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1.
Elife ; 112022 05 19.
Article in English | MEDLINE | ID: covidwho-1870091

ABSTRACT

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of Bloc1s1 mRNA encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Inactivation of the Bloc1s1 gene impaired the ability to assemble BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Bloc1s1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus murine hepatitis virus also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.


Subject(s)
Brucella , Brucellosis , Animals , Brucellosis/metabolism , Brucellosis/microbiology , Endoribonucleases/metabolism , Endosomes/metabolism , Mice
2.
Cell Signal ; 94: 110325, 2022 06.
Article in English | MEDLINE | ID: covidwho-1767965

ABSTRACT

Efforts to discover antiviral drugs and diagnostic platforms have intensified to an unprecedented level since the outbreak of COVID-19. Nano-sized endosomal vesicles called exosomes have gained considerable attention from researchers due to their role in intracellular communication to regulate the biological activity of target cells through cargo proteins, nucleic acids, and lipids. According to recent studies, exosomes play a vital role in viral diseases including covid-19, with their interaction with the host immune system opening the door to effective antiviral treatments. Utilizing the intrinsic nature of exosomes, it is imperative to elucidate how exosomes exert their effect on the immune system or boost viral infectivity. Exosome biogenesis machinery is hijacked by viruses to initiate replication, spread infection, and evade the immune response. Exosomes, however, also participate in protective mechanisms by triggering the innate immune system. Besides that, exosomes released from the cells can carry a robust amount of information about the diseased state, serving as a potential biomarker for detecting viral diseases. This review describes how exosomes increase virus infectivity, act as immunomodulators, and function as a potential drug delivery carrier and diagnostic biomarker for diseases caused by HIV, Hepatitis, Ebola, and Epstein-Barr viruses. Furthermore, the review analyzes various applications of exosomes within the context of COVID-19, including its management.


Subject(s)
COVID-19 , Exosomes , Virus Diseases , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Biomarkers/metabolism , COVID-19/diagnosis , Endosomes/metabolism , Exosomes/metabolism , Humans , Virus Diseases/diagnosis , Virus Diseases/metabolism
3.
J Virol ; 96(2): e0106021, 2022 01 26.
Article in English | MEDLINE | ID: covidwho-1759286

ABSTRACT

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.


Subject(s)
Capsid/chemistry , Mutation/drug effects , Rhinovirus/physiology , Virus Uncoating/physiology , Antiviral Agents/pharmacology , Capsid/drug effects , Capsid Proteins/genetics , Capsid Proteins/metabolism , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/genetics , Endosomes/chemistry , Endosomes/drug effects , Endosomes/metabolism , HeLa Cells , Humans , Hydrogen-Ion Concentration , Intercellular Adhesion Molecule-1/metabolism , Protein Conformation , Rhinovirus/chemistry , Rhinovirus/drug effects , Rhinovirus/genetics , Virion/chemistry , Virion/genetics , Virion/metabolism , Virus Internalization/drug effects , Virus Uncoating/drug effects , Virus Uncoating/genetics
6.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Article in English | MEDLINE | ID: covidwho-1621335

ABSTRACT

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27-retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Endosomes/metabolism , SARS-CoV-2 , Sorting Nexins/chemistry , COVID-19/virology , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , Crystallography, X-Ray , Cytosol/metabolism , Endocytosis , Gene Expression Profiling , HEK293 Cells , HeLa Cells , Homeostasis , Humans , Lentivirus , Lysosomes/metabolism , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Domains , Sorting Nexins/metabolism , Virus Internalization
8.
Commun Biol ; 4(1): 1076, 2021 09 14.
Article in English | MEDLINE | ID: covidwho-1550352

ABSTRACT

Lysine-selective molecular tweezers are promising drug candidates against proteinopathies, viral infection, and bacterial biofilm. Despite demonstration of their efficacy in multiple cellular and animal models, important questions regarding their mechanism of action, including cell penetrance and intracellular distribution, have not been answered to date. The main impediment to answering these questions has been the low intrinsic fluorescence of the main compound tested to date, called CLR01. Here, we address these questions using new fluorescently labeled molecular tweezers derivatives. We show that these compounds are internalized in neurons and astrocytes, at least partially through dynamin-dependent endocytosis. In addition, we demonstrate that the molecular tweezers concentrate rapidly in acidic compartments, primarily lysosomes. Accumulation of molecular tweezers in lysosomes may occur both through the endosomal-lysosomal pathway and via the autophagy-lysosome pathway. Moreover, by visualizing colocalization of molecular tweezers, lysosomes, and tau aggregates we show that lysosomes likely are the main site for the intracellular anti-amyloid activity of molecular tweezers. These findings have important implications for the mechanism of action of molecular tweezers in vivo, explaining how administration of low doses of the compounds achieves high effective concentrations where they are needed, and supporting the development of these compounds as drugs for currently cureless proteinopathies.


