The immune-evasive proline-283 substitution in influenza nucleoprotein increases aggregation propensity without altering the native structure.

Nucleoprotein (NP) is a key structural protein of influenza ribonucleoprotein complexes and is central to viral RNA packing and trafficking. NP also determines the sensitivity of influenza to myxovirus resistance protein 1 (MxA), an innate immunity factor that restricts influenza replication. A few critical MxA-resistant mutations have been identified in NP, including the highly conserved proline-283 substitution. This essential proline-283 substitution impairs influenza growth, a fitness defect that becomes particularly prominent at febrile temperature (39°C) when host chaperones are depleted. Here, we biophysically characterize proline-283 NP and serine-283 NP to test whether the fitness defect is caused by the proline-283 substitution introducing folding defects. We show that the proline-283 substitution changes the folding pathway of NP, making NP more aggregation prone during folding, but does not alter the native structure of the protein. These findings suggest that influenza has evolved to hijack host chaperones to promote the folding of otherwise biophysically incompetent viral proteins that enable innate immune system escape.

How does severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) achieve immune evasion?: A narrative review.

COVID-19 caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2, (SARS-CoV-2) is a highly contagious disease known for its significant lung damage. Although the impact of the COVID-19 pandemic on our daily lives has been limited, the virus has not vanished entirely and continues to undergo mutations. This calls for a concentrated focus on the matter of SARS-CoV-2 immune evasion. Drawing on observations of immune escape mechanisms in other viruses, some scholars have proposed that liquid-liquid phase separation might play a crucial role in SARS-CoV-2's ability to evade the immune system. Within the structure of SARS-CoV-2, the nucleocapsid protein plays a pivotal role in RNA replication and transcription. Concurrently, this protein can engage in phase separation with RNA. A thorough examination of the phase separation related to the nucleocapsid protein may unveil the mechanism by which SARS-CoV-2 accomplishes immune evasion. Moreover, this analysis may provide valuable insights for future development of innovative antiviral drugs or vaccines.

Modular design of bi- and multi-specific knob domain fusions.

Introduction: The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively. Methods: Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights. Results: Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference. Discussion: Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.

Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibiting TRIM25 oligomerization and functional activation of RIG-I/TRIM25.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-ß) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-ß production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

Impact of mutations on the stability of SARS-CoV-2 nucleocapsid protein structure.

The nucleocapsid (N) protein of SARS-CoV-2 is known to participate in various host cellular processes, including interferon inhibition, RNA interference, apoptosis, and regulation of virus life cycles. Additionally, it has potential as a diagnostic antigen and/or immunogen. Our research focuses on examining structural changes caused by mutations in the N protein. We have modeled the complete tertiary structure of native and mutated forms of the N protein using Alphafold2. Notably, the N protein contains 3 disordered regions. The focus was on investigating the impact of mutations on the stability of the protein's dimeric structure based on binding free energy calculations (MM-PB/GB-SA) and RMSD fluctuations after MD simulations. The results demonstrated that 28 mutations out of 37 selected mutations analyzed, compared with wild-type N protein, resulted in a stable dimeric structure, while 9 mutations led to destabilization. Our results are important to understand the tertiary structure of the N protein dimer of SARS-CoV-2 and the effect of mutations on it, their behavior in the host cell, as well as for the research of other viruses belonging to the same genus additionally, to anticipate potential strategies for addressing this viral illness․.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.

Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

The coordinated packaging of the segmented genome of the influenza A virus (IAV) into virions is an essential step of the viral life cycle. This process is controlled by the interaction of packaging signals present in all eight viral RNA (vRNA) segments and the viral nucleoprotein (NP), which binds vRNA via a positively charged binding groove. However, mechanistic models of how the packaging signals and NP work together to coordinate genome packaging are missing. Here, we studied genome packaging in influenza A/SC35M virus mutants that carry mutated packaging signals as well as specific amino acid substitutions at the highly conserved lysine (K) residues 184 and 229 in the RNA-binding groove of NP. Because these lysines are acetylated and thus neutrally charged in infected host cells, we replaced them with glutamine to mimic the acetylated, neutrally charged state or arginine to mimic the non-acetylated, positively charged state. Our analysis shows that the coordinated packaging of eight vRNAs is influenced by (i) the charge state of the replacing amino acid and (ii) its location within the RNA-binding groove. Accordingly, we propose that lysine acetylation induces different charge states within the RNA-binding groove of NP, thereby supporting the activity of specific packaging signals during coordinated genome packaging. IMPORTANCE: Influenza A viruses (IAVs) have a segmented viral RNA (vRNA) genome encapsidated by multiple copies of the viral nucleoprotein (NP) and organized into eight distinct viral ribonucleoprotein complexes. Although genome segmentation contributes significantly to viral evolution and adaptation, it requires a highly sophisticated genome-packaging mechanism. How eight distinct genome complexes are incorporated into the virion is poorly understood, but previous research suggests an essential role for both vRNA packaging signals and highly conserved NP amino acids. By demonstrating that the packaging process is controlled by charge-dependent interactions of highly conserved lysine residues in NP and vRNA packaging signals, our study provides new insights into the sophisticated packaging mechanism of IAVs.

The Nucleocapsid Protein of SARS-CoV-2, Combined with ODN-39M, Is a Potential Component for an Intranasal Bivalent Vaccine with Broader Functionality.

Despite the rapid development of vaccines against COVID-19, they have important limitations, such as safety issues, the scope of their efficacy, and the induction of mucosal immunity. The present study proposes a potential component for a new generation of vaccines. The recombinant nucleocapsid (N) protein from the SARS-CoV-2 Delta variant was combined with the ODN-39M, a synthetic 39 mer unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), used as an adjuvant. The evaluation of its immunogenicity in Balb/C mice revealed that only administration by intranasal route induced a systemic cross-reactive, cell-mediated immunity (CMI). In turn, this combination was able to induce anti-N IgA in the lungs, which, along with the specific IgG in sera and CMI in the spleen, was cross-reactive against the nucleocapsid protein of SARS-CoV-1. Furthermore, the nasal administration of the N + ODN-39M preparation, combined with RBD Delta protein, enhanced the local and systemic immune response against RBD, with a neutralizing capacity. Results make the N + ODN-39M preparation a suitable component for a future intranasal vaccine with broader functionality against Sarbecoviruses.

Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments.

Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.

Ultrasensitive Detection of SARS-CoV-2 Nucleocapsid Protein Based on Porphyrin-Based Metal-Organic Gels with Highly Efficient Electrochemiluminescence at Low Potential.

Metal-organic gels (MOGs) are a new type of intelligent soft material, which are bridged by metal ions and organic ligands through noncovalent interactions. In this paper, we prepared highly stable P-MOGs, using the classical organic electrochemiluminescence (ECL) luminescence meso-tetra(4-carboxyphenyl)porphine as the organic ligand and Fe3+ as the metal ion. Surprisingly, P-MOGs can stably output ECL signals at a low potential. We introduced P-MOGs into the ECL resonance energy transfer strategy (ECL-RET) and constructed a quenched ECL immunosensor for the detection of the SARS-CoV-2 nucleocapsid protein (SARS-CoV-2-N). In the ECL-RET system, P-MOGs were used as energy donors, and Au@Cu2O@Fe3O4 were selected as energy acceptors. The ultraviolet-visible spectrum of Au@Cu2O@Fe3O4 partially overlaps with the ECL spectrum of P-MOGs, which can effectively touch off the ECL-RET behavior between the donors and receptors. Under the ideal experimental situation, the linear detection range of the SARS-CoV-2-N concentration was 10 fg/mL to 100 ng/mL, and the limit of detection was 1.5 fg/mL. This work has broad application prospects for porphyrin-MOGs in ECL sensing.

TRIM6 facilitates SARS-CoV-2 proliferation by catalyzing the K29-typed ubiquitination of NP to enhance the ability to bind viral genomes.

