Coronaviruses (CoV) are enveloped viruses and rely on their nucleocapsid N protein to incorporate the positive-stranded genomic RNA into the virions. CoV N proteins form oligomers but the mechanism and relevance underlying their multimerization remain to be fully understood. Using in vitro pull-down experiments and density glycerol gradients, we found that at least 3 regions distributed over its entire length mediate the self-interaction of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) N protein. The fact that these regions can bind reciprocally between themselves provides a possible molecular basis for N protein oligomerization. Interestingly, cytoplasmic N molecules of MHV-infected cells constitutively assemble into oligomers through a process that does not require binding to genomic RNA. Based on our data, we propose a model where constitutive N protein oligomerization allows the optimal loading of the genomic viral RNA into a ribonucleoprotein complex via the presentation of multiple viral RNA binding motifs.

The homotrimeric fusion protein nucleocapsid (NC)-dUTPase combines domains that participate in RNA/DNA folding, reverse transcription, and DNA repair in Mason-Pfizer monkey betaretrovirus infected cells. The structural organization of the fusion protein remained obscured by the N- and C-terminal flexible segments of dUTPase and the linker region connecting the two domains that are invisible in electron density maps. Small-angle X-ray scattering reveals that upon oligonucleotide binding the NC domains adopt the trimeric symmetry of dUTPase. High-resolution X-ray structures together with molecular modeling indicate that fusion with NC domains dramatically alters the conformation of the flexible C-terminus by perturbing the orientation of a critical beta-strand. Consequently, the C-terminal segment is capable of double backing upon the active site of its own monomer and stabilized by non-covalent interactions formed with the N-terminal segment. This co-folding of the dUTPase terminal segments, not observable in other homologous enzymes, is due to the presence of the fused NC domain. Structural and genomic advantages of fusing the NC domain to a shortened dUTPase in betaretroviruses and the possible physiological consequences are envisaged.

The positive transcription elongation factor b (P-TEFb) is an essential factor that induces transcription elongation and is also negatively regulated by the cellular factor HEXIM1. Previously, the chimeric protein HEXIM1-Tat (HT) was demonstrated to inhibit human immunodeficiency virus-1 (HIV)-1 transcription. In this study, we attempted to develop an improved antiviral protein that specifically binds viral RNA (vRNA) by fusing HT to HIV-1 nucleocapsid (NC). Thus, we synthesized NC-HEXIM1-Tat (NHT) and HEXIM1-Tat-NC (HTN). NHT and HTN inhibited virus proliferation more effectively than HT, and they did not attenuate the function of HT. Notably, NHT and HTN inhibited the infectivity of the progeny virus, whereas HT had no such effect. NHT and HTN selectively and effectively interacted with vRNA and inhibited the proper packaging of the HIV-1 genome. Taken together, our results illustrated that the novel NC-fused chimeric proteins NHT and HTN display novel mechanisms of anti-HIV effects by inhibiting both HIV-1 transcription and packaging.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) share tropism for swine intestinal epithelial cells. Whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. In this study, we investigated how different coronavirus nucleocapsid (CoV N) proteins interact and affect PEDV replication. We found that PDCoV N and TGEV N can competitively interact with PEDV N. However, the presence of PDCoV or TGEV N led to very different outcomes on PEDV replication. While PDCoV N significantly suppresses PEDV replication, overexpression of TGEV N, like that of PEDV N, increases production of PEDV RNA and virions. Despite partial interchangeability in nucleocapsid oligomerization and viral RNA synthesis, endogenous PEDV N cannot be replaced in the production of infectious PEDV particles. Results from this study give insights into functional compatibilities and evolutionary relationship between CoV viral proteins during viral co-infection and co-evolution.

COVID-19 is increasingly affecting human health and global economy. Understanding the fundamental mechanisms of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is highly demanded to develop treatments for COVID-19. SARS-CoV and SARS-CoV-2 share 92.06% identity in their N protein RBDs' sequences, which results in very similar structures. However, the SARS-CoV-2 is more easily to spread. Utilizing multi-scale computational approaches, this work studied the fundamental mechanisms of the nucleocapsid (N) proteins of SARS-CoV and SARS-CoV-2, including their stabilities and binding strengths with RNAs at different pH values. Electrostatic potential on the surfaces of N proteins show that both the N proteins of SARS-CoV and SARS-CoV-2 have dominantly positive potential to attract RNAs. The binding forces between SARS-CoV N protein and RNAs at different distances are similar to that of SARS-CoV-2, both in directions and magnitudes. The electric filed lines between N proteins and RNAs are also similar for both SARS-CoV and SARS-CoV-2. The folding energy and binding energy dependence on pH revealed that the best environment for N proteins to perform their functions with RNAs is the weak acidic environment.

