembly/packaging signals in the genomes of the coronaviruses SARS-CoV and SARS-CoV-2: detection, comparison and implications for therapeutic targeting

The genomic ssRNA of coronaviruses is packaged within a helical nucleocapsid. Due to transitional symmetry of a helix, weakly specific cooperative interaction between ssRNA and nucleocapsid proteins leads to the natural selection of specific quasi-periodic assembly/packaging signals in the related genomic sequence. Such signals coordinated with the nucleocapsid helical structure were detected and reconstructed in the genomes of the coronaviruses SARS-CoV and SARS-CoV-2. The main period of the signals for both viruses was about 54 nt, that implies 6.75 nt per N protein. The complete coverage of the ssRNA genome of length about 30,000 nt by the nucleocapsid would need 4.4 × 103 N proteins, that makes them the most abundant among the structural proteins. The repertoires of motifs for SARS-CoV and SARS-CoV-2 were divergent but nearly coincided for different isolates of SARS-CoV-2. We obtained the distributions of assembly/packaging signals over the genomes with nonoverlapping windows of width 432 nt. Finally, using the spectral entropy, we compared the load from point mutations and indels during virus age for SARS-CoV and SARS-CoV-2. We found the higher mutational load on SARS-CoV. In this sense, SARS-CoV-2 can be treated as a ‘newborn’ virus. These observations may be helpful in practical medical applications and are of basic interest. Communicated by Ramaswamy H. Sarma

Clinical characteristics of COVID-19 and personal protective equipment for trauma surgeons

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by a new virus from the coronavirus family (1) regarded as enveloped, positive-sense, single-stranded ribonucleic acid (RNA) viruses with helical symmetric nucleocapsid (2). According to the whole genome sequencing and phylogenetic analysis of coronavirus strains, a distinct clade of betacoronavirus is shown to be associated with severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (3). This novel clade is called the 2019 novel coronavirus (2019-nCoV) and due to more similarity to the SARS virus is also called severe acute respiratory syndrome coronavirus 2. The disease is known as COVID-19.

New Insights from Chemical Biology: Molecular Basis of Transmission, Diagnosis, and Therapy of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly s...

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus.

The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

The Nucleoprotein and Phosphoprotein of Measles Virus.

Measles virus is a negative strand virus and the genomic and antigenomic RNA binds to the nucleoprotein (N), assembling into a helical nucleocapsid. The polymerase complex comprises two proteins, the Large protein (L), that both polymerizes RNA and caps the mRNA, and the phosphoprotein (P) that co-localizes with L on the nucleocapsid. This review presents recent results about N and P, in particular concerning their intrinsically disordered domains. N is a protein of 525 residues with a 120 amino acid disordered C-terminal domain, Ntail. The first 50 residues of Ntail extricate the disordered chain from the nucleocapsid, thereby loosening the otherwise rigid structure, and the C-terminus contains a linear motif that binds P. Recent results show how the 5′ end of the viral RNA binds to N within the nucleocapsid and also show that the bases at the 3′ end of the RNA are rather accessible to the viral polymerase. P is a tetramer and most of the protein is disordered; comprising 507 residues of which around 380 are disordered. The first 37 residues of P bind N, chaperoning against non-specific interaction with cellular RNA, while a second interaction site, around residue 200 also binds N. In addition, there is another interaction between C-terminal domain of P (XD) and Ntail. These results allow us to propose a new model of how the polymerase binds to the nucleocapsid and suggests a mechanism for initiation of transcription.

Self-capping of nucleoprotein filaments protects the Newcastle disease virus genome.

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. This tightly packed structure functions as a seed for the assembly of a nucleocapsid from both directions, facilitating the growth of double-headed filaments with two separate RNA strings inside. Disruption of this structure by mutations in its loop interface yielded a single-headed unfunctional filament.

Cryo EM structure of the rabies virus ribonucleoprotein complex

Rabies virus is an important zoonotic pathogen. Its bullet shaped particle contains a helical nucleocapsid. We used cryo-electron tomography and subsequent subtomogram averaging to determine the structure of its ribonucleoprotein. The resulting electron density map allowed for confident fitting of the N-protein crystal structure, indicating that interactions between neighbouring N-proteins are only mediated by N- and C-terminal protruding subdomains (aa 1–27 and aa 355–372). Additional connecting densities, likely stabilizing the ribonucleoprotein complex, are present between neighbouring M-protein densities on the same helical turn and between M- and N-protein densities located on neighbouring helical turns, but not between M-proteins of different turns, as is observed for the related Vesicular stomatitis virus (VSV). This insight into the architecture of the rabies virus nucleocapsid highlights the surprising structural divergence of large biological assemblies even if the building blocks – here exemplified by VSV M- and N-protein – are structurally closely related.

