ABSTRACT
Protein aggregation caused by the disruption of proteostasis will lead to cellular cytotoxicity and even cell death, which is implicated in multiple neurodegenerative diseases. The elimination of aggregated proteins is mediated by selective macroautophagy receptors, which is termed aggrephagy. However, the identity and redundancy of aggrephagy receptors in recognizing substrates remain largely unexplored. Here, we find that CCDC50, a highly expressed autophagy receptor in brain, is recruited to proteotoxic stresses-induced polyubiquitinated protein aggregates and ectopically expressed aggregation-prone proteins. CCDC50 recognizes and further clears these cytotoxic aggregates through autophagy. The ectopic expression of CCDC50 increases the tolerance to stress-induced proteotoxicity and hence improved cell survival in neuron cells, whereas CCDC50 deficiency caused accumulation of lipid deposits and polyubiquitinated protein conjugates in the brain of one-year-old mice. Our study illustrates how aggrephagy receptor CCDC50 combats proteotoxic stress for the benefit of neuronal cell survival, thus suggesting a protective role in neurotoxic proteinopathy.Abbreviations: AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; ATG5: autophagy related 5; BODIPY: boron-dipyrromethene; CASP3: caspase 3; CCDC50: coiled-coil domain containing 50; CCT2: chaperonin containing TCP1 subunit 2; CHX: cycloheximide; CQ: chloroquine; CRISPR: clustered regulatory interspaced short palindromic repeat; Cas9: CRISPR-associated system 9; DAPI: 4',6-diamidino-2-phenylindole; FK2: Anti-ubiquitinylated proteins antibody, clone FK2; FUS: FUS RNA binding protein; GFP: green fluorescent protein; HD: Huntington disease; HTT: huntingtin; KEGG: Kyoto Encyclopedia of Genes and Genomes; LDS: LIR-docking site; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPT/tau: microtubule associated protein tau; MIU: motif interacting with ubiquitin; NBR1: NBR1, autophagy cargo receptor; OPTN: optineurin; PD: Parkinson disease; PI: propidium iodide; ROS: reactive oxygen species; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; Ub: ubiquitin; UDS: UIM-docking site; UIM: ubiquitin interacting motif; UPS: ubiquitin-proteasome system.
ABSTRACT
The ongoing COVID-19 pandemic still threatens human health around the world. The methyltransferases (MTases) of SARS-CoV-2, specifically nsp14 and nsp16, play crucial roles in the methylation of the N7 and 2'-O positions of viral RNA, making them promising targets for the development of antiviral drugs. In this work, we performed structure-based virtual screening for nsp14 and nsp16 using the screening workflow (HTVS, SP, XP) of Schrödinger 2019 software, and we carried out biochemical assays and molecular dynamics simulation for the identification of potential MTase inhibitors. For nsp14, we screened 239,000 molecules, leading to the identification of three hits A1-A3 showing N7-MTase inhibition rates greater than 60% under a concentration of 50 µM. For the SAM binding and nsp10-16 interface sites of nsp16, the screening of 210,000 and 237,000 molecules, respectively, from ZINC15 led to the discovery of three hit compounds B1-B3 exhibiting more than 45% of 2'-O-MTase inhibition under 50 µM. These six compounds with moderate MTase inhibitory activities could be used as novel candidates for the further development of anti-SARS-CoV-2 drugs.
Subject(s)
Antiviral Agents , Enzyme Inhibitors , Methyltransferases , Molecular Dynamics Simulation , SARS-CoV-2 , Viral Nonstructural Proteins , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Viral Nonstructural Proteins/chemistry , Methyltransferases/antagonists & inhibitors , Methyltransferases/metabolism , Methyltransferases/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Humans , Molecular Docking Simulation , Drug Evaluation, Preclinical , COVID-19 Drug Treatment , COVID-19/virology , Binding Sites , ExoribonucleasesABSTRACT
Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Here, using a meta-transcriptomic approach, we determined the viromes of 2,438 individual mosquitoes (81 species), spanning ~4,000 km along latitudes and longitudes in China. From these data we identified 393 viral species associated with mosquitoes, including 7 (putative) species of arthropod-borne viruses (that is, arboviruses). We identified potential mosquito species and geographic hotspots of viral diversity and arbovirus occurrence, and demonstrated that the composition of individual mosquito viromes was strongly associated with host phylogeny. Our data revealed a large number of viruses shared among mosquito species or genera, enhancing our understanding of the host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, perhaps reflecting long-distance mosquito dispersal. Together, these results greatly expand the known mosquito virome, linked viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the biogeography and diversity of viruses in insect vectors.