The avian influenza viruses (AIVs) can be highly contagious to poultry and a zoonotic threat to humans. Since the memory CD8(+) T lymphocyte responses in chickens to AIV proteins have not been defined, these responses to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins were evaluated by ex vivo stimulation with virus infected non-professional antigen presenting cells. Secretion of IFNgamma by activated T lymphocytes was evaluated through macrophage induction of nitric oxide. AIV specific, MHC-I restricted memory CD8(+) T lymphocyte responses to NP and HA were observed 3 to 9 weeks post-inoculation (p.i.). The responses specific to NP were greater than those to HA with maximum responses being observed at 5 weeks p.i. followed by a decline to weakly detectable levels by 9 weeks p.i. The cross-reaction of T lymphocytes to a heterologous H7N2 AIV strain demonstrated their ability to respond to a broader range of AIV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic of the Coronavirus disease in late 2019 (COVID-19). Vaccine development efforts have predominantly been aimed at 'Extra-viral' Spike (S) protein as vaccine vehicles, but there are concerns regarding 'viral immune escape' since multiple mutations may enable the mutated virus strains to escape from immunity against S protein. The 'Intra-viral' Nucleocapsid (N-protein) is relatively conserved among mutant strains of coronaviruses during spread and evolution. Herein, we demonstrate novel vaccine candidates against SARS-CoV-2 by using the whole conserved N-protein or its fragment/peptides. Using ELISA assay, we showed that high titers of specific anti-N antibodies (IgG, IgG1, IgG2a, IgM) were maintained for a reasonably long duration (> 5 months), suggesting that N-protein is an excellent immunogen to stimulate host immune system and robust B-cell activation. We synthesized three peptides located at the conserved regions of N-protein among CoVs. One peptide showed as a good immunogen for vaccination as well. Cytokine arrays on post-vaccination mouse sera showed progressive up-regulation of various cytokines such as IFN-γ and CCL5, suggesting that TH1 associated responses are also stimulated. Furthermore, vaccinated mice exhibited an elevated memory T cells population. Here, we propose an unconventional vaccine strategy targeting the conserved N-protein as an alternative vaccine target for coronaviruses. Moreover, we generated a mouse monoclonal antibody specifically against an epitope shared between SARS-CoV and SARS-CoV-2, and we are currently developing the First-in-Class humanized anti-N-protein antibody to potentially treat patients infected by various CoVs in the future.

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.

Patients with severe coronavirus disease 2019 (COVID-19) demonstrate dysregulated immune responses including exacerbated neutrophil functions. Massive neutrophil infiltrations accompanying neutrophil extracellular trap (NET) formations are also observed in patients with severe COVID-19. However, the mechanism underlying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation has not yet been elucidated. Here we show that 2 viral proteins encoded by SARS-CoV-2, the nucleocapsid protein and the whole spike protein, induce NET formation from neutrophils. NET formation was ROS-independent and was completely inhibited by the spleen tyrosine kinase inhibition. The inhibition of p38 MAPK, protein kinase C, and JNK signaling pathways also inhibited viral protein-induced NET formation. Our findings demonstrate one method by which SARS-CoV-2 evades innate immunity and provide a potential target for therapeutics to treat patients with severe COVID-19.

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARS-CoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha- but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals.

