Review

Authors: Louis-Marie Bloyet

Viruses of the 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.

Topics: Gene Expression Regulation, Viral; Humans; Nucleocapsid; Nucleocapsid Proteins; Paramyxoviridae Infections; Paramyxovirinae; Phylogeny; RNA, Viral

PubMed: 34960734
DOI: 10.3390/v13122465

Authors: Jin Hu, Liudi Tang, Junjun Cheng...

Upon infection of hepatocyte, Hepatitis B virus (HBV) genomic DNA in nucleocapsid is transported into the nucleus and converted into a covalently closed circular (ccc) DNA to serve as the template for transcription of viral RNAs. Viral DNA in the cytoplasmic progeny nucleocapsid is another resource to fuel cccDNA amplification. Apparently, nucleocapsid disassembly, or viral genomic DNA uncoating, is an essential step for cccDNA synthesis from both infection and intracellular amplification pathways, and has a potential to activate DNA sensors and induce an innate immune response in infected hepatocytes. However, where and how the nucleocapsid disassembly occurs is not well understood. The work reported herein showed that the enhanced disassembly of progeny mature nucleocapsids in the cytoplasm supported cccDNA intracellular amplification, but failed to activate the cGAS-STING-mediated innate immune response in hepatocytes. Interestingly, while expression of a cytoplasmic exonuclease TREX1 in human hepatoma cells supporting HBV replication significantly reduced the amounts of cccDNA as well as its precursor, deproteinized relaxed circular (rc) DNA, expression of TREX1 in sodium taurocholate cotransporting polypeptide-expressing human hepatoma cells did not inhibit cccDNA synthesis from HBV infection. The results from this cytoplasmic nuclease protection assay imply that the disassembly of progeny mature nucleocapsids and removal of viral DNA polymerase covalently linked to the 5' end of minus strand of rcDNA take place in the cytoplasm. On the contrary, the disassembly of virion-derived nucleocapsids during infection may occur at a different subcellular compartment and possibly distinct mechanisms.

Topics: Cell Line; Cytoplasm; DNA, Circular; DNA, Viral; Exodeoxyribonucleases; Hep G2 Cells; Hepatitis B virus; Hepatocytes; Humans; Immunity, Innate; Mutation; Nucleocapsid; Nucleotidyltransferases; Phosphoproteins

PubMed: 33870849
DOI: 10.1080/22221751.2021.1919034

Comparative Study

Authors: Brad Poore, Robert D Nerenz, Dina Brodis...

INTRODUCTION

Commercially available serological assays for SARS-CoV-2 detect antibodies to either the nucleocapsid or spike protein. Here we compare the performance of the Beckman-Coulter SARS-CoV-2 spike IgG assay to that of the Abbott SARS-CoV-2 nucleocapsid IgG and Roche Anti-SARS-CoV-2 nucleocapsid total antibody assays. In addition, we document the trend in nucleocapsid and spike antibodies in sequential samples collected from convalescent plasma donors.

METHODS

Plasma or serum samples from 20 individual SARS-CoV-2 RT-PCR-positive inpatients (n = 172), 20 individual convalescent donors with a previous RT-PCR-confirmed SARS-CoV-2 infection (n = 20), were deemed positive SARS-CoV-2 samples. RT-PCR-negative inpatients (n = 24), and 109 pre-SARS-CoV-2 samples were determined to be SARS-CoV-2 negative. Samples were assayed by the Abbott, Roche, and Beckman assays.

RESULTS

All three assays demonstrated 100% specificity. Abbott, Beckman, and Roche platforms had sensitivities of 98%, 93%, and 90% respectively, with the difference in sensitivity attributed primarily to samples from immunocompromised patients. After the exclusion of samples immunocompromised patients, all assays exhibited ≥ 95% sensitivity. In sequential samples collected from the same individuals, the Roche nucleocapsid antibody assay demonstrated continually increasing signal intensity, with maximal values observed at the last time point examined. In contrast, the Beckman spike IgG antibody signal peaked between 14 and 28 days post positive SARS-CoV-2 PCR and steadily declined in subsequent samples. Subsequent collections 51-200 days (median of 139 days) post positive SARS-CoV-2 RT-PCR from five inpatients and five convalescent donors revealed that spike and nucleocapsid antibodies remained detectable for several months after confirmed infection.