Subject(s)
Culicidae , Mosquito Vectors , Virome , Animals , Culicidae/virology , China , Mosquito Vectors/virology , Metagenomics , Arboviruses/genetics , Arboviruses/classification , Phylogeny , BiodiversityABSTRACT
Cap RNA methylations play important roles in the replication, evasion of host RNA sensor recognition, and pathogenesis. Coronaviruses possess both guanine N7- and 2'-O-ribose methyltransferases (N7-MTase and 2'-O-MTase) encoded by nonstructural protein (nsp) 14 and nsp16/10 complex, respectively. In this study, we reconstituted the two-step RNA methylations of N7-MTase and 2'-O-MTase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and demonstrated its common and different features in comparison with that of SARS-CoV. We revealed that the nsp16/10 2'-O-MTase of SARS-CoV-2 has a broader substrate selectivity than the counterpart of SARS-CoV and can accommodate both unmethylated and uncapped RNA substrates in a sequence-independent manner. Most intriguingly, the substrate selectivity of nsp16/10 complex is not determined by the apoenzyme of nsp16 MTase but by its cofactor nsp10. These results provide insight into the unique features of SARS-CoV-2 MTases and may help develop strategies to precisely intervene in the methylation pathway and pathogenesis of SARS-CoV-2.
Subject(s)
COVID-19 , Methyltransferases , Humans , Methyltransferases/genetics , SARS-CoV-2/genetics , RNA Methylation , RNA CapsSubject(s)
COVID-19 , Humans , SARS-CoV-2 , Cyclophilin A/genetics , Protein Binding , Virion/geneticsABSTRACT
Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Using a meta-transcriptomic approach, we analysed the virome of 2,438 individual mosquitos (79 species), spanning ~4000 km along latitudes and longitudes in China. From these data we identified 393 core viral species associated with mosquitos, including seven (putative) arbovirus species. We identified potential species and geographic hotspots of viral richness and arbovirus occurrence, and demonstrated that host phylogeny had a strong impact on the composition of individual mosquito viromes. Our data revealed a large number of viruses shared among mosquito species or genera, expanding our knowledge of host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, possibly facilitated by long-distance mosquito migrations. Together, our results greatly expand the known mosquito virome, linked the viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the ecology of viruses of insect vectors.
ABSTRACT
The maintenance of lysosome homeostasis is crucial for cell growth. Lysosome-dependent degradation and metabolism sustain tumor cell survival. Here, we demonstrate that CCDC50 serves as a lysophagy receptor, promoting tumor progression and invasion by controlling lysosomal integrity and renewal. CCDC50 monitors lysosomal damage, recognizes galectin-3 and K63-linked polyubiquitination on damaged lysosomes, and specifically targets them for autophagy-dependent degradation. CCDC50 deficiency causes the accumulation of ruptured lysosomes, impaired autophagic flux, and superfluous reactive oxygen species, consequently leading to cell death and tumor suppression. CCDC50 expression is associated with malignancy, progression to metastasis, and poor overall survival in human melanoma. Targeting CCDC50 suppresses tumor growth and lung metastasis, and enhances the effect of BRAFV600E inhibition. Thus, we demonstrate critical roles of CCDC50-mediated clearance of damaged lysosomes in supporting tumor growth, hereby identifying a potential therapeutic target of melanoma.
ABSTRACT
Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within individual bats, and hence the frequency of virus co-infection and spillover among them. We characterize the mammal-associated viruses in 149 individual bats sampled from Yunnan province, China, using an unbiased meta-transcriptomics approach. This reveals a high frequency of virus co-infection (simultaneous infection of bat individuals by multiple viral species) and spillover among the animals studied, which may in turn facilitate virus recombination and reassortment. Of note, we identify five viral species that are likely to be pathogenic to humans or livestock, based on phylogenetic relatedness to known pathogens or in vitro receptor binding assays. This includes a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV and SARS-CoV-2. In vitro assays indicate that this recombinant virus can utilize the human ACE2 receptor such that it is likely to be of increased emergence risk. Our study highlights the common occurrence of co-infection and spillover of bat viruses and their implications for virus emergence.
