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Summary 1. Kindlin-3 but not talin-1 contributes to integrin inside-out signaling induced β2 integrin clustering. 2. The Pleckstrin homology domain of kindlin-3 was critical for its mediated β2 integrin clustering.

Abstract Modeling and predicting mutations are critical for COVID-19 and similar pandemic preparedness. However, existing predictive models have yet to integrate the regularity and randomness of viral mutations with minimal data requirements. Here, we develop a non-demanding language model utilizing both regularity and randomness to predict candidate SARS-CoV-2 variants and mutations that might prevail. We constructed the “grammatical frameworks” of the available S1 sequences for dimension reduction and semantic representation to grasp the model’s latent regularity. The mutational profile, defined as the frequency of mutations, was introduced into the model to incorporate randomness. With this model, we successfully identified and validated several variants with significantly enhanced viral infectivity and immune evasion by wet-lab experiments. By inputting the sequence data from three different time points, we detected circulating strains or vital mutations for XBB.1.16, EG.5, JN.1, and BA.2.86 strains before their emergence. In addition, our results also predicted the previously unknown variants that may cause future epidemics. With both the data validation and experiment evidence, our study represents a fast-responding, concise, and promising language model, potentially generalizable to other viral pathogens, to forecast viral evolution and detect crucial hot mutation spots, thus warning the emerging variants that might raise public health concern.

Summary: Background: Global dissemination of SARS-CoV-2 Omicron sublineages has provided a sufficient opportunity for natural selection, thus enabling beneficial mutations to emerge. Characterisation of these mutations uncovers the underlying machinery responsible for the fast transmission of Omicron variants and guides vaccine development for combating the COVID-19 pandemic. Methods: Through systematic bioinformatics analysis of 496,606 sequences of Omicron variants, we obtained 40 amino acid substitutions that occurred with high frequency in the S protein. Utilising pseudoviruses and a trans-complementation system of SARS-CoV-2, we identified the effect of high-frequency mutations on viral infectivity and elucidated the molecular mechanisms. Finally, we evaluated the impact of a key emerging mutation on the immune protection induced by the SARS-CoV-2 VLP mRNA vaccine in a murine model. Findings: We identified a proline-to-leucine substitution at the 1263rd residue of the Spike protein, and upon investigating the relative frequencies across multiple Omicron sublineages, we found a trend of increasing frequency for P1263L. The substitution significantly enhances the capacity for S-mediated viral entry and improves the immunogenicity of a virus-like particle mRNA vaccine. Mechanistic studies showed that this mutation is located in the FERM binding motif of the cytoplasmic tail and impairs the interaction between the S protein and the Ezrin/Radixin/Moesin proteins. Additionally, this mutation facilitates the incorporation of S proteins into SARS-CoV-2 virions. Interpretation: This study offers mechanistic insight into the constantly increasing transmissibility of SARS-CoV-2 Omicron variants and provides a meaningful optimisation strategy for vaccine development against SARS-CoV-2. Funding: This study was supported by grants from the National Key Research and Development Plan of China (2021YFC2302405, 2022YFC2303200, 2021YFC2300200 and 2022YFC2303400), the National Natural Science Foundation of China (32188101, 32200772, 82422049, 82241082, 32270182, 82372254, 82271872, 82341046, 32100755 and 82102389), Shenzhen Medical Research Fund (B2404002, A2303036), the Shenzhen Bay Laboratory Startup Fund (21330111), Shenzhen San-Ming Project for Prevention and Research on Vector-borne Diseases (SZSM202211023), Yunnan Provincial Science and Technology Project at Southwest United Graduate School (202302AO370010). The New Cornerstone Science Foundation through the New Cornerstone Investigator Program, and the Xplorer Prize from Tencent Foundation.

Summary: Background: The ubiquitin regulatory X (UBX) domain-containing proteins (UBXNs) are putative adaptors for ubiquitin ligases and valosin-containing protein; however, their in vivo physiological functions remain poorly characterised. We recently showed that UBXN3B is essential for activating innate immunity to DNA viruses and controlling DNA/RNA virus infection. Herein, we investigate its role in adaptive immunity. Methods: We evaluated the antibody responses to multiple viruses and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza in tamoxifen-inducible global and constitutive B cell-specific Ubxn3b knockout mice; quantified various immune populations, B lineage progenitors/precursors, B cell receptor (BCR) signalling and apoptosis by flow cytometry, immunoblotting and immunofluorescence microscopy. We also performed bone marrow transfer, single-cell and bulk RNA sequencing. Findings: Both global and B cell-specific Ubxn3b knockout mice present a marked reduction in small precursor B-II (>60%), immature (>70%) and mature B (>95%) cell numbers. Transfer of wildtype bone marrow to irradiated global Ubxn3b knockouts restores normal B lymphopoiesis, while reverse transplantation does not. The mature B population shrinks rapidly with apoptosis and higher pro and activated caspase-3 protein levels were observed following induction of Ubxn3b knockout. Mechanistically, Ubxn3b deficiency leads to impaired pre-BCR signalling and cell cycle arrest. Ubxn3b knockout mice are highly vulnerable to respiratory viruses, with increased viral loads and prolonged immunopathology in the lung, and reduced production of virus-specific IgM/IgG. Interpretation: UBXN3B is essential for B lymphopoiesis by maintaining constitutive pre-BCR signalling and cell survival in a cell-intrinsic manner. Funding: United States National Institutes of Health grants, R01AI132526 and R21AI155820.

