Host Innate Immunity Research Articles

Host immunity involvement in the outcome of phage therapy against hypervirulent Klebsiella pneumoniae infections.

Highly encapsulated hypervirulent Klebsiella pneumoniae (hvKp) causes severe infections. Bacteriophage therapy, an antibiotic alternative, effectively treats bacterial infections. Phage φFK1979 encoding polysaccharide depolymerases can target and disarm the capsule of hvKp FK1979, showing promise against FK1979 infection. Resistant strains induced by φFK1979 are possibly eliminated by host immunity and new phage phiR3 targeting them. We constructed varied immunocompromised FK1979 infection mouse models to assess the therapy efficacy of φFK1979 alone or in combination with phiR3. Survival rates, bacterial loads, histopathology, inflammation, and immune cell distribution of mice were studied. Prompt and adequate administration of φFK1979, rather than phiR3, significantly improved survival rates in mice with different immune statuses. However, immunocompromised mice showed lower efficacy due to reduced tolerance to low-virulence φFK1979-resistant bacteria compared to immunocompetent mice. Adding phiR3 sequentially greatly enhanced therapy efficacy for them, leading to increased survival rates and notable improvements in pathology and inflammation. Immunocompetent mice exhibited the most favorable response to φFK1979 monotherapy, as their immune system cleared φFK1979-resistant bacteria while avoiding a robust response to phiR3 combating φFK1979-resistant bacteria. This study revealed host immunity involvement in the outcome of phage therapy against infections and introduced, for the first time, personalized phage therapy strategies for hvKp-infected mice with varying immune statuses.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp), with high capsular polysaccharide production, can cause severe invasive infections. Capsule-targeting phage poses the potential to fight against hvKp. We previously elucidated that the capsule-targeting phage induces resistance in hvKp, while phage-resistant strains exhibit sensitivity to host innate immunity and new phages targeting them. This indicated that phage-resistant strains can be eliminated by the immune system in immunocompetent patients, whereas they may require treatment with phages targeting resistant bacteria in immunocompromised patients. HvKp can infect individuals with varying immune statuses, including both immunocompetent and immunocompromised/deficient patients. This study, for the first time, developed personalized phage therapy strategies for hvKp-infected mice with different immune statuses, optimizing phage therapy against hvKp infections. This research is expected to provide a theoretical foundation and novel insights for clinical phage therapy against hvKp infections, offering significant societal benefits and clinical value.

Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

Bacterial community analysis of the skin microbiota of cultured Chinese giant salamander infected with Ranavirus.

Skin microorganisms are an important component of host innate immunity and serve as the first line of defense against pathogenic infections. The relative abundance of bacterial species, microbial community assembly, and secretion of specific bacterial metabolites are closely associated with host health. In this study, we investigated the association between the skin microbiome and Ranavirus, and compared the bacterial community assemblage, alpha and beta diversity, and functional predictions of the skin bacterial assemblage in cultured healthy Chinese giant salamanders (Andrias davidianus) and individuals infected with Chinese giant salamander iridovirus (GSIV or ADRV). To achieve this, we employed 16S rRNA amplicon sequencing. The results identified Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota as the dominant phyla in the diseased and healthy groups. Alpha diversity analysis indicated that the skin bacterial community in the diseased group exhibited no significant differences in bacterial species diversity and lower species richness compared to the healthy group. Beta diversity suggested that the two group bacterial community was quite different. Kyoto encyclopedia of genes and genomes (KEGG) pathway analyze and clusters of orthologous groups of proteins (COG) function predictions revealed that changes and variations occurred in the metabolic pathways and function distribution of skin bacterial communities in two groups.

Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ Tcell-depleted mice.

IFN-γ plays a critical role in host defense against intracellular pathogens. IFN-γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN-γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN-γ has beneficial effects on eradication of Pneumocystis, endogenous IFN-γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell-depleted IFN-γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin-1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN-γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis-phagocytic activity. These results indicate that endogenous IFN-γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin-1.

