Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding.

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

The Serological Cross-Detection of Bat-Borne Hantaviruses: A Valid Strategy or Taking Chances?

Bats are hosts of a range of viruses, and their great diversity and unique characteristics that distinguish them from all other mammals have been related to the maintenance, evolution, and dissemination of these pathogens. Recently, very divergent hantaviruses have been discovered in distinct species of bats worldwide, but their association with human disease remains unclear. Considering the low success rates of detecting hantavirus RNA in bat tissues and that to date no hantaviruses have been isolated from bat samples, immunodiagnostic tools could be very helpful to understand pathogenesis, epidemiology, and geographic range of bat-borne hantaviruses. In this sense, we aimed to identify in silico immunogenic B-cell epitopes present on bat-borne hantaviruses nucleoprotein (NP) and verify if they are conserved among them and other selected members of Mammantavirinae, using a combination of (the three most used) different prediction algorithms, ELLIPRO, Discotope 2.0, and PEPITO server. To support our data, we in silico modeled 3D structures of NPs from representative members of bat-borne hantaviruses, using comparative and ab initio methods due to the absence of crystallographic structures of studied proteins or similar models in the Protein Data Bank. Our analysis demonstrated the antigenic complexity of the bat-borne hantaviruses group, showing a low sequence conservation of epitopes among members of its own group and a minor conservation degree in comparison to Orthohantavirus, with a recognized importance to public health. Our data suggest that the use of recombinant rodent-borne hantavirus NPs to cross-detect antibodies against bat- or shrew-borne viruses could underestimate the real impact of this virus in nature.

The 1H, 15N, and 13C resonance assignments of the N-terminal domain of the nucleocapsid protein from the Middle East respiratory syndrome coronavirus.

During the past 17 years, the coronaviruses have become a global public emergency, with the first appearance in 2012 in Saudi Arabia of the Middle East respiratory syndrome. Among the structural proteins encoded in the viral genome, the nucleocapsid protein is the most abundant in infected cells. It is a multifunctional phosphoprotein involved in the capsid formation, in the modulation and regulation of the viral life cycle. The N-terminal domain of N protein specifically interacts with transcriptional regulatory sequence (TRS) and is involved in the discontinuous transcription through the melting activity of double-stranded TRS (dsTRS).

An evolutionary insight into emerging Ebolavirus strains isolated in Africa.

On July 19, 2019, the World Health Organization declared the current Ebolavirus (EBOV) outbreak in Congo Democratic Republic (COD) a public health emergency of international concern. To address the potential threat of EBOV evolution outpacing antibody treatment and vaccine efforts, a detailed evolutionary analysis of EBOV strains circulating in different African countries was performed. Genome composition of EBOV strains was studied using multivariate statistical analysis. To investigate the patterns of evolution of EBOV strains, a Bayesian Markov Chain Monte Carlo approach was used. Two different genetic lineages, with a distinct genome composition gave rise to the recent EBOV outbreaks in central and western Africa. Strains isolated in COD in 2018 fall into two different genetic clusters, according to their geographical location of isolation. Different amino acid substitutions among strains from these two clusters have been found, particularly in NP, GP, and L proteins. Significant differences in codon and amino acid usage among clusters were found. Strains isolated in COD in 2018 belong to two distinct genetic clusters, with distinct codon and amino acid usage. Geographical diversity plays an important role in shaping the molecular evolution of EBOV populations.

Characterization of cross-reactive and serotype-specific epitopes on the nucleocapsid proteins of hantaviruses.

The hantavirus nucleocapsid (N) protein fulfills several key roles in virus replication and assembly and is the major antigen in humoral immune responses in humans and mice. Here we report on epitopes involved in serotype-specific and cross-reactive recognition of the N proteins of hantaviruses using monoclonal antibodies (mAbs) against the N proteins of Andes virus (ANDV) and Sin Nombre virus (SNV). The mAbs define at least twelve different epitopic patterns which span eight sequences, including amino acids 17-59, 66-78, 79-91, 157-169, 222-234, 244-263, 274-286 and 326-338 on the SNV and ANDV N proteins. Studies on the cross-reactivity of these mAbs with different hantavirus N proteins indicated that epitopes located within amino acids 244-286 are related to serotype specificity. We analyzed further the location of epitopes with available three-dimensional structure information including the N-terminal coiled-coil and derived exposed and hidden residues of these epitopes. The generated recombinant N proteins and the characterized mAbs are functional tools being now available for hantavirus diagnostics and replication studies.