Subject(s)
Astrocytes/metabolism , Bridged-Ring Compounds/metabolism , Endosomes/metabolism , Lysine/metabolism , Lysosomes/metabolism , Neurons/metabolism , Organophosphates/metabolism , Animals , Autophagy/drug effects , Cell Line, Tumor , Humans , Mice , Mice, Inbred C57BL
9.
PLoS Pathog ; 17(11): e1009820, 2021 11.
Article in English | MEDLINE | ID: covidwho-1528735

ABSTRACT

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.


Subject(s)
COVID-19/virology , Furin/metabolism , Nuclear Receptor Coactivators/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Cell Line , Endosomes/metabolism , Furin/genetics , Gene Expression , Humans , Immune Evasion , Interferons/metabolism , Lysosomes/enzymology , Nuclear Receptor Coactivators/genetics , Protein Isoforms , Proteolysis , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
10.
mBio ; 12(5): e0254221, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1462902

ABSTRACT

Damage in COVID-19 results from both the SARS-CoV-2 virus and its triggered overactive host immune responses. Therapeutic agents that focus solely on reducing viral load or hyperinflammation fail to provide satisfying outcomes in all cases. Although viral and cellular factors have been extensively profiled to identify potential anti-COVID-19 targets, new drugs with significant efficacy remain to be developed. Here, we report the potent preclinical efficacy of ALD-R491, a vimentin-targeting small molecule compound, in treating COVID-19 through its host-directed antiviral and anti-inflammatory actions. We found that by altering the physical properties of vimentin filaments, ALD-491 affected general cellular processes as well as specific cellular functions relevant to SARS-CoV-2 infection. Specifically, ALD-R491 reduced endocytosis, endosomal trafficking, and exosomal release, thus impeding the entry and egress of the virus; increased the microcidal capacity of macrophages, thus facilitating the pathogen clearance; and enhanced the activity of regulatory T cells, therefore suppressing the overactive immune responses. In cultured cells, ALD-R491 potently inhibited the SARS-CoV-2 spike protein and human ACE2-mediated pseudoviral infection. In aged mice with ongoing, productive SARS-CoV-2 infection, ALD-R491 reduced disease symptoms as well as lung damage. In rats, ALD-R491 also reduced bleomycin-induced lung injury and fibrosis. Our results indicate a unique mechanism and significant therapeutic potential for ALD-R491 against COVID-19. We anticipate that ALD-R491, an oral, fast-acting, and non-cytotoxic agent targeting the cellular protein with multipart actions, will be convenient, safe, and broadly effective, regardless of viral mutations, for patients with early- or late-stage disease, post-COVID-19 complications, and other related diseases. IMPORTANCE With the Delta variant currently fueling a resurgence of new infections in the fully vaccinated population, developing an effective therapeutic drug is especially critical and urgent in fighting COVID-19. In contrast to the many efforts to repurpose existing drugs or address only one aspect of COVID-19, we are developing a novel agent with first-in-class mechanisms of action that address both the viral infection and the overactive immune system in the pathogenesis of the disease. Unlike virus-directed therapeutics that may lose efficacy due to viral mutations, and immunosuppressants that require ideal timing to be effective, this agent, with its unique host-directed antiviral and anti-inflammatory actions, can work against all variants of the virus, be effective during all stages of the disease, and even resolve post-disease damage and complications. Further development of the compound will provide an important tool in the fight against COVID-19 and its complications, as well as future outbreaks of new viruses.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/metabolism , Organic Chemicals/therapeutic use , Spike Glycoprotein, Coronavirus/metabolism , Vimentin/metabolism , Animals , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Exosomes/drug effects , Exosomes/metabolism , HEK293 Cells , Humans , Mice , RAW 264.7 Cells
12.
Biochimie ; 179: 237-246, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1326916