A conformational switch balances viral RNA accessibility and protection in a nucleocapsid ring model

Virus from the Mononegavirales order share common features ranging from virion structure arrangement to mechanisms of replication and transcription. One of them is the way the nucleoprotein (N) wraps and protects the RNA genome from degradation by forming a highly ordered helical nucleocapsid. However, crystal structures from numerous Mononegavirales reveal that binding to the nucleoprotein results in occluded nucleotides that hinder base pairing necessary for transcription and replication. This hints at the existence of alternative conformations of the N protein that would impact on the protein-RNA interface, allowing for transient exposure of the nucleotides without complete RNA release. Moreover, the regulation between the alternative conformations should be finely tuned. Recombinant expression of N from the respiratory syncytial virus form regular N/RNA common among all Mononegavirales, and these constitute an ideal minimal unit for investigating the mechanisms through which these structures protect RNA so efficiently while allowing for partial accessibility during transcription and replication. Neither pH nor high ionic strength could dissociate the RNA but led to irreversible aggregation of the nucleoprotein. Low concentrations of guanidine chloride dissociated the RNA moiety but leading to irreversible aggregation of the protein moiety. On the other hand, high concentrations of urea and long incubation periods were required to remove bound RNA. Both denaturants eventually led to unfolding but converged in the formation of an RNA-free β-enriched intermediate species that remained decameric even at high denaturant concentrations. Although the N-RNA rings interact with the phosphoprotein P, the scaffold of the RNA polymerase complex, this interaction did not lead to RNA dissociation from the rings in vitro. Thus, we have uncovered complex equilibria involving changes in secondary structure of N and RNA loosening, processes that must take place in the context of RNA transcription and replication, whose detailed mechanisms and cellular and viral participants need to be established.

NMR chemical shift assignment of the C-terminal region of the Menangle virus phosphoprotein.

Menangle virus is a bat-borne paramyxovirus with zoonotic potential. The single-stranded RNA genome of the virus is encapsidated in a helical nucleocapsid which is the template for both transcription and genome replication. Each of these operations is performed by the viral RNA polymerase. The phosphoprotein is the non-catalytic subunit of the polymerase, and its C-terminal region enables the polymerase to engage with the nucleocapsid. Here, we report the 1H, 15N, and 13C chemical shift assignments of the C-terminal region (amino acids 267-388) of the Menangle virus phosphoprotein. This region has a bipartite character, with a highly flexible and structurally disordered sequence preceding a structured nucleocapsid-binding domain. NMR chemical shift assignment will enable the detailed characterization of the dynamic behavior of the phosphoprotein, and its functional linkage with polymerase translocation.

High resolution cryo-EM structure of the helical RNA-bound Hantaan virus nucleocapsid reveals its assembly mechanisms.

Negative-strand RNA viruses condense their genome into helical nucleocapsids that constitute essential templates for viral replication and transcription. The intrinsic flexibility of nucleocapsids usually prevents their full-length structural characterisation at high resolution. Here, we describe purification of full-length recombinant metastable helical nucleocapsid of Hantaan virus (Hantaviridae family, Bunyavirales order) and determine its structure at 3.3 Å resolution by cryo-electron microscopy. The structure reveals the mechanisms of helical multimerisation via sub-domain exchanges between protomers and highlights nucleotide positions in a continuous positively charged groove compatible with viral genome binding. It uncovers key sites for future structure-based design of antivirals that are currently lacking to counteract life-threatening hantavirus infections. The structure also suggests a model of nucleoprotein-polymerase interaction that would enable replication and transcription solely upon local disruption of the nucleocapsid.

ICTV Virus Taxonomy Profile: Tristromaviridae.

Tristromaviridae is a family of viruses with linear, double-stranded DNA genomes of 16-18 kbp. The flexible, filamentous virions (400±20 nm×30±3 nm) consist of an envelope and an inner core constructed from two structural units: a rod-shaped helical nucleocapsid and a nucleocapsid-encompassing matrix protein layer. Tristromaviruses are lytic and infect hyperthermophilic archaea of the order Thermoproteales. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Tristromaviridae, which is available at www.ictv.global/report/tristromaviridae.

Filovirus Filament Proteins.