Hantavirus infection in Brazil: development and evaluation of an enzyme immunoassay and immunoblotting based on N recombinant protein.

The symptoms of hantavirus pulmonary syndrome may resemble those of other febrile illnesses. The development of an accurate diagnostic test should therefore improve clinical prognosis and be useful in epidemiologic studies. We evaluated the use of a recombinant antigen (rNDelta(85)) based on the S-segment sequences of a Brazilian hantavirus for detecting immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against hantavirus in an indirect enzyme immunoassay (EIA). We assayed 613 serum samples (570 from humans and 43 from rodents). IgM EIA had a sensitivity of 94.1% and a specificity of 99.1%. IgG EIA had a sensitivity of 95.2% and a specificity of 98.4%. This evaluation confirms that rNDelta(85) IgM and IgG EIA tests are potentially useful rapid, sensitive, and cost-effective tools for detecting antibodies against hantaviruses indigenous to Brazil and other South American countries, in patients with acute or convalescent hantavirus infection, and in rodent reservoirs.

Geographical distribution of vampire bat-related cattle rabies in Brazil.

Seventy-seven rabies virus (RV) isolates originating from Brazilian cattle were genetically characterized. Partial nucleoprotein gene sequences of these isolates were phylogenetically and geographically analyzed. Cattle isolates, which clustered with the vampire bat-related RV group, were further subdivided into nine genetic subgroups. These subgroups were distributed widely in lowland regions, with some subgroups separated from each other by mountain ranges. In addition, separation of the groups in mountainous regions was correlated with altitude. These results indicate that cattle rabies is derived from several regionally-defined variants, which suggests that its geographical distribution is related to that of the vampire bat population.

Could the homologous sequence of anti-inflammatory pentapeptide (MLIF) produced by Entamoeba histolytica in the N protein of rabies virus affect the inflammatory process?

Amebiasis and rabies are public health problems, and they have in common a poor inflammatory effect in the target organs that they affect. In the GenBank, it was found that the anti-inflammatory peptide monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica homologates 80%, with a fragment of the N protein of the rabies virus. We speculated if the N protein could contribute to the scant inflammatory reaction produced by rabies virus in central nervous system. The N protein was obtained and studied in vitro and in vivo. The N protein, as MLIF, inhibited the respiratory burst in human mononuclear phagocytes (43%, p<0.05), but in contrast to MLIF, it increased chemotaxis and it did not significantly inhibit delayed hypersensitivity skin reaction to 1-chloro-2-4-dinitrobenzene in guinea pigs. Therefore, the full peptide sequence has to be present or it has to be cleaved-free from the large recombinant N protein molecule (55 kDa) to become active.

Studies on antigenic and genomic properties of Brazilian rabies virus isolates.

Despite the recognized stability of rabies virus, differences among isolates from different species have been found. This work was carried out with the aim to identify antigenic and genomic differences in Brazilian rabies virus isolates and to verify whether such alterations would bear any relationship with the different hosts for the virus in nature. For that, 79 Brazilian rabies viruses isolated from different host species and from distinct regions within Brazil were submitted to antigenic characterization with a panel of 11 monoclonal antibodies (Mabs) directed to lyssavirus antigens and to genomic analyses by the reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the N gene followed by restriction endonuclease analysis (REA). In addition, the nucleotide sequences of part of the N gene (225 bp) of seven isolates, taken as representative of the majority of the viruses under study, were determined. The analyses with the Mabs and RT-PCR/REA allowed the identification of two major groups of variants, the first formed by most isolates of cattle and bats and the second formed by viruses of dog origin. Partial sequencing of the N gene confirmed the similarity among isolates from cattle origin and those of vampire bats. However, viruses from non-haematophagous bats exhibited consistent differences from those of vampire bat isolates. Such findings suggest that the variants have evolved fairly stable modifications, which are not altered after passage in a dead-end host of a distinct species. No association could be established between antigenic or genomic alterations and geographic distribution of the isolates, which suggests that evolution of the virus has been directed to adaptation to the host species.

Nucleic acid binding properties and intermediates of HCV core protein multimerization in Pichia pastoris.