NS7a of SADS-CoV promotes viral infection via inducing apoptosis to suppress type III interferon production.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered swine coronavirus with potential cross-species transmission risk. Although SADS-CoV-induced host cell apoptosis and innate immunity antagonization has been revealed, underlying signaling pathways remain obscure. Here, we demonstrated that infection of SADS-CoV induced apoptosis in vivo and in vitro, and that viral protein NS7a is mainly responsible for SADS-CoV-induced apoptosis in host cells. Furthermore, we found that NS7a interacted with apoptosis-inducing factor mitochondria associated 1 (AIFM1) to activate caspase-3 via caspase-6 in SADS-CoV-infected cells, and enhanced SADS-CoV replication. Importantly, NS7a suppressed poly(I:C)-induced expression of type III interferon (IFN-λ) via activating caspase-3 to cleave interferon regulatory factor 3 (IRF3), and caspase-3 inhibitor protects piglets against SADS-CoV infection in vivo. These findings reveal how SADS-CoV induced apoptosis to inhibit innate immunity and provide a valuable clue to the development of effective drugs for the clinical control of SADS-CoV infection.IMPORTANCEOver the last 20 years, multiple animal-originated coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused millions of deaths, seriously jeopardized human health, and hindered social development, indicating that the study of animal-originated coronaviruses with potential for cross-species transmission is particularly important. Bat-originated swine acute diarrhea syndrome coronavirus (SADS-CoV), discovered in 2017, can not only cause fatal diarrhea in piglets, but also infect multiple human cells, with a potential risk of cross-species transmission, but its pathogenesis is unclear. In this study, we demonstrated that NS7a of SADS-CoV suppresses IFN-λ production via apoptosis-inducing factor mitochondria associated 1 (AIFM1)-caspase-6-caspase-3-interferon regulatory factor 3 (IRF3) pathway, and caspase-3 inhibitor (Z-DEVD-FMK) can effectively inhibit SADS-CoV replication and protect infected piglets. Our findings in this study contribute to a better understanding of SADS-CoV-host interactions as a part of the coronaviruses pathogenesis and using apoptosis-inhibitor as a drug as potential therapeutic approaches for prevention and control of SADS-CoV infection.

Bosutinib Stimulates Macrophage Survival, Phagocytosis, and Intracellular Killing of Bacteria.

Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.

Novel mutation N588 residue in the NS1 protein of feline parvovirus greatly augments viral replication.

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.

When ASFV Infection Meets the cGAS-STING Signaling Pathway

The African swine fever virus (ASFV) has the ability to infect both wild boars and domestic pigs, regardless of their breeds or ages, often resulting in a mortality rate of 100%. Host innate immunity is the most important defense weapon against invasion of pathogenic microbial infection. cGAS-STING signaling pathway is one of the greatest discoveries of the twenty-first century, which is crucial in host’s innate immune response. Recent studies have found that the interaction between cGAS/STING pathway and ASFV plays a key role during ASFV infection. At the same time, ASFV has also evolved different strategies to evade the killing of the host cGAS/STING pathway and promote its survival. Here, we review the latest progress in the interaction between ASFV infection, cGAS/STING pathways, and their related molecular mechanisms, aiming to provide new ideas for further research on the pathogenesis of ASFV, the development of vaccines and therapeutic drugs.

The death domain-associated protein suppresses porcine epidemic diarrhea virus replication by interacting with signal transducer and activator of transcription 1 and inducing downstream ISG15 expression

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute enteric disease in piglets and severely threatens the pig industry all over the world. Death domain-associated protein (DAXX) is a classical chaperone protein involved in multiple biological processes, such as cell apoptosis, transcriptional regulation, DNA damage repair, and host innate immunity. However, whether DAXX functions in the anti-PEDV innate immune responses remains unclear. In this study, we found that PEDV infection upregulated DAXX expression and induced its nucleocytoplasmic translocation in IPEC-J2 cells. Furthermore, we found that DAXX overexpression was inhibitory to PEDV replication, while downregulation of DAXX by RNA interference facilitated PEDV replication. The antiviral activity of DAXX was due to its positive effect on IFN-λ3-STAT1 signaling, as DAXX positively regulated STAT1 activation through their interaction in cytoplasm and enhancing the downstream ISG15 expression. Mutation of tryptophan at 621 to alanine in DAXX increased its abundance in the cytoplasm, leading to the upregulation of STAT1 phosphorylation and ISG15 expression. It indicated that cytoplasmic fraction of DAXX was advantageous for the STAT1-ISG15 signaling axis and PEDV inhibition. In summary, these results show that DAXX inhibits PEDV infection by increasing IFN-λ3-induced STAT1 phosphorylation and the downstream ISG15 expression.