ABSTRACT

The anti-malarial drug Chloroquine (CQ) and its derivative hydroxychloroquine have shown antiviral activities in vitro against many viruses, including coronaviruses, dengue virus and the biosafety level 4 Nipah and Hendra paramyxoviruses. The in vivo efficacy of CQ in the treatment of COVID-19 is currently a matter of debate. CQ is a lysosomotrophic compound that accumulates in lysosomes, as well as in food vacuoles of Plasmodium falciparum. In the treatment of malaria, CQ impairs the digestion and growth of the parasite by increasing the pH of the food vacuole. Similarly, it is assumed that the antiviral effects of CQ results from the increase of lysosome pH and the inhibition of acidic proteases involved in the maturation of virus fusion protein. CQ has however other effects, among which phospholipidosis, characterized by the accumulation of multivesicular bodies within the cell. The increase in phospholipid species particularly concerns bis(monoacylglycero)phosphate (BMP), a specific lipid of late endosomes involved in vesicular trafficking and pH-dependent vesicle budding. It was shown previously that drugs like progesterone, the cationic amphiphile U18666A and the phospholipase inhibitor methyl arachidonyl fluoro phosphonate (MAFP) induce the accumulation of BMP in THP-1 cells and decrease cell infection by human immunodeficiency virus. HIV viral particles were found to be retained into large endosomal-type vesicles, preventing virus spreading. Since BMP was also reported to favour virus entry through hijacking of the endocytic pathway, we propose here that BMP could play a dual role in viral infection, with its antiviral effects triggered by lysosomotropic drugs like CQ.


Subject(s)
Antiviral Agents/pharmacology , Chloroquine/pharmacology , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Lysophospholipids/metabolism , Monoglycerides/metabolism , SARS-CoV-2/drug effects , Humans , SARS-CoV-2/physiology
13.
PLoS Pathog ; 17(7): e1009706, 2021 07.
Article in English | MEDLINE | ID: covidwho-1305581

ABSTRACT

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.


Subject(s)
COVID-19/drug therapy , COVID-19/virology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Virus Internalization/drug effects , Ammonium Chloride/pharmacology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/physiology , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Chloroquine/pharmacology , Clathrin/metabolism , Drug Synergism , Endocytosis/drug effects , Endocytosis/physiology , Endosomes/drug effects , Endosomes/metabolism , Humans , Hydrogen-Ion Concentration/drug effects , Hydroxychloroquine/administration & dosage , Macrolides/pharmacology , Niclosamide/administration & dosage , Niclosamide/pharmacology , Protein Binding/drug effects , Protein Domains , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/physiology , Vero Cells
14.
Sci Immunol ; 6(60)2021 06 18.
Article in English | MEDLINE | ID: covidwho-1276879

ABSTRACT

The nutrient-sensing mammalian target of rapamycin (mTOR) is integral to cell fate decisions after T cell activation. Sustained mTORC1 activity favors the generation of terminally differentiated effector T cells instead of follicular helper and memory T cells. This is particularly pertinent for T cell responses of older adults who have sustained mTORC1 activation despite dysfunctional lysosomes. Here, we show that lysosome-deficient T cells rely on late endosomes rather than lysosomes as an mTORC1 activation platform, where mTORC1 is activated by sensing cytosolic amino acids. T cells from older adults have an increased expression of the plasma membrane leucine transporter SLC7A5 to provide a cytosolic amino acid source. Hence, SLC7A5 and VPS39 deficiency (a member of the HOPS complex promoting early to late endosome conversion) substantially reduced mTORC1 activities in T cells from older but not young individuals. Late endosomal mTORC1 is independent of the negative-feedback loop involving mTORC1-induced inactivation of the transcription factor TFEB that controls expression of lysosomal genes. The resulting sustained mTORC1 activation impaired lysosome function and prevented lysosomal degradation of PD-1 in CD4+ T cells from older adults, thereby inhibiting their proliferative responses. VPS39 silencing of human T cells improved their expansion to pertussis and to SARS-CoV-2 peptides in vitro. Furthermore, adoptive transfer of CD4+ Vps39-deficient LCMV-specific SMARTA cells improved germinal center responses, CD8+ memory T cell generation, and recall responses to infection. Thus, curtailing late endosomal mTORC1 activity is a promising strategy to enhance T cell immunity.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Endosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , SARS-CoV-2/metabolism , Signal Transduction/genetics , Adoptive Transfer/methods , Adult , Aged , Aged, 80 and over , Animals , Autophagy-Related Proteins/deficiency , Autophagy-Related Proteins/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , COVID-19/virology , Cells, Cultured , Female , Forkhead Box Protein O1/deficiency , Forkhead Box Protein O1/genetics , Healthy Volunteers , Humans , Large Neutral Amino Acid-Transporter 1/metabolism , Lysosomes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Signal Transduction/immunology , Transfection , Vesicular Transport Proteins/deficiency , Vesicular Transport Proteins/genetics , Young Adult
15.
Cells ; 10(5)2021 05 07.
Article in English | MEDLINE | ID: covidwho-1223961

ABSTRACT

The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a blocker of an emerging class of human intracellular channels, namely the two-pore channel (TPC) family, whose role has been established in several diseases. Indeed, Nar was shown to be effective against neoangiogenesis, a process essential for solid tumor progression, by specifically impairing TPC activity. The goal of the present review is to illustrate the rationale that links TPC channels to the mechanism of coronavirus infection, and how their inhibition by Nar could be an efficient pharmacological strategy to fight the current pandemic plague COVID-19.