Abstract The Retinoic acid-Inducible Gene I (RIG-I) like receptors (RLRs) are the major viral RNA sensors essential for the initiation of antiviral immune responses. RLRs are subjected to stringent transcriptional and posttranslational regulations, of which ubiquitination is one of the most important. However, the role of ubiquitination in RLR transcription is unknown. Here, we screen 375 definite ubiquitin ligase knockout cell lines and identify Ubiquitin Protein Ligase E3 Component N-Recognin 5 (UBR5) as a positive regulator of RLR transcription. UBR5 deficiency reduces antiviral immune responses to RNA viruses, while increases viral replication in primary cells and mice. Ubr5 knockout mice are more susceptible to lethal RNA virus infection than wild type littermates. Mechanistically, UBR5 mediates the Lysine 63-linked ubiquitination of Tripartite Motif Protein 28 (TRIM28), an epigenetic repressor of RLRs. This modification prevents intramolecular SUMOylation of TRIM28, thus disengages the TRIM28-imposed brake on RLR transcription. In sum, UBR5 enables rapid upregulation of RLR expression to boost antiviral immune responses by ubiquitinating and de-SUMOylating TRIM28.

Abstract Herd immunity achieved through mass vaccination is an effective approach to prevent contagious diseases. Nonetheless, emerging SARS-CoV-2 variants with frequent mutations largely evaded humoral immunity induced by Spike-based COVID-19 vaccines. Herein, we develop a lipid nanoparticle (LNP)-formulated mRNA-based T-cell-inducing antigen, which targeted three SARS-CoV-2 proteome regions that enriched human HLA-I epitopes (HLA-EPs). Immunization of HLA-EPs induces potent cellular responses to prevent SARS-CoV-2 infection in humanized HLA-A*02:01/DR1 and HLA-A*11:01/DR1 transgenic mice. Of note, the sequences of HLA-EPs are highly conserved among SARS-CoV-2 variants of concern. In humanized HLA-transgenic mice and female rhesus macaques, dual immunization with the LNP-formulated mRNAs encoding HLA-EPs and the receptor-binding domain of the SARS-CoV-2 B.1.351 variant (RBDbeta) is more efficacious in preventing infection of SARS-CoV-2 Beta and Omicron BA.1 variants than single immunization of LNP-RBD beta . This study demonstrates the necessity to strengthen the vaccine effectiveness by comprehensively stimulating both humoral and cellular responses, thereby offering insight for optimizing the design of COVID-19 vaccines.

Innate immune signaling plays a significant role in the rapid cellular responses against foreign entities. An inflammasome is a large cytosolic polymer of a pattern recognition receptor with/without an adaptor protein, formed in response to these entities. Canonically, an inflammasome can recruit and lead to auto-activation of caspase-1, subsequent maturation and secretion of inflammatory cytokines, and pyroptosis. One particular inflammasome, the noncanonical inflammasome, is formed by caspase-4 or -5 (mouse caspase-11) upon binding of lipopolysaccharide and is essential for controlling gram-negative bacterial infection. However, prolonged hyper-activation of the non-canonical inflammasome has been implicated in the pathogenesis of inflammatory diseases and endotoxemia sepsis. This review will summarize the recent advances on the noncanonical inflammasome, its mechanism of activation, key cellular regulators and role in health and disease.

Yang et al report that intrinsic cardiac adrenergic cell-derived norepinephrine promotes myocardial dysfunction in response to LPS. They demonstrate norepinephrine produced by these cells activated β1-adrenergic receptors on cardiomyocytes increasing the expression of tumor necrosis factor-a in a Ca2+/calmodulin-dependent protein kinase II dependent manner.

ABSTRACT Ubiquitin regulatory X domain-containing proteins (UBXN) might be involved in diverse cellular processes. However, their in vivo physiological functions remain largely elusive. We recently showed that UBXN3B positively regulated stimulator-of-interferon-genes (STING)-mediated innate immune responses to DNA viruses. Herein, we reported the essential role of UBXN3B in the control of infection and immunopathogenesis of two arthritogenic RNA viruses, Chikungunya (CHIKV) and O’nyong’nyong (ONNV) viruses. Ubxn3b deficient (Ubxn3b−/−) mice presented higher viral loads, more severe foot swelling and immune infiltrates, and slower clearance of viruses and resolution of inflammation than the Ubxn3b+/+ littermates. While the serum cytokine levels were intact, the virus-specific immunoglobulin G and neutralizing antibody levels were lower in the Ubxn3b−/− mice. The Ubxn3b−/− mice had more neutrophils and macrophages, but much fewer B cells in the ipsilateral feet. Of note, this immune dysregulation was also observed in the spleens and blood of uninfected Ubxn3b−/− mice. UBXN3B restricted CHIKV replication in a cell-intrinsic manner but independent of type I IFN signaling. These results demonstrated a dual role of UBXN3B in the maintenance of immune homeostasis and control of RNA virus replication. IMPORTANCE The human genome encodes 13 ubiquitin regulatory X (UBX) domain-containing proteins (UBXN) that might participate in diverse cellular processes. However, their in vivo physiological functions remain largely elusive. Herein, we reported an essential role of UBXN3B in the control of infection and immunopathogenesis of arthritogenic alphaviruses, including Chikungunya virus (CHIKV), which causes acute and chronic crippling arthralgia, long-term neurological disorders, and poses a significant public health problem in the tropical and subtropical regions worldwide. However, there are no approved vaccines or specific antiviral drugs. This was partly due to a poor understanding of the protective and detrimental immune responses elicited by CHIKV. We showed that UBXN3B was critical for the control of CHIKV replication in a cell-intrinsic manner in the acute phase and persistent immunopathogenesis in the post-viremic stage. Mechanistically, UBXN3B was essential for the maintenance of hematopoietic homeostasis during viral infection and in steady-state.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the global pandemic and life-threatening coronavirus disease 2019 (COVID-19). Although vaccines and therapeutic antibodies are available, their efficacy is continuously undermined by rapidly emerging SARS-CoV-2 variants. Here, we found that all-trans retinoic acid (ATRA), a vitamin A (retinol) derivative, showed potent antiviral activity against all SARS-CoV-2 variants in both human cell lines and human organoids of the lower respiratory tract. Mechanistically, ATRA directly binds in a deep hydrophobic pocket of the receptor binding domain (RBD) located on the top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the “down” RBDs and locks most of the S trimers in an RBD “all-down” and ACE2-inaccessible inhibitory conformation. In summary, our results reveal the pharmacological biotargets and structural mechanism of ATRA and other retinoids in SARS-CoV-2 infection and suggest that ATRA and its derivatives could be potential hit compounds against a broad spectrum of coronaviruses. IMPORTANCE Retinoids, a group of compounds including vitamin A and its active metabolite all-trans retinoic acid (ATRA), regulate serial physiological activity in multiple organ systems, such as cell growth, differentiation, and apoptosis. The ATRA analogues reported to date include more than 4,000 natural and synthetic molecules that are structurally and/or functionally related to ATRA. Here, we found that ATRA showed potent antiviral activity against all SARS-CoV-2 variants by directly binding in a deep hydrophobic pocket of the receptor binding domain (RBD) located on top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the “down” RBDs and locks most of the S trimers in an RBD “all-down” and ACE2-inaccessible inhibitory conformation, suggesting the pharmacological feasibility of using ATRA or its derivatives as a remedy for and prevention of COVID-19 disease.

