Deep mutational scanning identifies SARS-CoV-2 Nucleocapsid escape mutations of currently available rapid antigen tests.

The effects of mutations in continuously emerging variants of SARS-CoV-2 are a major concern for the performance of rapid antigen tests. To evaluate the impact of mutations on 17 antibodies used in 11 commercially available antigen tests with emergency use authorization, we measured antibody binding for all possible Nucleocapsid point mutations using a mammalian surface-display platform and deep mutational scanning. The results provide a complete map of the antibodies' epitopes and their susceptibility to mutational escape. Our data predict no vulnerabilities for detection of mutations found in variants of concern. We confirm this using the commercial tests and sequence-confirmed COVID-19 patient samples. The antibody escape mutational profiles generated here serve as a valuable resource for predicting the performance of rapid antigen tests against past, current, as well as any possible future variants of SARS-CoV-2, establishing the direct clinical and public health utility of our system.

Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses.

The SARS-CoV-2 pandemic has resulted in millions of infections, yet the role of host immune responses in early COVID-19 pathogenesis remains unclear. By investigating 17 acute and 24 convalescent patients, we found that acute SARS-CoV-2 infection resulted in broad immune cell reduction including T, natural killer, monocyte, and dendritic cells (DCs). DCs were significantly reduced with functional impairment, and ratios of conventional DCs to plasmacytoid DCs were increased among acute severe patients. Besides lymphocytopenia, although neutralizing antibodies were rapidly and abundantly generated in patients, there were delayed receptor binding domain (RBD)- and nucleocapsid protein (NP)-specific T cell responses during the first 3 weeks after symptoms onset. Moreover, acute RBD- and NP-specific T cell responses included relatively more CD4 T cells than CD8 T cells. Our findings provided evidence that impaired DCs, together with timely inverted strong antibody but weak CD8 T cell responses, could contribute to acute COVID-19 pathogenesis and have implications for vaccine development.

Orthogonal SARS-CoV-2 Serological Assays Enable Surveillance of Low-Prevalence Communities and Reveal Durable Humoral Immunity.

We conducted a serological study to define correlates of immunity against SARS-CoV-2. Compared to those with mild coronavirus disease 2019 (COVID-19) cases, individuals with severe disease exhibited elevated virus-neutralizing titers and antibodies against the nucleocapsid (N) and the receptor binding domain (RBD) of the spike protein. Age and sex played lesser roles. All cases, including asymptomatic individuals, seroconverted by 2 weeks after PCR confirmation. Spike RBD and S2 and neutralizing antibodies remained detectable through 5-7 months after onset, whereas α-N titers diminished. Testing 5,882 members of the local community revealed only 1 sample with seroreactivity to both RBD and S2 that lacked neutralizing antibodies. This fidelity could not be achieved with either RBD or S2 alone. Thus, inclusion of multiple independent assays improved the accuracy of antibody tests in low-seroprevalence communities and revealed differences in antibody kinetics depending on the antigen. We conclude that neutralizing antibodies are stably produced for at least 5-7 months after SARS-CoV-2 infection.

HSP90AB1 is a host factor that promotes porcine deltacoronavirus replication.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus. It causes mortality in neonatal piglets and is of growing concern because of its broad host range, including humans. To date, the mechanism of PDCoV infection remains poorly understood. Here, based on a genome-wide CRISPR screen of PDCoV-infected cells, we found that HSP90AB1 (heat shock protein 90 alpha family class B1) promotes PDCoV infection. Knockdown or KO of HSP90AB1 in LLC-PK cells resulted in a significantly suppressed PDCoV infection. Infected cells treated with HSP90 inhibitors 17-AAG and VER-82576 also showed a significantly suppressed PDCoV infection, although KW-2478, which does not affect the ATPase activity of HSP90AB1, had no effect on PDCoV infection. We found that HSP90AB1 interacts with the N, NS7, and NSP10 proteins of PDCoV. We further evaluated the interaction between N and HSP90AB1 and found that the C-tail domain of the N protein is the HSP90AB1-interacting domain. Further studies showed that HSP90AB1 protects N protein from degradation via the proteasome pathway. In summary, our results reveal a key role for HSP90AB1 in the mechanism of PDCoV infection and contribute to provide new host targets for PDCoV antiviral research.

Structural basis for the participation of the SARS-CoV-2 nucleocapsid protein in the template switch mechanism and genomic RNA reorganization.