The Nucleocapsid Protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not only the core structural protein required for viral packaging, but also participates in the regulation of viral replication, and its post-translational modifications such as phosphorylation have been shown to be an important strategy for regulating virus proliferation. Our previous work identified NP could be ubiquitinated, as confirmed by two independent studies. But the function of NP ubiquitination is currently unknown. In this study, we first pinpointed TRIM6 as the E3 ubiquitin ligase responsible for NP ubiquitination, binding to NP's CTD via its RING and B-box-CCD domains. TRIM6 promotes the K29-typed polyubiquitination of NP at K102, K347, and K361 residues, increasing its binding to viral genomic RNA. Consistently, functional experiments such as the use of the reverse genetic tool trVLP model and gene knockout of TRIM6 further confirmed that blocking the ubiquitination of NP by TRIM6 significantly inhibited the proliferation of SARS-CoV-2. Notably, the NP of coronavirus is relatively conserved, and the NP of SARS-CoV can also be ubiquitinated by TRIM6, indicating that NP could be a broad-spectrum anti-coronavirus target. These findings shed light on the intricate interaction between SARS-CoV-2 and the host, potentially opening new opportunities for COVID-19 therapeutic development.

Intrinsic factors behind long COVID: IV. Hypothetical roles of the SARS-CoV-2 nucleocapsid protein and its liquid-liquid phase separation.

When the SARS-CoV-2 virus infects humans, it leads to a condition called COVID-19 that has a wide spectrum of clinical manifestations, from no symptoms to acute respiratory distress syndrome. The virus initiates damage by attaching to the ACE-2 protein on the surface of endothelial cells that line the blood vessels and using these cells as hosts for replication. Reactive oxygen species levels are increased during viral replication, which leads to oxidative stress. About three-fifths (~60%) of the people who get infected with the virus eradicate it from their body after 28 days and recover their normal activity. However, a large fraction (~40%) of the people who are infected with the virus suffer from various symptoms (anosmia and/or ageusia, fatigue, cough, myalgia, cognitive impairment, insomnia, dyspnea, and tachycardia) beyond 12 weeks and are diagnosed with a syndrome called long COVID. Long-term clinical studies in a group of people who contracted SARS-CoV-2 have been contrasted with a noninfected matched group of people. A subset of infected people can be distinguished by a set of cytokine markers to have persistent, low-grade inflammation and often self-report two or more bothersome symptoms. No medication can alleviate their symptoms efficiently. Coronavirus nucleocapsid proteins have been investigated extensively as potential drug targets due to their key roles in virus replication, among which is their ability to bind their respective genomic RNAs for incorporation into emerging virions. This review highlights basic studies of the nucleocapsid protein and its ability to undergo liquid-liquid phase separation. We hypothesize that this ability of the nucleocapsid protein for phase separation may contribute to long COVID. This hypothesis unlocks new investigation angles and could potentially open novel avenues for a better understanding of long COVID and treating this condition.

Multiplexed Capillary-Flow Driven Immunoassay for Respiratory Illnesses.

Multiplexed analysis in medical diagnostics is widely accepted as a more thorough and complete method compared to single-analyte detection. While analytical methods like polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) exist for multiplexed detection of biomarkers, they remain time-consuming and expensive. Lateral flow assays (LFAs) are an attractive option for point-of-care testing, and examples of multiplexed LFAs exist. However, these devices are limited by spatial resolution of test lines, large sample volume requirements, cross-reactivity, and poor sensitivity. Recent work has developed capillary-flow microfluidic ELISA platforms as a more sensitive alternative to LFAs; however, multiplexed detection on these types of devices has yet to be demonstrated. In the aftermath of the initial SARS-CoV-2 pandemic, the need for rapid, sensitive point-of-care devices has become ever clearer. Moving forward, devices that can distinguish between diseases with similar presenting symptoms would be the ideal home diagnostic. Here, the first example of a multiplexed capillary-flow immunoassay device for the simultaneous detection of multiple biomarkers is reported. From a single sample addition step, the reagents and washing steps required for two simultaneous ELISAs are delivered to spatially separated test strips. Visual results can be obtained in <15 min, and images captured with a smartphone can be analyzed for quantitative data. This device was used to distinguish between and quantify H1N1 hemagglutinin (HA) and SARS-CoV-2 nucleocapsid protein (N-protein). Using this device, analytical detection limits of 840 and 133 pg/mL were obtained for hemagglutinin and nucleocapsid protein, respectively. The presence of one target in the device did not increase the signal on the other test line, indicating no cross-reactivity between the assays. Additionally, simultaneous detection of both N-protein and HA was performed as well as simultaneous detection of N-protein and human C-reactive protein (CRP). Elevated levels of CRP in a patient infected with SARS-CoV-2 have been shown to correlate with more severe outcomes and a greater risk of death as well. To further expand on the simultaneous detection of two biomarkers, CRP and N-protein were detected simultaneously, and the presence of SARS-CoV-2 N-protein did not interfere with the detection of CRP when both targets were present in the sample.

Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication.

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

In planta expression of specific single chain fragment antibody (scFv) against nucleocapsid protein of fig mosaic virus (FMV).

Fig mosaic virus (FMV) is recognized as the main viral agent associated with the mosaic disease (MD) of fig trees (Ficus carica). Due to its worldwide occurrence, FMV represents the most significant global threat to the production of fig fruit. A disease management strategy against the MD in fig orchards has never been effective; and therefore, expression of recombinant antibody in plant cells could provide an alternative approach to suppress FMV infections. In this study we focused on expressing a specific recombinant antibody, a single-chain variable fragment (scFv), targeting the nucleocapsid protein (NP) of FMV in planta. To accomplish this objective, we inserted the scFv gene into a plant expression vector and conducted transient expression in leaves of Nicotiana tabacum cv. Samson plants. The construct was transiently expressed in tobacco plants by agroinfiltration, and antibody of the anticipated size was detected by immunoblotting. The produced plantibody was then assessed for specificity using ELISA and confirmed by Western blot analysis. In this study, the plantibody developed against FMV could be considered as a potential countermeasure to the infection by conferring resistance to MD.

RBM14 inhibits the replication of porcine epidemic diarrhea virus by recruiting p62 to degrade nucleocapsid protein through the activation of autophagy and interferon pathway.

Porcine epidemic diarrhea virus (PEDV) results in PED, which is an infectious intestinal disease with the representative features of diarrhea, vomiting, and dehydration. PEDV infects neonatal piglets, causing high mortality rates. Therefore, elucidating the interaction between the virus and host in preventing and controlling PEDV infection is of immense significance. We found a new antiviral function of the host protein, RNA-binding motif protein 14 (RBM14), which can inhibit PEDV replication via the activation of autophagy and interferon (IFN) signal pathways. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV nucleocapsid (N) protein through the RBM14-p62-autophagosome pathway. Furthermore, RBM14 can also improve the antiviral ability of the hosts through interacting with mitochondrial antiviral signaling protein to induce IFN expression. These results highlight the novel mechanism underlying RBM14-induced viral restriction. This mechanism leads to the degradation of viral N protein via the autophagy pathway and upregulates IFN for inhibiting PEDV replication; thus, offering new ways for preventing and controlling PED.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a vital reason for diarrhea in neonatal piglets, which causes high morbidity and mortality rates. There is currently no effective vaccine or drug to treat and prevent infection with the PEDV. During virus infection, the host inhibits virus replication through various antiviral factors, and at the same time, the virus antagonizes the host's antiviral reaction through its own encoded protein, thus completing the process of virus replication. Our study has revealed that the expression of RNA-binding motif protein 14 (RBM14) was downregulated in PEDV infection. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV N protein via the RBM14-p62-autophagosome pathway and interacted with mitochondrial antiviral signaling protein and TRAF3 to activate the interferon signal pathway, resulting in the inhibition of PEDV replication.

Nucleoprotein reassortment enhanced transmissibility of H3 1990.4.a clade influenza A virus in swine.