SARS-CoV-2 is a new member of the genus Betacoronavirus, responsible for the COVID-19 pandemic. The virus crossed the species barrier and established in the human population taking advantage of the spike protein high affinity for the ACE receptor to infect the lower respiratory tract. The Nucleocapsid (N) and Spike (S) are highly immunogenic structural proteins and most commercial COVID-19 diagnostic assays target these proteins. In an unpredictable epidemic, it is essential to know about their genetic variability. The objective of this study was to describe the substitution frequency of the S and N proteins of SARS-CoV-2 in South America. A total of 504 amino acid and nucleotide sequences of the S and N proteins of SARS-CoV-2 from seven South American countries (Argentina, Brazil, Chile, Ecuador, Peru, Uruguay, and Colombia), reported as of June 3, and corresponding to samples collected between March and April 2020, were compared through substitution matrices using the Muscle algorithm. Forty-three sequences from 13 Colombian departments were obtained in this study using the Oxford Nanopore and Illumina MiSeq technologies, following the amplicon-based ARTIC network protocol. The substitutions D614G in S and R203K/G204R in N were the most frequent in South America, observed in 83% and 34% of the sequences respectively. Strikingly, genomes with the conserved position D614 were almost completely replaced by genomes with the G614 substitution between March to April 2020. A similar replacement pattern was observed with R203K/G204R although more marked in Chile, Argentina and Brazil, suggesting similar introduction history and/or control strategies of SARS-CoV-2 in these countries. It is necessary to continue with the genomic surveillance of S and N proteins during the SARS-CoV-2 pandemic as this information can be useful for developing vaccines, therapeutics and diagnostic tests.

Most coronaviruses infecting humans cause mild diseases, whereas severe acute respiratory syndrome (SARS)-associated coronavirus is an extremely dangerous pathogen. Here, we report the development of a serologic assay for detection of antibodies to human coronaviruses (HCoVs) based on recombinant nucleocapsid (N) proteins of all known pathogenic strains (229E, NL63, OC43, HKU1, SARS). The novel immunoassay is highly useful for epidemiologic surveys, where use of nucleic acid diagnostics often is limited. Purified recombinant antigens were immobilized on nitrocellulose membranes and applied in a line immunoassay, which allows rapid detection of antibodies to 5 different HCoVs in a single experiment. For assay evaluation, serum samples from persons infected with 229E or OC43 (acute/convalescent), recovered SARS patients and healthy donors were analyzed. Screening for nucleocapsid (N)-specific immunoglobulin G (IgG) in convalescent sera reached 100% sensitivity. With this new technique, we found that recently identified NL63 and HKU1 contribute significantly to the overall spectrum of coronavirus infections. Possibly, cross-reactive antibody responses were observed using 229E and OC43 serum pairs. However, the potential of this assay could clearly be demonstrated employing SARS-positive serum samples, where nonspecific binding to nucleocapsids of other HCoVs was not observed. This coronavirus strain-specific line immunoassay represents a powerful tool for serologic diagnostics.

The APOBEC3 (A3) cytidine deaminases are antiretroviral proteins, whose targets include human immunodeficiency virus type-1 (HIV-1). Their incorporation into viral particles is critical for antiviral activity and is driven by interactions with the RNA molecules that are packaged into virions. However, it is unclear whether A3 proteins preferentially target RNA molecules that are destined to be packaged and if so, how. Using cross-linking immunoprecipitation sequencing (CLIP-seq), we determined the RNA binding preferences of the A3F, A3G and A3H proteins. We found that A3 proteins bind preferentially to RNA segments with particular properties, both in cells and in virions. Specifically, A3 proteins target RNA sequences that are G-rich and/or A-rich and are not scanned by ribosomes during translation. Comparative analyses of HIV-1 Gag, nucleocapsid (NC) and A3 RNA binding to HIV-1 RNA in cells and virions revealed the striking finding that A3 proteins partially mimic the RNA binding specificity of the HIV-1 NC protein. These findings suggest a model for A3 incorporation into HIV-1 virions in which an NC-like RNA binding specificity is determined by nucleotide composition rather than sequence. This model reconciles the promiscuity of A3 RNA binding that has been observed in previous studies with a presumed advantage that would accompany selective binding to RNAs that are destined to be packaged into virions.

Successful vaccine efforts countering the COVID-19 pandemic are centralized around the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein as viral antigen and have greatly reduced the morbidity and mortality associated with COVID-19. Since the start of this pandemic, SARS-CoV-2 has evolved resulting in new variants of concern (VOC) challenging the vaccine-established immunologic memory. We show that vaccination with a vesicular stomatitis virus (VSV)-based vaccine expressing the SARS-CoV-2 S plus the conserved nucleocapsid (N) protein was protective in a hamster challenge model when a single dose was administered 28 or 10 days prior to challenge, respectively. In this study, only intranasal vaccination resulted in protection against challenge with multiple VOC highlighting that the addition of the N protein indeed improved protective efficacy. This data demonstrates the ability of a VSV-based dual-antigen vaccine to reduce viral shedding and protect from disease caused by SARS-CoV-2 VOC.