The COVID-19 pandemic has resulted in a significant toll of deaths worldwide, exceeding seven million individuals, prompting intensive research efforts aimed at elucidating the molecular mechanisms underlying the pathogenesis of SARS-CoV-2 infection. Despite the rapid development of effective vaccines and therapeutic interventions, COVID-19 remains a threat to humans due to the emergence of novel variants and largely unknown long-term consequences. Among the viral proteins, the nucleocapsid protein (N) stands out as the most conserved and abundant, playing the primary role in nucleocapsid assembly and genome packaging. The N protein is promiscuous for the recognition of RNA, yet it can perform specific functions. Here, we discuss the structural basis of specificity, which is directly linked to its regulatory role. Notably, the RNA chaperone activity of N is central to its multiple roles throughout the viral life cycle. This activity encompasses double-stranded RNA (dsRNA) annealing and melting and facilitates template switching, enabling discontinuous transcription. N also promotes the formation of membraneless compartments through liquid‒liquid phase separation (LLPS), thereby facilitating the congregation of the replication and transcription complex (RTC). Considering the information available regarding the catalytic activities and binding signatures of the N protein‒RNA interaction, this review focuses on the regulatory role of the SARS-CoV‒2 N protein. We emphasize the participation of the N protein in discontinuous transcription, template switching, and RNA chaperone activity, including double-stranded RNA melting and annealing activities.

N-terminus of SARS-CoV-2 nonstructural protein 3 interrupts RNA-driven phase separation of N protein by displacing RNA.

The connection between SARS-CoV-2 replication-transcription complexes (RTCs) and nucleocapsid (N) protein is critical for regulating genomic RNA replication and virion packaging over the viral life cycle. However, the mechanism that dynamically regulates genomic RNA packaging and replication remains elusive. Here, we demonstrate that the N-terminal domain (NTD) of SARS-CoV-2 nonstructural protein 3 (Nsp3), a core component of viral RTCs, binds N protein and displaces RNA in a concentration-dependent manner. This interaction disrupts liquid-liquid phase separation of N protein driven by N protein-RNA interactions which is crucial for virion packaging and viral replication. We also report a high-resolution crystal structure of the Nsp3 ubiquitin-like domain 1 (Ubl1) at 1.49 Å, which reveals abundant negative charges on the protein surface. Sequence and structural analyses identify several conserved motifs at the Ubl1-N protein interface and a previously unexplored highly negative groove, providing insights into the molecular mechanism of Ubl1-mediated modulation of N protein-RNA binding. Our findings elucidate the mechanism of dynamic regulation of SARS-CoV-2 genomic RNA replication and packaging over the viral life cycle. Targeting the conserved Ubl1-N protein interaction hotspots also promises to aid in the development of broad-spectrum antivirals against pathogenic coronaviruses.

Unraveling the assembly mechanism of SADS-CoV virus nucleocapsid protein: insights from RNA binding, dimerization, and epitope diversity profiling.

The swine acute diarrhea syndrome coronavirus (SADS-CoV) has caused significant disruptions in porcine breeding and raised concerns about potential human infection. The nucleocapsid (N) protein of SADS-CoV plays a vital role in viral assembly and replication, but its structure and functions remain poorly understood. This study utilized biochemistry, X-ray crystallography, and immunization techniques to investigate the N protein's structure and function in SADS-CoV. Our findings revealed distinct domains within the N protein, including an RNA-binding domain, two disordered domains, and a dimerization domain. Through biochemical assays, we confirmed that the N-terminal domain functions as an RNA-binding domain, and the C-terminal domain is involved in dimerization, with the crystal structure analysis providing visual evidence of dimer formation. Immunization experiments demonstrated that the disordered domain 2 elicited a significant antibody response. These identified domains and their interactions are crucial for viral assembly. This comprehensive understanding of the N protein in SADS-CoV enhances our knowledge of its assembly and replication mechanisms, enabling the development of targeted interventions and therapeutic strategies. IMPORTANCE: SADS-CoV is a porcine coronavirus that originated from a bat HKU2-related coronavirus. It causes devastating swine diseases and poses a high risk of spillover to humans. The coronavirus N protein, as the most abundant viral protein in infected cells, likely plays a key role in viral assembly and replication. However, the structure and function of this protein remain unclear. Therefore, this study employed a combination of biochemistry and X-ray crystallography to uncover distinct structural domains in the N protein, including RNA-binding domains, two disordered domains, and dimerization domains. Additionally, we made the novel discovery that the disordered domain elicited a significant antibody response. These findings provide new insights into the structure and functions of the SADS-CoV N protein, which have important implications for future studies on SADS-CoV diagnosis, as well as the development of vaccines and anti-viral drugs.