Subject(s)
COVID-19 , Chiroptera , Coinfection , Severe acute respiratory syndrome-related coronavirus , Animals , Humans , Phylogeny , SARS-CoV-2 , Virome , China/epidemiology , Severe acute respiratory syndrome-related coronavirus/geneticsABSTRACT
The protein activator of protein kinase R (PKR) (PACT) has been shown to play a crucial role in stimulating the host antiviral response through the activation of PKR, retinoic acid-inducible gene I, and melanoma differentiation-associated protein 5. Whether PACT can inhibit viral replication independent of known mechanisms is still unrevealed. In this study, we show that, like many viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks GSK-3ß to facilitate its replication. GSK-3ß-induced phosphorylation on N protein increased the interaction between N protein and nsp3. Thus, GSK-3ß-N-nsp3 cascade promotes viral replication. Although SARS-CoV-2 can sabotage the activation of AKT, the upstream proteins suppressing the activation of GSK-3ß, we found that the host can use PACT, another protein kinase, instead of AKT to decrease the activity of GSK-3ß and the interaction between PACT and GSK-3ß is enhanced upon viral infection. Moreover, PACT inhibited the activity of GSK-3ß independent of its well-studied double-stranded RNA binding and PKR activating ability. In summary, this study identified an unknown function of PACT in inhibiting SARS-CoV-2 replication through the blockage of GSK-3ß-N-nsp3 cascade.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , SARS-CoV-2/metabolism , Cell Line , Proto-Oncogene Proteins c-akt/metabolism , PhosphorylationABSTRACT
Viral infection-induced cell death has long been considered as a double-edged sword in the inhibition or exacerbation of viral infections. Patients with severe Coronavirus Disease 2019 (COVID-19) are characterized by multiple organ dysfunction syndrome and cytokine storm, which may result from SARS-CoV-2-induced cell death. Previous studies have observed enhanced ROS level and signs of ferroptosis in SARS-CoV-2 infected cells or specimens of patients with COVID-19, but the exact mechanism is not clear yet. Here, we find SARS-CoV-2 ORF3a sensitizes cells to ferroptosis via Keap1-NRF2 axis. SARS-CoV-2 ORF3a promotes the degradation of NRF2 through recruiting Keap1, thereby attenuating cellular resistance to oxidative stress and facilitated cells to ferroptotic cell death. Our study uncovers that SARS-CoV-2 ORF3a functions as a positive regulator of ferroptosis, which might explain SARS-CoV-2-induced damage in multiple organs in COVID-19 patients and imply the potential of ferroptosis inhibition in COVID-19 treatment.
Subject(s)
COVID-19 , Ferroptosis , Humans , SARS-CoV-2 , Kelch-Like ECH-Associated Protein 1 , NF-E2-Related Factor 2/genetics , COVID-19 Drug TreatmentABSTRACT
S-217622 (Ensitrelvir) is a reversible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3-chymotrypsin-like protease (3CLpro) inhibitor which obtained emergency regulatory approval in Japan for the treatment of SARS-CoV-2 infection on Nov 22, 2022. Herein, analogs of S-271622 with deuterium-for-hydrogen replacement were synthesized for comparison of the antiviral activities and pharmacokinetic (PK) profiles. Compared to the parent compound, C11-d2-S-217622 compound YY-278 retained in vitro activity against 3CLpro and SARS-CoV-2. X-ray crystal structural studies showed similar interactions of SARS-CoV-2 3CLpro with YY-278 and S-271622. The PK profiling revealed the relatively favorable bioavailability and plasma exposure of YY-278. In addition, YY-278, as well as S-217622, displayed broadly anti-coronaviral activities against 6 other coronaviruses that infect humans and animals. These results laid the foundation for further research on the therapeutic potential of YY-278 against COVID-19 and other coronaviral diseases.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Humans , Antiviral Agents/therapeutic use , Japan , Protease Inhibitors/chemistryABSTRACT
5-Methylcytosine (m5C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m5C methyltransferase, negatively regulates type I interferon responses during various viral infections, including SARS-CoV-2. NSUN2 specifically mediates m5C methylation of IRF3 mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-ß production. Knockout or knockdown of NSUN2 enhanced type I interferon and downstream ISGs during various viral infection in vitro. And in vivo, the antiviral innate response is more dramatically enhanced in Nsun2+/- mice than in Nsun2+/+ mice. The highly m5C methylated cytosines in IRF3 mRNA were identified, and their mutation enhanced cellular IRF3 mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), or Zika virus (ZIKV) resulted in a reduction of endogenous NSUN2 levels. Especially, SARS-CoV-2 infection (WT strain and BA.1 omicron variant) also decreased endogenous levels of NSUN2 in COVID-19 patients and K18-hACE2 KI mice, further increasing type I interferon and downstream ISGs. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of IRF3 mRNA, while endogenous NSUN2 levels decrease during SARS-CoV-2 and various viral infections to boost antiviral responses for effective elimination of viruses.
