An evolutionarily conserved ubiquitin ligase drives infection and transmission of flaviviruses.

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.

A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

A mosquito salivary protein-driven influx of myeloid cells facilitates flavivirus transmission.

Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein (AaNRP), facilitates the transmission of Zika and dengue viruses. AaNRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, AaNRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces AaNRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.

UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors.

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.

A Bayesian spatiotemporal approach to modelling arboviral diseases in Mexico.

BACKGROUND: The objective of this study was to evaluate the spatial and temporal patterns of disease prevalence clusters of dengue (DENV), chikungunya (CHIKV) and Zika (ZIKV) virus and how socio-economic and climatic variables simultaneously influence the risk and rate of occurrence of infection in Mexico. METHODS: To determine the spatiotemporal clustering and the effect of climatic and socio-economic covariates on the rate of occurrence of disease and risk in Mexico, we applied correlation methods, seasonal and trend decomposition using locally estimated scatterplot smoothing, hotspot analysis and conditional autoregressive Bayesian models. RESULTS: We found cases of the disease are decreasing and a significant association between DENV, CHIKV and ZIKV cases and climatic and socio-economic variables. An increment of cases was identified in the northeastern, central west and southeastern regions of Mexico. Climatic and socio-economic covariates were significantly associated with the rate of occurrence and risk of the three arboviral disease cases. CONCLUSION: The association of climatic and socio-economic factors is predominant in the northeastern, central west and southeastern regions of Mexico. DENV, CHIKV and ZIKV cases showed an increased risk in several states in these regions and need urgent attention to allocate public health resources to the most vulnerable regions in Mexico.

Baseline Gut Microbiome Signatures Correlate with Immunogenicity of SARS-CoV-2 mRNA Vaccines.

The powerful immune responses elicited by the mRNA vaccines targeting the SARS-CoV-2 Spike protein contribute to their high efficacy. Yet, their efficacy can vary greatly between individuals. For vaccines not based on mRNA, cumulative evidence suggests that differences in the composition of the gut microbiome, which impact vaccine immunogenicity, are some of the factors that contribute to variations in efficacy. However, it is unclear if the microbiome impacts the novel mode of immunogenicity of the SARS-CoV-2 mRNA vaccines. We conducted a prospective longitudinal cohort study of individuals receiving SARS-CoV-2 mRNA vaccines where we measured levels of anti-Spike IgG and characterized microbiome composition, at pre-vaccination (baseline), and one week following the first and second immunizations. While we found that microbial diversity at all timepoints correlated with final IgG levels, only at baseline did microbial composition and predicted function correlate with vaccine immunogenicity. Specifically, the phylum Desulfobacterota and genus Bilophila, producers of immunostimulatory LPS, positively correlated with IgG, while Bacteroides was negatively correlated. KEGG predicted pathways relating to SCFA metabolism and sulfur metabolism, as well as structural components such as flagellin and capsular polysaccharides, also positively correlated with IgG levels. Consistent with these findings, depleting the microbiome with antibiotics reduced the immunogenicity of the BNT162b2 vaccine in mice. These findings suggest that gut microbiome composition impacts the immunogenicity of the SARS-CoV-2 mRNA vaccines.

An mRNA-based T-cell-inducing antigen strengthens COVID-19 vaccine against SARS-CoV-2 variants.

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-RBDbeta. 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.

Jumonji domain-containing protein-3 (JMJD3) promotes myeloid fibroblast activation and macrophage polarization in kidney fibrosis.