Pemetrexed alleviates piglet diarrhea by blocking the interaction between porcine epidemic diarrhea virus nucleocapsid protein and Ezrin.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes high mortality in piglets, thus posing a serious threat to the world pig industry. Porcine epidemic diarrhea (PED) is related to the imbalance of sodium absorption by small intestinal epithelial cells; however, the etiology of sodium imbalanced diarrhea caused by PEDV remains unclear. Herein, we first proved that PEDV can cause a significant decrease in Na+/H+ exchanger 3 (NHE3) expression on the cell membrane, in a viral dose-dependent manner. Further study showed that the PEDV nucleocapsid (N) protein participates in the regulation of NHE3 activity through interacting with Ezrin. Flame atomic absorption spectroscopy results indicated a serious imbalance in Na+ concentration inside and outside cells following overexpression of PEDV N. Meanwhile, molecular docking technology identified that the small molecule drug Pemetrexed acts on the PEDV N-Ezrin interaction region. It was confirmed that Pemetrexed can alleviate the imbalanced Na+ concentration in IPEC-J2 cells and the diarrhea symptoms of Rongchang pigs caused by PEDV infection. Overall, our data suggest that the interaction between PEDV N and Ezrin reduces the level of phosphorylated Ezrin, resulting in a decrease in the amount of NHE3 protein on the cell membrane. This leads to an imbalance of intracellular and extracellular Na+, which causes diarrhea symptoms in piglets. Pemetrexed is effective in relieving diarrhea caused by PEDV. Our results provide a reference to screen for anti-PEDV targets and to develop drugs to prevent PED.IMPORTANCEPorcine epidemic diarrhea (PED) has caused significant economic losses to the pig industry since its initial outbreak, and the pathogenic mechanism of porcine epidemic diarrhea virus (PEDV) is still under investigation. Herein, we found that the PEDV nucleocapsid protein interacts with Ezrin to regulate Na+/H+ exchanger 3 activity. In addition, we screened out Pemetrexed, a small molecule drug, which can effectively alleviate pig diarrhea caused by PEDV. These results provide support for further exploration of the pathogenesis of PEDV and the development of drugs to prevent PED.

A nanobody inhibiting porcine reproductive and respiratory syndrome virus replication via blocking self-interaction of viral nucleocapsid protein.

Porcine reproductive and respiratory syndrome (PRRS) is a serious global pig industry disease. Understanding the mechanism of viral replication and developing efficient antiviral strategies are necessary for combating with PRRS virus (PRRSV) infection. Recently, nanobody is considered to be a promising antiviral drug, especially for respiratory viruses. The present study evaluated two nanobodies against PRRSV nucleocapsid (N) protein (PRRSV-N-Nb1 and -Nb2) for their anti-PRRSV activity in vitro and in vivo. The results showed that intracellularly expressed PRRSV-N-Nb1 significantly inhibited PRRSV-2 replication in MARC-145 cells (approximately 100%). Then, the PRRSV-N-Nb1 fused with porcine IgG Fc (Nb1-pFc) as a delivering tag was produced and used to determine its effect on PRRSV-2 replication in porcine alveolar macrophages (PAMs) and pigs. The inhibition rate of Nb1-pFc against PRRSV-2 in PAMs could reach >90%, and it can also inhibit viral replication in vivo. Epitope mapping showed that the motif Serine 105 (S105) in PRRSV-2 N protein was the key amino acid binding to PRRSV-N-Nb1, which is also pivotal for the self-interaction of N protein via binding to Arginine 97. Moreover, viral particles were not successfully rescued when the S105 motif was mutated to Alanine (S105A). Attachment, entry, genome replication, release, docking model analysis, and blocking enzyme-linked immunosorbent assay (ELISA) indicated that the binding of PRRSV-N-Nb1 to N protein could block its self-binding, which prevents the viral replication of PRRSV. PRRSV-N-Nb1 may be a promising drug to counter PRRSV-2 infection. We also provided some new insights into the molecular basis of PRRSV N protein self-binding and assembly of viral particles.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes serious economic losses to the swine industry worldwide, and there are no highly effective strategies for prevention. Nanobodies are considered a promising novel approach for treating diseases because of their ease of production and low costing. Here, we showed that PRRSV-N-Nb1 against PRRSV-N protein significantly inhibited PRRSV-2 replication in vitro and in vivo. Furthermore, we demonstrated that the motif Serine 105 (S105) in PRRSV-N protein was the key amino acid to interact with PRRSV-N-Nb1 and bond to its motif R97, which is important for the self-binding of N protein. The PRRSV-N-Nb1 could block the self-interaction of N protein following viral assembly. These findings not only provide insights into the molecular basis of PRRSV N protein self-binding as a key factor for viral replication for the first time but also highlight a novel target for the development of anti-PRRSV replication drugs.