Subject(s)
COVID-19 , Interferon Type I , Virus Diseases , Zika Virus Infection , Zika Virus , Animals , Mice , Interferon Type I/genetics , Interferon Type I/metabolism , Interferon-beta/genetics , Interferon-beta/metabolism , Methylation , Zika Virus/metabolism , Mice, Knockout , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Antiviral Agents , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolismSubject(s)
COVID-19 , Prodrugs , Humans , Nucleosides , SARS-CoV-2 , Prodrugs/pharmacology , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic useABSTRACT
The assembly of the NLRP3 inflammasome can be initiated by a wide range of stimuli including exogenous infection as well as endogenous damage. Therefore, the tight regulation of the NLRP3 inflammasome is crucial for the host to resist microbial invasion and maintain homeostasis. Our recent work has identified a negative regulator of NLRP3-mediated inflammation, namely CCDC50 (coiled-coil domain containing protein 50). CCDC50 can be induced by NLRP3 agonists and then functions as a macroautophagy/autophagy cargo receptor to recognize K63-polyubiquitinated NLRP3 and deliver it to MAP1LC3/LC3-conjugated phagophores for degradation. CCDC50 inhibits the polymerization of NLRP3 and the recruitment of PYCARD/ASC, consequently suppressing the assembly of inflammasomes. ccdc50-knockout mice are more susceptible to dextran-sulfate (DSS)-induced colitis and exhibit more severe gut inflammation with elevated NLRP3 inflammasome activity, suggesting a protective role of CCDC50 in the pathology and progression of inflammatory bowel disease (IBD). Our finding reveals a function of autophagy-related proteins in the regulation of NLRP3-mediated inflammation, thus demonstrating the intricate crosstalk between autophagy and inflammation.
Subject(s)
Colitis , Inflammasomes , Intracellular Signaling Peptides and Proteins , NLR Family, Pyrin Domain-Containing 3 Protein , Animals , Mice , Autophagy , Colitis/chemically induced , Colitis/metabolism , Inflammasomes/metabolism , Inflammation/metabolism , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Intracellular Signaling Peptides and Proteins/metabolismABSTRACT
Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma. However, the function and mechanism of the effect of HBV on host protein ubiquitination remain largely unknown. We aimed at characterizing whether and how HBV promotes self-replication by affecting host protein ubiquitination. In this study, we identified UBXN7, a novel inhibitor for nuclear factor kappa B (NF-κB) signaling, was degraded via interaction with HBV X protein (HBx) to activate NF-κB signaling and autophagy, thereby affecting HBV replication. The expression of UBXN7 was analyzed by Western blot and quantitative reverse transcription polymerase chain reaction in HBV-transfected hepatoma cells and HBV-infected primary human hepatocytes (PHHs). The effects of UBXN7 on HBV replication were analyzed by using in vitro and in vivo assays, including stable isotope labeling by amino acids in cell culture (SILAC) analysis. Changes in HBV replication and the associated molecular mechanisms were analyzed in hepatoma cell lines. SILAC analyses showed that the ubiquitination of UBXN7 was significantly increased in HepG2.2.15 cells compared with control cells. After HBV infection, HBx protein interacted with UBXN7 to promote K48-linked ubiquitination of UBXN7 at K99, leading to UBXN7 degradation. On the other hand, UBXN7 interacted with the ULK domain of IκB kinase ß through its ubiquitin-associating domain to facilitate its degradation. This in turn reduced NF-κB signaling, leading to reduced autophagy and consequently decreased HBV replication.