Anexelekto (AXL) no more: microRNA-155 (miR-155) controls the "Uncontrolled" in SARS-CoV-2.

AXL is the gene that encodes the Anexelekto (AXL) receptor tyrosine kinase that demonstrates significant roles in various cellular processes, including cell growth, survival, and migration. Anexelekto is a Greek word meaning excessive and uncontrolled, semantically implying the crucial involvement of AXL in cancer and immune biology, and in promoting cancer metastasis. AXL overexpression appears to drive epithelial to mesenchymal transition, tumor angiogenesis, decreased antitumor immune response, and resistance to therapeutic agents. Recently, AXL has been reported to play important roles in several viral infections, including SARS-CoV-2. We have previously outlined the importance of microRNAs (miRNAs, miRs) and especially miR-155 in SARS-CoV-2 pathophysiology through regulation of the Renin-AngiotensinAldosterone System (RAAS) and influence on several aspects of host innate immunity. MiRNAs are negative regulators of gene expression, decreasing the stability of target RNAs or limiting their translation and, enthrallingly, miR-155 is also involved in AXL homeostasis-both endogenously and pharmaceutically using repurposed drugs (e.g., metformin)-highlighting thrifty evolutionary host innate immunity mechanisms that successfully can thwart viral entry and replication. Cancer, infections, and immune system disturbances will increasingly involve miRNA diagnostics and therapeutics in the future.

SARS-CoV-2 virus-like particle variants alpha and delta mimic the native viruses in their differential inflammasome activating potential

The emerging SARS-CoV-2 variants are evolving to evade human immunity and differ in their pathogenicity. While evasion of the variants from adaptive immunity is widely investigated, there is a paucity of knowledge about their interactions with innate immunity. Inflammasome assembly is one of the most potent mechanisms of the early innate response to viruses, but when it is inappropriate, it can perpetuate tissue damage. In this study, we focused on the capacity of SARS-CoV-2 Alpha and Delta variants to activate the NLRP3 inflammasome. We compared the macrophage activation, particularly the inflammasome formation, using Alpha- and Delta-spike virus-like particles (VLPs). We found that VLPs of both variants activated the inflammasome even without a priming step. Delta-spike VLPs had a significantly stronger effect on triggering pyroptosis and inflammasome assembly in THP-1 macrophages than did Alfa-spike VLPs. Cells treated with Delta VLPs showed greater cleavage of caspase-1 and IL-1β release. Furthermore, Delta VLPs induced stronger cytokine secretion from macrophages and caused essential impairment of mitochondrial respiration in comparison to Alpha VLPs. Additionally, infection of primary human monocyte-derived macrophages with the SARS-CoV-2 variants confirmed the observations in VLPs. Collectively, we revealed that SARS-CoV-2 Delta had a greater impact on the inflammasome activation, cell death and mitochondrial respiration in macrophages than did the Alpha variant. Importantly, the differential response to the SARS-CoV-2 variants can influence the efficacy of therapies targeting the host's innate immunity.

Molecular characterization, antibacterial and immunoregulatory activities of liver-expressed antimicrobial peptide 2 in black rockfish, Sebastes schlegelii

LEAP2 (liver expression antimicrobial peptide 2), is an antimicrobial peptide widely found in vertebrates and mainly expressed in liver. LEAP2 plays a vital role in host innate immunity. In teleosts, a number of LEAP2 homologs have been reported, but their in vivo effects on host defense are still limited. In this study, a LEAP2 homolog (SsLEAP2) was identified from black rockfish, Sebastes schlegelii, and its structure, expression as well as biological functions were analyzed. The results showed that the open reading frame of SsLEAP2 is 300 bp, with a 5′- untranslated region (UTR) of 375 bp and a 3’ - UTR of 238 bp. The deduced amino acid sequence of SsLEAP2 shares the highest overall identity (96.97%) with LEAP2 of Sebastes umbrosus. SsLEAP2 possesses conserved LEAP2 features, including a signal peptide sequence, a prodomain and a mature peptide, in which four well-conserved cysteines formed two intrachain disulphide domain. The expression of SsLEAP2 was highest in liver and could be induced by experimental infection with Listonella anguillarum, Edwardsiealla piscicida and Rock bream iridovirus C1 (RBIV-C1). Recombinant SsLEAP2 (rSsLEAP2) purified from Escherichia coli was able to bind with various Gram-positive and Gram-negative bacteria. Further analysis showed that rSsLEAP2 could enhance the respiratory burst activity, and induce the expression of immune genes including interleukin 1-β (IL-1β) and serum amyloid A (SAA) in macrophages; additionally, rSsLEAP2 could also promote the proliferation and chemotactic of peripheral blood lymphocytes (PBLs). In vivo experiments indicated that overexpression of SsLEAP2 could inhibit bacterial infection, and increase the expression level of immune genes including IL-1β, tumor necrosis factor ligand superfamily member 13B (TNF13B) and haptoglobin (HP); conversely, knock down of SsLEAP2 promoted bacterial infection and decreased the expression level of above genes. Taken together, these results suggest that SsLEAP2 is a novel LEAP2 homolog that possesses apparent antibacterial activity and immunoregulatory property, thus plays a critical role in host defense against pathogens invasion.

