Association of SARS-CoV-2 Nucleocapsid Protein Mutations with Patient Demographic and Clinical Characteristics during the Delta and Omicron Waves.

SARS-CoV-2 genomic mutations outside the spike protein that may increase transmissibility and disease severity have not been well characterized. This study identified mutations in the nucleocapsid protein and their possible association with patient characteristics. We analyzed 695 samples from patients with confirmed COVID-19 in Saudi Arabia between 1 April 2021, and 30 April 2022. Nucleocapsid protein mutations were identified through whole genome sequencing. 𝜒2 tests and t tests assessed associations between mutations and patient characteristics. Logistic regression estimated the risk of intensive care unit (ICU) admission or death. Of the 60 mutations identified, R203K was the most common, followed by G204R, P13L, E31del, R32del, and S33del. These mutations were associated with reduced risk of ICU admission. P13L, E31del, R32del, and S33del were also associated with reduced risk of death. By contrast, D63G, R203M, and D377Y were associated with increased risk of ICU admission. Most mutations were detected in the SR-rich region, which was associated with low risk of death. The C-tail and central linker regions were associated with increased risk of ICU admission, whereas the N-arm region was associated with reduced ICU admission risk. Consequently, mutations in the N protein must be observed, as they may exacerbate viral infection and disease severity. Additional research is needed to validate the mutations' associations with clinical outcomes.

A New Cellular Interactome of SARS-CoV-2 Nucleocapsid Protein and Its Biological Implications.

There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.

A review of the effects of ATP and hydroxychloroquine on the phase separation of the SARS-CoV-2 nucleocapsid protein.

SARS-CoV-2 is the coronavirus causing the ongoing pandemic with > 460 millions of infections and > 6 millions of deaths. SARS-CoV-2 nucleocapsid (N) is the only structural protein which plays essential roles in almost all key steps of the viral life cycle with its diverse functions depending on liquid-liquid phase separation (LLPS) driven by interacting with various nucleic acids. The 419-residue N protein is highly conserved in all variants including delta and omicron, and composed of both folded N-/C-terminal domains (NTD/CTD) as well as three long intrinsically disordered regions (IDRs). Recent results have suggested that its CTD and IDRs are also cryptic nucleic acid-binding domains. In this context, any small molecules capable of interfering in its interaction with nucleic acids are anticipated to modulate its LLPS and associated functions. Indeed, ATP, the energy currency existing at very high concentrations (2-12 mM) in all living cells but absent in viruses, modulates LLPS of N protein, and consequently appears to be evolutionarily hijacked by SARS-CoV-2 to promote its life cycle. Hydroxychloroquine (HCQ) has been also shown to specifically bind NTD and CTD to inhibit their interactions with nucleic acids, as well as to disrupt LLPS. Particularly, the unique structure of the HCQ-CTD complex offers a promising strategy for further design of anti-SARS-CoV-2 drugs with better affinity and specificity. The finding may indicate that LLPS is indeed druggable by small molecules, thus opening up a promising direction for drug discovery/design by targeting LLPS in general.

Study of Fluorescence Immunochromatography Based on Quantum Dots for the Detection of SARS-CoV-2 anti-N Protein IgG Antibody

Background: Based on the current prevalence of Coronavirus Disease 2019 (COVID-19), early diagnosis, isolation, and treatment are important methods to prevent and control infectious diseases. The establishment of convenient and efficient immunochromatographic detection techniques is essential for the prevention and control of COVID-19 epidemic. Objective: To establish a method for the detection of SARS-CoV-2 anti-N protein IgG antibody by immun of luorescence chromatography method based on quantum dots labeling technology in August, 2020. In order to determine whether the detected persons had been infected with COVID-19 or been injected with SARS-CoV-2 inactivated vaccine. Methods The prepared rat anti-human secondary antibody and anti-N protein antibody were immobilized on a Nitrocellulose (NC) membrane as detection line (T) and quality control line (C), respectively. Then the SARS-CoV-2 N protein labeled by quantum dots was evenly sprayed on glass fiber, which was assembled, cut and packaged into test strips after drying. The test strips were used to detect the clinical serum of 35 COVID-19 patients and 50 healthy individuals, the results of the initial screening of the ELISA kit were used as a control to calculate the detection specificity and sensitivity of quantum dots fluorescence immunochromatography. The sensitivity of the test strip was detected by using the N protein antibody standard. Results The specificity and sensitivity of the strip were 100.00%, 94.29%, and the susceptibility was 8.53-17.06 ng/ml antibody concentration. Conclusion: The detection of anti-N protein IgG antibody in serum by quantum dots labeling is simple, fast, with strong sensitivity and specificity. Copyright © 2022 by the Chinese General Practice.