Subject(s)
COVID-19/drug therapy , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Flavanones/pharmacology , Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Arabidopsis/metabolism , COVID-19/epidemiology , COVID-19/pathology , COVID-19/virology , Calcium Channel Blockers/therapeutic use , Drug Evaluation, Preclinical , Endosomes/drug effects , Endosomes/metabolism , Endosomes/virology , Flavanones/therapeutic use , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Lysosomes/virology , Neoplasms/blood supply , Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Pandemics/prevention & control , SARS-CoV-2/pathogenicity , Vacuoles/metabolism , Virus Internalization/drug effects
16.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1214021

ABSTRACT

To realize RNA interference (RNAi) therapeutics, it is necessary to deliver therapeutic RNAs (such as small interfering RNA or siRNA) into cell cytoplasm. A major challenge of RNAi therapeutics is the endosomal entrapment of the delivered siRNA. In this study, we developed a family of delivery vehicles called Janus base nanopieces (NPs). They are rod-shaped nanoparticles formed by bundles of Janus base nanotubes (JBNTs) with RNA cargoes incorporated inside via charge interactions. JBNTs are formed by noncovalent interactions of small molecules consisting of a base component mimicking DNA bases and an amino acid side chain. NPs presented many advantages over conventional delivery materials. NPs efficiently entered cells via macropinocytosis similar to lipid nanoparticles while presenting much better endosomal escape ability than lipid nanoparticles; NPs escaped from endosomes via a "proton sponge" effect similar to cationic polymers while presenting significant lower cytotoxicity compared to polymers and lipids due to their noncovalent structures and DNA-mimicking chemistry. In a proof-of-concept experiment, we have shown that NPs are promising candidates for antiviral delivery applications, which may be used for conditions such as COVID-19 in the future.


Subject(s)
DNA/chemistry , Drug Delivery Systems , Endosomes/metabolism , Nanostructures/administration & dosage , Amino Acids/chemistry , Cell Survival , Endocytosis , Humans , Nanostructures/chemistry , Nanotubes, Peptide/chemistry , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , RNAi Therapeutics
17.
Nat Microbiol ; 6(7): 899-909, 2021 07.
Article in English | MEDLINE | ID: covidwho-1205445

ABSTRACT

SARS-CoV-2 entry requires sequential cleavage of the spike glycoprotein at the S1/S2 and the S2' cleavage sites to mediate membrane fusion. SARS-CoV-2 has a polybasic insertion (PRRAR) at the S1/S2 cleavage site that can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture-adapted SARS-CoV-2 virus with an S1/S2 deletion, we show that the polybasic insertion endows SARS-CoV-2 with a selective advantage in lung cells and primary human airway epithelial cells, but impairs replication in Vero E6, a cell line used for passaging SARS-CoV-2. Using engineered spike variants and live virus competition assays and by measuring growth kinetics, we find that the selective advantage in lung and primary human airway epithelial cells depends on the expression of the cell surface protease TMPRSS2, which enables endosome-independent virus entry by a route that avoids antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. Analysis of 100,000 SARS-CoV-2 sequences derived from patients and 24 human postmortem tissues showed low frequencies of naturally occurring mutants that harbour deletions at the polybasic site. Taken together, our findings reveal that the furin cleavage site is an important determinant of SARS-CoV-2 transmission.