BACKGROUND AND PURPOSE: Renal fibrosis is a common feature of chronic kidney disease. Myeloid fibroblasts and macrophages contribute significantly to the pathogenesis of renal fibrosis. However, the molecular mechanisms underlying myeloid fibroblast activation and macrophage polarization are not fully understood. In this study, we examined the role of Jumonji domain-containing protein-3 (JMJD3) in myeloid fibroblast activation, macrophage polarization, and renal fibrosis development in a preclinical model of obstructive nephropathy. EXPERIMENTAL APPROACH: To examine the role of JMJD3 in renal fibrosis, we generated mice with global or myeloid cell-specific deletion of JMJD3, and we treated wild-type mice with vehicle or GSK-J4 (selective JMJD3 inhibitor). Mice were subjected to unilateral ureteral obstructive injury to induce renal fibrosis. KEY RESULTS: JMJD3 expression was significantly increased in the kidneys during the development of renal fibrosis, which was associated with an increase in H3K27 dimethylation. Mice with either global or myeloid JMJD3 deficiency exhibited significantly reduced total collagen deposition and extracellular matrix protein production, myeloid fibroblast activation and M2 macrophage polarization in the obstructed kidney. Moreover, IFN regulatory factor 4, a mediator of M2 macrophage polarization, was significantly induced in the obstructed kidneys, which was abolished by JMJD3 deficiency. Furthermore, pharmacological inhibition of JMJD3 with GSK-J4 attenuated kidney fibrosis, reduced myeloid fibroblast activation and suppressed M2 macrophage polarization in the obstructed kidney. CONCLUSION AND IMPLICATIONS: Our study identifies JMJD3 as a critical regulator of myeloid fibroblast activation, macrophage polarization, and renal fibrosis development. Therefore, JMJD3 may represent a promising therapeutic target for chronic kidney disease.

Autophagy ameliorates Pseudomonas aeruginosa-infected diabetic wounds by regulating the toll-like receptor 4/myeloid differentiation factor 88 pathway.

Diabetic foot ulcers (DFUs) are among the most common complications in patients with diabetes and a leading cause of lower extremity amputation. DFUs are exacerbated by prolonged bacterial infection; therefore, there is an urgent need for effective treatments to alleviate the burden associated with this condition. Although autophagy plays a unique role in pathogen phagocytosis and inflammation, its role in diabetic foot infections (DFIs) remains unclear. Pseudomonas aeruginosa (PA) is the most frequently isolated gram-negative bacterium from DFUs. Here, we evaluated the role of autophagy in ameliorating PA infection in wounds in a diabetic rat model and a bone marrow-derived macrophage (BMDM) hyperglycemia model. Both models were pretreated with or without rapamycin (RAPA) and then infected with or without PA. Pretreatment of rats with RAPA significantly enhanced PA phagocytosis, suppressed wound inflammation, reduced the M1:M2 macrophage ratio, and improved wound healing. In vitro investigation of the underlying mechanisms revealed that enhanced autophagy resulted in decreased macrophage secretion of inflammatory factors such as TNF-α, IL-6, and IL-1ß but increased that of IL-10 in response to PA infection. Additionally, RAPA treatment significantly enhanced autophagy in macrophages by increasing LC3 and beclin-1 levels, which led to altered macrophage function. Furthermore, RAPA blocked the PA-induced TLR4/MyD88 pathway to regulate macrophage polarisation and inflammatory cytokine production, which was validated by RNA interference and use of the autophagy inhibitor 3-methyladenine (3-MA). These findings suggest enhancing autophagy as a novel therapeutic strategy against PA infection to ultimately improve diabetic wound healing.

Quantitative Analysis of B-Cell Subpopulations in Bone Marrow by Flow Cytometry.

Flow cytometry is a technology that rapidly detects and measures physical and chemical characteristics of single cells or particles. It is a powerful tool for many areas of research, in particular, immunology, which allows for simultaneous analysis of different immune cell populations in a tissue. Here we describe the procedures to quantify and/or purify various B fractions in mouse bone marrows by flow cytometry using their signature surface markers. This method is useful to study B-cell development during steady-state or emergency hematopoiesis such as viral infections.

UBXN3B Controls Immunopathogenesis of Arthritogenic Alphaviruses by Maintaining Hematopoietic Homeostasis.

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.

Neighboring mutation-mediated enhancement of dengue virus infectivity and spread.

Frequent turnover of dengue virus (DENV) clades is one of the major forces driving DENV persistence and prevalence. In this study, we assess the fitness advantage of nine stable substitutions within the envelope (E) protein of DENV serotypes. Two tandem neighboring substitutions, threonine to lysine at the 226th (T226K) and glycine to glutamic acid at the 228th (G228E) residues in the DENV2 Asian I genotype, enhance virus infectivity in either mosquitoes or mammalian hosts, thereby promoting clades turnover and dengue epidemics. Mechanistic studies indicate that the substitution-mediated polarity changes in these two residues increase the binding affinity of E for host C-type lectins. Accordingly, we predict that a G228E substitution could potentially result in a forthcoming epidemic of the DENV2 Cosmopolitan genotype. Investigations into the substitutions associated with DENV fitness in hosts may offer mechanistic insights into dengue prevalence, thus providing a warning of potential epidemics in the future.