Arboviruses are etiological agents of various severe human diseases that place a tremendous burden on global public health and the economy; compounding this issue is the fact that effective prophylactics and therapeutics are lacking for most arboviruses. Herein, we identified 2 bacterial lipases secreted by a Chromobacterium bacterium isolated from Aedes aegypti midgut, Chromobacterium antiviral effector-1 (CbAE-1) and CbAE-2, with broad-spectrum virucidal activity against mosquito-borne viruses, such as dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), yellow fever virus (YFV) and Sindbis virus (SINV). The CbAEs potently blocked viral infection in the extracellular milieu through their lipase activity. Mechanistic studies showed that this lipase activity directly disrupted the viral envelope structure, thus inactivating infectivity. A mutation in the lipase motif of CbAE-1 fully abrogated the virucidal ability. Furthermore, CbAEs also exert lipase-dependent entomopathogenic activity in mosquitoes. The anti-arboviral and entomopathogenic properties of CbAEs render them potential candidates for the development of novel transmission control strategies against vector-borne diseases.

Summary: Hematophagous arthropods, such as mosquitoes, naturally carry and transmit hundreds of arboviruses to humans. Blood meal is a predominant physical interface that shapes cross-species communications among humans, bloodsuckers, and arboviruses. Here, we identify a human-blood-derived microRNA, hsa-miR-150-5p, that interferes with a mosquito antiviral system to facilitate flavivirus infection and transmission. hsa-miR-150-5p is acquired with a blood meal into the mosquito hemocoel and persists for a prolonged time there. The agomir of hsa-miR-150-5p enhances, whereas the antagomir represses flaviviral infection in mosquitoes and transmission from mice to mosquitoes. Mechanistic studies indicate that hsa-miR-150-5p hijacks the mosquito Argonaute-1-mediated RNA interference system to suppress the expression of some chymotrypsins with potent virucidal activity. Mosquito chymotrypsins are essential for resisting systemic flavivirus infection in hemocoel tissues. Chymotrypsin homologs potentially targeted by miR-150-5p are also found in other hematophagous arthropods, demonstrating a conserved miR-150-5p-mediated cross-species RNAi mechanism that might determine flaviviral transmissibility in nature.

A hallmark of chronic kidney disease is renal fibrosis, which can result in progressive loss of kidney function. Currently, there is no effective therapy for renal fibrosis. Therefore, there is an urgent need to identify potential drug targets for renal fibrosis. In this study, we examined the effect of a selective STAT6 inhibitor, AS1517499, on myeloid fibroblast activation, macrophage polarization, and development of renal fibrosis in two experimental murine models. To investigate the effect of STAT6 inhibition on myeloid fibroblast activation, macrophage polarization, and kidney fibrosis, wild-type mice were subjected to unilateral ureteral obstruction or folic acid administration and treated with AS1517499. Mice treated with vehicle were used as control. At the end of experiments, kidneys were harvested for analysis of myeloid fibroblast activation, macrophage polarization, and renal fibrosis and function. Unilateral ureteral obstruction or folic acid administration induced STAT6 activation in interstitial cells of the kidney, which was significantly abolished by AS1517499 treatment. Mice treated with AS1517499 accumulated fewer myeloid fibroblasts and myofibroblasts in the kidney with ureteral obstruction or folic acid nephropathy compared with vehicle-treated mice. Moreover, AS1517499 significantly suppressed M2 macrophage polarization in the injured kidney. Furthermore, AS1517499 markedly reduced the expression levels of extracellular matrix proteins, and development of kidney fibrosis and dysfunction. These findings suggest that AS1517499 inhibits STAT6 activation, suppresses myeloid fibroblast activation, reduces M2 macrophage polarization, attenuates extracellular matrix protein production, and preserves kidney function. Therefore, targeting STAT6 with AS1517499 is a novel therapeutic approach for chronic kidney disease.

Mosquito saliva affects transmission of flaviviruses, but underlying mechanisms are incompletely understood. Here, the authors show that Aedes aegypti venom allergen-1 (AaVA-1) promotes dengue and Zika virus transmission by activating autophagy in host immune cells of the monocyte lineage.

Here the authors show that Aedes mosquitoes can acquire ZIKV by breeding in contaminated aquatic systems, and that these infected mosquitoes can transmit ZIKV to susceptible mice. This suggests that human urine containing aquatic environments could contribute to ZIKV transmission.