Subject(s)
COVID-19/transmission , Furin/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Animals , COVID-19/virology , Cathepsins/metabolism , Chlorocebus aethiops , Endosomes/metabolism , Epithelial Cells , Ferrets , Humans , Immune Evasion , Membrane Proteins/metabolism , RNA-Binding Proteins/metabolism , Respiratory System/cytology , Respiratory System/virology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Viral Genome Packaging , Virus Internalization , Virus Replication , Virus Shedding
18.
Cells ; 10(4)2021 03 28.
Article in English | MEDLINE | ID: covidwho-1154291

ABSTRACT

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons in the substantia nigra, intraneuronal deposition of misfolded proteins known as Lewy bodies, and chronic neuroinflammation. PD can arise from monogenic mutations, but in most cases, the etiology is unclear. Viral infection is gaining increasing attentions as a trigger of PD. In this study, we investigated whether the PD-causative 620 aspartate (D) to asparagine (N) mutation in the vacuolar protein sorting 35 ortholog (Vps35) precipitated herpes simplex virus (HSV) infection. We observed that ectopic expression of Vps35 significantly reduced the proliferation and release of HSV-1 virions; the D620N mutation rendered Vps35 a partial loss of such inhibitory effects. Tetherin is a host cell protein capable of restricting the spread of encapsulated viruses including HSV-1 and SARS-Cov-2, both of which are implicated in the development of parkinsonism. Compared with cells overexpressing wildtype Vps35, cells expressing mutant Vps35 with D620N had less Tetherin on cell surfaces. Real-time and static cell imaging revealed that Tetherin recycled through Vps35-positive endosomes. Expression of Vps35 with D620N reduced endosomal dynamics and frequency of motile Tetherin-containing vesicles, a sign of defective production of recycling carriers. Our study suggests that the D620N mutation in Vps35 hinders Tetherin trafficking to cell surfaces and facilitates virus spread.


Subject(s)
Bone Marrow Stromal Antigen 2/metabolism , Parkinson Disease/metabolism , Parkinson Disease/virology , Simplexvirus/metabolism , Vesicular Transport Proteins/metabolism , COVID-19/virology , Cell Line, Tumor , Endosomes/metabolism , Humans , Mutation , Parkinson Disease/genetics , Protein Transport/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Simplexvirus/pathogenicity , Transfection , Vesicular Transport Proteins/genetics , Virus Replication/genetics
19.
J Biol Chem ; 296: 100306, 2021.
Article in English | MEDLINE | ID: covidwho-1152462

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Clathrin Heavy Chains/genetics , Endocytosis/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization/drug effects , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , Clathrin Heavy Chains/antagonists & inhibitors , Clathrin Heavy Chains/metabolism , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Endosomes/virology , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Host-Pathogen Interactions/genetics , Humans , Hydrazones/pharmacology , Lentivirus/genetics , Lentivirus/metabolism , Protein Binding/drug effects , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/metabolism , Sulfonamides/pharmacology , Thiazolidines/pharmacology , Vero Cells
20.
Virol J ; 18(1): 46, 2021 02 27.
Article in English | MEDLINE | ID: covidwho-1105717

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and broke out as a global pandemic in late 2019. The acidic pH environment of endosomes is believed to be essential for SARS-CoV-2 to be able to enter cells and begin replication. However, the clinical use of endosomal acidification inhibitors, typically chloroquine, has been controversial with this respect. METHODS: In this study, RT-qPCR method was used to detect the SARS-CoV-2N gene to evaluate viral replication. The CCK-8 assay was also used to evaluate the cytotoxic effect of SARS-CoV-2. In situ hybridization was used to examine the distribution of the SARS-CoV-2 gene in lung tissues. Hematoxylin and eosin staining was also used to evaluate virus-associated pathological changes in lung tissues. RESULTS: In this study, analysis showed that endosomal acidification inhibitors, including chloroquine, bafilomycin A1 and NH4CL, significantly reduced the viral yields of SARS-CoV-2 in Vero E6, Huh-7 and 293T-ACE2 cells. Chloroquine and bafilomycin A1 also improved the viability and proliferation of Vero E6 cells after SARS-CoV-2 infection. Moreover, in the hACE2 transgenic mice model of SARS-CoV-2 infection, chloroquine and bafilomycin A1 reduced viral replication in lung tissues and alleviated viral pneumonia with reduced inflammatory exudation and infiltration in peribronchiolar and perivascular tissues, as well as improved structures of alveolar septum and pulmonary alveoli. CONCLUSIONS: Our research investigated the antiviral effects of endosomal acidification inhibitors against SARS-CoV-2 in several infection models and provides an experimental basis for further mechanistic studies and drug development.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Endosomes/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication/drug effects , Ammonium Chloride/pharmacology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/pathology , Cell Survival/drug effects , Chlorocebus aethiops , Chloroquine/pharmacology , Endosomes/metabolism , Female , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Lung/pathology , Macrolides/pharmacology , Mice , Mice, Transgenic , Random Allocation , SARS-CoV-2/genetics , Vero Cells
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