Host factor MxA restricts Dabie bandavirus infection by targeting the viral NP protein to inhibit NP-RdRp interaction and ribonucleoprotein activity.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high case mortality rates, which is caused by Dabie bandavirus (DBV), a novel pathogen also termed as SFTS virus (SFTSV). Currently, no specific therapeutic drugs or vaccines are available for SFTS. Myxovirus resistance protein A (MxA) has been shown to inhibit multiple viral pathogens; however, the role of MxA in DBV infection is unknown. Here, we demonstrated that DBV stimulates MxA expression which, in turn, restricts DBV infection. Mechanistic target analysis revealed that MxA specifically interacts with the viral nucleocapsid protein (NP) in a manner independent of RNA. Minigenome reporter assay showed that in agreement with its targeting of NP, MxA inhibits DBV ribonucleoprotein (RNP) activity. In detail, MxA interacts with the NP N-terminal and disrupts the interaction of NP with the viral RNA-dependent RNA polymerase (RdRp) but not NP multimerization, the critical activities of NP for RNP formation and function. Furthermore, MxA N-terminal domain was identified as the functional domain inhibiting DBV infection, and, consistently, then was shown to interact with NP and obstruct the NP-RdRp interaction. Additionally, threonine 103 within the N-terminal domain is important for MxA inhibition to DBV, and its mutation (T103A) attenuates MxA binding to NP and obstruction of the NP-RdRp interaction. This study uncovers MxA inhibition of DBV with a series of functional and mechanistical analyses, providing insights into the virus-host interactions and probably helping inform the development of antiviral agents in the future.IMPORTANCEDBV/SFTSV is an emerging high-pathogenic virus. Since its first identification in China in 2009, cases of DBV infection have been reported in many other countries, posing a significant threat to public health. Uncovering the mechanisms of DBV-host interactions is necessary to understand the viral pathogenesis and host response and may advance the development of antiviral therapeutics. Here, we found that host factor MxA whose expression is induced by DBV restricts the virus infection. Mechanistically, MxA specifically interacts with the viral NP and blocks the NP-RdRp interaction, inhibiting the viral RNP activity. Further studies identified the key domain and amino acid residue required for MxA inhibition to DBV. Consistently, they were then shown to be important for MxA targeting of NP and obstruction of the NP-RdRp association. These findings unravel the restrictive role of MxA in DBV infection and the underlying mechanism, expanding our knowledge of the virus-host interactions.

Porcine deltacoronavirus nucleocapsid protein antagonizes JAK-STAT signaling pathway by targeting STAT1 through KPNA2 degradation.

Porcine deltacoronavirus (PDCoV) is an enteric pathogenic coronavirus that causes acute and severe watery diarrhea in piglets and has the ability of cross-species transmission, posing a great threat to swine production and public health. The interferon (IFN)-mediated signal transduction represents an important component of virus-host interactions and plays an essential role in regulating viral infection. Previous studies have suggested that multifunctional viral proteins encoded by coronaviruses antagonize the production of IFN via various means. However, the function of these viral proteins in regulating IFN-mediated signaling pathways is largely unknown. In this study, we demonstrated that PDCoV and its encoded nucleocapsid (N) protein antagonize type I IFN-mediated JAK-STAT signaling pathway. We identified that PDCoV infection stimulated but delayed the production of IFN-stimulated genes (ISGs). In addition, PDCoV inhibited JAK-STAT signal transduction by targeting the nuclear translocation of STAT1 and ISGF3 formation. Further evidence showed that PDCoV N is the essential protein involved in the inhibition of type I IFN signaling by targeting STAT1 nuclear translocation via its C-terminal domain. Mechanistically, PDCoV N targets STAT1 by interacting with it and subsequently inhibiting its nuclear translocation. Furthermore, PDCoV N inhibits STAT1 nuclear translocation by specifically targeting KPNA2 degradation through the lysosomal pathway, thereby inhibiting the activation of downstream sensors in the JAK-STAT signaling pathway. Taken together, our results reveal a novel mechanism by which PDCoV N interferes with the host antiviral response.IMPORTANCEPorcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus that receives increased attention and seriously threatens the pig industry and public health. Understanding the underlying mechanism of PDCoV evading the host defense during infection is essential for developing targeted drugs and effective vaccines against PDCoV. This study demonstrated that PDCoV and its encoded nucleocapsid (N) protein antagonize type I interferon signaling by targeting STAT1, which is a crucial signal sensor in the JAK-STAT signaling pathway. Further experiments suggested that PDCoV N-mediated inhibition of the STAT1 nuclear translocation involves the degradation of KPNA2, and the lysosome plays a role in KPNA2 degradation. This study provides new insights into the regulation of PDCoV N in the JAK-STAT signaling pathway and reveals a novel mechanism by which PDCoV evades the host antiviral response. The novel findings may guide us to discover new therapeutic targets and develop live attenuated vaccines for PDCoV infection.