CONCLUSIONS

The three assays are sensitive and specific for SARS-CoV-2 antibodies. Nucleocapsid and spike antibodies were detectable for up to 200 days post-positive SARS-CoV-2 PCR but demonstrated markedly different trends in signal intensity.

Topics: Antibodies, Viral; COVID-19; COVID-19 Serological Testing; Humans; Immunoassay; Longitudinal Studies; Nucleocapsid; SARS-CoV-2

PubMed: 34118242
DOI: 10.1016/j.clinbiochem.2021.05.011

Authors: Christiane Iserman, Christine A Roden, Mark A Boerneke...

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

Topics: Animals; Antiviral Agents; COVID-19; Chlorocebus aethiops; Coronavirus Nucleocapsid Proteins; Drug Evaluation, Preclinical; Genome, Viral; HEK293 Cells; Humans; Nucleocapsid; Phosphoproteins; RNA, Viral; SARS-CoV-2; Vero Cells

PubMed: 33290746
DOI: 10.1016/j.molcel.2020.11.041

Authors: De-Sheng Ker, Huw T Jenkins, Sandra J Greive...

Nipah and its close relative Hendra are highly pathogenic zoonotic viruses, storing their ssRNA genome in a helical nucleocapsid assembly formed by the N protein, a major viral immunogen. Here, we report the first cryoEM structure for a Henipavirus RNA-bound nucleocapsid assembly, at 3.5 Å resolution. The helical assembly is stabilised by previously undefined N- and C-terminal segments, contributing to subunit-subunit interactions. RNA is wrapped around the nucleocapsid protein assembly with a periodicity of six nucleotides per protomer, in the "3-bases-in, 3-bases-out" conformation, with protein plasticity enabling non-sequence specific interactions. The structure reveals commonalities in RNA binding pockets and in the conformation of bound RNA, not only with members of the Paramyxoviridae family, but also with the evolutionarily distant Filoviridae Ebola virus. Significant structural differences with other Paramyxoviridae members are also observed, particularly in the position and length of the exposed α-helix, residues 123-139, which may serve as a valuable epitope for surveillance and diagnostics.

Topics: Cryoelectron Microscopy; Models, Molecular; Molecular Conformation; Nipah Virus; Nucleocapsid; Nucleocapsid Proteins; RNA, Viral

PubMed: 34270629
DOI: 10.1371/journal.ppat.1009740

Review

Authors: Ming Luo, James Ross Terrell, Shelby Ashlyn Mcmanus...

Negative strand RNA viruses (NSVs) include many important human pathogens, such as influenza virus, Ebola virus, and rabies virus. One of the unique characteristics that NSVs share is the assembly of the nucleocapsid and its role in viral RNA synthesis. In NSVs, the single strand RNA genome is encapsidated in the linear nucleocapsid throughout the viral replication cycle. Subunits of the nucleocapsid protein are parallelly aligned along the RNA genome that is sandwiched between two domains composed of conserved helix motifs. The viral RNA-dependent-RNA polymerase (vRdRp) must recognize the protein-RNA complex of the nucleocapsid and unveil the protected genomic RNA in order to initiate viral RNA synthesis. In addition, vRdRp must continuously translocate along the protein-RNA complex during elongation in viral RNA synthesis. This unique mechanism of viral RNA synthesis suggests that the nucleocapsid may play a regulatory role during NSV replication.

Topics: Genome, Viral; Models, Molecular; Negative-Sense RNA Viruses; Nucleocapsid; Nucleocapsid Proteins; Protein Conformation; Protein Folding; RNA, Viral; RNA-Dependent RNA Polymerase

PubMed: 32751700
DOI: 10.3390/v12080835

Authors: Chao Wu, Abraham J Qavi, Austin B Moyle...