The increased detection of H3 C-IVA (1990.4.a) clade influenza A viruses (IAVs) in US swine in 2019 was associated with a reassortment event to acquire an H1N1pdm09 lineage nucleoprotein (pdmNP) gene, replacing a TRIG lineage NP (trigNP). We hypothesized that acquiring the pdmNP conferred a selective advantage over prior circulating H3 viruses with a trigNP. To investigate the role of NP reassortment in transmission, we identified two contemporary 1990.4.a representative strains (NC/19 and MN/18) with different evolutionary origins of the NP gene. A reverse genetics system was used to generate wild-type (wt) strains and swap the pdm and TRIG lineage NP genes, generating four viruses: wtNC/19-pdmNP, NC/19-trigNP, wtMN/18-trigNP, and MN/18-pdmNP. The pathogenicity and transmission of the four viruses were compared in pigs. All four viruses infected 10 primary pigs and transmitted to five indirect contact pigs per group. Pigs infected via contact with MN/18-pdmNP shed virus 2 days earlier than pigs infected with wtMN/18-trigNP. The inverse did not occur for wtNC/19-pdmNP and NC/19-trigNP. This suggests that pdmNP reassortment resulted in a combination of genes that improved transmission efficiency when paired with the 1990.4.a hemagglutinin (HA). This is likely a multigenic trait, as replacing the trigNP gene did not diminish the transmission of a wild-type IAV in swine. This study demonstrates how reassortment and evolutionary change of internal genes can result in more transmissible viruses that influence HA clade detection frequency. Thus, rapidly identifying novel reassortants paired with dominant hemagglutinin/neuraminidase may improve the prediction of strains to include in vaccines.IMPORTANCEInfluenza A viruses (IAVs) are composed of eight non-continuous gene segments that can reassort during coinfection of a host, creating new combinations. Some gene combinations may convey a selective advantage and be paired together preferentially. A reassortment event was detected in swine in the United States that involved the exchange of two lineages of nucleoprotein (NP) genes (trigNP to pdmNP) that became a predominant genotype detected in surveillance. Using a transmission study, we demonstrated that exchanging the trigNP for a pdmNP caused the virus to shed from the nose at higher levels and transmit to other pigs more rapidly. Replacing a pdmNP with a trigNP did not hinder transmission, suggesting that transmission efficiency depends on interactions between multiple genes. This demonstrates how reassortment alters IAV transmission and that reassortment events can provide an explanation for why genetically related viruses with different internal gene combinations experience rapid fluxes in detection frequency.

Aptamers targeting SARS-CoV-2 nucleocapsid protein exhibit potential anti pan-coronavirus activity.

Emerging and recurrent infectious diseases caused by human coronaviruses (HCoVs) continue to pose a significant threat to global public health security. In light of this ongoing threat, the development of a broad-spectrum drug to combat HCoVs is an urgently priority. Herein, we report a series of anti-pan-coronavirus ssDNA aptamers screened using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). These aptamers have nanomolar affinity with the nucleocapsid protein (NP) of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and also show excellent binding efficiency to the N proteins of both SARS, MERS, HCoV-OC43 and -NL63 with affinity KD values of 1.31 to 135.36 nM. Such aptamer-based therapeutics exhibited potent antiviral activity against both the authentic SARS-CoV-2 prototype strain and the Omicron variant (BA.5) with EC50 values at 2.00 nM and 41.08 nM, respectively. The protein docking analysis also evidenced that these aptamers exhibit strong affinities for N proteins of pan-coronavirus and other HCoVs (-229E and -HKU1). In conclusion, we have identified six aptamers with a high pan-coronavirus antiviral activity, which could potentially serve as an effective strategy for preventing infections by unknown coronaviruses and addressing the ongoing global health threat.

Immunological mechanisms of the nucleocapsid protein in COVID-19.

The emergence of corona virus disease 2019 (COVID-19), resulting from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has left an indelible mark on a global scale, causing countless infections and fatalities. This investigation delves into the role of the SARS-CoV-2 nucleocapsid (N) protein within the HEK293 cells, shedding light on its influence over apoptosis, interferon signaling, and cytokines production. The N gene was amplified, inserted into the pAdTrack-CMV vector, and then transfected to the HEK293 cells. Changes in the expression of IRF3, IRF7, IFN-ß, BAK, BAX, and BCL-2 genes were evaluated. The levels of proinflammatory cytokines of IL-6, IL-12, IL-1ß, and TNF-α were also determined. The N protein exhibited an anti-apoptotic effect by modulating critical genes associated with apoptosis, including BAK, BAX, and BCL-2. This effect potentially prolonged the survival of infected cells. The N protein also played a role in immune evasion by suppressing the interferon pathway, evidenced by the downregulation of essential interferon regulatory factors of IRF3 and IRF7, and IFN-ß expression. The N protein expression led to a substantial increase in the production of proinflammatory cytokines of IL-6, IL-12, IL-1ß, and TNF-α. The N protein emerged as a versatile factor and was exerted over apoptosis, interferon signaling, and cytokine production. These findings carry potential implications for the development of targeted therapies to combat COVID-19 and mitigate its global health impact.