Single-Cell Profiling of the Differential In Vivo Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Infection Among Lung Tissue Cell Subtypes at the Protein Level.

The complexity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its variants in lung cells can truly be characterized only at the tissue and protein levels among unique cell subtypes. However, in vivo data are limited due to lack of accessible human tissues. Using a transgenic mouse model of SARS-CoV-2 infection and flow cytometry, we provide in vivo novel insight at the protein level that the differential impact of SARS-CoV-2 (Wuhan strain) and its B.1.617.2 (Delta) and BA.1 (Omicron) variants on lung may be attributed to differential patterns of viral protein levels among ciliated airway cells, alveolar types 1 and 2 cells, immune cells, and endothelial lung cells.

A novel compound targets the feline infectious peritonitis virus nucleocapsid protein and inhibits viral replication in cell culture.

Feline infectious peritonitis (FIP) is a serious viral illness in cats, caused by feline coronavirus. Once a cat develops clinical FIP, the prognosis is poor. The effective treatment strategy for coronavirus infections with immunopathological complications such as SARS-CoV-2, MERS, and FIP is focused on antiviral and immunomodulatory agents to inhibit virus replication and enhance the protective immune response. In this article we report the binding and conformational alteration of feline alphacoronavirus (FCoV) nucleocapsid protein by a novel compound K31. K31 noncompetitively inhibited the interaction between the purified nucleocapsid protein and the synthetic 5' terminus of viral genomic RNA in vitro. K31 was well tolerated by cells and inhibited FCoV replication in cell culture with a selective index of 115. A single dose of K31inhibited FCoV replication to an undetectable level in 24 h post treatment. K31 did not affect the virus entry to the host cell but inhibited the postentry steps of virus replication. The nucleocapsid protein forms ribonucleocapsid in association with the viral genomic RNA that serves as a template for transcription and replication of the viral genome. Our results show that K31 treatment disrupted the structural integrity of ribonucleocapsid in virus-infected cells. After the COVID-19 pandemic, most of the antiviral drug development strategies have focused on RdRp and proteases encoded by the viral genome. Our results have shown that nucleocapsid protein is a druggable target for anticoronavirus drug discovery.

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.

Peste Des Petits Ruminants Virus Nucleocapsid Protein Interacts with Protein Kinase R-Activating Protein and Induces Stress Granules To Promote Viral Replication.