Nucleocapsid proteins are essential for SARS-CoV-2 life cycle. Here, we describe protocols to gather domain-specific insights about essential properties of nucleocapsids. These assays include dynamic light scattering to characterize oligomerization, fluorescence polarization to quantify RNA binding, hydrogen-deuterium exchange mass spectrometry to map RNA binding regions, negative-stain electron microscopy to visualize oligomeric species, interferon reporter assay to evaluate interferon signaling modulation, and a serology assay to reveal insights for improved sensitivity and specificity. These assays are broadly applicable to RNA-encapsidated nucleocapsids. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).

Topics: Antiviral Agents; COVID-19; Coronavirus Nucleocapsid Proteins; Humans; Interferons; Nucleocapsid; Phosphoproteins; Protein Binding; RNA, Viral; SARS-CoV-2

PubMed: 34642671
DOI: 10.1016/j.xpro.2021.100906

Authors: Maria Tutukina, Anna Kaznadzey, Maria Kireeva...

Since SARS-CoV-2 appeared in late 2019, many studies on the immune response to COVID-19 have been conducted, but the asymptomatic or light symptom cases were somewhat understudied as respective individuals often did not seek medical help. Here, we analyze the production of the IgG antibodies to viral nucleocapsid (N) protein and receptor-binding domain (RBD) of the spike protein and assess the serum neutralization capabilities in a cohort of patients with different levels of disease severity. In half of light or asymptomatic cases the antibodies to the nucleocapsid protein, which serve as the main target in many modern test systems, were not detected. They were detected in all cases of moderate or severe symptoms, and severe lung lesions correlated with respective higher signals. Antibodies to RBD were present in the absolute majority of samples, with levels being sometimes higher in light symptom cases. We thus suggest that the anti-RBD/anti-N antibody ratio may serve as an indicator of the disease severity. Anti-RBD IgG remained detectable after a year or more since the infection, even with a slight tendency to raise over time, and the respective signal correlated with the serum capacity to inhibit the RBD interaction with the ACE-2 receptor.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antibodies; Antibodies, Neutralizing; Antibodies, Viral; Asymptomatic Infections; COVID-19; Coronavirus Nucleocapsid Proteins; Female; Humans; Immunoglobulin G; Immunoglobulin M; Male; Middle Aged; Nucleocapsid; Nucleocapsid Proteins; Phosphoproteins; Russia; SARS-CoV-2; Spike Glycoprotein, Coronavirus

PubMed: 34696374
DOI: 10.3390/v13101945

Authors: Anne Monette, Andrew J Mouland

Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases.

Topics: Computational Biology; Copper; Meta-Analysis as Topic; Molecular Dynamics Simulation; Neurodegenerative Diseases; Nucleocapsid; Nucleocapsid Proteins; Prions; Protein Domains; Viral Proteins; Zinc

PubMed: 33081049
DOI: 10.3390/v12101179

Authors: Chaoyi Xu, Nidhi Katyal, Tanya Nesterova...

Ebola virus (EBOV) is a human pathogen with the ability to cause hemorrhagic fever and bleeding diathesis in hosts. The life cycle of EBOV depends on its nucleocapsid. The Ebola nucleocapsid consists of a helical assembly of nucleoproteins (NPs) encapsidating single-stranded viral RNA (ssRNA). Knowledge of the molecular determinants of Ebola nucleocapsid stability is essential for the development of therapeutics against EBOV. However, large degrees of freedom associated with the Ebola nucleocapsid helical assembly pose a computational challenge, thereby limiting the previous simulation studies to the level of monomers. In the present work, we have performed all atom molecular dynamics (MD) simulations of the helical assembly of EBOV nucleoproteins in the absence and presence of ssRNA. We found that ssRNA is essential for maintaining structural integrity of the nucleocapsid. Other molecular determinants observed to stabilize the nucleocapsid include NP-RNA and NP-NP interactions and ion distributions. Additionally, the structural and dynamical behavior of the nucleocapsid monomer depends on its position in the helical assembly. NP monomers present on the longitudinal edges of the helical tube are more exposed, flexible, and have weaker NP-NP interactions than those residing in the center. This work provides key structural features stabilizing the nucleocapsid that may serve as therapeutic targets.

Topics: Ebolavirus; Humans; Molecular Dynamics Simulation; Nucleocapsid

PubMed: 33092380
DOI: 10.1063/5.0021491