Filoviruses are highly filamentous enveloped animal viruses that can cause severe haemorrhagic fevers. The filovirus ribonucleoprotein forms a highly organized double-layered helical nucleocapsid (NC) containing five different virally encoded proteins. The inner layer consists of NP, the RNA binding protein, complexed with the monopartite linear genome. A distinctive outer layer links individual NP subunits with bridges composed of VP24-VP35 heterodimers, which achieves condensation of the NP-RNA into tight helical coils. There are no vertical connections between the outer helical layers, explaining the flexibility of the NC and its ability to bend into tight curves without breaking the genomic RNA. These properties allow the formation of enveloped virions with varying polymorphisms, including single, linear, continuous, linked, comma-shaped and torroidal forms. Virion length is modular so that just one, or two or more genome copies may be present in each virion, producing polyploid particles. The matrix protein VP40, which drives budding and envelopment, is found in a layer adjacent to the inner cytoplasmic side of viral envelope and is arranged in a 5 nm lattice structure, but its exact symmetry is unclear. There is a constant low density gap between VP40 and the nucleocapsid, so that the latter is held rigidly centred on the long axis of the viral filament. This gap likely contains a region of flexible contacts between VP40 and the NC. The unique morphology of filoviruses may be related to high titre replication, their ease of transmission, and abilities to invade a wide range of host cells and tissues.

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications.IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets.

Structure and assembly of the Ebola virus nucleocapsid.

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause hemorrhagic fever1. Filoviruses are within the order Mononegavirales2 which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein (NP) and other viral proteins to form a helical nucleocapsid (NC). NC acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into NP-NP interactions have been derived from structural studies of oligomerized, RNA-encapsidating NP3–6 and cryo-electron microscopy (cryo-EM) of NC7–12 or NC-like structures11–13. There have been no high-resolution reconstructions of complete mononegavirus NCs. Here, we have applied cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus NC within intact viruses and recombinant NC-like assemblies. These structures reveal the identity and arrangement of the NC components, and suggest that the formation of an extended alpha-helix from the disordered C-terminal region of NP-core links NP oligomerization, NC condensation, RNA encapsidation, and accessory protein recruitment.

RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription.

The template for Ebola virus (EBOV) transcription and replication is the helical viral nucleocapsid composed of the viral negative-sense (-) RNA genome, which is complexed by the nucleoprotein (NP), VP35, polymerase L, VP24, and VP30. While viral replication is exerted by polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the viral nucleocapsid protein VP30, an essential EBOV-specific transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP30's function in transcriptional activation. Using a novel mobility shift assay and the 3'-terminal 154 nucleotides of the EBOV genome as a standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein's capability to form homohexamers, (ii) disrupt the integrity of its zinc finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA dependent, while formation of VP30 homohexamers and VP35 homotetramers is not. Our data indicate that RNA binding of VP30 is essential for its transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (-) RNA template to favor productive transcriptional initiation in the presence of termination-active RNA secondary structures. Ebola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance viral transcription and replication in the infected cells. This study shows that transcriptional support activity of the Ebola virus transcription factor VP30 is highly correlated with its ability to bind viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the viral RNA template in order to promote viral RNA synthesis.

Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence.

Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo‐proteins (N) that bind the entire genome. N‐RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self‐organization of N into NC‐like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real‐time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA‐sequence, with the genomic 5′ end and poly‐Adenine sequences assembling efficiently, while sequences such as poly‐Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process.

Self‐Assembly of Measles Virus Nucleocapsid‐like Particles: Kinetics and RNA Sequence Dependence

AbstractMeasles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo‐proteins (N) that bind the entire genome. N‐RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self‐organization of N into NC‐like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real‐time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA‐sequence, with the genomic 5′ end and poly‐Adenine sequences assembling efficiently, while sequences such as poly‐Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process.

A virus of hyperthermophilic archaea with a unique architecture among DNA viruses

Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle.

Rabies in Nigeria: A review of literature

Rabies, also known as hydrophobia is an acute, viral disease of all warm blooded animals including man. It is caused by the rabies virus (RABV), a bullet–shaped, enveloped RNA virus, 45-100 nm in diameter & 100-430 nm in length with projections and helical nucleocapsid, one of the better known encephalitis viruses of the family Rhabdoviridae and genus Lyssavirus type 1It is a major public-health problem in most parts of the developing world. The domestic dog (Canis familiaris) plays a principal role (accounting for over 99%) as a reservoir and transmitter of the disease to humans. Developing countries account for almost all the reported human deaths (99.9%) and most cases of human post-exposure treatments. Rabies is an important public health problem especially in the developing countries and this articles aims to draw attention to this neglected disease.Keywords: rhabdoviridae, rabies