Emerging Role of Platelet-Endothelium Interactions in the Pathogenesis of Severe SARS-CoV-2 Infection-Associated Myocardial Injury.

Cardiovascular dysfunction and disease are common and frequently fatal complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Indeed, from early on during the SARS-CoV-2 virus pandemic it was recognized that cardiac complications may occur, even in patients with no underlying cardiac disorders, as part of the acute infection, and that these were associated with more severe disease and increased morbidity and mortality. The most common cardiac complication is acute cardiac injury, defined by significant elevation of cardiac troponins. The potential mechanisms of cardiovascular complications include direct viral myocardial injury, systemic inflammation induced by the virus, sepsis, arrhythmia, myocardial oxygen supply-demand mismatch, electrolyte abnormalities, and hypercoagulability. This review is focused on the prevalence, risk factors and clinical course of COVID-19-related myocardial injury, as well as on current data with regard to disease pathogenesis, specifically the interaction of platelets with the vascular endothelium. The latter section includes consideration of the role of SARS-CoV-2 proteins in triggering development of a generalized endotheliitis that, in turn, drives intense activation of platelets. Most prominently, SARS-CoV-2-induced endotheliitis involves interaction of the viral spike protein with endothelial angiotensin-converting enzyme 2 (ACE2) together with alternative mechanisms that involve the nucleocapsid and viroporin. In addition, the mechanisms by which activated platelets intensify endothelial activation and dysfunction, seemingly driven by release of the platelet-derived calcium-binding proteins, SA100A8 and SA100A9, are described. These events create a SARS-CoV-2-driven cycle of intravascular inflammation and coagulation, which contributes significantly to a poor clinical outcome in patients with severe disease.

SARS-CoV-2 N Protein Induces Acute Lung Injury in Mice via NF-ĸB Activation.

Background: Infection of SARS-CoV-2 may cause acute respiratory syndrome. It has been reported that SARS-CoV-2 nucleocapsid protein (N-protein) presents early in body fluids during infection. The direct involvement of N-protein in lung injury is poorly understood. Methods: Recombinant N-protein was pretreated with polymyxin B, a lipopolysaccharide (LPS)-neutralizing agent. C57BL/6, C3H/HeJ (resistant to LPS), and C3H/HeN (control for C3H/HeJ) mice were exposed to N-protein via intratracheal administration to examine acute lung injury. In vitro, bone marrow-derived macrophages (BMDMs) were cultured with N-protein to study phosphorylation of nuclear factor kappa B (NF-ĸB) p65, macrophage polarization, and expression of proinflammatory cytokines. Results: N-protein produced acute lung injury in C57BL/6 mice, with elevated protein permeability, total cell count, neutrophil infiltration, and proinflammatory cytokines in the bronchioalveolar lavage. N-protein also induced lung injury in both C3H/HeJ and C3H/HeN mice, indicating that the effect could not be attributed to the LPS contamination. N-protein triggered phosphorylation of NF-ĸB p65 in vitro, which was abolished by both N-protein denaturation and treatment with an antibody for N-protein, demonstrating that the effect is N-protein specific. In addition, N-protein promoted M1 macrophage polarization and the expression of proinflammatory cytokines, which was also blocked by N-protein denaturation and antibody for N-protein. Furthermore, N-protein induced NF-ĸB p65 phosphorylation in the lung, while pyrrolidine dithiocarbamate, an NF-ĸB inhibitor, alleviated the effect of N-protein on acute lung injury. Conclusions: SARS-CoV-2 N-protein itself is toxic and induces acute lung injury in mice. Both N-protein and NF-ĸB pathway may be therapeutic targets for treating multi-organ injuries in Coronavirus disease 2019 (COVID-19).