NLRP1 restricts porcine deltacoronavirus infection via IL-11 inhibiting the phosphorylation of the ERK signaling pathway.

Continuously emerging highly pathogenic coronaviruses remain a major threat to human and animal health. Porcine deltacoronavirus (PDCoV) is a newly emerging enterotropic swine coronavirus that causes large-scale outbreaks of severe diarrhea disease in piglets. Unlike other porcine coronaviruses, PDCoV has a wide range of species tissue tropism, including primary human cells, which poses a significant risk of cross-species transmission. Nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 1 (NLRP1) has a key role in linking host innate immunity to microbes and the regulation of inflammatory pathways. We now report a role for NLRP1 in the control of PDCoV infection. Overexpression of NLRP1 remarkably suppressed PDCoV infection, whereas knockout of NLRP1 led to a significant increase in PDCoV replication. A mechanistic study revealed that NLRP1 suppressed PDCoV replication in cells by upregulating IL-11 expression, which in turn inhibited the phosphorylation of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor U0126 effectively hindered PDCoV replication in pigs. Together, our results demonstrated that NLRP1 exerted an anti-PDCoV effect by IL-11-mediated inhibition of the phosphorylation of the ERK signaling pathway, providing a novel antiviral signal axis of NLRP1-IL-11-ERK. This study expands our understanding of the regulatory network of NLRP1 in the host defense against virus infection and provides a new insight into the treatment of coronaviruses and the development of corresponding drugs.IMPORTANCECoronavirus, which mainly infects gastrointestinal and respiratory epithelial cells in vivo, poses a huge threat to both humans and animals. Although porcine deltacoronavirus (PDCoV) is known to primarily cause fatal diarrhea in piglets, reports detected in plasma samples from Haitian children emphasize the potential risk of animal-to-human spillover. Finding effective therapeutics against coronaviruses is crucial for controlling viral infection. Nucleotide-binding oligomerization-like receptor (NLR) family pyrin domain-containing 1 (NLRP1), a key regulatory factor in the innate immune system, is highly expressed in epithelial cells and associated with the pathogenesis of viruses. We demonstrate here that NLRP1 inhibits the infection of the intestinal coronavirus PDCoV through IL-11-mediated phosphorylation inhibition of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor can control the infection of PDCoV in pigs. Our study emphasizes the importance of NLRP1 as an immune regulatory factor and may open up new avenues for the treatment of coronavirus infection.

Strategies of Helicobacter pylori in evading host innate and adaptive immunity: insights and prospects for therapeutic targeting.

Helicobacter pylori (H. pylori) is the predominant pathogen causing chronic gastric mucosal infections globally. During the period from 2011 to 2022, the global prevalence of H. pylori infection was estimated at 43.1%, while in China, it was slightly higher at approximately 44.2%. Persistent colonization by H. pylori can lead to gastritis, peptic ulcers, and malignancies such as mucosa-associated lymphoid tissue (MALT) lymphomas and gastric adenocarcinomas. Despite eliciting robust immune responses from the host, H. pylori thrives in the gastric mucosa by modulating host immunity, particularly by altering the functions of innate and adaptive immune cells, and dampening inflammatory responses adverse to its survival, posing challenges to clinical management. The interaction between H. pylori and host immune defenses is intricate, involving evasion of host recognition by modifying surface molecules, manipulating macrophage functionality, and modulating T cell responses to evade immune surveillance. This review analyzes the immunopathogenic and immune evasion mechanisms of H. pylori, underscoring the importance of identifying new therapeutic targets and developing effective treatment strategies, and discusses how the development of vaccines against H. pylori offers new hope for eradicating such infections.