To inform seroepidemiological studies, we characterized the IgG- responses in COVID-19 patients against the two major SARS-CoV-2 viral proteins, spike (S) and nucleocapsid (N). We tested 70 COVID-19 sera collected up to 85 days post-symptom onset and 230 non-COVID-19 sera, including 27 SARS sera from 2003. Although the average SARS-CoV-2 S and N-IgG titers were comparable, N-responses were more variable among individuals. S- and N-assay specificity tested with non-COVID-19 sera were comparable at 97.5% and 97.0%, respectively. Therefore, S will make a better target due to its lower cross-reactive potential and its' more consistent frequency of detection compared to N.

Host-virus protein interactions are critical for intracellular viral propagation. Understanding the interactions between cellular and viral proteins may help us develop new antiviral strategies. Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe damage to the global swine industry. Here, we employed co-immunoprecipitation and liquid chromatography-mass spectrometry to characterize 426 unique PEDV nucleocapsid (N) protein-binding proteins in infected Vero cells. A protein-protein interaction network (PPI) was created, and gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses revealed that the PEDV N-bound proteins belong to different cellular pathways, such as nucleic acid binding, ribonucleoprotein complex binding, RNA methyltransferase, and polymerase activities. Interactions of the PEDV N protein with 11 putative proteins: tripartite motif containing 21, DEAD-box RNA helicase 24, G3BP stress granule assembly factor 1, heat shock protein family A member 8, heat shock protein 90 alpha family class B member 1, YTH domain containing 1, nucleolin, Y-box binding protein 1, vimentin, heterogeneous nuclear ribonucleoprotein A2/B1, and karyopherin subunit alpha 1, were further confirmed by in vitro co-immunoprecipitation assay. In summary, studying an interaction network can facilitate the identification of antiviral therapeutic strategies and novel targets for PEDV infection.

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.

γδ T cells are thought to contribute to immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanisms by which they are activated by the virus are unknown. Using flow cytometry, we investigated if the two most abundant viral structural proteins, spike and nucleocapsid, can activate human γδ T cell subsets, directly or in the presence of dendritic cells (DC). Both proteins failed to induce interferon-γ production by Vδ1 or Vδ2 T cells within fresh mononuclear cells or lines of expanded γδ T cells generated from healthy donors, but the same proteins stimulated CD3+ cells from COVID-19 patients. The nucleocapsid protein stimulated interleukin-12 production by DC and downstream interferon-γ production by co-cultured Vδ1 and Vδ2 T cells, but protease digestion and use of an alternative nucleocapsid preparation indicated that this activity was due to contaminating non-protein material. Thus, SARS-CoV-2 spike and nucleocapsid proteins do not have stimulatory activity for DC or γδ T cells. We propose that γδ T cell activation in COVID-19 patients is mediated by immune recognition of viral RNA or other structural proteins by γδ T cells, or by other immune cells, such as DC, that produce γδ T cell-stimulatory ligands or cytokines.

This study assesses the clinical performance of three anti-SARS-CoV-2 assays, namely EUROIMMUN anti-SARS-CoV-2 nucleocapsid (IgG) ELISA, Elecsys anti-SARS-CoV-2 nucleocapsid (total antibodies) assay, and LIAISON anti-SARS-CoV-2 spike proteins S1 and S2 (IgG) assay. One hundred and thirty-seven coronavirus disease 2019 (COVID-19) samples from 96 reverse-transcription polymerase chain reaction confirmed patients were chosen to perform the sensitivity analysis. Non-SARS-CoV-2 sera (n = 141) with a potential cross-reaction to SARS-CoV-2 immunoassays were included in the specificity analysis. None of these tests demonstrated a sufficiently high clinical sensitivity to diagnose acute infection. Fourteen days since symptom onset, we did not find any significant difference between the three techniques in terms of sensitivities. However, Elecsys performed better in terms of specificity. All three anti-SARS-CoV-2 assays had equivalent sensitivities 14 days from symptom onset to diagnose past-COVID-19 infection. We also confirmed that anti-SARS-CoV-2 determination before Day 14 is of less clinical interest.

The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC. Non-human primates are frequently used as a platform to study retroviral infection in vivo; thus, it is important to understand underlying mechanistic differences between HIV-1 and SIV NC.