The pathogenic mechanisms of peste des petits ruminants virus (PPRV) infection remain poorly understood, leaving peste des petits ruminants (PPR) control and eradication especially difficult. Here, we determined that PPRV nucleocapsid (N) protein triggers formation of stress granules (SGs) to benefit viral replication. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N protein interacted with protein kinase R (PKR)-activating protein (PACT), and this interaction was confirmed in the context of PPRV infection. PACT was essential for PPRV replication. Besides, the ectopic expression of N activated the PKR/eIF2α (α subunit of eukaryotic initiation factor 2) pathway through induction of PKR phosphorylation, but it did not induce PKR phosphorylation in PACT-deficient (PACT-/-) cells. PPRV N interacted with PACT, impairing the interaction between PACT and a PKR inhibitor, transactivation response RNA-binding protein (TRBP), which subsequently enhanced the interaction between PACT and PKR and thus promoted the activation of PKR and eIF2α phosphorylation, resulting in formation of stress granules (SGs). Consistently, PPRV infection induced SG formation through activation of the PKR/eIF2α pathway, and knockdown of N impaired PPRV-induced SG formation. PPRV-induced SG formation significantly decreased in PACT-/- cells as well. The role of SG formation in PPRV replication was subsequently investigated, which showed that SG formation plays a positive role in PPRV replication. By using an RNA fluorescence in situ hybridization assay, we found that PPRV-induced SGs hid cellular mRNA rather than viral mRNA. Altogether, our data provide the first evidence that PPRV N protein plays a role in modulating the PKR/eIF2α/SG axis and promotes virus replication through targeting PACT. IMPORTANCE Stress granule (SG) formation is a conserved cellular strategy to reduce stress-related damage regulating cell survival. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N interacted with PACT to regulate the assembly of SGs. N protein inhibited the interaction between PACT and a PKR inhibitor, TRBP, through binding to the M1 domain of PACT, which enhanced the interaction between PACT and PKR and thus promoted PKR activation and subsequent eIF2α phosphorylation as well as SG formation. The regulatory function of N protein was strikingly abrogated in PACT-/- cells. SGs induced by PPRV infection through the PKR/eIF2α pathway are PACT dependent. The loss-of-function assay indicated that PPRV-induced SGs were critical for PPRV replication. We concluded that the PPRV N protein manipulates the host PKR/eIF2α/SG axis to favor virus replication.

EGR1 functions as a new host restriction factor for SARS-CoV-2 to inhibit virus replication through the E3 ubiquitin ligase MARCH8.

IMPORTANCE: Emerging vaccine-breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight an urgent need for novel antiviral therapies. Understanding the pathogenesis of coronaviruses is critical for developing antiviral drugs. Here, we demonstrate that the SARS-CoV-2 N protein suppresses interferon (IFN) responses by reducing early growth response gene-1 (EGR1) expression. The overexpression of EGR1 inhibits SARS-CoV-2 replication by promoting IFN-regulated antiviral protein expression, which interacts with and degrades SARS-CoV-2 N protein via the E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. The MARCH8 mutants without ubiquitin ligase activity are no longer able to degrade SARS-CoV-2 N proteins, indicating that MARCH8 degrades SARS-CoV-2 N proteins dependent on its ubiquitin ligase activity. This study found a novel immune evasion mechanism of SARS-CoV-2 utilized by the N protein, which is helpful for understanding the pathogenesis of SARS-CoV-2 and guiding the design of new prevention strategies against the emerging coronaviruses.

Identification of differences in the magnitude and specificity of SARS-CoV-2 nucleocapsid antibody responses in naturally infected and vaccinated individuals.

As there are limited data on B-cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron, and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine. Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n = 30), WT infected (n = 30), delta infected (n = 30), omicron infected + vaccinated (n = 20) and Sinopharm (BBIBP-CorV) vaccinees (n = 30). We then investigated the sensitivity and specificity of these immunodominant regions and analyzed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses, and bat Sarbecoviruses. We identified four immunodominant regions aa 29-52, aa 155-178, aa 274-297, and aa 365-388, which were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines.

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.

Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

The SARS-CoV-2 nucleocapsid protein suppresses innate immunity by remodeling stress granules to atypical foci.

Viruses deploy multiple strategies to suppress the host innate immune response to facilitate viral replication and pathogenesis. Typical G3BP1+ stress granules (SGs) are usually formed in host cells after virus infection to restrain viral translation and to stimulate innate immunity. Thus, viruses have evolved various mechanisms to inhibit SGs or to repurpose SG components such as G3BP1. Previous studies showed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection inhibited host immunity during the early stage of COVID-19. However, the precise mechanism is not yet well understood. Here we showed that the SARS-CoV-2 nucleocapsid (SARS2-N) protein suppressed the double-stranded RNA (dsRNA)-induced innate immune response, concomitant with inhibition of SGs and the induction of atypical SARS2-N+ /G3BP1+ foci (N+ foci). The SARS2-N protein-induced formation of N+ foci was dependent on the ability of its ITFG motif to hijack G3BP1, which contributed to suppress the innate immune response. Importantly, SARS2-N protein facilitated viral replication by inducing the formation of N+ foci. Viral mutations within SARS2-N protein that impair the formation of N+ foci are associated with the inability of the SARS2-N protein to suppress the immune response. Taken together, our study has revealed a novel mechanism by which SARS-CoV-2 suppresses the innate immune response via induction of atypical N+ foci. We think that this is a critical strategy for viral pathogenesis and has potential therapeutic implications.