Inhibition of SARS-CoV-2 replication by a ssDNA aptamer targeting the nucleocapsid protein.

The nucleocapsid protein of SARS-CoV-2 plays significant roles in viral assembly, immune evasion, and viral stability. Due to its immunogenicity, high expression levels during COVID-19, and conservation across viral strains, it represents an attractive target for antiviral treatment. In this study, we identified and characterized a single-stranded DNA aptamer, N-Apt17, which effectively disrupts the liquid-liquid phase separation (LLPS) mediated by the N protein. To enhance the aptamer's stability, a circular bivalent form, cb-N-Apt17, was designed and evaluated. Our findings demonstrated that cb-N-Apt17 exhibited improved stability, enhanced binding affinity, and superior inhibition of N protein LLPS; thus, it has the potential inhibition ability on viral replication. These results provide valuable evidence supporting the potential of cb-N-Apt17 as a promising candidate for the development of antiviral therapies against COVID-19.IMPORTANCEVariants of SARS-CoV-2 pose a significant challenge to currently available COVID-19 vaccines and therapies due to the rapid epitope changes observed in the viral spike protein. However, the nucleocapsid (N) protein of SARS-CoV-2, a highly conserved structural protein, offers promising potential as a target for inhibiting viral replication. The N protein forms complexes with genomic RNA, interacts with other viral structural proteins during virion assembly, and plays a critical role in evading host innate immunity by impairing interferon production during viral infection. In this investigation, we discovered a single-stranded DNA aptamer, designated as N-Apt17, exhibiting remarkable affinity and specificity for the N protein. Notably, N-Apt17 disrupts the liquid-liquid phase separation (LLPS) of the N protein. To enhance the stability and molecular recognition capabilities of N-Apt17, we designed a circular bivalent DNA aptamer termed cb-N-Apt17. In both in vivo and in vitro experiments, cb-N-Apt17 exhibited increased stability, enhanced binding affinity, and superior LLPS disrupting ability. Thus, our study provides essential proof-of-principle evidence supporting the further development of cb-N-Apt17 as a therapeutic candidate for COVID-19.

Ubiquitin-specific proteinase 1 stabilizes PRRSV nonstructural protein Nsp1β to promote viral replication by regulating K48 ubiquitination.

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1β (Nsp1β) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1β protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense.IMPORTANCEDeubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1β (Nsp1β) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1β, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1β, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.

CFTR is required for zinc-mediated antibacterial defense in human macrophages

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion transporter required for epithelial homeostasis in the lung and other organs, with CFTR mutations leading to the autosomal recessive genetic disease CF. Apart from excessive mucus accumulation and dysregulated inflammation in the airways, people with CF (pwCF) exhibit defective innate immune responses and are susceptible to bacterial respiratory pathogens such as Pseudomonas aeruginosa. Here, we investigated the role of CFTR in macrophage antimicrobial responses, including the zinc toxicity response that is used by these innate immune cells against intracellular bacteria. Using both pharmacological approaches, as well as cells derived from pwCF, we show that CFTR is required for uptake and clearance of pathogenic Escherichia coli by CSF-1-derived primary human macrophages. CFTR was also required for E. coli-induced zinc accumulation and zinc vesicle formation in these cells, and E. coli residing in macrophages exhibited reduced zinc stress in the absence of CFTR function. Accordingly, CFTR was essential for reducing the intramacrophage survival of a zinc-sensitive E. coli mutant compared to wild-type E. coli. Ectopic expression of the zinc transporter SLC30A1 or treatment with exogenous zinc was sufficient to restore antimicrobial responses against E. coli in human macrophages. Zinc supplementation also restored bacterial killing in GM-CSF-derived primary human macrophages responding to P. aeruginosa, used as an in vitro macrophage model relevant to CF. Thus, restoration of the zinc toxicity response could be pursued as a therapeutic strategy to restore innate immune function and effective host defense in pwCF.

Roles of autophagy in killing of mycobacterial pathogens by host macrophages - Effects of some medicinal plants.