Summary: Macrophage scavenger receptor 1 (MSR1) plays an important role in host defense to bacterial infections, M2 macrophage polarization, and lipid homeostasis. However, its physiological function in viral pathogenesis remains poorly defined. Herein, we report that MSR1 facilitates vesicular stomatitis virus (VSV) infection in the central nervous system. Msr1-deficient (Msr1−/−) mice presented reduced morbidity, mortality, and viral loads in the spinal cord following lethal VSV infection, along with normal viremia and innate immune responses, compared to Msr1+/− littermates and wild-type mice. Msr1 expression was most significantly upregulated in the spinal cord, the predominant target of VSV. Mechanistically, through its extracellular domains, MSR1 interacted with VSV surface glycoprotein and facilitated its cellular entry in a low-density lipoprotein receptor-dependent manner. In conclusion, our results demonstrate that MSR1 serves as a cofactor for VSV cellular entry and facilitates its infection preferentially in the spinal cord.

Summary: Plant-nectar-derived sugar is the major energy source for mosquitoes, but its influence on vector competence for malaria parasites remains unclear. Here, we show that Plasmodium berghei infection of Anopheles stephensi results in global metabolome changes, with the most significant impact on glucose metabolism. Feeding on glucose or trehalose (the main hemolymph sugars) renders the mosquito more susceptible to Plasmodium infection by alkalizing the mosquito midgut. The glucose/trehalose diets promote proliferation of a commensal bacterium, Asaia bogorensis, that remodels glucose metabolism in a way that increases midgut pH, thereby promoting Plasmodium gametogenesis. We also demonstrate that the sugar composition from different natural plant nectars influences A. bogorensis growth, resulting in a greater permissiveness to Plasmodium. Altogether, our results demonstrate that dietary glucose is an important determinant of mosquito vector competency for Plasmodium, further highlighting a key role for mosquito-microbiota interactions in regulating the development of the malaria parasite.

The UBXN proteins are likely involved in a diverse range of biological processes, but their physiological functions remain largely unknown. Here the authors show that UBXN3B positively regulates STING-mediated immune responses in the context of viral infections.

Renal fibrosis is a pathologic feature of chronic kidney disease, which can lead to end-stage kidney disease. Myeloid fibroblasts play a central role in the pathogenesis of renal fibrosis. However, the molecular mechanisms pertaining to myeloid fibroblast activation remain to be elucidated. In the present study, we examine the role of signal transducer and activator of transcription 6 (STAT6) in myeloid fibroblast activation, macrophage polarization, and renal fibrosis development in a mouse model of folic acid nephropathy. STAT6 is activated in the kidney with folic acid nephropathy. Compared with folic-acid-treated wild-type mice, STAT6 knockout mice had markedly reduced myeloid fibroblasts and myofibroblasts in the kidney with folic acid nephropathy. Furthermore, STAT6 knockout mice exhibited significantly less CD206 and PDGFR-β dual-positive fibroblast accumulation and M2 macrophage polarization in the kidney with folic acid nephropathy. Consistent with these findings, STAT6 knockout mice produced less extracellular matrix protein, exhibited less severe interstitial fibrosis, and preserved kidney function in folic acid nephropathy. Taken together, these results have shown that STAT6 plays a critical role in myeloid fibroblasts activation, M2 macrophage polarization, extracellular matrix protein production, and renal fibrosis development in folic acid nephropathy. Therefore, targeting STAT6 may provide a novel therapeutic strategy for fibrotic kidney disease.

Transmission of many human viruses depends on replication in their mosquito vectors. Here, Zhu et al. show that glutamic acid digested from the blood meal activates GABA signaling, resulting in suppression of antiviral innate immunity and increased virus replication in mosquitoes.

Abstract The phosphatidyl-inositol-3 kinases (PI3K) pathway regulates a variety of cellular processes, including cell proliferation, RNA processing, protein translation, autophagy, apoptosis and antiviral immunity. Many viruses depend on PI3K signaling for replication. However, its role in flaviviral infection has not been clearly defined. Here we report that PI3K signaling is critical for the control of West Nile virus (WNV) infection by regulating type I IFN (IFN-I) response. Inhibition of PI3K activity by 3-methyl adenine (3-MA), Wortmannin (WM) and LY294002 (LY) increased viral titers by 3–16 folds in primary mouse macrophages, embryonic fibroblasts and human cell lines. Both 3-MA and LY repressed IFN-I mRNA and protein expression significantly. Surprisingly, WM enhanced the mRNA expression of IFN-I and TNF-α, and TNF-α protein production modestly, while dramatically decreased the secreted IFN-I. Further studies showed that the catalytic subunit p110δ of class I PI3K played a role in induction of antiviral immune responses. Lastly translocation of interferon regulatory factor 7(IRF7) from the cytosol to the nuclei was effectively blocked in the presence of PI3K inhibitors. Our results clearly define an antiviral role of PI3K by modulating immune responses and demonstrate differential mode of action of three PI3K inhibitors on IFN-I.

Objective To describe a new strategy for the whole genome resequencing of small parasite samples. Methods Whole genome resequencing was based on a multiple displacement amplification (MDA) method. Sequencing reads were aligned with the reference genome, and a Bayesian model was used to calculate genotype probabilities. De novo genome assembly was conducted, and single nucleotide polymorphisms (SNPs) were detected. Gene ontology (GO) analysis was used to determine connections between SNPs and genes. Results In total, 64.12% of the parasite genome sequence was mapped to Necator americanus . fa, and 125,553 SNPs were detected. GO analysis revealed that most SNPs in coding regions were probably associated with common drug targets. Conclusion These results reveal the feasibility of a new strategy to detect genetic variations of small parasites. This study also provides a proof-of-principle for the molecular classification and epidemiological analysis of other parasites.