Characterization of IgG Antibody Response against SARS-CoV-2 (COVID-19) in the Cypriot Population.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has hit its second year and continues to damage lives and livelihoods across the globe. There continues to be a global effort to present serological data on SARS-CoV-2 antibodies in different individuals. As such, this study aimed to characterize the seroprevalence of SARS-CoV-2 antibodies in the Cypriot population for the first time since the pandemic started. Our results show that a majority of people infected with SARS-CoV-2 developed IgG antibodies against the virus, whether anti-NP, anti-S1RBD, or both, at least 20 days after their infection. Additionally, the percentage of people with at least one antibody against SARS-CoV-2 in the group of volunteers deemed SARS-CoV-2 negative via RT-PCR or who remain untested/undetermined (14.43%) is comparable to other reported percentages worldwide, ranging anywhere from 0.2% to 24%. We postulate that these percentages reflect the underreporting of true infections in the population, and also show the steady increase of herd immunity. Additionally, we showed a significantly marked decrease in anti-NP IgG antibodies in contrast to relatively stable levels of anti-S1RBD IgG antibodies in previously infected individuals across time.

Structural basis of anti-SARS-CoV-2 activity of HCQ: Specific binding to N protein to disrupt its interaction with nucleic acids and LLPS

SARS-CoV-2 nucleocapsid (N) protein plays the essential roles in key steps of the viral life cycle, thus representing a top drug target. Functionality of N protein including liquid-liquid phase separation (LLPS) depends on its interaction with nucleic acids. Only the variants with N proteins functional in binding nucleic acids might survive and spread in evolution and indeed, the residues critical for binding nucleic acids are highly conserved. Hydroxychloroquine (HCQ) was shown to prevent the transmission in a large-scale clinical study in Singapore but so far, no specific SARS-CoV-2 protein was experimentally identified to be targeted by HCQ. Here by NMR, we unambiguously decode that HCQ specifically binds NTD and CTD of N protein with Kd of 112.1 and 57.1 ?M respectively to inhibit their interaction with nucleic acid, as well as to disrupt LLPS. Most importantly, HCQ-binding residues are identical in SARS-CoV-2 variants and therefore HCQ is likely effective to different variants. The results not only provide a structural basis for the anti-SARS-CoV-2 activity of HCQ, but also renders HCQ to be the first known drug capable of targeting LLPS. Furthermore, the unique structure of the HCQ-CTD complex suggests a promising strategy for design of better anti-SARS-CoV-2 drugs from HCQ. Copyright © 2021 The Author(s). Published by Cambridge University Press.

CTD of SARS-CoV-2 N protein is a cryptic domain for binding ATP and nucleic acid that interplay in modulating phase separation.

SARS-CoV-2 nucleocapsid (N) protein plays essential roles in many steps of the viral life cycle, thus representing a key drug target. N protein contains the folded N-/C-terminal domains (NTD/CTD) and three intrinsically disordered regions, while its functions including liquid-liquid phase separation (LLPS) depend on the capacity in binding various viral/host-cell RNA/DNA of diverse sequences. Previously NTD was established to bind various RNA/DNA while CTD to dimerize/oligomerize for forming high-order structures. By NMR, here for the first time we decrypt that CTD is not only capable of binding S2m, a specific probe derived from SARS-CoV-2 gRNA but with the affinity even higher than that of NTD. Very unexpectedly, ATP, the universal energy currency for all living cells with high cellular concentrations (2-16 mM), specifically binds CTD with Kd of 1.49 ± 0.28 mM. Strikingly, the ATP-binding residues of NTD/CTD are identical in the SARS-CoV-2 variants while ATP and S2m interplay in binding NTD/CTD, as well as in modulating LLPS critical for the viral life cycle. Results together not only define CTD as a novel binding domain for ATP and nucleic acid, but enforce our previous proposal that ATP has been evolutionarily exploited by SARS-CoV-2 to complete its life cycle in the host cell. Most importantly, the unique ATP-binding pockets on NTD/CTD may offer promising targets for design of specific anti-SARS-CoV-2 molecules to fight the pandemic. Fundamentally, ATP emerges to act at mM as a cellular factor to control the interface between the host cell and virus lacking the ability to generate ATP.

Structural Basis for SARS-CoV-2 Nucleocapsid Protein Recognition by Single-Domain Antibodies.

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.

Arginine methylation of SARS-Cov-2 nucleocapsid protein regulates RNA binding, its ability to suppress stress granule formation, and viral replication.

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5'-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein-mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs.

In-vitro acetylation of SARS-CoV and SARS-CoV-2 nucleocapsid proteins by human PCAF and GCN5.