A Retinol Derivative Inhibits SARS-CoV-2 Infection by Interrupting Spike-Mediated Cellular Entry.

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.

A volatile from the skin microbiota of flavivirus-infected hosts promotes mosquito attractiveness.

The host-seeking activity of hematophagous arthropods is essential for arboviral transmission. Here, we demonstrate that mosquito-transmitted flaviviruses can manipulate host skin microbiota to produce a scent that attracts mosquitoes. We observed that Aedes mosquitoes preferred to seek and feed on mice infected by dengue and Zika viruses. Acetophenone, a volatile compound that is predominantly produced by the skin microbiota, was enriched in the volatiles from the infected hosts to potently stimulate mosquito olfaction for attractiveness. Of note, acetophenone emission was higher in dengue patients than in healthy people. Mechanistically, flaviviruses infection suppressed the expression of RELMα, an essential antimicrobial protein on host skin, thereby leading to the expansion of acetophenone-producing commensal bacteria and, consequently, a high acetophenone level. Given that RELMα can be specifically induced by a vitamin A derivative, the dietary administration of isotretinoin to flavivirus-infected animals interrupted flavivirus life cycle by reducing mosquito host-seeking activity, thus providing a strategy of arboviral control.

Lipases secreted by a gut bacterium inhibit arbovirus transmission in mosquitoes.

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.

A glucose-like metabolite deficient in diabetes inhibits cellular entry of SARS-CoV-2.

The severity and mortality of COVID-19 are associated with pre-existing medical comorbidities such as diabetes mellitus. However, the underlying causes for increased susceptibility to viral infection in patients with diabetes is not fully understood. Here we identify several small-molecule metabolites from human blood with effective antiviral activity against SARS-CoV-2, one of which, 1,5-anhydro-D-glucitol (1,5-AG), is associated with diabetes mellitus. The serum 1,5-AG level is significantly lower in patients with diabetes. In vitro, the level of SARS-CoV-2 replication is higher in the presence of serum from patients with diabetes than from healthy individuals and this is counteracted by supplementation of 1,5-AG to the serum from patients. Diabetic (db/db) mice undergo SARS-CoV-2 infection accompanied by much higher viral loads and more severe respiratory tissue damage when compared to wild-type mice. Sustained supplementation of 1,5-AG in diabetic mice reduces SARS-CoV-2 loads and disease severity to similar levels in nondiabetic mice. Mechanistically, 1,5-AG directly binds the S2 subunit of the SARS-CoV-2 spike protein, thereby interrupting spike-mediated virus-host membrane fusion. Our results reveal a mechanism that contributes to COVID-19 pathogenesis in the diabetic population and suggest that 1,5-AG supplementation may be beneficial to diabetic patients against severe COVID-19.

Intrinsic cardiac adrenergic cells contribute to LPS-induced myocardial dysfunction.

Intrinsic cardiac adrenergic (ICA) cells regulate both developing and adult cardiac physiological and pathological processes. However, the role of ICA cells in septic cardiomyopathy is unknown. Here we show that norepinephrine (NE) secretion from ICA cells is increased through activation of Toll-like receptor 4 (TLR4) to aggravate myocardial TNF-α production and dysfunction by lipopolysaccharide (LPS). In ICA cells, LPS activated TLR4-MyD88/TRIF-AP-1 signaling that promoted NE biosynthesis through expression of tyrosine hydroxylase, but did not trigger TNF-α production due to impairment of p65 translocation. In a co-culture consisting of LPS-treated ICA cells and cardiomyocytes, the upregulation and secretion of NE from ICA cells activated cardiomyocyte ß1-adrenergic receptor driving Ca2+/calmodulin-dependent protein kinase II (CaMKII) to crosstalk with NF-κB and mitogen-activated protein kinase pathways. Importantly, blockade of ICA cell-derived NE prevented LPS-induced myocardial dysfunction. Our findings suggest that ICA cells may be a potential therapeutic target for septic cardiomyopathy.

The noncanonical inflammasome in health and disease.

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.