ABSTRACT Endogenous retroviruses (ERVs) are transposable elements that cause host genome instability and usually play deleterious roles in disease such as tumorigenesis. Recent advances also suggest that this “enemy within” may encode a viral mimic to induce antiviral immune responses through viral sensors. Here, through whole-genome transcriptome analysis with RNA sequencing (RNA-Seq), we discovered that a full-length ERV-derived long noncoding RNA (lncRNA), designated lnc-EPAV (ERV-derived lncRNA positively regulates antiviral responses), was a positive regulator of NF-κB signaling. lnc-EPAV expression was rapidly upregulated by viral RNA mimics or RNA viruses to facilitate the expression of RELA, an NF-κB subunit that plays a crucial role in antiviral responses. Transcriptome analysis of lnc-EPAV-silenced macrophages showed that lnc-EPAV was critical for RELA target gene expression and innate immune responses. Consistently, lnc-EPAV-deficient mice exhibited reduced expression of type I interferons (IFNs) and, consequently, increased viral loads and mortality following lethal RNA virus infection. Mechanistically, lnc-EPAV promoted expression of RELA by competitively binding to and displacing SFPQ, a transcriptional repressor of Rela. Altogether, our work demonstrates an alternative mechanism by which ERVs regulate antiviral immune responses. IMPORTANCE Endogenous retroviruses are transposable genetic elements comprising 8% to 10% of the human and mouse genomes. Although most ERVs have been inactivated due to deleterious mutations, some are still transcribed. However, the biological functions of transcribed ERVs are largely unknown. Here, we identified a full-length ERV-derived lncRNA, designated lnc-EPAV, as a positive regulator of host innate immune responses. We found that silencing lnc-EPAV impaired virus-induced cytokine production, resulting in increased viral replication in cells. The lnc-EPAV-deficient mice exhibited enhanced susceptibility to viral challenge. We also found that lnc-EPAV regulated expression of RELA, an NF-κB subunit that plays a critical role in antiviral responses. ERV-derived lncRNA coordinated with a transcription repressor, SFPQ, to control Rela transcription. Our report provides new insights into the previously unrecognized immune gene regulatory mechanism of ERV-derived lncRNAs.

Emerging and re-emerging arthritogenic alphaviruses, such as Chikungunya virus (CHIKV) and O’nyong nyong virus, cause acute and chronic crippling arthralgia associated with inflammatory immune responses. Approximately 50% of CHIKV-infected patients suffer from rheumatic manifestations that last 6 months to years. However, the physiological functions of individual immune signaling pathways in the pathogenesis of alphaviral arthritis remain poorly understood. Here, we report that a deficiency in CXCL10, which is a chemoattractant for monocytes/macrophages/T cells, led to the same viremia as wild-type animals, but fewer immune infiltrates and lower viral loads in footpads at the peak of arthritic disease (6–8 days post infection). Macrophages constituted the largest immune cell population in footpads following infection, and were significantly reduced in Cxcl10−/− mice. The viral RNA loads in neutrophils and macrophages were reduced in Cxcl10−/− compared to wild-type mice. In summary, our results demonstrate that CXCL10 signaling promotes the pathogenesis of alphaviral disease and suggest that CXCL10 may be a therapeutic target for mitigating alphaviral arthritis.

In order to eliminate viral infections, hundreds of interferon-stimulated genes (ISGs) are induced via type I interferons (IFNs). However, the functions and mechanisms of most ISGs are largely unclear. A tripartite motif (TRIM) protein encoding gene TRIM69 is induced by dengue virus (DENV) infection as an ISG. TRIM69 restricts DENV replication, and its RING domain, which has the E3 ubiquitin ligase activity, is critical for its antiviral activity. An in vivo study further confirmed that TRIM69 contributes to the control of DENV infection in immunocompetent mice. Unlike many other TRIM family members, TRIM69 is not involved in modulation of IFN signaling. Instead, TRIM69 interacts with DENV Nonstructural Protein 3 (NS3) directly and mediates its polyubiquitination and degradation. Finally, Lys104 of NS3 is identified as the target of TRIM69-mediated ubiquitination. Our study demonstrates that TRIM69 restricts DENV replication by specifically ubiquitinating a viral nonstructural protein.

Objectives: Human Urinary Kallidinogenase (HUK) is a tissue kallikrein that plays neuroprotective role in ischemic conditions via different mechanisms. Mild hypothermia (MH) is another robust neuroprotectant that reduces mortality but does not profoundly ameliorate the neurological outcome in hypoxic-ischemic encephalopathy (HIE) patients. However, whether the combination of HUK and MH can be used as a promising neuroprotective treatment in HIE is unknown.Methods: One-hundred and forty-four adult Wistar rats were randomly divided into five groups: Sham, HIE, HUK, MH and a combination of HUK and MH treatment. The HIE rat model was established by right carotid dissection followed by hypoxia aspiration. The survival curve was created within 7 days, and the neurological severity scores (NSS) were assessed at days 0, 1, 3, and 7. Nissl staining, Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), immunofluorescent staining and western blotting were used to evaluate neuronal survival, apoptosis and necrosis, tight-junction proteins Claudin-1 and Zonula occludens-1 (ZO-1), vascular endothelial growth factor (VEGF), doublecortex (DCX), bradykinin receptor B1 (BDKRB1), BDKRB2 and Ki67 staining.Results: The combined treatment rescued all HIE rats from death and had a best survival curve compared to HIE. The Combination also reduced the NSS scores after HIE at days 7, better than HUK or MH alone. The combination of HUK and MH reserved more cells in Nissl staining and inhibited neuronal apoptosis and necrosis as well as significantly attenuated HIE-induced decreases in claudin-1, ZO-1, cyclin D1 and BDKRB1/B2 in comparison to HUK or MH treatment alone. Moreover, the combined treatment increased the expression of VEGF and DCX as well as the number of Ki67-labeled cells.Conclusions: This study demonstrates that both HUK and MH are neuroprotective after HIE insult; however, the combined therapy with HUK and MH enhanced the efficiency and efficacy of either therapy alone in the treatment of HIE, at least partially by promoting angiogenesis and regeneration and rescuing tight-junction loss. The combination of HUK and MH seems to be a feasible and promising clinical strategy to alleviate cerebral injury following HIE insult.Highlights: -The combination of HUK and MH distinctly reduces neurological dysfunction in HIE rats.-HUK enhances the neuroprotective effects of MH in HIE.-MH attenuates tight-junction disruption, upregulates the BDKR B1/2, DCX and cyclin D1.-The combination of MH and HUK enhances the expressions of MH/HUK mediated-BDKR B1/2, DCX, cyclin D1 and Ki67 positive cells.