Recently, the novel coronavirus (SARS-CoV-2), which has spread from China to the world, was declared a global public health emergency, which causes lethal respiratory infections. Acetylation of several proteins plays essential roles in various biological processes, such as viral infections. We reported that the nucleoproteins of influenza virus and Zaire Ebolavirus were acetylated, suggesting that these modifications contributed to the molecular events involved in viral replication. Similar to influenza virus and Ebolavirus, the coronavirus also contains single-stranded RNA, as its viral genome interacts with the nucleocapsid (N) proteins. In this study, we report that SARS-CoV and SARS-CoV-2 N proteins are strongly acetylated by human histone acetyltransferases, P300/CBP-associated factor (PCAF), and general control nonderepressible 5 (GCN5), but not by CREB-binding protein (CBP) in vitro. Liquid chromatography-mass spectrometry analyses identified 2 and 12 acetyl-lysine residues from SARS-CoV and SARS-CoV-2 N proteins, respectively. Particularly in the SARS-CoV-2 N proteins, the acetyl-lysine residues were localized in or close to several functional sites, such as the RNA interaction domains and the M-protein interacting site. These results suggest that acetylation of SARS-CoV-2 N proteins plays crucial roles in their functions.

ATP biphasically modulates LLPS of SARS-CoV-2 nucleocapsid protein and specifically binds its RNA-binding domain.

SARS-CoV-2 is a highly contagious coronavirus causing the ongoing pandemic. Very recently its genomic RNA of ∼30 kb was decoded to be packaged with nucleocapsid (N) protein into phase separated condensates. Interestingly, viruses have no ability to generate ATP but host cells have very high ATP concentrations of 2-12 mM. A key question thus arises whether ATP modulates liquid-liquid phase separation (LLPS) of the N protein. Here we discovered that ATP not only biphasically modulates LLPS of the viral N protein as we previously found on human FUS and TDP-43, but also dissolves the droplets induced by oligonucleic acid. Residue-specific NMR characterization showed ATP specifically binds the RNA-binding domain (RBD) of the N protein with the average Kd of 3.3 ± 0.4 mM. The ATP-RBD complex structure was constructed by NMR-derived constraints, in which ATP occupies a pocket within the positive-charged surface utilized for binding nucleic acids. Our study suggests that ATP appears to be exploited by SARS-CoV-2 to promote its life cycle by facilitating the uncoating, localizing and packing of its genomic RNA. Therefore the interactions of ATP with the viral RNA and N protein might represent promising targets for design of drugs and vaccines to terminate the pandemic.

A computational study to disclose potential drugs and vaccine ensemble for COVID-19 conundrum.

The nucleocapsid (N) protein of SARS-COV-2, a virus responsible for the current COVID-19 pandemic, is considered a potential candidate for the design of new drugs and vaccines. The protein is central to several critical events in virus production, with its highly druggable nature and rich antigenic determinants making it an excellent anti-viral biomolecule. Docking-based virtual screening using the Asinex anti-viral library identified binding of drug molecules at three specific positions: loop 1 region, loop 2 region and ß-sheet core pockets, the loop 2 region being the most common binding and stable site for the bulk of the molecules. In parallel, the protein was characterized by vaccine design perspective and harboured three potential B cell-derived T cell epitopes: PINTNSSPD, GVPINTNSS, and DHIGTRNPA. The epitopes are highly antigenic, virulent, non-allergic, non-toxic, bind with good affinity to the highly prevalent DRB*0101 allele and show an average population coverage of 95.04%. A multi-epitope vaccine ensemble which was 83 amino acids long was created. This was highly immunogenic, robust in generating both humoral and cellular immune responses, thermally stable, and had good physicochemical properties that could be easily analyzed in in vivo and in vitro studies. Conformational dynamics of both drug and vaccine ensemble with respect to the receptors are energetically stable, shedding light on favourable conformation and chemical interactions. These facts were validated by subjecting the complexes to relative and absolute binding free energy methods of MMGB/PBSA and WaterSwap. A strong agreement on the system stability was disclosed that supported ligand high affinity potential for the receptors. Collectively, this work sought to provide preliminary experimental data of existing anti-viral drugs as a possible therapy for COVID-19 infections and a new peptide-based vaccine for protection against this pandemic virus.

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle.

Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis.IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.