Little is known about the in vivo assembly pathway or structure of the hepatitis C virus nucleocapsid. In this work the intermediates of HCcAg multimerization in Pichia pastoris cells and the nucleic acid binding properties of structured nucleocapsid-like particles (NLPs) were studied. Extensive cross-linking was observed for HCcAg after glutaraldehyde treatment. Data suggest that HCcAg exists in dimeric forms probably representing P21-P21, P21-P23, and P23-P23 dimers. In addition, the presence of HCcAg species that might represent trimers and multimers was observed. After sucrose equilibrium density gradient purification and nuclease digestion, NLPs were shown to contain both RNA and DNA molecules. Finally, the analysis by electron microscopy indicated that native NLPs were resistant to nuclease treatment. These results indicated that HCcAg assembles through dimers, trimers, and multimers' intermediates into capsids in P. pastoris cells. Assembly of NLPs in its natural environment might confer stability to these particles by adopting a compact structure.

Functional domains in the feline immunodeficiency virus nucleocapsid protein.

Retroviral nucleocapsid (NC) proteins are small Gag-derived products containing one or two zinc finger motifs that mediate genomic RNA packaging into virions. In this study, we addressed the role of the feline immunodeficiency virus (FIV) NC protein in the late stages of virus replication by analyzing the assembly phenotype of FIV NC mutant viruses and the RNA binding activity of a panel of recombinant FIV NC mutant proteins. Substitution of serine for the first cysteine residue in the NC proximal zinc finger was sufficient to impair both virion assembly and genomic RNA binding. A similar defective phenotype with respect to particle formation and RNA binding was observed when the basic residues Lys28 and Lys29 in the region connecting both zinc fingers were replaced by alanine. In contrast, mutation of the first cysteine residue in the distal zinc finger had no effect on virion production and allowed substantial RNA binding activity of the mutant NC protein. Moreover, this NC mutant virus exhibited wild-type replication kinetics in the feline MYA-1 T-cell line. Interestingly, amino acid substitutions disrupting the highly conserved PSAP and LLDL motifs present in the C-terminus of the FIV NC abrogated virion formation without affecting the NC RNA binding activity. Our results indicate that the proximal zinc finger of the FIV NC is more important for virion production and genomic RNA binding than the distal motif. In addition, this study suggests that assembly domains in the FIV NC C-terminus may be functionally equivalent to those present in the p6 domain of the Gag polyprotein of primate lentiviruses.

In vitro self-assembled HCV core virus-like particles induce a strong antibody immune response in sheep.

The in vitro self-assembly properties of the entire hepatitis C virus core protein (HCcAg) obtained from Pichia pastoris cells and the induction of specific antibody immune response were studied. HCcAg was purified as a low-molecular-weight species by electroelution under denaturing conditions for confirmation of its self-assembly properties. After renaturalization, electron microscopy showed that HCcAg assembled into spherical particles of 30 nm. HCcAg also showed homogeneity and was specifically recognized by serum from a chronic HCV carrier patient. The data indicated that in vitro assembly of HCcAg, into virus-like particles resembling HCV nucleocapsid particles at a mature stage, is an intrinsic quality of this protein. Finally, HCcAg generated a strong antibody immune response in sheep, suggesting its usefulness for stimulating the host immune response against HCV.

The metastable state of nucleocapsids of enveloped viruses as probed by high hydrostatic pressure.

Enveloped viruses fuse their membranes with cellular membranes to transfer their genomes into cells at the beginning of infection. What is not clear, however, is the role of the envelope (lipid bilayer and glycoproteins) in the stability of the viral particle. To address this question, we compared the stability between enveloped and nucleocapsid particles of the alphavirus Mayaro using hydrostatic pressure and urea. The effects were monitored by intrinsic fluorescence, light scattering, and binding of fluorescent dyes, including bis(8-anilinonaphthalene-1-sulfonate) and ethidium bromide. Pressure caused a drastic dissociation of the nucleocapsids as determined by tryptophan fluorescence, light scattering, and gel filtration chromatography. Pressure-induced dissociation of the nucleocapsids was poorly reversible. In contrast, when the envelope was present, pressure effects were much less marked and were highly reversible. Binding of ethidium bromide occurred when nucleocapsids were dissociated under pressure, indicating exposure of the nucleic acid, whereas enveloped particles underwent no changes. Overall, our results demonstrate that removal of the envelope with the glycoproteins leads the particle to a metastable state and, during infection, may serve as the trigger for disassembly and delivery of the genome. The envelope acts as a "Trojan horse," gaining entry into the host cell to allow release of a metastable nucleocapsid prone to disassembly.