Flaviviruses have evolved complex mechanisms to evade the mammalian host immune systems including the RIG-I (retinoic acid-inducible gene I) like receptor (RLR) signaling. Zika virus (ZIKV) is a re-emerging flavivirus that is associated with severe neonatal microcephaly and adult Guillain-Barre syndrome. However, the molecular mechanisms underlying ZIKV pathogenesis remain poorly defined. Here we report that ZIKV non-structural protein 4A (NS4A) impairs the RLR-mitochondrial antiviral-signaling protein (MAVS) interaction and subsequent induction of antiviral immune responses. In human trophoblasts, both RIG-I and melanoma differentiation-associated protein 5 (MDA5) contribute to type I interferon (IFN) induction and control ZIKV replication. Type I IFN induction by ZIKV is almost completely abolished in MAVS-/- cells. NS4A represses RLR-, but not Toll-like receptor-mediated immune responses. NS4A specifically binds the N-terminal caspase activation and recruitment domain (CARD) of MAVS and thus blocks its accessibility by RLRs. Our study provides in-depth understanding of the molecular mechanisms of immune evasion by ZIKV and its pathogenesis.

UBXN proteins likely participate in the global regulation of protein turnover, and we have shown that UBXN1 interferes with RIG-I-like receptor (RLR) signaling by interacting with MAVS and impeding its downstream effector functions. Here we demonstrate that over-expression of multiple UBXN family members decreased lentivirus and retrovirus production by several orders-of-magnitude in single cycle assays, at the level of long terminal repeat-driven transcription, and three family members, UBXN1, N9, and N11 blocked the canonical NFκB pathway by binding to Cullin1 (Cul1), inhibiting IκBα degradation. Multiple regions of UBXN1, including its UBA domain, were critical for its activity. Elimination of UBXN1 resulted in early murine embryonic lethality. shRNA-mediated knockdown of UBXN1 enhanced human immunodeficiency virus type 1 (HIV) production up to 10-fold in single cycle assays. In primary human fibroblasts, knockdown of UBXN1 caused prolonged degradation of IκBα and enhanced NFκB signaling, which was also observed after CRISPR-mediated knockout of UBXN1 in mouse embryo fibroblasts. Knockout of UBXN1 significantly up- and down-regulated hundreds of genes, notably those of several cell adhesion and immune signaling pathways. Reduction in UBXN1 gene expression in Jurkat T cells latently infected with HIV resulted in enhanced HIV gene expression, consistent with the role of UBXN1 in modulating the NFκB pathway. Based upon co-immunoprecipitation studies with host factors known to bind Cul1, models are presented as to how UBXN1 could be inhibiting Cul1 activity. The ability of UBXN1 and other family members to negatively regulate the NFκB pathway may be important for dampening the host immune response in disease processes and also re-activating quiescent HIV from latent viral reservoirs in chronically infected individuals.

Alphaviruses belong to a family of positive sense, single-stranded RNA viruses that are transmitted mainly by mosquitoes through a blood meal and cause arthritis and/or encephalitis in humans and animals [...]

Mosquito-borne diseases constitute a large portion of infectious diseases, causing more than 700,000 deaths annually. Mosquito-transmitted viruses, such as yellow fever, dengue, West Nile, chikungunya, and Zika viruses, have re-emerged recently and remain a public health threat worldwide. Global climate change, rapid urbanization, burgeoning international travel, expansion of mosquito populations, vector competence, and host and viral genetics may all together contribute to the re-emergence of arboviruses. In this brief review, we summarize the host and viral genetic determinants that may enhance infectivity in the host, viral fitness in mosquitoes and viral transmission by mosquitoes.

RNA viruses are sensed by RIG-I-like receptors (RLRs), which signal through a mitochondria-associated adaptor molecule, MAVS, resulting in systemic antiviral immune responses. Although RLR signaling is essential for limiting RNA virus replication, it must be stringently controlled to prevent damage from inflammation. We demonstrate here that among all tested UBX-domain-containing protein family members, UBXN1 exhibits the strongest inhibitory effect on RNA-virus-induced type I interferon response. UBXN1 potently inhibits RLR- and MAVS-induced, but not TLR3-, TLR4-, or DNA-virus-induced innate immune responses. Depletion of UBXN1 enhances virus-induced innate immune responses, including those resulting from RNA viruses such as vesicular stomatitis, Sendai, West Nile, and dengue virus infection, repressing viral replication. Following viral infection, UBXN1 is induced, binds to MAVS, interferes with intracellular MAVS oligomerization, and disrupts the MAVS/TRAF3/TRAF6 signalosome. These findings underscore a critical role of UBXN1 in the modulation of a major antiviral signaling pathway.