Subject(s)
Hepatitis B virus , Trans-Activators , Viral Regulatory and Accessory Proteins , Virus Replication , Humans , Hepatitis B virus/physiology , Hepatitis B, Chronic , NF-kappa B/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Trans-Activators/metabolism , Adaptor Proteins, Signal Transducing/metabolismABSTRACT
Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.
ABSTRACT
Zika virus (ZIKV) targets the neural progenitor cells (NPCs) in brain during intrauterine infections and consequently causes severe neurological disorders, such as microcephaly in neonates. Although replicating in the cytoplasm, ZIKV dysregulates the expression of thousands of host genes, yet the detailed mechanism remains elusive. Herein, we report that ZIKV encodes a unique DNA-binding protein to regulate host gene transcription in the nucleus. We found that ZIKV NS5, the viral RNA polymerase, associates tightly with host chromatin DNA through its methyltransferase domain and this interaction could be specifically blocked by GTP. Further study showed that expression of ZIKV NS5 in human NPCs markedly suppressed the transcription of its target genes, especially the genes involved in neurogenesis. Mechanistically, ZIKV NS5 binds onto the gene body of its target genes and then blocks their transcriptional elongation. The utero electroporation in pregnant mice showed that NS5 expression significantly disrupts the neurogenesis by reducing the number of Sox2- and Tbr2-positive cells in the fetal cortex. Together, our findings demonstrate a molecular clue linking to the abnormal neurodevelopment caused by ZIKV infection and also provide intriguing insights into the interaction between the host cell and the pathogenic RNA virus, where the cytoplasmic RNA virus encodes a DNA-binding protein to control the transcription of host cell in the nuclei.
Subject(s)
Zika Virus Infection , Zika Virus , Humans , Female , Pregnancy , Animals , Mice , Chromatin/genetics , Zika Virus/genetics , Zika Virus Infection/genetics , DNA , DNA-Directed RNA Polymerases/genetics , Transcription, GeneticABSTRACT
Promyelocytic leukemia protein (PML) bodies are implicated in one of the key pathways in the establishment of antiviral status in response to interferon (IFN), yet the molecular mechanisms bridging the cross talk remain elusive. Herein, we report that a major constitutive component of the PML body, Sp100A, is ubiquitously located in the cytosol of various cell types and is an immediate responder to multiple extracellular stimuli, including virus infection, IFN, epidermal growth factor (EGF), glial cell-derived nerve factor (GDNF), etc., signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. IFN-ß induces phosphorylation of Sp100A on Ser188, which fortifies the binding of Sp100A to pyruvate kinase 2 (PKM2) and facilitates its nuclear importation through the extracellular signal-regulated kinase 1/2 (ERK1/2)-PKM2-PIN1-importin axes. Blocking PI3K pathway signaling or interference with the ERK1/2-PKM2-PIN1-importin axes independently hampers nuclear translocation of Sp100A in response to IFN, reflecting a dual-regulation mechanism governing this event. In the nucleus, Sp100A is enriched in the promoter regions of essential antiviral interferon-stimulated genes (ISGs), such as those coding for IFI16, OAS2, and RIG-I, and activates their transcription. Importantly, nuclear importation of Sp100A, but not accumulation of a mutant Sp100A that failed to respond to IFN, during infection potently enhanced transcription of these antiviral ISGs and restricted virus propagation. These findings depict a novel IFN response mechanism by PML bodies in the cytosol and shed light on the complex sensing-regulatory network of PML bodies. IMPORTANCE PML bodies sit at the center stage of various important biological processes; however, the signal transduction networks of these macromolecular protein complexes remain enigmatic. The present study illustrates, in detail and for the first time, the course of signal receiving, processing, and implementation by PML bodies in response to IFN and virus infection. It shows that PML body constitutive component Sp100A was phosphorylated on Ser188 by IFN signaling through the PI3K pathway in the cytosol, cotranslocated into the nucleus with PKM2, enriched on the promoter regions of essential antiviral ISGs such as those coding for IFI16, RIG-I, OAS2, etc., and mediating their transcriptional activation.