Autophagy is a cellular stress-induced intracellular process, through which damaged cellular components are decomposed via lysosomal degradation. This process plays important roles in host innate immunity, particularly the elimination of intracellular pathogens inside host macrophages. A more detailed understanding of the roles of autophagic events in the effective manifestation of macrophagic antimycobacterial activity is needed. Furthermore, the effects of medicinal plants on macrophagic autophagy response to mycobacterial infection need to be clarified. We herein examined the significance of autophagic events in the manifestation of host immunity during mycobacterial infection, by performing a literature search using PubMed. Recent studies demonstrated that autophagy up-regulated macrophage functions related to the intracellular killing of mycobacteria, even when pathogens were residing within the cytoplasm of macrophages. The majority of medicinal plants potentiated macrophagic autophagy, thereby enhancing their antimycobacterial functions. In contrast, most medicinal plants down-regulate the development and activation of the Th17 cell population, which reduces macrophage antimycobacterial activity. These opposing effects of medicinal plants on macrophage autophagy (enhancement) and Th17 cell functions (inhibition) may provide a plausible explanation for the clinical observation of their modest efficacy in the treatment of mycobacterial infections.

HCV-induced autophagy and innate immunity.

The interplay between autophagy and host innate immunity has been of great interest. Hepatitis C virus (HCV) impedes signaling pathways initiated by pattern-recognition receptors (PRRs) that recognize pathogens-associated molecular patterns (PAMPs). Autophagy, a cellular catabolic process, delivers damaged organelles and protein aggregates to lysosomes for degradation and recycling. Autophagy is also an innate immune response of cells to trap pathogens in membrane vesicles for removal. However, HCV controls the autophagic pathway and uses autophagic membranes to enhance its replication. Mitophagy, a selective autophagy targeting mitochondria, alters the dynamics and metabolism of mitochondria, which play important roles in host antiviral responses. HCV also alters mitochondrial dynamics and promotes mitophagy to prevent premature cell death and attenuate the interferon (IFN) response. In addition, the dysregulation of the inflammasomal response by HCV leads to IFN resistance and immune tolerance. These immune evasion properties of HCV allow HCV to successfully replicate and persist in its host cells. In this article, we discuss HCV-induced autophagy/mitophagy and its associated immunological responses and provide a review of our current understanding of how these processes are regulated in HCV-infected cells.

Infectious bursal disease virus VP5 triggers host shutoff in a transcription-dependent manner.

Viruses have evolved intricate mechanisms to evade host antiviral responses and exploit cellular resources by manipulating the expression profile of host genes. During infection, viruses encode proteins with shutoff activity to globally inhibit host protein synthesis, which is an effective strategy for immune evasion. In this study, compelling evidence shows that infectious bursal disease virus (IBDV) infection triggers the suppression of host protein synthesis. Furthermore, using both in vitro and in vivo viral infection models, we have identified that IBDV specifically impedes the transcription of host genes via the shutoff activity of viral VP5, simultaneously conferring advantages to IBDV infection in these circumstances. The proposed mechanism suggests that VP5 competitively binds to RanBP1, disrupting the RanGDP/GTP gradient. This disruption interferes with cellular nucleocytoplasmic transport, impairing the nuclear import of proteins bearing nuclear localization signals. The nuclear transport of pivotal transcriptional regulatory factors, such as p65 and IFN regulatory factor 7, is also compromised, leading to the inhibition of pro-inflammatory cytokines and interferon expression. This newly discovered strategy employed by IBDV enables them to manipulate host gene expression, providing novel insights into how viruses evade host immune responses and establish infections.IMPORTANCEViruses manipulate host processes at various levels to regulate or evade both innate and adaptive immune responses, promoting self-survival and efficient transmission. The "host shutoff," a global suppression of host gene expression mediated by various viruses, is considered a critical mechanism for evading immunity. In this study, we have validated the presence of host shutoff during infectious bursal disease virus (IBDV) infection and additionally uncovered that the viral protein VP5 plays a pivotal role in inhibiting the overall synthesis of host proteins, including cytokines, through a transcription-dependent pathway. VP5 competitively binds with RanBP1, leading to disruption of the Ran protein cycle and consequently interfering with nucleocytoplasmic transport, which ultimately results in the suppression of host gene transcription. These findings unveil a novel strategy employed by IBDV to evade host innate immunity and rapidly establish infection. This study also suggests a novel supplement to understanding the pathway through which viruses inhibit host protein synthesis.