Mosquitoes are natural vectors for many etiologic agents of human viral diseases. Mosquito-borne flaviviruses can persistently infect the mosquito central nervous system without causing dramatic pathology or influencing the mosquito behavior and lifespan. The mechanism by which the mosquito nervous system resists flaviviral infection is still largely unknown. Here we report that an Aedes aegypti homologue of the neural factor Hikaru genki (AaHig) efficiently restricts flavivirus infection of the central nervous system. AaHig was predominantly expressed in the mosquito nervous system and localized to the plasma membrane of neural cells. Functional blockade of AaHig enhanced Dengue virus (DENV) and Japanese encephalitis virus (JEV), but not Sindbis virus (SINV), replication in mosquito heads and consequently caused neural apoptosis and a dramatic reduction in the mosquito lifespan. Consistently, delivery of recombinant AaHig to mosquitoes reduced viral infection. Furthermore, the membrane-localized AaHig directly interfaced with a highly conserved motif in the surface envelope proteins of DENV and JEV, and consequently interrupted endocytic viral entry into mosquito cells. Loss of either plasma membrane targeting or virion-binding ability rendered AaHig nonfunctional. Interestingly, Culex pipien pallens Hig also demonstrated a prominent anti-flavivirus activity, suggesting a functionally conserved function for Hig. Our results demonstrate that an evolutionarily conserved antiviral mechanism prevents lethal flaviviral infection of the central nervous system in mosquitoes, and thus may facilitate flaviviral transmission in nature.

Flaviviruses including Zika, Dengue and Hepatitis C virus cause debilitating diseases in humans, and the former are emerging as global health concerns with no antiviral treatments. We investigated Sophora Flavecens, used in Chinese medicine, as a source for antiviral compounds. We isolated Sophoraflavenone G and found that it inhibited Hepatitis C replication, but not Sendai or Vesicular Stomatitis Virus. Pre- and post-infection treatments demonstrated anti-flaviviral activity against Dengue and Zika virus, via viral RNA polymerase inhibition. These data suggest that Sophoraflavenone G represents a promising candidate regarding anti-Flaviviridae research.

The complement system functions during the early phase of infection and directly mediates pathogen elimination. The recent identification of complement-like factors in arthropods indicates that this system shares common ancestry in vertebrates and invertebrates as an immune defense mechanism. Thioester (TE)-containing proteins (TEPs), which show high similarity to mammalian complement C3, are thought to play a key role in innate immunity in arthropods. Herein, we report that a viral recognition cascade composed of two complement-related proteins limits the flaviviral infection of Aedes aegypti. An A. aegypti macroglobulin complement-related factor (AaMCR), belonging to the insect TEP family, is a crucial effector in opposing the flaviviral infection of A. aegypti. However, AaMCR does not directly interact with DENV, and its antiviral effect requires an A. aegypti homologue of scavenger receptor-C (AaSR-C), which interacts with DENV and AaMCR simultaneously in vitro and in vivo. Furthermore, recognition of DENV by the AaSR-C/AaMCR axis regulates the expression of antimicrobial peptides (AMPs), which exerts potent anti-DENV activity. Our results both demonstrate the existence of a viral recognition pathway that controls the flaviviral infection by inducing AMPs and offer insights into a previously unappreciated antiviral function of the complement-like system in arthropods.

C-type lectins are a family of proteins with carbohydrate-binding activity. Several C-type lectins in mammals or arthropods are employed as receptors or attachment factors to facilitate flavivirus invasion. We previously identified a C-type lectin in Aedes aegypti, designated as mosquito galactose specific C-type lectin-1 (mosGCTL-1), facilitating the attachment of West Nile virus (WNV) on the cell membrane. Here, we first identified that 9 A. aegypti mosGCTL genes were key susceptibility factors facilitating DENV-2 infection, of which mosGCTL-3 exhibited the most significant effect. We found that mosGCTL-3 was induced in mosquito tissues with DENV-2 infection, and that the protein interacted with DENV-2 surface envelop (E) protein and virions in vitro and in vivo. In addition, the other identified mosGCTLs interacted with the DENV-2 E protein, indicating that DENV may employ multiple mosGCTLs as ligands to promote the infection of vectors. The vectorial susceptibility factors that facilitate pathogen invasion may potentially be explored as a target to disrupt the acquisition of microbes from the vertebrate host. Indeed, membrane blood feeding of antisera against mosGCTLs dramatically reduced mosquito infective ratio. Hence, the immunization against mosGCTLs is a feasible approach for preventing dengue infection. Our study provides a future avenue for developing a transmission-blocking vaccine that interrupts the life cycle of dengue virus and reduces disease burden.

The Th17 cytokine, IL-22, regulates host immune responses to extracellular pathogens. Whether IL-22 plays a role in viral infection, however, is poorly understood. We report here that Il22(-/-) mice were more resistant to lethal West Nile virus (WNV) encephalitis, but had similar viral loads in the periphery compared to wild type (WT) mice. Viral loads, leukocyte infiltrates, proinflammatory cytokines and apoptotic cells in the central nervous system (CNS) of Il22(-/-) mice were also strikingly reduced. Further examination showed that Cxcr2, a chemokine receptor that plays a non-redundant role in mediating neutrophil migration, was significantly reduced in Il22(-/-) compared to WT leukocytes. Expression of Cxcr2 ligands, cxcl1 and cxcl5, was lower in Il22(-/-) brains than wild type mice. Correspondingly, neutrophil migration from the blood into the brain was attenuated following lethal WNV infection of Il22(-/-) mice. Our results suggest that IL-22 signaling exacerbates lethal WNV encephalitis likely by promoting WNV neuroinvasion.

West Nile (WNV), dengue (DENV) and yellow fever (YFV) viruses are (re)emerging, mosquito-borne flaviviruses that cause human disease and mortality worldwide. Alterations in mosquito gene expression common and unique to individual flaviviral infections are poorly understood. Here, we present a microarray analysis of the Aedes aegypti transcriptome over time during infection with DENV, WNV or YFV. We identified 203 mosquito genes that were ≥ 5-fold differentially up-regulated (DUR) and 202 genes that were ≥ 10-fold differentially down-regulated (DDR) during infection with one of the three flaviviruses. Comparative analysis revealed that the expression profile of 20 DUR genes and 15 DDR genes was quite similar between the three flaviviruses on D1 of infection, indicating a potentially conserved transcriptomic signature of flaviviral infection. Bioinformatics analysis revealed changes in expression of genes from diverse cellular processes, including ion binding, transport, metabolic processes and peptidase activity. We also demonstrate that virally-regulated gene expression is tissue-specific. The overexpression of several virally down-regulated genes decreased WNV infection in mosquito cells and Aedes aegypti mosquitoes. Among these, a pupal cuticle protein was shown to bind WNV envelope protein, leading to inhibition of infection in vitro and the prevention of lethal WNV encephalitis in mice. This work provides an extensive list of targets for controlling flaviviral infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection.

Mosquitoes transmit pathogens that cause infectious diseases of global importance. Techniques to easily introduce genes into mosquitoes, however, limit investigations of the interaction between microbes and their arthropod vectors. We now show that a cationic liposome significantly enhances delivery and expression of plasmid DNA in Aedes aegypti and Anopheles gambiae mosquitoes. We then introduced the genes for Ae. aegypti thioester-containing proteins (AeTEPs), which are involved in the control of flaviviral infection, into mosquitoes using this technique. In vivo transfection of AeTEP-1 into Ae. aegypti significantly reduced dengue virus infection, suggesting that the approach can further our understanding of pathogen-mosquito interactions.

Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Effective vaccines against most tick-borne pathogens are not currently available. In this study, we characterized a tick histamine release factor (tHRF) from Ixodes scapularis and addressed the vaccine potential of this antigen in the context of tick engorgement and B. burgdorferi transmission. Results from western blotting and quantitative Reverse Transcription-PCR showed that tHRF is secreted in tick saliva, and upregulated in Borrelia burgdorferi-infected ticks. Further, the expression of tHRF was coincident with the rapid feeding phase of the tick, suggesting a role for tHRF in tick engorgement and concomitantly, for efficient B. burgdorferi transmission. Silencing tHRF by RNA interference (RNAi) significantly impaired tick feeding and decreased B. burgdorferi burden in mice. Interfering with tHRF by actively immunizing mice with recombinant tHRF, or passively transferring tHRF antiserum, also markedly reduced the efficiency of tick feeding and B. burgdorferi burden in mice. Recombinant tHRF was able to bind to host basophils and stimulate histamine release. Therefore, we speculate that tHRF might function in vivo to modulate vascular permeability and increase blood flow to the tick bite-site, facilitating tick engorgement. These findings suggest that blocking tHRF might offer a viable strategy to complement ongoing efforts to develop vaccines to block tick feeding and transmission of tick-borne pathogens.

West Nile virus (WNV), a mosquito-borne single-stranded RNA flavivirus, can cause significant human morbidity and mortality. Our data show that interleukin-10 (IL-10) is dramatically elevated both in vitro and in vivo following WNV infection. Consistent with an etiologic role of IL-10 in WNV pathogenesis, we find that WNV infection is markedly diminished in IL-10 deficient (IL-10(-/-)) mice, and pharmacologic blockade of IL-10 signaling by IL-10 neutralizing antibody increases survival of WNV-infected mice. Increased production of antiviral cytokines in IL-10(-/-) mice is associated with more efficient control of WNV infection. Moreover, CD4(+) T cells produce copious amounts of IL-10, and may be an important cellular source of IL-10 during WNV infection in vivo. In conclusion, IL-10 signaling plays a negative role in immunity against WNV infection, and blockade of IL-10 signaling by genetic or pharmacologic means helps to control viral infection, suggesting a novel anti-WNV therapeutic strategy.

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1beta mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.

Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission. [ABSTRACT FROM AUTHOR]

Pathogen strain diversity is an important driver of the trajectory of epidemics. The role of bioclimatic factors on the spatial distribution of dengue virus (DENV) serotypes has, however, not been previously studied. Hence, we developed municipality-scale environmental suitability maps for the four dengue virus serotypes using maximum entropy modeling. We fit climatic variables to municipality presence records from 2012 to 2020 in Mexico. Bioclimatic variables were explored for their environmental suitability to different DENV serotypes, and the different distributions were visualized using three cutoff probabilities representing 90%, 95%, and 99% sensitivity. Municipality-level results were then mapped in ArcGIS. The overall accuracy for the predictive models was 0.69, 0.68, 0.75, and 0.72 for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. Important predictors of all DENV serotypes were the growing degree days for December, January, and February, which are an indicator of higher temperatures and the precipitation of the wettest month. The minimum temperature of the coldest month between -5°C and 20°C was found to be suitable for DENV-1 and DENV-2 serotypes. Respectively, above 700-900mm of rainfall, the suitability for DENV-1 and DENV-2 begins to decline, while higher humidity still favors DENV-3 and DENV-4. The sensitivity concerning the suitability map was developed for Mexico. DENV-1, DENV-2, DENV-3, and DENV-4 serotypes will be found more commonly in the municipalities classified as suitable based on their respective sensitivity of 91%, 90%, 89%, and 85% in Mexico. As the microclimates continue to change, specific bioclimatic indices may be used to monitor potential changes in DENV serotype distribution. The suitability for DENV-1 and DENV-2 is expected to increase in areas with lower minimum temperature ranges, while DENV-3 and DENV-4 will likely increase in areas that experience higher humidity. Ongoing surveillance of municipalities with predicted suitability of 89% and 85% should be expanded to account for the accurate DENV serotype prevalence and association between bioclimatic parameters. [ABSTRACT FROM AUTHOR]